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MicroRNA-17~92 Is Required for Nephrogenesis and Renal Function April K. Marrone,* Donna B. Stolz,† Sheldon I. Bastacky,‡§ Dennis Kostka,| Andrew J. Bodnar,* and Jacqueline Ho* *Division of Nephrology, Department of Pediatrics, †Department of Cell Biology, ‡Department of Pathology, and | Departments of Developmental Biology and Computational Systems Biology, University of Pittsburgh School of Medicine, and §University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania
ABSTRACT Deletion of all microRNAs (miRNAs) in nephron progenitors leads to premature loss of these cells, but the roles of specific miRNAs in progenitors have not been identified. Deletions in the MIR17HG cluster (miR17~92 in mice), detected in a subset of patients with Feingold syndrome, represent the first miRNA mutations to be associated with a developmental defect in humans. Although MIR17HG is expressed in the developing kidney, and patients with Feingold syndrome caused by MYCN mutations have renal anomalies, it remains unclear to what extent MIR17HG contributes to renal development and function. To define the role of miR-17~92, we generated mice with a conditional deletion of miR-17~92 in nephron progenitors and their derivatives. The nephron progenitor population was preserved in these mice; however, this deletion impaired progenitor cell proliferation and reduced the number of developing nephrons. Postnatally, mutant mice developed signs of renal disease, including albuminuria by 6 weeks and focal podocyte foot process effacement and glomerulosclerosis at 3 months. Taken together, these data support a role for this miRNA cluster in renal development, specifically in the regulation of nephron development, with subsequent consequences for renal function in adult mice. J Am Soc Nephrol 25: 1440–1452, 2014. doi: 10.1681/ASN.2013040390
Congenital anomalies of the kidney and urinary tract (CAKUTs) are among the most frequent birth defects in humans and are the major cause of childhood CKD.1 Although CAKUTs represent a diverse group of developmental anomalies, the risk of CKD is related to decreased renal reserve as a result of the formation of fewer and/or abnormal nephrons. Kidney development occurs largely by reciprocal signaling between nephron progenitors and the ureteric bud (reviewed by Little and McMahon2). Nephron progenitors are capable of self-renewal and differentiation into the multiple cell types of the mature nephron to generate a sufficient number of nephrons for the adult animal. The nephron progenitors undergo a mesenchymeto-epithelial transition during nephrogenesis to sequentially form renal vesicles, comma-shaped bodies, S-shaped bodies, and, ultimately, functional nephrons consisting of glomeruli and tubules. Despite the prevalence of pediatric renal disease 1440
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associated with abnormal kidney development, many of the molecular mechanisms underlying nephron endowment remain unclear. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by repressing their target mRNAs (reviewed by Bartel 3). Several recent studies have demonstrated that miRNAs are critical in kidney development and function by blocking the miRNA biogenesis pathway in
Received April 18, 2013. Accepted November 15, 2013. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Jacqueline Ho, Division of Nephrology, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Rangos Research Center, University of Pittsburgh School of Medicine, 4401 Penn Avenue, Pittsburgh, PA 15224. Email: [email protected] Copyright © 2014 by the American Society of Nephrology
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specific cell lineages.4–12 We, and others, have reported that loss of functional miRNAs in nephron progenitors results in a premature depletion of progenitors related to increased apoptosis, leading to the formation of fewer nephrons.4,7 However, the requirement for individual miRNAs in survival or other roles in nephron progenitors remains unclear. The human MIR17 (MIR17HG) cluster has been linked to developmental, apoptotic, and oncogenic pathways in other organs,13–16 and the locus is conserved between mouse and human. Heterozygous deletion of the MIR17HG cluster is the first reported miRNA mutation that results in a developmental defect, in humans with Feingold syndrome.17 Although patients with Feingold syndrome associated with MIR17HG mutations have not yet been reported to have kidney defects, this syndrome has also been associated with MYCN mutations and, in that context, has up to an 18% incidence of CAKUT.18,19 Deletion of the miR-17~92 cluster in mice results in perinatal lethality, and multiple congenital anomalies, including pulmonary hypoplasia, growth retardation, and cardiac defects.20 While members of the cluster are expressed in the developing kidney, including in nephron progenitors, the function of miR-17~92 in renal development and function is unknown.4 Here, we demonstrate that ablation of miR-17~92 in nephron progenitors and their derivatives results in renal hypodysplasia. We show that the nephron progenitor population is preserved but cell proliferation is impaired, ultimately leading to fewer developing nephron structures. Postnatally, these mice develop evidence of glomerular kidney disease with albuminuria, glomerulosclerosis, and podocyte foot process effacement. Collectively, our data demonstrate an essential requirement for miR-17~92 in nephron development that affects renal function postnatally. To our knowledge, this is the first report of an miRNA mutation associated with developmental renal anomalies.
RESULTS Conditional Deletion of miR-17~92 from Nephron Progenitors Results in Kidney Hypodysplasia
To define the role of the miR-17~92 cluster in kidney development and function, we generated mice with conditional deletion of the miR-17~92 cluster using the Six2-TGC line, which drives Cre expression in nephron progenitors,21 and a floxed allele of the miR-17~92 cluster.20 RT quantitative PCR for the primary miR-17~92 transcript (pri-miR-17~92) confirmed a reduction in mutant embryonic day 16.5 (E16.5) kidneys (=Six2-TGCtg/+; miR-17~92flx/flx) relative to littermate controls (Figure 1A). Locked nucleic acid (LNA) in situ hybridization analysis of miR-17–5p in E14.5 kidneys produced a signal in nephron progenitors, developing nephrons, ureteric buds, and the ureter (Figure 1B), as previously reported for E16.5 kidneys. 4 As expected, the Six2TGC tg/+ ; miR-17~92 flx/flx mutant kidney showed reduced J Am Soc Nephrol 25: 1440–1452, 2014
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expression in nephron progenitors and their derivatives (Figure 1B). Mutant mice were born at normal Mendelian ratios and have been aged up to 6 months. Postnatal day 0 (P0) Six2-TGC tg/+ ; miR-17~92 flx/flx kidneys were significantly smaller than those of their littermates, while overall body sizes were equivalent (Figure 1, C and D, Supplemental Table 1). This finding persisted at 3 months, with a smaller kidney-to-body weight ratio in Six2-TGCtg/+; miR-17~92flx/flx animals (Supplemental Figure 1). Furthermore, heterozygous animals with a one-allele loss of miR-17~92 (Six2-TGCtg/+; miR-17~92flx/+) in nephron progenitors had kidneys that were intermediate in size compared with their control and homozygous mutant littermates (Figure 1, C and D, Supplemental Figure 1, Supplemental Table 1). This finding is in accordance with previously published work that global one-allele loss of miR-17~92 resulted in developmental defects in both mice and humans.17 miR-17~92 belongs to a highly conserved family of three miRNA clusters that includes miR-106a~363 and miR106b~25. Previous work has suggested functional redundancy between miR-17~92 and miR-106b~25 (but not miR106a~363), resulting in a worsening embryonic phenotype in global miR-17~92 2 /2 ; miR-106b~25 2 /2 double mutants.20 Therefore, we generated animals lacking miR-17~92 in nephron progenitors in a miR-106b~252/2 background (Six2-TGCtg/+; miR-17~92flx/flx; miR-106b~252/2) and observed no change in the degree of kidney hypoplasia compared with Six2-TGCtg/+; miR-17~92flx/flx mutants (Figure 1C); histologic features also did not differ (data not shown). The sizes of mutant miR-106b~252/2 kidneys were also unchanged relative to control kidneys (Figure 1C). Moreover, the expression of miR-106b~25 and miR-106a~363 primary transcripts were not affected by miR-17~92 loss (Figure 1A). These results imply that the role of miR-17~92 in nephron progenitors and their derivatives is unique and that there is no compensatory increase in miR-106a~363 and miR-106b~25 expression in mutants. Nephrons are continually induced at ureteric bud tips just below the renal capsule throughout kidney development, resulting in a gradient of differentiating nephrons with the most immature nephrons in the periphery of the renal cortex. 2 Histologic sections of P0 kidneys revealed fewer developing nephrons in the renal cortex of Six2-TGC tg/+ ; miR-17~92 flx/flx kidneys (Figure 1, E–G9). Furthermore, at P30 when nephrogenesis is complete, there were markedly fewer observable glomeruli in Six2-TGC tg/+; miR-17~92 flx/flx kidney sections (yellow stars) compared with controls (Figure 1, H and I). Thus, loss of miR17~92 in nephron progenitors and their derivatives results in renal hypodysplasia. miR-17~92 Is Required in Early Kidney Development
To assess the developmental time points at which the miR17~92 cluster is active, RT-quantitative PCR was performed MiR-17~92 in Kidney Development
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Figure 1. Loss of miR-17~92 in nephron progenitors results in renal hypodysplasia. (A) RT-quantitative PCR for the miR-17~92 primary transcript in E16.5 kidneys shows a reduction in mutant kidneys relative to controls. In contrast, the levels of the miR-106b~25 and miR106a~363 primary transcripts are not significantly different. (B) LNA in situ hybridization for miR-17–5p in E14.5 kidneys reveals expression in nephron progenitors, their derivatives, and ureteric epithelium in control littermates, which is reduced in nephron
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to measure relative miRNA levels in E12.5, E14.5, E16.5, and E18.5 kidneys (Figure 2A). The primary miR-17~92 transcript was present in embryonic kidney throughout these time points, but its expression was lower at E16.5 and E18.5. Of the miRNAs expressed by this cluster, miR-17–5p followed a pattern similar to the primary transcript; however, the expression of miR-18a-5p, miR-19b-1–5p, and miR-20a-5p each peaked at E14.5 (Figure 2A). Expression of miR-92a1–5p decreased steadily throughout, whereas miR-19a-5p levels increased approximately 5-fold in E14.5 kidneys and remained relatively high (Figure 2A). These results suggest that the effects of miR-17~92 loss from nephron progenitors could begin to manifest early, and, furthermore, that there may be differential regulation of the individual miRNAs within this cluster during kidney development. Histologic analysis of E14.5, E16.5, and E18.5 kidneys was used to define the onset of impaired nephrogenesis in Six2TGCtg/+; miR-17~92flx/flx mutants relative to controls (Figure 2B-C). At E14.5, there were no gross histologic differences in mutants, with an appropriate number of developing nephrons observed at various stages: renal vesicles, comma-shaped bodies, S-shaped bodies, and capillary loop stage glomeruli (Figure 2, B and C). By E16.5, it became apparent that fewer nephron structures were developing, and this persisted in E18.5 and P0 mutant kidneys (Figures 1, E–G, and 2, B9 and C99). Deletion of miR-17~92 in Nephron Progenitors Impairs Nephrogenesis
To more clearly define the effect on nephrogenesis, immunofluorescence was performed for markers specific to nephron progenitors and developing nephron structures. P0 Six2-TGCtg/+; miR-17~92flx/flx kidneys expressed several nephron progenitor markers, including Six2, Pax2, Wt1, Cited1, and neural cell adhesion molecule (NCAM),2,22–25 although it appears that Cited1 expression was decreased (Figure 3, B and B9). Several of these markers continue to be present in differentiating nephrons, including in the renal vesicle (Pax2 and NCAM) and developing podocytes of the glomerulus (Wt1). Six2-TGCtg/+; miR-17~92flx/flx kidneys have fewer Pax2- and NCAM-positive renal vesicles (yellow arrows in Figure 3, C, C9, D9 and D0), the earliest epithelial derivatives of nephron progenitors. There were also fewer Wt1-positive glomeruli present in Six2-TGCtg/+; miR-17~92flx/flx kidneys (yellow stars in Figure
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3, E and –E9). There were no gross differences in the formation of proximal or distal nephron tubules of Six2-TGCtg/+; miR17~92flx/flx kidneys, as noted by lotus tetragonolobus lectin (LTL) staining for proximal tubules or E-cadherin–positive (dolichos biflorus agglutinin [DBA]-negative) staining for distal tubules, respectively (Figure 3, E and F9). Despite the abnormalities seen in nephron development, the ureteric bud lineage appeared normal on the basis of calbindin staining of ureteric buds (Figure 3, B and B9) and DBA labeling of collecting ducts (Figure 3, F and F9) in the mutant kidneys. Thus, there appears to be normal initial nephron progenitor specification in Six2-TGCtg/+; miR-17~92flx/flx kidneys. Immunofluorescence was subsequently conducted on E14.5 kidneys, a time point that coincides with the highest expression of most miRNAs in miR-17~92. In mutant E14.5 kidneys, nephron progenitors appeared to express Six2 normally; however, Cited1 expression was reduced, similar to what was observed at P0 (Figure 4, A–D9). To evaluate whether renal vesicles formed normally, staining for Lim1 and Jagged1 was performed.26,27 Both Jagged1 and Lim1 were unchanged in renal vesicles (Figure 4, E and H9), but Jagged1 expression appeared to be decreased in the nephron progenitors of Six2TGCtg/+; miR-17~92flx/flx mutants relative to littermate controls (Figure 4, E versus F). Jagged1 and Lim1 expression in renal vesicles at E16.5 persisted in mutants compared with controls (Figure 4, I–L). These data suggest that the signals required to form renal vesicles are unaffected by the loss of miR-17~92 from nephron progenitors but that the nephron progenitors themselves may not be completely functionally normal. Lineage tracing experiments were performed by crossing a CAG reporter allele28 with the Six2-Cre transgene in both control and mutant mice (Six2-TGCtg/+; RosaCAG-tdTomato/+ and Six2-TGCtg/+; RosaCAG-tdTomato/+; miR-17~92flx/flx respectively) to assess the cell fate of nephron progenitors. All tissues examined in both controls and mutants showed efficient Cre-recombination, and Cre-expressing cells adopted cell fates consistent with those normally derived from the progenitor population in Six2-TGC t g / + ; Rosa C AG - t d To m a t o / + ; miR-17~92flx/flx compared with controls at P0 (Supplemental Figure 2). Together, these results are consistent with a partial block in nephrogenesis and a relative preservation of nephron patterning, resulting in fewer nephrons in Six2-TGCtg/+ ; miR-17~92flx/flx kidneys.
progenitors and their derivatives in Six2-TGCtg/+; miR-17~92flx/flx kidneys. Blue dotted lines: ureteric bud; red dotted lines: S-shape body (left) and renal vesicle (right); red arrow: nephron progenitors. (C) Kidney surface area normalized to crown rump length at P0 demonstrates that loss of a single copy of miR-17~92 results in mild hypoplasia, whereas loss of both alleles results in more severe hypoplasia. The crownrump length was similar among all genotypes. (D) Sample photos of newborn pups and their kidneys. The adrenal gland is attached to the left Six2-TGCtg/+; miR-17~92flx/+ kidney. (E–G’) Hematoxylin and eosin sections show fewer developing nephron structures in P0 Six2TGCtg/+; miR-17~92flx/flx animals when compared with controls. (H and I) At P30, methenamine silver–periodic acid-Schiff stain with hematoxylin and eosin reveals more glomeruli (yellow stars) in controls than in Six2-TGCtg/+; miR-17~92flx/flx kidneys. Mutant kidneys also have dilated tubules (blue arrow) and proteinaceous casts (blue star). Scale bars, 100 mm. *P#0.05; **P#0.01; ***P#0.001; ****P#0.0001; n.s., nonsignificant.
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Figure 2. miR-17~92 is expressed throughout kidney development and is required for early development. (A) Expression levels of the primary miR-17~92 transcript and miRNAs expressed by this cluster (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a) at E12.5, E14.5, E16.5, and E18.5 by RT quantitative PCR. Time points are compared against E12.5 expression levels, after normalization to an endogenous control. (B–B’’) Hematoxylin and eosin sections of control E14.5, E16.5, and E18.5 kidneys. (C–C’’) Hematoxylin and eosin sections of Six2-TGCtg/+; miR-17~92flx/flx E14.5, E16.5, and E18.5 kidneys. At E16.5 and E18.5, fewer developing nephron structures were observed. Scale bar, 100 mm.
miR-17~92 Regulates Nephron Progenitor Proliferation
The miR-17~92 cluster regulates both cell proliferation and apoptosis.29–32 The partial block in nephrogenesis in mutants could be caused by alterations in either of these processes. Proliferation was analyzed using bromodeoxyuridine (BrdU) incorporation into dividing cells in E14.5, E16.5, and E18.5 embryos, and coimmunolabeling for BrdU and Six2 (progenitor marker) or Pax2 (progenitor, renal vesicle, and ureteric bud marker—ureteric bud cells were not counted). Similar to the histologic analysis, no significant difference was noted in the number of double BrdU-positive, Six2- (or Pax2-) positive cells relative to single-positive Six2- or Pax2-positive cells at E14.5. However, Six2-TGCtg/+; miR-17~92flx/flx kidneys had a significantly smaller percentage of double-positive cells than controls at E16.5 and E18.5 (Figure 5, A and B, Supplemental Table 2). These data suggest that the loss of miR-17~92 in nephron progenitors results in impaired proliferation. miR-17–5p and miR-20a directly target the cell cycle inhibitor p21.30,33–35 Interestingly, RT-qPCR for p21 transcripts demonstrated an approximately 1.5-fold increase in E16.5 Six2-TGC tg/+ ; miR-17~92 flx/flx kidneys compared with 1444
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littermate controls (Figure 5C). This finding was corroborated by RNA sequencing that showed an approximately 20% increase in p21 mRNA levels in mutant kidneys (data not shown). Thus, the defect in cellular proliferation in Six2-TGCtg/+;miR-17~92flx/flx kidneys may be related, at least in part, to misregulation of p21 by miR-17~92 miRNAs in nephron progenitors. The loss of all mature miRNAs in nephron progenitors has previously been reported to result in increased cell death, in association with elevated expression of the proapoptotic protein Bim.4,7 Apoptosis was assessed with the TUNEL assay or immunostaining for activated Caspase-3 (an early marker of apoptosis), followed by colabeling with Six2 (progenitors) or NCAM (progenitors and renal vesicles). There was no statistically significant difference detected in the number of double TUNEL-positive/Six2-positive or activated Caspase3–positive/NCAM-positive cells relative to single positive Six2- or NCAM-positive cells at E14.5, E16.5, or E.18.5 in control and mutant kidneys, despite a trend toward increased TUNEL-positive cells at E16.5, and decreased TUNEL-positive and activated Caspase-3 positive cells at E16.5 and E18.5 in miR-17~92 mutant nephron progenitors (Figure 5, E and F, J Am Soc Nephrol 25: 1440–1452, 2014
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Figure 3. miR-17~92 is required in nephron progenitors for normal nephron development. (A–B’) Immunofluorescence for nephron progenitor markers demonstrates preserved Six2 expression (A and A’), but decreased Cited1 expression (B and B’) in Six2-TGCtg/+; miR-17~92flx/flx P0 kidneys (pink). Calbindin (blue) labels ureteric bud and LTL (blue) marks proximal tubules, both of which are unchanged. (C–D’) Immunofluorescence for Pax2 (C and C’) and NCAM (D and D’) shows decreased formation of renal vesicles in mutant newborn kidneys (pink; yellow arrows). (E and E’) Wt1 staining demonstrates fewer glomeruli in the Six2-TGCtg/+; miR-17~92flx/flx kidneys (pink, yellow stars). (F and F’) Distal nephron tubules were labeled with E-cadherin (and are DBA negative), showing that distal tubules form in the absence of miR-17~92 (E-cadherin, pink; DBA blue). Scale bar, 100 mm.
Supplemental Table 3). Moreover, there was no change in Bim expression by immunofluorescence, Western blot, or RNA-sequencing in control and mutant kidneys (Figure 5, D and E). Together, these data suggest that miR-17~92 loss does not markedly affect cell death in nephron progenitors. Deletion of miR-17~92 from the Progenitor Population Results in CKD
The mutant animals were then evaluated for the functional consequences of miR-17~92 loss in the kidney. At 6 weeks and 3 months of age, heterozygous (Six2-TGCtg/+; miR-17~92flx/+) and homozygous (Six2-TGCtg/+; miR-17~92flx/flx) mice had increased albumin excretion in their urine, in comparison to Cre-negative controls (Figure 6, A and B, Supplemental Table 4). BUN and serum creatinine levels were significantly elevated in Six2-TGCtg/+; miR-17~92flx/flx mice relative to controls at 3 months (Figure 6C). In contrast, urine volume and osmolality were unchanged, suggesting that the animals did not possess a urinary concentrating defect (as can be seen in patients with renal dysplasia) (Supplemental Figure 3). Histologic analysis of 3-month-old kidneys demonstrated FSGS and hyalinosis (Figure 6, D0 and E0, Supplemental Figure 4), focal J Am Soc Nephrol 25: 1440–1452, 2014
tubular dilation with hyaline and proteinaceous casts (Figure 6, E0, F9, and G0), patchy inflammation in the renal interstitium (Figure 6, D9 and G0), and patchy areas of fibrosis in the mutant kidneys (Figure 6F0). Ultrastructural analysis demonstrated basement membrane wrinkling in heterozygous animals (Figure 6H9), a sign of early glomerular collapse. Partial podocyte foot process effacement was noted in both Six2-TGC tg/+; miR17~92flx/+ and Six2-TGCtg/+; miR-17~92flx/flx kidneys at 3 months of age (Figure 6, H9 and H0); the podocyte foot process effacement is probably responsible for the proteinuria. Together, these data provide evidence that the loss of miR-17~92 from nephron progenitors results in proteinuric kidney disease, histologic injury, and renal dysfunction. Given the proteinuria and glomerular changes in mutant animals, immunostaining was performed for markers of the three cell types in the glomerulus: podocytes (which arise from nephron progenitors) and endothelial and mesangial cells (which are not derived from nephron progenitors). The podocyte markers, Wt1 and podocin, continued to be expressed normally in Six2-TGCtg/+; miR-17~92flx/flx animals (Figure 7, A–D9),36 suggesting that podocytes are normally specified. The mesangial cell marker platelet-derived growth MiR-17~92 in Kidney Development
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Figure 4. miR-17~92 loss from nephron progenitors does not significantly affect the initial formation of renal vesicles. (A–B’) Immunofluorescence demonstrates preserved Six2 (pink) expression at E14.5 in mutant kidneys. (C–D’) In contrast, decreased Cited1 (pink) expression is observed in Six2-TGCtg/+; miR-17~92flx/flx kidneys, relative to controls. (E–F’, I–J’) Immunofluorescent staining for Jagged1 (Jag1) and (G–H’, K–L’) Lim1 shows that there is unchanged expression of these proteins in the renal vesicle in Six2-TGCtg/+; miR-17~92flx/flx kidneys, relative to littermate controls at E14.5 and E16.5. Calbindin (blue) labels ureteric buds; NCAM (blue) marks progenitors and epithelial derivatives; and Pax2 (blue) labels progenitors, ureteric buds, and progenitors. Scale bar, 100 mm.
factor receptor-b and the endothelial cell marker, platelet endothelial cell adhesion molecule, were also present in mutant glomeruli (Figure 7, E–H9).37 Taken with the histologic data 1446
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showing podocyte effacement, these findings are in keeping with previously published reports in which loss of all mature miRNAs in the podocyte results in marked proteinuria and J Am Soc Nephrol 25: 1440–1452, 2014
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renal failure, in the setting of normal podocyte specification but abnormal podocyte function.5,8,9
DISCUSSION
We show that nephron development is exquisitely sensitive to miR-17~92 gene dosage in nephron progenitors, with evidence for renal hypodysplasia in both heterozygous and homozygous conditional mutants. The initial specification of nephron progenitors appears to occur normally in Six2TGC tg/+ ; miR-17~92 flx/flx kidneys, and there are no gross differences in the formation of renal vesicles or nephron segmentation. We present evidence that the reduction in congenital nephron endowment is related to impaired progenitor proliferation that occurs by midgestation, which results in the formation of fewer developing nephrons. Postnatally, the animals develop glomerular dysfunction and proteinuric kidney disease. Despite growing evidence that links miRNAs to numerous biologic processes, comparatively few miRNA mutations result in developmental defects, with most published deletions having no overt phenotype.38–41 This has been attributed to data suggesting that most miRNAs act by fine-tuning gene expression. Thus, the observation that loss of a single miR17~92 allele in nephron progenitors is sufficient to result in renal hypodysplasia and albuminuria is striking. This is in accord with several recent studies that have implicated both loss and gain of function of miR-17~92 miRNAs in regulating the cell cycle and in oncogenesis.30,31,33–35,42,43 In addition, heterozygous deletion of MIR17HG was recently reported in Feingold syndrome (although the renal phenotype in these patients remains undefined).17 The miR-17~92 cluster has pleiotropic functions in normal development and malignant transformation and has been
Figure 5. Decreased proliferation (and no change in cell death) in nephron progenitors and their derivatives in Six2-TGCtg/+; miR17~92flx/flx kidneys. (A) The percentage of BrdU-positive Six2expressing cells (top) and BrdU-positive Pax2-expressing cells
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(bottom) in E14.5, E16.5, and E18.5 mutant and control kidneys. (B) Representative images of E18.5 kidneys demonstrating fewer BrdU-/Six2-positive cells, as well as fewer Six2-positive cells in Six2TGCtg/+; miR-17~92flx/flx kidneys than in control littermates. BrdU (blue); Six2 (pink). (C) RT quantitative PCR analysis demonstrates increased p21 mRNA in Six2-TGCtg/+; miR-17~92flx/flx E16.5 kidneys relative to controls. (D) No significant difference in Bim expression, normalized to b-tubulin, is noted in E16.5 control and mutant kidneys by Western blot, and no significant change in Bim mRNA was detected via RNA sequencing (log2FC=20.177, false discovery rate=0.233). (E) Representative images of E14.5 kidneys showing TUNEL (blue) and Six2 (pink) colabeling (top), Bim (pink) and NCAM (blue) colabeling (middle), and Bim (gray) expression (bottom) in Six2-TGCtg/+; miR-17~92flx/flx animals compared with control littermates at E14.5. (F) The percentages of TUNEL-positive Six2-positive cells (top) and activated Caspase-3–positive NCAMpositive cells (bottom) at E14.5, E16.5, and E18.5 in mutant and control kidneys are not significantly different.
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Figure 6. miR-17~92 is required for normal kidney function. (A) Urine from 6-week-old mutants had increased albumin excretion versus controls on an SDS-PAGE gel stained with Coomassie (control was BSA; molecular mass is 70 kD). (B) ELISAs demonstrate that animals with decreased miR-17~92 expression have markedly increased albumin-to-creatinine ratios at 6 weeks and 3 months of age. (C) Six2TGCtg/+; miR-17~92flx/flx animals have significantly elevated BUN and serum creatinine levels. (D–G’’) Periodic acid-Schiff and hematoxylin (D and G), methenamine silver–periodic acid-Schiff and hematoxylin and eosin (E), and Masson trichrome staining (F) of 3-month-old kidney tissues reveals that loss of miR-17~92 results in focal glomerulosclerosis (yellow stars, D’’ and E’’), dilated tubules (blue arrows, G’’), proteinaceous casts (blue stars, E’’, F’, and G’’), interstitial inflammation (green arrows, D’, G’, and G’’), and fibrosis (blue color, F–F’’). Scale bars = 100 mm. (H–H’’) Electron micrographs (original magnification, 320,000) reveal that mutants have partial podocyte foot process effacement and wrinkling of the glomerular basement membrane (green arrows). Scale bar, 25 mm. *P#0.05; **P#0.01; ***P#0.001;****P#0.0001; n.s., nonsignificant.
shown to promote proliferation, inhibit differentiation, and sustain cell survival, depending on the cellular context.44 One potential mechanism is via inhibition of the miR-17–5p and miR-20a target, the cyclin-dependent kinase (cdk) inhibitor, p21.30,33–35 Generally, p21 is thought to negatively regulate cell cycle progression, which would be consistent with impaired proliferation due to increased p21 expression in Six2-TGCtg/+; miR-17~92flx/flx kidneys. However, the precise means by which p21 functions remains unclear; p21 has previously been shown to inhibit the activity of cyclin/cdk2 complexes, to bind proliferating cell nuclear antigen to block DNA synthesis, 1448
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or to stabilize the interaction between cdk4/cdk6 and Dcyclins.45–48 In addition, because the miR-17~92 cluster regulates the cell cycle at multiple levels, it is likely that other, as yet undefined, miRNA targets contribute to the observed decrease in proliferation of nephron progenitors. The absence of miR-17~92 in nephron progenitors results in a phenotype distinct from that previously reported for Dicer deletion.4,7 In that model, nephron progenitors are prematurely depleted because of excessive apoptosis and expression of the proapoptotic protein Bim increases.4,7 In contrast, there is no evidence for misregulated Bim expression in Six2-TGCtg/+; J Am Soc Nephrol 25: 1440–1452, 2014
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Future studies will define the mechanisms by which miR-17~92 regulates progenitor proliferation and the determination of congenital nephron endowment. Collectively, our results demonstrate an essential requirement for miR-17~92 in nephron development and renal function postnatally and represent the first report of an miRNA null mutation associated with a renal developmental defect. Furthermore, our data suggests that individuals with MIR17HG mutations may be susceptible to congenital kidney disease. Concise Methods The methods are described in detail in the Supplemental Methods.
Mouse Strains
Figure 7. Normal specification of glomerular cell types in kidneys that are deficient for miR-17~92 in nephron progenitors and their derivatives. (A–D’) Immunofluorescent staining for the podocyte markers, podocin and WT1, demonstrates normal podocyte expression in 3-month-old Six2-TGCtg/+; miR-17~92flx/flx kidneys. (E and F’) The presence of the mesangial marker platelet-derived growth factor receptor-b, was unchanged in mutant kidneys at 3 months. (G and H’) Expression of the endothelial marker, platelet endothelial cell adhesion molecule, was also similar in control and mutant kidneys at 3 months. Scale bar, 25 mm.
miR-17~92flx/flx kidneys, even though Bim is a known target of miR-92a.49 In addition, no statistically significant difference in apoptosis was detected in Six2-TGCtg/+; miR-17~92flx/flx nephron progenitors, unlike the almost 6-fold increase reported in Six2-TGCtg/+; Dicerflx/flx progenitors (although smaller changes cannot be entirely excluded given the sample size and inherent variability in apoptosis of normal nephron progenitors) (Supplemental Table 3).7 This discrepancy is probably related to the effect of Dicer deletion on the many miRNAs expressed in nephron progenitors and the importance of cellular context in determining the functional readout for miR-17~92. Dicer deletion in podocytes results in marked proteinuria, abnormal expression of podocin, foot process effacement, and renal failure.5,8,9 The phenotype in Six2-TGCtg/+; miR-17~92flx/flx kidneys is less severe, with no apparent change in podocin expression and prolonged survival. Moreover, conditional deletion of miR-17~92 in podocytes alone results in no proteinuria or histologic changes (data not shown).50 Thus, the major role for miR-17~92 during kidney development appears to lie within nephron progenitors. J Am Soc Nephrol 25: 1440–1452, 2014
The conditional miR-17~92 (miR-17~92flx/flx) and Six2-TGC transgenic mouse strains were obtained from The Jackson Laboratory.20,21 Animal experiments were carried out in accordance with the policies of the Institutional Animal Care and Use Committee at the University of Pittsburgh School of Medicine.
Proliferation and Apoptosis
Pregnant dams were injected with 30 mg/g body weight BrdU (Invitrogen) 2 hours before harvesting of embryos. The embryonic kidneys were embedded in paraffin, sectioned, and coimmunolabeled with BrdU and with Six2 or Pax2. The percentage of BrdU-positive cells as a proportion of Six2- or Pax2-positive cells were counted per image. The two-tailed t test was used to determine statistical significance. TUNEL staining was done using the Apoptag Plus Fluorescein Apoptosis Detection kit (Millipore) on paraffin-embedded sectioned kidney tissues. Tissue sections were coimmunolabeled with Six2 or activated Caspase-3 and NCAM, and the percentage of TUNEL- or activated Caspase-3–positive cells as a proportion of Six2- or NCAMpositive cells, respectively, were counted as detailed above.
Histopathology and Electron Microscopy Kidneys were processed for Masson trichrome, methenamine silver– periodic acid-Schiff stain, or periodic acid-Schiff histologic staining by the Children’s Hospital of Pittsburgh of UPMC Histology Core Laboratory. Kidney tissues were processed, embedded, and visualized at the Center for Biologic Imaging, University of Pittsburgh, for electron microscopy.
Blood and Urine Measurements Urine and sera were sent to the Kansas State University Veterinary Diagnostic Laboratories, Clinical Pathology Laboratory. Urine samples MiR-17~92 in Kidney Development
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were analyzed on a Coomassie-stained SDS-PAGE gel tovisualize protein excretion. Albumin and creatinine excretion was quantified using the Albuwell murine microalbuminuria ELISA kit and Creatinine Companion kit, respectively (Exocell, Philadelphia, PA).
Immunohistochemistry E14.5, E16.5, E18.5, and P0 kidneys were embedded in paraffin and immunohistochemically labeled as outlined in the Supplemental Methods.
RT Quantitative PCR Total RNA from embryonic kidneys was isolated using the TRIzol Reagent (Invitrogen), and Taqman miRNA, primary miRNA transcript, and gene expression assays (Life Technologies) were performed to determine relative expression levels, per the manufacturer’s instructions. Expression levels were normalized to that of endogenous controls using the cycle threshold value (Cq). The two-tailed t test was used to determine statistical significance.
LNA In Situ Hybridization LNA in situ hybridization was conducted on 10-mm cryosections of E14.5 embryos using an LNA probe (Exiqon) complementary to the miR-17–5p mature miRNA, as published previously4 and as described in the Supplemental Methods.
ACKNOWLEDGMENTS This work was supported by a National Institutes of Health grant to J. H. (DK087922) and a Children’s Hospital of Pittsburgh of UPMC Research Advisory Committee startup grant. AKM was supported by a Children’s Hospital of Pittsburgh of UPMC Research Advisory Committee fellowship award. The Kidney Imaging Core of the Pittsburgh Center for Kidney Research provided expert pathological and electron microscopy support (funded by National Institutes of Health grant P30-DK079307). We thank the Children’s Hospital of Pittsburgh of UPMC Histology Core Laboratory and the Kansas State University Veterinary Diagnostic Laboratories for technical assistance. We would also like to thank Carlton Bates, Sunder Sims-Lucas, Kenneth Walker, and Valeria Di Giovanni for thoughtful discussion and review of the manuscript. The BrdU antibody developed by Stephen J. Kaufman and the 4F2 (Lim1/2) antibody developed by Thomas M. Jessel and Susan Brenner-Morton was obtained from the Developmental Studies Hybridoma Bank, which is under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA. This work was previously presented, in part, at the annual meeting of the American Society for Nephrology (November 7–10, 2013, Atlanta, GA) and published in abstract form.
RNA Sequencing Total RNA for high-throughput RNA sequencing was extracted using Trizol (Invitrogen). Two control and two mutant E16.5 kidneys were pooled per litter across three litters, resulting in six litter-matched biologic samples (three mutants and three controls). Library generation and 100-bp paired end high-throughput sequencing were performed at the Tuft University Genomics Core (http://TUCFGenomics.tufts.edu/) using the Illumina HiSEquation 2000 system yielding approximately 80,000,000 reads per sample. Read alignment and expression abundance quantification were performed using the tophat51 and DESeq52 programs, respectively (Gene Expression Omnibus Series accession number GSE52514; http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE52514).
Western Blot Protein from control and mutant E16.5 kidneys was isolated, run on an SDS-PAGE gel, and transferred to a nitrocellulose membrane (BioRad). The membrane was incubated with primary antibodies against Bim and b-tubulin, and the signals were detected using horseradish peroxidase–conjugated secondary antibodies and the SuperSignal West Femto kit (ThermoScientific).
Statistical Analyses The two-tailed t test was used to determine statistical significance where applicable: *P#0.05; **P#0.01; ***P#0.001; ****P#0.0001. In graphs, the mean6SEM is presented. Open symbols in graphs represent data derived from a female animal, and closed symbols represent data derived from a male animal. All animal experiments were performed with at least four animals across three different litters.
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DISCLOSURES J.H.’s laboratory is supported in part by a Norman S. Coplon Extramural Grant from Satellite Healthcare (for work unrelated to this current manuscript).
REFERENCES 1. Smith JM, Stablein DM, Munoz R, Hebert D, McDonald RA: Contributions of the Transplant Registry: The 2006 Annual Report of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS). Pediatr Transplant 11: 366–373, 2007 2. Little MH, McMahon AP: Mammalian kidney development: Principles, progress, and projections. Cold Spring Harb Perspect Biol 4: 4, 2012 3. Bartel DP: MicroRNAs: Target recognition and regulatory functions. Cell 136: 215–233, 2009 4. Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA: The pro-apoptotic protein Bim is a microRNA target in kidney progenitors. J Am Soc Nephrol 22: 1053–1063, 2011 5. Ho J, Ng KH, Rosen S, Dostal A, Gregory RI, Kreidberg JA: Podocytespecific loss of functional microRNAs leads to rapid glomerular and tubular injury. J Am Soc Nephrol 19: 2069–2075, 2008 6. Patel V, Hajarnis S, Williams D, Hunter R, Huynh D, Igarashi P: MicroRNAs regulate renal tubule maturation through modulation of Pkd1. J Am Soc Nephrol 23: 1941–1948, 2012 7. Nagalakshmi VK, Ren Q, Pugh MM, Valerius MT, McMahon AP, Yu J: Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney. Kidney Int 79: 317–330, 2011 8. Harvey SJ, Jarad G, Cunningham J, Goldberg S, Schermer B, Harfe BD, McManus MT, Benzing T, Miner JH: Podocyte-specific deletion of dicer
J Am Soc Nephrol 25: 1440–1452, 2014
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9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
alters cytoskeletal dynamics and causes glomerular disease. J Am Soc Nephrol 19: 2150–2158, 2008 Shi S, Yu L, Chiu C, Sun Y, Chen J, Khitrov G, Merkenschlager M, Holzman LB, Zhang W, Mundel P, Bottinger EP: Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis. J Am Soc Nephrol 19: 2159–2169, 2008 Sequeira-Lopez ML, Weatherford ET, Borges GR, Monteagudo MC, Pentz ES, Harfe BD, Carretero O, Sigmund CD, Gomez RA: The microRNA-processing enzyme dicer maintains juxtaglomerular cells. J Am Soc Nephrol 21: 460–467, 2010 Wei Q, Bhatt K, He HZ, Mi QS, Haase VH, Dong Z: Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 21: 756–761, 2010 Zhdanova O, Srivastava S, Di L, Li Z, Tchelebi L, Dworkin S, Johnstone DB, Zavadil J, Chong MM, Littman DR, Holzman LB, Barisoni L, Skolnik EY: The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy. Kidney Int 80: 719–730, 2011 Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL: Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol 310: 442–453, 2007 Danielson LS, Park DS, Rotllan N, Chamorro-Jorganes A, Guijarro MV, Fernandez-Hernando C, Fishman GI, Phoon CK, Hernando E: Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis. FASEB J 27: 1460–1467, 2013 Li Y, Vecchiarelli-Federico LM, Li YJ, Egan SE, Spaner D, Hough MR, Ben-David Y: The miR-17-92 cluster expands multipotent hematopoietic progenitors whereas imbalanced expression of its individual oncogenic miRNAs promotes leukemia in mice. Blood 119: 4486–4498, 2012 Conkrite K, Sundby M, Mukai S, Thomson JM, Mu D, Hammond SM, MacPherson D: miR-17~92 cooperates with RB pathway mutations to promote retinoblastoma. Genes Dev 25: 1734–1745, 2011 de Pontual L, Yao E, Callier P, Faivre L, Drouin V, Cariou S, Van Haeringen A, Geneviève D, Goldenberg A, Oufadem M, Manouvrier S, Munnich A, Vidigal JA, Vekemans M, Lyonnet S, Henrion-Caude A, Ventura A, Amiel J: Germline deletion of the miR-17;92 cluster causes skeletal and growth defects in humans. Nat Genet 43: 1026–1030, 2011 Celli J, van Bokhoven H, Brunner HG: Feingold syndrome: Clinical review and genetic mapping. Am J Med Genet A 122A: 294–300, 2003 Marcelis CL, Hol FA, Graham GE, Rieu PN, Kellermayer R, Meijer RP, Lugtenberg D, Scheffer H, van Bokhoven H, Brunner HG, de Brouwer AP: Genotype-phenotype correlations in MYCN-related Feingold syndrome. Hum Mutat 29: 1125–1132, 2008 Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, Newman J, Bronson RT, Crowley D, Stone JR, Jaenisch R, Sharp PA, Jacks T: Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 132: 875–886, 2008 Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP: Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell 3: 169–181, 2008 Self M, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G: Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. EMBO J 25: 5214–5228, 2006 Torres M, Gómez-Pardo E, Dressler GR, Gruss P: Pax-2 controls multiple steps of urogenital development. Development 121: 4057–4065, 1995 Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R: WT-1 is required for early kidney development. Cell 74: 679–691, 1993
J Am Soc Nephrol 25: 1440–1452, 2014
BASIC RESEARCH
25. Boyle S, Shioda T, Perantoni AO, de Caestecker M: Cited1 and Cited2 are differentially expressed in the developing kidney but are not required for nephrogenesis. Dev Dyn 236: 2321–2330, 2007 26. McCright B, Lozier J, Gridley T: A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development 129: 1075–1082, 2002 27. Fujii T, Pichel JG, Taira M, Toyama R, Dawid IB, Westphal H: Expression patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system. Dev Dyn 199: 73–83, 1994 28. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H: A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13: 133–140, 2010 29. Ranji N, Sadeghizadeh M, Shokrgozar MA, Bakhshandeh B, Karimipour M, Amanzadeh A, Azadmanesh K: MiR-17-92 cluster: An apoptosis inducer or proliferation enhancer. Mol Cell Biochem 380: 229–238, 2013 30. Cloonan N, Brown MK, Steptoe AL, Wani S, Chan WL, Forrest AR, Kolle G, Gabrielli B, Grimmond SM: The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition. Genome Biol 9: R127, 2008 31. Yin R, Bao W, Xing Y, Xi T, Gou S: MiR-19b-1 inhibits angiogenesis by blocking cell cycle progression of endothelial cells. Biochem Biophys Res Commun 417: 771–776, 2012 32. Patel V, Williams D, Hajarnis S, Hunter R, Pontoglio M, Somlo S, Igarashi P: miR-17~92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease. Proc Natl Acad Sci U S A 110: 10765–10770, 2013 33. Fontana L, Fiori ME, Albini S, Cifaldi L, Giovinazzi S, Forloni M, Boldrini R, Donfrancesco A, Federici V, Giacomini P, Peschle C, Fruci D: Antagomir17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM. PLoS ONE 3: e2236, 2008 34. Wong P, Iwasaki M, Somervaille TC, Ficara F, Carico C, Arnold C, Chen CZ, Cleary ML: The miR-17-92 microRNA polycistron regulates MLL leukemia stem cell potential by modulating p21 expression. Cancer Res 70: 3833–3842, 2010 35. Gibcus JH, Kroesen BJ, Koster R, Halsema N, de Jong D, de Jong S, Poppema S, Kluiver J, Diepstra A, van den Berg A: MiR-17/106b seed family regulates p21 in Hodgkin’s lymphoma. J Pathol 225: 609–617, 2011 36. Roselli S, Gribouval O, Boute N, Sich M, Benessy F, Attié T, Gubler MC, Antignac C: Podocin localizes in the kidney to the slit diaphragm area. Am J Pathol 160: 131–139, 2002 37. Vaughan MR, Quaggin SE: How do mesangial and endothelial cells form the glomerular tuft? J Am Soc Nephrol 19: 24–33, 2008 38. Bejarano F, Bortolamiol-Becet D, Dai Q, Sun K, Saj A, Chou YT, Raleigh DR, Kim K, Ni JQ, Duan H, Yang JS, Fulga TA, Van Vactor D, Perrimon N, Lai EC: A genome-wide transgenic resource for conditional expression of Drosophila microRNAs. Development 139: 2821–2831, 2012 39. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH: Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol 5: e203, 2007 40. Smibert P, Lai EC: Lessons from microRNA mutants in worms, flies and mice. Cell Cycle 7: 2500–2508, 2008 41. Miska EA, Alvarez-Saavedra E, Abbott AL, Lau NC, Hellman AB, McGonagle SM, Bartel DP, Ambros VR, Horvitz HR: Most Caenorhabditis elegans microRNAs are individually not essential for development or viability. PLoS Genet 3: e215, 2007 42. Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J, Henderson JM, Kutok JL, Rajewsky K: Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol 9: 405–414, 2008 43. Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada K: MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes. Blood 113: 396–402, 2009
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44. Olive V, Jiang I, He L: mir-17-92, a cluster of miRNAs in the midst of the cancer network. Int J Biochem Cell Biol 42: 1348–1354, 2010 45. Waga S, Hannon GJ, Beach D, Stillman B: The p21 inhibitor of cyclindependent kinases controls DNA replication by interaction with PCNA. Nature 369: 574–578, 1994 46. Brugarolas J, Moberg K, Boyd SD, Taya Y, Jacks T, Lees JA: Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation. Proc Natl Acad Sci U S A 96: 1002–1007, 1999 47. Kavurma MM, Khachigian LM: Sp1 inhibits proliferation and induces apoptosis in vascular smooth muscle cells by repressing p21WAF1/ Cip1 transcription and cyclin D1-Cdk4-p21WAF1/Cip1 complex formation. J Biol Chem 278: 32537–32543, 2003 48. LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E: New functional activities for the p21 family of CDK inhibitors. Genes Dev 11: 847–862, 1997
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49. Tsuchida A, Ohno S, Wu W, Borjigin N, Fujita K, Aoki T, Ueda S, Takanashi M, Kuroda M: miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer. Cancer Sci 102: 2264–2271, 2011 50. Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB: Podocytespecific expression of cre recombinase in transgenic mice. Genesis 35: 39–42, 2003 51. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25: 1105–1111, 2009 52. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol 11: R106, 2010
This article contains supplemental material online at http://jasn.asnjournals. org/lookup/suppl/doi:10.1681/ASN.2013040390/-/DCSupplemental.
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MicroRNA-17~92 Is Required for Nephrogenesis and Renal Function April K. Marrone,* Donna B. Stolz,† Sheldon I. Bastacky,‡§ Dennis Kostka,| Andrew J. Bodnar,* and Jacqueline Ho* *Division of Nephrology, Department of Pediatrics, †Department of Cell Biology, ‡Department of Pathology, and | Departments of Developmental Biology and Computational Systems Biology, University of Pittsburgh School of Medicine, and §University of Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania
ABSTRACT Deletion of all microRNAs (miRNAs) in nephron progenitors leads to premature loss of these cells, but the roles of specific miRNAs in progenitors have not been identified. Deletions in the MIR17HG cluster (miR17~92 in mice), detected in a subset of patients with Feingold syndrome, represent the first miRNA mutations to be associated with a developmental defect in humans. Although MIR17HG is expressed in the developing kidney, and patients with Feingold syndrome caused by MYCN mutations have renal anomalies, it remains unclear to what extent MIR17HG contributes to renal development and function. To define the role of miR-17~92, we generated mice with a conditional deletion of miR-17~92 in nephron progenitors and their derivatives. The nephron progenitor population was preserved in these mice; however, this deletion impaired progenitor cell proliferation and reduced the number of developing nephrons. Postnatally, mutant mice developed signs of renal disease, including albuminuria by 6 weeks and focal podocyte foot process effacement and glomerulosclerosis at 3 months. Taken together, these data support a role for this miRNA cluster in renal development, specifically in the regulation of nephron development, with subsequent consequences for renal function in adult mice. J Am Soc Nephrol 25: 1440–1452, 2014. doi: 10.1681/ASN.2013040390
Congenital anomalies of the kidney and urinary tract (CAKUTs) are among the most frequent birth defects in humans and are the major cause of childhood CKD.1 Although CAKUTs represent a diverse group of developmental anomalies, the risk of CKD is related to decreased renal reserve as a result of the formation of fewer and/or abnormal nephrons. Kidney development occurs largely by reciprocal signaling between nephron progenitors and the ureteric bud (reviewed by Little and McMahon2). Nephron progenitors are capable of self-renewal and differentiation into the multiple cell types of the mature nephron to generate a sufficient number of nephrons for the adult animal. The nephron progenitors undergo a mesenchymeto-epithelial transition during nephrogenesis to sequentially form renal vesicles, comma-shaped bodies, S-shaped bodies, and, ultimately, functional nephrons consisting of glomeruli and tubules. Despite the prevalence of pediatric renal disease 1440
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associated with abnormal kidney development, many of the molecular mechanisms underlying nephron endowment remain unclear. MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by repressing their target mRNAs (reviewed by Bartel 3). Several recent studies have demonstrated that miRNAs are critical in kidney development and function by blocking the miRNA biogenesis pathway in
Received April 18, 2013. Accepted November 15, 2013. Published online ahead of print. Publication date available at www.jasn.org. Correspondence: Dr. Jacqueline Ho, Division of Nephrology, Department of Pediatrics, Children’s Hospital of Pittsburgh of UPMC, Rangos Research Center, University of Pittsburgh School of Medicine, 4401 Penn Avenue, Pittsburgh, PA 15224. Email: [email protected] Copyright © 2014 by the American Society of Nephrology
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specific cell lineages.4–12 We, and others, have reported that loss of functional miRNAs in nephron progenitors results in a premature depletion of progenitors related to increased apoptosis, leading to the formation of fewer nephrons.4,7 However, the requirement for individual miRNAs in survival or other roles in nephron progenitors remains unclear. The human MIR17 (MIR17HG) cluster has been linked to developmental, apoptotic, and oncogenic pathways in other organs,13–16 and the locus is conserved between mouse and human. Heterozygous deletion of the MIR17HG cluster is the first reported miRNA mutation that results in a developmental defect, in humans with Feingold syndrome.17 Although patients with Feingold syndrome associated with MIR17HG mutations have not yet been reported to have kidney defects, this syndrome has also been associated with MYCN mutations and, in that context, has up to an 18% incidence of CAKUT.18,19 Deletion of the miR-17~92 cluster in mice results in perinatal lethality, and multiple congenital anomalies, including pulmonary hypoplasia, growth retardation, and cardiac defects.20 While members of the cluster are expressed in the developing kidney, including in nephron progenitors, the function of miR-17~92 in renal development and function is unknown.4 Here, we demonstrate that ablation of miR-17~92 in nephron progenitors and their derivatives results in renal hypodysplasia. We show that the nephron progenitor population is preserved but cell proliferation is impaired, ultimately leading to fewer developing nephron structures. Postnatally, these mice develop evidence of glomerular kidney disease with albuminuria, glomerulosclerosis, and podocyte foot process effacement. Collectively, our data demonstrate an essential requirement for miR-17~92 in nephron development that affects renal function postnatally. To our knowledge, this is the first report of an miRNA mutation associated with developmental renal anomalies.
RESULTS Conditional Deletion of miR-17~92 from Nephron Progenitors Results in Kidney Hypodysplasia
To define the role of the miR-17~92 cluster in kidney development and function, we generated mice with conditional deletion of the miR-17~92 cluster using the Six2-TGC line, which drives Cre expression in nephron progenitors,21 and a floxed allele of the miR-17~92 cluster.20 RT quantitative PCR for the primary miR-17~92 transcript (pri-miR-17~92) confirmed a reduction in mutant embryonic day 16.5 (E16.5) kidneys (=Six2-TGCtg/+; miR-17~92flx/flx) relative to littermate controls (Figure 1A). Locked nucleic acid (LNA) in situ hybridization analysis of miR-17–5p in E14.5 kidneys produced a signal in nephron progenitors, developing nephrons, ureteric buds, and the ureter (Figure 1B), as previously reported for E16.5 kidneys. 4 As expected, the Six2TGC tg/+ ; miR-17~92 flx/flx mutant kidney showed reduced J Am Soc Nephrol 25: 1440–1452, 2014
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expression in nephron progenitors and their derivatives (Figure 1B). Mutant mice were born at normal Mendelian ratios and have been aged up to 6 months. Postnatal day 0 (P0) Six2-TGC tg/+ ; miR-17~92 flx/flx kidneys were significantly smaller than those of their littermates, while overall body sizes were equivalent (Figure 1, C and D, Supplemental Table 1). This finding persisted at 3 months, with a smaller kidney-to-body weight ratio in Six2-TGCtg/+; miR-17~92flx/flx animals (Supplemental Figure 1). Furthermore, heterozygous animals with a one-allele loss of miR-17~92 (Six2-TGCtg/+; miR-17~92flx/+) in nephron progenitors had kidneys that were intermediate in size compared with their control and homozygous mutant littermates (Figure 1, C and D, Supplemental Figure 1, Supplemental Table 1). This finding is in accordance with previously published work that global one-allele loss of miR-17~92 resulted in developmental defects in both mice and humans.17 miR-17~92 belongs to a highly conserved family of three miRNA clusters that includes miR-106a~363 and miR106b~25. Previous work has suggested functional redundancy between miR-17~92 and miR-106b~25 (but not miR106a~363), resulting in a worsening embryonic phenotype in global miR-17~92 2 /2 ; miR-106b~25 2 /2 double mutants.20 Therefore, we generated animals lacking miR-17~92 in nephron progenitors in a miR-106b~252/2 background (Six2-TGCtg/+; miR-17~92flx/flx; miR-106b~252/2) and observed no change in the degree of kidney hypoplasia compared with Six2-TGCtg/+; miR-17~92flx/flx mutants (Figure 1C); histologic features also did not differ (data not shown). The sizes of mutant miR-106b~252/2 kidneys were also unchanged relative to control kidneys (Figure 1C). Moreover, the expression of miR-106b~25 and miR-106a~363 primary transcripts were not affected by miR-17~92 loss (Figure 1A). These results imply that the role of miR-17~92 in nephron progenitors and their derivatives is unique and that there is no compensatory increase in miR-106a~363 and miR-106b~25 expression in mutants. Nephrons are continually induced at ureteric bud tips just below the renal capsule throughout kidney development, resulting in a gradient of differentiating nephrons with the most immature nephrons in the periphery of the renal cortex. 2 Histologic sections of P0 kidneys revealed fewer developing nephrons in the renal cortex of Six2-TGC tg/+ ; miR-17~92 flx/flx kidneys (Figure 1, E–G9). Furthermore, at P30 when nephrogenesis is complete, there were markedly fewer observable glomeruli in Six2-TGC tg/+; miR-17~92 flx/flx kidney sections (yellow stars) compared with controls (Figure 1, H and I). Thus, loss of miR17~92 in nephron progenitors and their derivatives results in renal hypodysplasia. miR-17~92 Is Required in Early Kidney Development
To assess the developmental time points at which the miR17~92 cluster is active, RT-quantitative PCR was performed MiR-17~92 in Kidney Development
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Figure 1. Loss of miR-17~92 in nephron progenitors results in renal hypodysplasia. (A) RT-quantitative PCR for the miR-17~92 primary transcript in E16.5 kidneys shows a reduction in mutant kidneys relative to controls. In contrast, the levels of the miR-106b~25 and miR106a~363 primary transcripts are not significantly different. (B) LNA in situ hybridization for miR-17–5p in E14.5 kidneys reveals expression in nephron progenitors, their derivatives, and ureteric epithelium in control littermates, which is reduced in nephron
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to measure relative miRNA levels in E12.5, E14.5, E16.5, and E18.5 kidneys (Figure 2A). The primary miR-17~92 transcript was present in embryonic kidney throughout these time points, but its expression was lower at E16.5 and E18.5. Of the miRNAs expressed by this cluster, miR-17–5p followed a pattern similar to the primary transcript; however, the expression of miR-18a-5p, miR-19b-1–5p, and miR-20a-5p each peaked at E14.5 (Figure 2A). Expression of miR-92a1–5p decreased steadily throughout, whereas miR-19a-5p levels increased approximately 5-fold in E14.5 kidneys and remained relatively high (Figure 2A). These results suggest that the effects of miR-17~92 loss from nephron progenitors could begin to manifest early, and, furthermore, that there may be differential regulation of the individual miRNAs within this cluster during kidney development. Histologic analysis of E14.5, E16.5, and E18.5 kidneys was used to define the onset of impaired nephrogenesis in Six2TGCtg/+; miR-17~92flx/flx mutants relative to controls (Figure 2B-C). At E14.5, there were no gross histologic differences in mutants, with an appropriate number of developing nephrons observed at various stages: renal vesicles, comma-shaped bodies, S-shaped bodies, and capillary loop stage glomeruli (Figure 2, B and C). By E16.5, it became apparent that fewer nephron structures were developing, and this persisted in E18.5 and P0 mutant kidneys (Figures 1, E–G, and 2, B9 and C99). Deletion of miR-17~92 in Nephron Progenitors Impairs Nephrogenesis
To more clearly define the effect on nephrogenesis, immunofluorescence was performed for markers specific to nephron progenitors and developing nephron structures. P0 Six2-TGCtg/+; miR-17~92flx/flx kidneys expressed several nephron progenitor markers, including Six2, Pax2, Wt1, Cited1, and neural cell adhesion molecule (NCAM),2,22–25 although it appears that Cited1 expression was decreased (Figure 3, B and B9). Several of these markers continue to be present in differentiating nephrons, including in the renal vesicle (Pax2 and NCAM) and developing podocytes of the glomerulus (Wt1). Six2-TGCtg/+; miR-17~92flx/flx kidneys have fewer Pax2- and NCAM-positive renal vesicles (yellow arrows in Figure 3, C, C9, D9 and D0), the earliest epithelial derivatives of nephron progenitors. There were also fewer Wt1-positive glomeruli present in Six2-TGCtg/+; miR-17~92flx/flx kidneys (yellow stars in Figure
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3, E and –E9). There were no gross differences in the formation of proximal or distal nephron tubules of Six2-TGCtg/+; miR17~92flx/flx kidneys, as noted by lotus tetragonolobus lectin (LTL) staining for proximal tubules or E-cadherin–positive (dolichos biflorus agglutinin [DBA]-negative) staining for distal tubules, respectively (Figure 3, E and F9). Despite the abnormalities seen in nephron development, the ureteric bud lineage appeared normal on the basis of calbindin staining of ureteric buds (Figure 3, B and B9) and DBA labeling of collecting ducts (Figure 3, F and F9) in the mutant kidneys. Thus, there appears to be normal initial nephron progenitor specification in Six2-TGCtg/+; miR-17~92flx/flx kidneys. Immunofluorescence was subsequently conducted on E14.5 kidneys, a time point that coincides with the highest expression of most miRNAs in miR-17~92. In mutant E14.5 kidneys, nephron progenitors appeared to express Six2 normally; however, Cited1 expression was reduced, similar to what was observed at P0 (Figure 4, A–D9). To evaluate whether renal vesicles formed normally, staining for Lim1 and Jagged1 was performed.26,27 Both Jagged1 and Lim1 were unchanged in renal vesicles (Figure 4, E and H9), but Jagged1 expression appeared to be decreased in the nephron progenitors of Six2TGCtg/+; miR-17~92flx/flx mutants relative to littermate controls (Figure 4, E versus F). Jagged1 and Lim1 expression in renal vesicles at E16.5 persisted in mutants compared with controls (Figure 4, I–L). These data suggest that the signals required to form renal vesicles are unaffected by the loss of miR-17~92 from nephron progenitors but that the nephron progenitors themselves may not be completely functionally normal. Lineage tracing experiments were performed by crossing a CAG reporter allele28 with the Six2-Cre transgene in both control and mutant mice (Six2-TGCtg/+; RosaCAG-tdTomato/+ and Six2-TGCtg/+; RosaCAG-tdTomato/+; miR-17~92flx/flx respectively) to assess the cell fate of nephron progenitors. All tissues examined in both controls and mutants showed efficient Cre-recombination, and Cre-expressing cells adopted cell fates consistent with those normally derived from the progenitor population in Six2-TGC t g / + ; Rosa C AG - t d To m a t o / + ; miR-17~92flx/flx compared with controls at P0 (Supplemental Figure 2). Together, these results are consistent with a partial block in nephrogenesis and a relative preservation of nephron patterning, resulting in fewer nephrons in Six2-TGCtg/+ ; miR-17~92flx/flx kidneys.
progenitors and their derivatives in Six2-TGCtg/+; miR-17~92flx/flx kidneys. Blue dotted lines: ureteric bud; red dotted lines: S-shape body (left) and renal vesicle (right); red arrow: nephron progenitors. (C) Kidney surface area normalized to crown rump length at P0 demonstrates that loss of a single copy of miR-17~92 results in mild hypoplasia, whereas loss of both alleles results in more severe hypoplasia. The crownrump length was similar among all genotypes. (D) Sample photos of newborn pups and their kidneys. The adrenal gland is attached to the left Six2-TGCtg/+; miR-17~92flx/+ kidney. (E–G’) Hematoxylin and eosin sections show fewer developing nephron structures in P0 Six2TGCtg/+; miR-17~92flx/flx animals when compared with controls. (H and I) At P30, methenamine silver–periodic acid-Schiff stain with hematoxylin and eosin reveals more glomeruli (yellow stars) in controls than in Six2-TGCtg/+; miR-17~92flx/flx kidneys. Mutant kidneys also have dilated tubules (blue arrow) and proteinaceous casts (blue star). Scale bars, 100 mm. *P#0.05; **P#0.01; ***P#0.001; ****P#0.0001; n.s., nonsignificant.
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Figure 2. miR-17~92 is expressed throughout kidney development and is required for early development. (A) Expression levels of the primary miR-17~92 transcript and miRNAs expressed by this cluster (miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a) at E12.5, E14.5, E16.5, and E18.5 by RT quantitative PCR. Time points are compared against E12.5 expression levels, after normalization to an endogenous control. (B–B’’) Hematoxylin and eosin sections of control E14.5, E16.5, and E18.5 kidneys. (C–C’’) Hematoxylin and eosin sections of Six2-TGCtg/+; miR-17~92flx/flx E14.5, E16.5, and E18.5 kidneys. At E16.5 and E18.5, fewer developing nephron structures were observed. Scale bar, 100 mm.
miR-17~92 Regulates Nephron Progenitor Proliferation
The miR-17~92 cluster regulates both cell proliferation and apoptosis.29–32 The partial block in nephrogenesis in mutants could be caused by alterations in either of these processes. Proliferation was analyzed using bromodeoxyuridine (BrdU) incorporation into dividing cells in E14.5, E16.5, and E18.5 embryos, and coimmunolabeling for BrdU and Six2 (progenitor marker) or Pax2 (progenitor, renal vesicle, and ureteric bud marker—ureteric bud cells were not counted). Similar to the histologic analysis, no significant difference was noted in the number of double BrdU-positive, Six2- (or Pax2-) positive cells relative to single-positive Six2- or Pax2-positive cells at E14.5. However, Six2-TGCtg/+; miR-17~92flx/flx kidneys had a significantly smaller percentage of double-positive cells than controls at E16.5 and E18.5 (Figure 5, A and B, Supplemental Table 2). These data suggest that the loss of miR-17~92 in nephron progenitors results in impaired proliferation. miR-17–5p and miR-20a directly target the cell cycle inhibitor p21.30,33–35 Interestingly, RT-qPCR for p21 transcripts demonstrated an approximately 1.5-fold increase in E16.5 Six2-TGC tg/+ ; miR-17~92 flx/flx kidneys compared with 1444
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littermate controls (Figure 5C). This finding was corroborated by RNA sequencing that showed an approximately 20% increase in p21 mRNA levels in mutant kidneys (data not shown). Thus, the defect in cellular proliferation in Six2-TGCtg/+;miR-17~92flx/flx kidneys may be related, at least in part, to misregulation of p21 by miR-17~92 miRNAs in nephron progenitors. The loss of all mature miRNAs in nephron progenitors has previously been reported to result in increased cell death, in association with elevated expression of the proapoptotic protein Bim.4,7 Apoptosis was assessed with the TUNEL assay or immunostaining for activated Caspase-3 (an early marker of apoptosis), followed by colabeling with Six2 (progenitors) or NCAM (progenitors and renal vesicles). There was no statistically significant difference detected in the number of double TUNEL-positive/Six2-positive or activated Caspase3–positive/NCAM-positive cells relative to single positive Six2- or NCAM-positive cells at E14.5, E16.5, or E.18.5 in control and mutant kidneys, despite a trend toward increased TUNEL-positive cells at E16.5, and decreased TUNEL-positive and activated Caspase-3 positive cells at E16.5 and E18.5 in miR-17~92 mutant nephron progenitors (Figure 5, E and F, J Am Soc Nephrol 25: 1440–1452, 2014
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Figure 3. miR-17~92 is required in nephron progenitors for normal nephron development. (A–B’) Immunofluorescence for nephron progenitor markers demonstrates preserved Six2 expression (A and A’), but decreased Cited1 expression (B and B’) in Six2-TGCtg/+; miR-17~92flx/flx P0 kidneys (pink). Calbindin (blue) labels ureteric bud and LTL (blue) marks proximal tubules, both of which are unchanged. (C–D’) Immunofluorescence for Pax2 (C and C’) and NCAM (D and D’) shows decreased formation of renal vesicles in mutant newborn kidneys (pink; yellow arrows). (E and E’) Wt1 staining demonstrates fewer glomeruli in the Six2-TGCtg/+; miR-17~92flx/flx kidneys (pink, yellow stars). (F and F’) Distal nephron tubules were labeled with E-cadherin (and are DBA negative), showing that distal tubules form in the absence of miR-17~92 (E-cadherin, pink; DBA blue). Scale bar, 100 mm.
Supplemental Table 3). Moreover, there was no change in Bim expression by immunofluorescence, Western blot, or RNA-sequencing in control and mutant kidneys (Figure 5, D and E). Together, these data suggest that miR-17~92 loss does not markedly affect cell death in nephron progenitors. Deletion of miR-17~92 from the Progenitor Population Results in CKD
The mutant animals were then evaluated for the functional consequences of miR-17~92 loss in the kidney. At 6 weeks and 3 months of age, heterozygous (Six2-TGCtg/+; miR-17~92flx/+) and homozygous (Six2-TGCtg/+; miR-17~92flx/flx) mice had increased albumin excretion in their urine, in comparison to Cre-negative controls (Figure 6, A and B, Supplemental Table 4). BUN and serum creatinine levels were significantly elevated in Six2-TGCtg/+; miR-17~92flx/flx mice relative to controls at 3 months (Figure 6C). In contrast, urine volume and osmolality were unchanged, suggesting that the animals did not possess a urinary concentrating defect (as can be seen in patients with renal dysplasia) (Supplemental Figure 3). Histologic analysis of 3-month-old kidneys demonstrated FSGS and hyalinosis (Figure 6, D0 and E0, Supplemental Figure 4), focal J Am Soc Nephrol 25: 1440–1452, 2014
tubular dilation with hyaline and proteinaceous casts (Figure 6, E0, F9, and G0), patchy inflammation in the renal interstitium (Figure 6, D9 and G0), and patchy areas of fibrosis in the mutant kidneys (Figure 6F0). Ultrastructural analysis demonstrated basement membrane wrinkling in heterozygous animals (Figure 6H9), a sign of early glomerular collapse. Partial podocyte foot process effacement was noted in both Six2-TGC tg/+; miR17~92flx/+ and Six2-TGCtg/+; miR-17~92flx/flx kidneys at 3 months of age (Figure 6, H9 and H0); the podocyte foot process effacement is probably responsible for the proteinuria. Together, these data provide evidence that the loss of miR-17~92 from nephron progenitors results in proteinuric kidney disease, histologic injury, and renal dysfunction. Given the proteinuria and glomerular changes in mutant animals, immunostaining was performed for markers of the three cell types in the glomerulus: podocytes (which arise from nephron progenitors) and endothelial and mesangial cells (which are not derived from nephron progenitors). The podocyte markers, Wt1 and podocin, continued to be expressed normally in Six2-TGCtg/+; miR-17~92flx/flx animals (Figure 7, A–D9),36 suggesting that podocytes are normally specified. The mesangial cell marker platelet-derived growth MiR-17~92 in Kidney Development
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Figure 4. miR-17~92 loss from nephron progenitors does not significantly affect the initial formation of renal vesicles. (A–B’) Immunofluorescence demonstrates preserved Six2 (pink) expression at E14.5 in mutant kidneys. (C–D’) In contrast, decreased Cited1 (pink) expression is observed in Six2-TGCtg/+; miR-17~92flx/flx kidneys, relative to controls. (E–F’, I–J’) Immunofluorescent staining for Jagged1 (Jag1) and (G–H’, K–L’) Lim1 shows that there is unchanged expression of these proteins in the renal vesicle in Six2-TGCtg/+; miR-17~92flx/flx kidneys, relative to littermate controls at E14.5 and E16.5. Calbindin (blue) labels ureteric buds; NCAM (blue) marks progenitors and epithelial derivatives; and Pax2 (blue) labels progenitors, ureteric buds, and progenitors. Scale bar, 100 mm.
factor receptor-b and the endothelial cell marker, platelet endothelial cell adhesion molecule, were also present in mutant glomeruli (Figure 7, E–H9).37 Taken with the histologic data 1446
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showing podocyte effacement, these findings are in keeping with previously published reports in which loss of all mature miRNAs in the podocyte results in marked proteinuria and J Am Soc Nephrol 25: 1440–1452, 2014
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renal failure, in the setting of normal podocyte specification but abnormal podocyte function.5,8,9
DISCUSSION
We show that nephron development is exquisitely sensitive to miR-17~92 gene dosage in nephron progenitors, with evidence for renal hypodysplasia in both heterozygous and homozygous conditional mutants. The initial specification of nephron progenitors appears to occur normally in Six2TGC tg/+ ; miR-17~92 flx/flx kidneys, and there are no gross differences in the formation of renal vesicles or nephron segmentation. We present evidence that the reduction in congenital nephron endowment is related to impaired progenitor proliferation that occurs by midgestation, which results in the formation of fewer developing nephrons. Postnatally, the animals develop glomerular dysfunction and proteinuric kidney disease. Despite growing evidence that links miRNAs to numerous biologic processes, comparatively few miRNA mutations result in developmental defects, with most published deletions having no overt phenotype.38–41 This has been attributed to data suggesting that most miRNAs act by fine-tuning gene expression. Thus, the observation that loss of a single miR17~92 allele in nephron progenitors is sufficient to result in renal hypodysplasia and albuminuria is striking. This is in accord with several recent studies that have implicated both loss and gain of function of miR-17~92 miRNAs in regulating the cell cycle and in oncogenesis.30,31,33–35,42,43 In addition, heterozygous deletion of MIR17HG was recently reported in Feingold syndrome (although the renal phenotype in these patients remains undefined).17 The miR-17~92 cluster has pleiotropic functions in normal development and malignant transformation and has been
Figure 5. Decreased proliferation (and no change in cell death) in nephron progenitors and their derivatives in Six2-TGCtg/+; miR17~92flx/flx kidneys. (A) The percentage of BrdU-positive Six2expressing cells (top) and BrdU-positive Pax2-expressing cells
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(bottom) in E14.5, E16.5, and E18.5 mutant and control kidneys. (B) Representative images of E18.5 kidneys demonstrating fewer BrdU-/Six2-positive cells, as well as fewer Six2-positive cells in Six2TGCtg/+; miR-17~92flx/flx kidneys than in control littermates. BrdU (blue); Six2 (pink). (C) RT quantitative PCR analysis demonstrates increased p21 mRNA in Six2-TGCtg/+; miR-17~92flx/flx E16.5 kidneys relative to controls. (D) No significant difference in Bim expression, normalized to b-tubulin, is noted in E16.5 control and mutant kidneys by Western blot, and no significant change in Bim mRNA was detected via RNA sequencing (log2FC=20.177, false discovery rate=0.233). (E) Representative images of E14.5 kidneys showing TUNEL (blue) and Six2 (pink) colabeling (top), Bim (pink) and NCAM (blue) colabeling (middle), and Bim (gray) expression (bottom) in Six2-TGCtg/+; miR-17~92flx/flx animals compared with control littermates at E14.5. (F) The percentages of TUNEL-positive Six2-positive cells (top) and activated Caspase-3–positive NCAMpositive cells (bottom) at E14.5, E16.5, and E18.5 in mutant and control kidneys are not significantly different.
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Figure 6. miR-17~92 is required for normal kidney function. (A) Urine from 6-week-old mutants had increased albumin excretion versus controls on an SDS-PAGE gel stained with Coomassie (control was BSA; molecular mass is 70 kD). (B) ELISAs demonstrate that animals with decreased miR-17~92 expression have markedly increased albumin-to-creatinine ratios at 6 weeks and 3 months of age. (C) Six2TGCtg/+; miR-17~92flx/flx animals have significantly elevated BUN and serum creatinine levels. (D–G’’) Periodic acid-Schiff and hematoxylin (D and G), methenamine silver–periodic acid-Schiff and hematoxylin and eosin (E), and Masson trichrome staining (F) of 3-month-old kidney tissues reveals that loss of miR-17~92 results in focal glomerulosclerosis (yellow stars, D’’ and E’’), dilated tubules (blue arrows, G’’), proteinaceous casts (blue stars, E’’, F’, and G’’), interstitial inflammation (green arrows, D’, G’, and G’’), and fibrosis (blue color, F–F’’). Scale bars = 100 mm. (H–H’’) Electron micrographs (original magnification, 320,000) reveal that mutants have partial podocyte foot process effacement and wrinkling of the glomerular basement membrane (green arrows). Scale bar, 25 mm. *P#0.05; **P#0.01; ***P#0.001;****P#0.0001; n.s., nonsignificant.
shown to promote proliferation, inhibit differentiation, and sustain cell survival, depending on the cellular context.44 One potential mechanism is via inhibition of the miR-17–5p and miR-20a target, the cyclin-dependent kinase (cdk) inhibitor, p21.30,33–35 Generally, p21 is thought to negatively regulate cell cycle progression, which would be consistent with impaired proliferation due to increased p21 expression in Six2-TGCtg/+; miR-17~92flx/flx kidneys. However, the precise means by which p21 functions remains unclear; p21 has previously been shown to inhibit the activity of cyclin/cdk2 complexes, to bind proliferating cell nuclear antigen to block DNA synthesis, 1448
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or to stabilize the interaction between cdk4/cdk6 and Dcyclins.45–48 In addition, because the miR-17~92 cluster regulates the cell cycle at multiple levels, it is likely that other, as yet undefined, miRNA targets contribute to the observed decrease in proliferation of nephron progenitors. The absence of miR-17~92 in nephron progenitors results in a phenotype distinct from that previously reported for Dicer deletion.4,7 In that model, nephron progenitors are prematurely depleted because of excessive apoptosis and expression of the proapoptotic protein Bim increases.4,7 In contrast, there is no evidence for misregulated Bim expression in Six2-TGCtg/+; J Am Soc Nephrol 25: 1440–1452, 2014
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Future studies will define the mechanisms by which miR-17~92 regulates progenitor proliferation and the determination of congenital nephron endowment. Collectively, our results demonstrate an essential requirement for miR-17~92 in nephron development and renal function postnatally and represent the first report of an miRNA null mutation associated with a renal developmental defect. Furthermore, our data suggests that individuals with MIR17HG mutations may be susceptible to congenital kidney disease. Concise Methods The methods are described in detail in the Supplemental Methods.
Mouse Strains
Figure 7. Normal specification of glomerular cell types in kidneys that are deficient for miR-17~92 in nephron progenitors and their derivatives. (A–D’) Immunofluorescent staining for the podocyte markers, podocin and WT1, demonstrates normal podocyte expression in 3-month-old Six2-TGCtg/+; miR-17~92flx/flx kidneys. (E and F’) The presence of the mesangial marker platelet-derived growth factor receptor-b, was unchanged in mutant kidneys at 3 months. (G and H’) Expression of the endothelial marker, platelet endothelial cell adhesion molecule, was also similar in control and mutant kidneys at 3 months. Scale bar, 25 mm.
miR-17~92flx/flx kidneys, even though Bim is a known target of miR-92a.49 In addition, no statistically significant difference in apoptosis was detected in Six2-TGCtg/+; miR-17~92flx/flx nephron progenitors, unlike the almost 6-fold increase reported in Six2-TGCtg/+; Dicerflx/flx progenitors (although smaller changes cannot be entirely excluded given the sample size and inherent variability in apoptosis of normal nephron progenitors) (Supplemental Table 3).7 This discrepancy is probably related to the effect of Dicer deletion on the many miRNAs expressed in nephron progenitors and the importance of cellular context in determining the functional readout for miR-17~92. Dicer deletion in podocytes results in marked proteinuria, abnormal expression of podocin, foot process effacement, and renal failure.5,8,9 The phenotype in Six2-TGCtg/+; miR-17~92flx/flx kidneys is less severe, with no apparent change in podocin expression and prolonged survival. Moreover, conditional deletion of miR-17~92 in podocytes alone results in no proteinuria or histologic changes (data not shown).50 Thus, the major role for miR-17~92 during kidney development appears to lie within nephron progenitors. J Am Soc Nephrol 25: 1440–1452, 2014
The conditional miR-17~92 (miR-17~92flx/flx) and Six2-TGC transgenic mouse strains were obtained from The Jackson Laboratory.20,21 Animal experiments were carried out in accordance with the policies of the Institutional Animal Care and Use Committee at the University of Pittsburgh School of Medicine.
Proliferation and Apoptosis
Pregnant dams were injected with 30 mg/g body weight BrdU (Invitrogen) 2 hours before harvesting of embryos. The embryonic kidneys were embedded in paraffin, sectioned, and coimmunolabeled with BrdU and with Six2 or Pax2. The percentage of BrdU-positive cells as a proportion of Six2- or Pax2-positive cells were counted per image. The two-tailed t test was used to determine statistical significance. TUNEL staining was done using the Apoptag Plus Fluorescein Apoptosis Detection kit (Millipore) on paraffin-embedded sectioned kidney tissues. Tissue sections were coimmunolabeled with Six2 or activated Caspase-3 and NCAM, and the percentage of TUNEL- or activated Caspase-3–positive cells as a proportion of Six2- or NCAMpositive cells, respectively, were counted as detailed above.
Histopathology and Electron Microscopy Kidneys were processed for Masson trichrome, methenamine silver– periodic acid-Schiff stain, or periodic acid-Schiff histologic staining by the Children’s Hospital of Pittsburgh of UPMC Histology Core Laboratory. Kidney tissues were processed, embedded, and visualized at the Center for Biologic Imaging, University of Pittsburgh, for electron microscopy.
Blood and Urine Measurements Urine and sera were sent to the Kansas State University Veterinary Diagnostic Laboratories, Clinical Pathology Laboratory. Urine samples MiR-17~92 in Kidney Development
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were analyzed on a Coomassie-stained SDS-PAGE gel tovisualize protein excretion. Albumin and creatinine excretion was quantified using the Albuwell murine microalbuminuria ELISA kit and Creatinine Companion kit, respectively (Exocell, Philadelphia, PA).
Immunohistochemistry E14.5, E16.5, E18.5, and P0 kidneys were embedded in paraffin and immunohistochemically labeled as outlined in the Supplemental Methods.
RT Quantitative PCR Total RNA from embryonic kidneys was isolated using the TRIzol Reagent (Invitrogen), and Taqman miRNA, primary miRNA transcript, and gene expression assays (Life Technologies) were performed to determine relative expression levels, per the manufacturer’s instructions. Expression levels were normalized to that of endogenous controls using the cycle threshold value (Cq). The two-tailed t test was used to determine statistical significance.
LNA In Situ Hybridization LNA in situ hybridization was conducted on 10-mm cryosections of E14.5 embryos using an LNA probe (Exiqon) complementary to the miR-17–5p mature miRNA, as published previously4 and as described in the Supplemental Methods.
ACKNOWLEDGMENTS This work was supported by a National Institutes of Health grant to J. H. (DK087922) and a Children’s Hospital of Pittsburgh of UPMC Research Advisory Committee startup grant. AKM was supported by a Children’s Hospital of Pittsburgh of UPMC Research Advisory Committee fellowship award. The Kidney Imaging Core of the Pittsburgh Center for Kidney Research provided expert pathological and electron microscopy support (funded by National Institutes of Health grant P30-DK079307). We thank the Children’s Hospital of Pittsburgh of UPMC Histology Core Laboratory and the Kansas State University Veterinary Diagnostic Laboratories for technical assistance. We would also like to thank Carlton Bates, Sunder Sims-Lucas, Kenneth Walker, and Valeria Di Giovanni for thoughtful discussion and review of the manuscript. The BrdU antibody developed by Stephen J. Kaufman and the 4F2 (Lim1/2) antibody developed by Thomas M. Jessel and Susan Brenner-Morton was obtained from the Developmental Studies Hybridoma Bank, which is under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biology, Iowa City, IA. This work was previously presented, in part, at the annual meeting of the American Society for Nephrology (November 7–10, 2013, Atlanta, GA) and published in abstract form.
RNA Sequencing Total RNA for high-throughput RNA sequencing was extracted using Trizol (Invitrogen). Two control and two mutant E16.5 kidneys were pooled per litter across three litters, resulting in six litter-matched biologic samples (three mutants and three controls). Library generation and 100-bp paired end high-throughput sequencing were performed at the Tuft University Genomics Core (http://TUCFGenomics.tufts.edu/) using the Illumina HiSEquation 2000 system yielding approximately 80,000,000 reads per sample. Read alignment and expression abundance quantification were performed using the tophat51 and DESeq52 programs, respectively (Gene Expression Omnibus Series accession number GSE52514; http://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE52514).
Western Blot Protein from control and mutant E16.5 kidneys was isolated, run on an SDS-PAGE gel, and transferred to a nitrocellulose membrane (BioRad). The membrane was incubated with primary antibodies against Bim and b-tubulin, and the signals were detected using horseradish peroxidase–conjugated secondary antibodies and the SuperSignal West Femto kit (ThermoScientific).
Statistical Analyses The two-tailed t test was used to determine statistical significance where applicable: *P#0.05; **P#0.01; ***P#0.001; ****P#0.0001. In graphs, the mean6SEM is presented. Open symbols in graphs represent data derived from a female animal, and closed symbols represent data derived from a male animal. All animal experiments were performed with at least four animals across three different litters.
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DISCLOSURES J.H.’s laboratory is supported in part by a Norman S. Coplon Extramural Grant from Satellite Healthcare (for work unrelated to this current manuscript).
REFERENCES 1. Smith JM, Stablein DM, Munoz R, Hebert D, McDonald RA: Contributions of the Transplant Registry: The 2006 Annual Report of the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS). Pediatr Transplant 11: 366–373, 2007 2. Little MH, McMahon AP: Mammalian kidney development: Principles, progress, and projections. Cold Spring Harb Perspect Biol 4: 4, 2012 3. Bartel DP: MicroRNAs: Target recognition and regulatory functions. Cell 136: 215–233, 2009 4. Ho J, Pandey P, Schatton T, Sims-Lucas S, Khalid M, Frank MH, Hartwig S, Kreidberg JA: The pro-apoptotic protein Bim is a microRNA target in kidney progenitors. J Am Soc Nephrol 22: 1053–1063, 2011 5. Ho J, Ng KH, Rosen S, Dostal A, Gregory RI, Kreidberg JA: Podocytespecific loss of functional microRNAs leads to rapid glomerular and tubular injury. J Am Soc Nephrol 19: 2069–2075, 2008 6. Patel V, Hajarnis S, Williams D, Hunter R, Huynh D, Igarashi P: MicroRNAs regulate renal tubule maturation through modulation of Pkd1. J Am Soc Nephrol 23: 1941–1948, 2012 7. Nagalakshmi VK, Ren Q, Pugh MM, Valerius MT, McMahon AP, Yu J: Dicer regulates the development of nephrogenic and ureteric compartments in the mammalian kidney. Kidney Int 79: 317–330, 2011 8. Harvey SJ, Jarad G, Cunningham J, Goldberg S, Schermer B, Harfe BD, McManus MT, Benzing T, Miner JH: Podocyte-specific deletion of dicer
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9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23. 24.
alters cytoskeletal dynamics and causes glomerular disease. J Am Soc Nephrol 19: 2150–2158, 2008 Shi S, Yu L, Chiu C, Sun Y, Chen J, Khitrov G, Merkenschlager M, Holzman LB, Zhang W, Mundel P, Bottinger EP: Podocyte-selective deletion of dicer induces proteinuria and glomerulosclerosis. J Am Soc Nephrol 19: 2159–2169, 2008 Sequeira-Lopez ML, Weatherford ET, Borges GR, Monteagudo MC, Pentz ES, Harfe BD, Carretero O, Sigmund CD, Gomez RA: The microRNA-processing enzyme dicer maintains juxtaglomerular cells. J Am Soc Nephrol 21: 460–467, 2010 Wei Q, Bhatt K, He HZ, Mi QS, Haase VH, Dong Z: Targeted deletion of Dicer from proximal tubules protects against renal ischemia-reperfusion injury. J Am Soc Nephrol 21: 756–761, 2010 Zhdanova O, Srivastava S, Di L, Li Z, Tchelebi L, Dworkin S, Johnstone DB, Zavadil J, Chong MM, Littman DR, Holzman LB, Barisoni L, Skolnik EY: The inducible deletion of Drosha and microRNAs in mature podocytes results in a collapsing glomerulopathy. Kidney Int 80: 719–730, 2011 Lu Y, Thomson JM, Wong HY, Hammond SM, Hogan BL: Transgenic over-expression of the microRNA miR-17-92 cluster promotes proliferation and inhibits differentiation of lung epithelial progenitor cells. Dev Biol 310: 442–453, 2007 Danielson LS, Park DS, Rotllan N, Chamorro-Jorganes A, Guijarro MV, Fernandez-Hernando C, Fishman GI, Phoon CK, Hernando E: Cardiovascular dysregulation of miR-17-92 causes a lethal hypertrophic cardiomyopathy and arrhythmogenesis. FASEB J 27: 1460–1467, 2013 Li Y, Vecchiarelli-Federico LM, Li YJ, Egan SE, Spaner D, Hough MR, Ben-David Y: The miR-17-92 cluster expands multipotent hematopoietic progenitors whereas imbalanced expression of its individual oncogenic miRNAs promotes leukemia in mice. Blood 119: 4486–4498, 2012 Conkrite K, Sundby M, Mukai S, Thomson JM, Mu D, Hammond SM, MacPherson D: miR-17~92 cooperates with RB pathway mutations to promote retinoblastoma. Genes Dev 25: 1734–1745, 2011 de Pontual L, Yao E, Callier P, Faivre L, Drouin V, Cariou S, Van Haeringen A, Geneviève D, Goldenberg A, Oufadem M, Manouvrier S, Munnich A, Vidigal JA, Vekemans M, Lyonnet S, Henrion-Caude A, Ventura A, Amiel J: Germline deletion of the miR-17;92 cluster causes skeletal and growth defects in humans. Nat Genet 43: 1026–1030, 2011 Celli J, van Bokhoven H, Brunner HG: Feingold syndrome: Clinical review and genetic mapping. Am J Med Genet A 122A: 294–300, 2003 Marcelis CL, Hol FA, Graham GE, Rieu PN, Kellermayer R, Meijer RP, Lugtenberg D, Scheffer H, van Bokhoven H, Brunner HG, de Brouwer AP: Genotype-phenotype correlations in MYCN-related Feingold syndrome. Hum Mutat 29: 1125–1132, 2008 Ventura A, Young AG, Winslow MM, Lintault L, Meissner A, Erkeland SJ, Newman J, Bronson RT, Crowley D, Stone JR, Jaenisch R, Sharp PA, Jacks T: Targeted deletion reveals essential and overlapping functions of the miR-17 through 92 family of miRNA clusters. Cell 132: 875–886, 2008 Kobayashi A, Valerius MT, Mugford JW, Carroll TJ, Self M, Oliver G, McMahon AP: Six2 defines and regulates a multipotent self-renewing nephron progenitor population throughout mammalian kidney development. Cell Stem Cell 3: 169–181, 2008 Self M, Lagutin OV, Bowling B, Hendrix J, Cai Y, Dressler GR, Oliver G: Six2 is required for suppression of nephrogenesis and progenitor renewal in the developing kidney. EMBO J 25: 5214–5228, 2006 Torres M, Gómez-Pardo E, Dressler GR, Gruss P: Pax-2 controls multiple steps of urogenital development. Development 121: 4057–4065, 1995 Kreidberg JA, Sariola H, Loring JM, Maeda M, Pelletier J, Housman D, Jaenisch R: WT-1 is required for early kidney development. Cell 74: 679–691, 1993
J Am Soc Nephrol 25: 1440–1452, 2014
BASIC RESEARCH
25. Boyle S, Shioda T, Perantoni AO, de Caestecker M: Cited1 and Cited2 are differentially expressed in the developing kidney but are not required for nephrogenesis. Dev Dyn 236: 2321–2330, 2007 26. McCright B, Lozier J, Gridley T: A mouse model of Alagille syndrome: Notch2 as a genetic modifier of Jag1 haploinsufficiency. Development 129: 1075–1082, 2002 27. Fujii T, Pichel JG, Taira M, Toyama R, Dawid IB, Westphal H: Expression patterns of the murine LIM class homeobox gene lim1 in the developing brain and excretory system. Dev Dyn 199: 73–83, 1994 28. Madisen L, Zwingman TA, Sunkin SM, Oh SW, Zariwala HA, Gu H, Ng LL, Palmiter RD, Hawrylycz MJ, Jones AR, Lein ES, Zeng H: A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13: 133–140, 2010 29. Ranji N, Sadeghizadeh M, Shokrgozar MA, Bakhshandeh B, Karimipour M, Amanzadeh A, Azadmanesh K: MiR-17-92 cluster: An apoptosis inducer or proliferation enhancer. Mol Cell Biochem 380: 229–238, 2013 30. Cloonan N, Brown MK, Steptoe AL, Wani S, Chan WL, Forrest AR, Kolle G, Gabrielli B, Grimmond SM: The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition. Genome Biol 9: R127, 2008 31. Yin R, Bao W, Xing Y, Xi T, Gou S: MiR-19b-1 inhibits angiogenesis by blocking cell cycle progression of endothelial cells. Biochem Biophys Res Commun 417: 771–776, 2012 32. Patel V, Williams D, Hajarnis S, Hunter R, Pontoglio M, Somlo S, Igarashi P: miR-17~92 miRNA cluster promotes kidney cyst growth in polycystic kidney disease. Proc Natl Acad Sci U S A 110: 10765–10770, 2013 33. Fontana L, Fiori ME, Albini S, Cifaldi L, Giovinazzi S, Forloni M, Boldrini R, Donfrancesco A, Federici V, Giacomini P, Peschle C, Fruci D: Antagomir17-5p abolishes the growth of therapy-resistant neuroblastoma through p21 and BIM. PLoS ONE 3: e2236, 2008 34. Wong P, Iwasaki M, Somervaille TC, Ficara F, Carico C, Arnold C, Chen CZ, Cleary ML: The miR-17-92 microRNA polycistron regulates MLL leukemia stem cell potential by modulating p21 expression. Cancer Res 70: 3833–3842, 2010 35. Gibcus JH, Kroesen BJ, Koster R, Halsema N, de Jong D, de Jong S, Poppema S, Kluiver J, Diepstra A, van den Berg A: MiR-17/106b seed family regulates p21 in Hodgkin’s lymphoma. J Pathol 225: 609–617, 2011 36. Roselli S, Gribouval O, Boute N, Sich M, Benessy F, Attié T, Gubler MC, Antignac C: Podocin localizes in the kidney to the slit diaphragm area. Am J Pathol 160: 131–139, 2002 37. Vaughan MR, Quaggin SE: How do mesangial and endothelial cells form the glomerular tuft? J Am Soc Nephrol 19: 24–33, 2008 38. Bejarano F, Bortolamiol-Becet D, Dai Q, Sun K, Saj A, Chou YT, Raleigh DR, Kim K, Ni JQ, Duan H, Yang JS, Fulga TA, Van Vactor D, Perrimon N, Lai EC: A genome-wide transgenic resource for conditional expression of Drosophila microRNAs. Development 139: 2821–2831, 2012 39. Kloosterman WP, Lagendijk AK, Ketting RF, Moulton JD, Plasterk RH: Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development. PLoS Biol 5: e203, 2007 40. Smibert P, Lai EC: Lessons from microRNA mutants in worms, flies and mice. Cell Cycle 7: 2500–2508, 2008 41. Miska EA, Alvarez-Saavedra E, Abbott AL, Lau NC, Hellman AB, McGonagle SM, Bartel DP, Ambros VR, Horvitz HR: Most Caenorhabditis elegans microRNAs are individually not essential for development or viability. PLoS Genet 3: e215, 2007 42. Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, Wang J, Henderson JM, Kutok JL, Rajewsky K: Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol 9: 405–414, 2008 43. Inomata M, Tagawa H, Guo YM, Kameoka Y, Takahashi N, Sawada K: MicroRNA-17-92 down-regulates expression of distinct targets in different B-cell lymphoma subtypes. Blood 113: 396–402, 2009
MiR-17~92 in Kidney Development
1451
BASIC RESEARCH
www.jasn.org
44. Olive V, Jiang I, He L: mir-17-92, a cluster of miRNAs in the midst of the cancer network. Int J Biochem Cell Biol 42: 1348–1354, 2010 45. Waga S, Hannon GJ, Beach D, Stillman B: The p21 inhibitor of cyclindependent kinases controls DNA replication by interaction with PCNA. Nature 369: 574–578, 1994 46. Brugarolas J, Moberg K, Boyd SD, Taya Y, Jacks T, Lees JA: Inhibition of cyclin-dependent kinase 2 by p21 is necessary for retinoblastoma protein-mediated G1 arrest after gamma-irradiation. Proc Natl Acad Sci U S A 96: 1002–1007, 1999 47. Kavurma MM, Khachigian LM: Sp1 inhibits proliferation and induces apoptosis in vascular smooth muscle cells by repressing p21WAF1/ Cip1 transcription and cyclin D1-Cdk4-p21WAF1/Cip1 complex formation. J Biol Chem 278: 32537–32543, 2003 48. LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E: New functional activities for the p21 family of CDK inhibitors. Genes Dev 11: 847–862, 1997
1452
Journal of the American Society of Nephrology
49. Tsuchida A, Ohno S, Wu W, Borjigin N, Fujita K, Aoki T, Ueda S, Takanashi M, Kuroda M: miR-92 is a key oncogenic component of the miR-17-92 cluster in colon cancer. Cancer Sci 102: 2264–2271, 2011 50. Moeller MJ, Sanden SK, Soofi A, Wiggins RC, Holzman LB: Podocytespecific expression of cre recombinase in transgenic mice. Genesis 35: 39–42, 2003 51. Trapnell C, Pachter L, Salzberg SL. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25: 1105–1111, 2009 52. Anders S, Huber W. Differential expression analysis for sequence count data. Genome Biol 11: R106, 2010
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