Animal Models Of Human Disease–Original Article
Nephronophthisis and Retinal Degeneration in Tmem218–/– Mice: A Novel Mouse Model for Senior-Løken Syndrome?
Veterinary Pathology 2015, Vol. 52(3) 580-595 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985814547392 vet.sagepub.com
P. Vogel1, C. M. Gelfman2, T. Issa2, B. J. Payne1, G. M. Hansen3, R. W. Read1, C. Jones2, M. R. Pitcher2, Z.-M. Ding4, C. M. DaCosta4, M. K. Shadoan4, R. B. Vance1, and D. R. Powell4
Abstract Mice deficient in TMEM218 (Tmem218–/–) were generated as part of an effort to identify and validate pharmaceutically tractable targets for drug development through large-scale phenotypic screening of knockout mice. Routine diagnostics, expression analysis, histopathology, and electroretinogram analyses completed on Tmem218–/– mice identified a previously unknown role for TMEM218 in the development and function of the kidney and eye. The major observed phenotypes in Tmem218–/– mice were progressive cystic kidney disease and retinal degeneration. The renal lesions were characterized by diffuse renal cyst development with tubulointerstitial nephropathy and disruption of tubular basement membranes in essentially normal-sized kidneys. The retinal lesions were characterized by slow-onset loss of photoreceptors, which resulted in reduced electroretinogram responses. These renal and retinal lesions are most similar to those associated with nephronophthisis (NPHP) and retinitis pigmentosa in humans. At least 10% of NPHP cases present with extrarenal conditions, which most often include retinal degeneration. Senior-Løken syndrome is characterized by the concurrent development of autosomal recessive NPHP and retinitis pigmentosa. Since mutations in the known NPHP genes collectively account for only about 30% of NPHP cases, it is possible that TMEM218 could be involved in the development of similar ciliopathies in humans. In reviewing all other reported mouse models of NPHP, we suggest that Tmem218–/– mice could provide a useful model for elucidating the pathogenesis of cilia-associated disease in both the kidney and the retina, as well as in developing and testing novel therapeutic strategies for Senior-Løken syndrome. Keywords Senior-Løken, nephronophthisis, cystic kidney disease, retina, retinal degeneration, retinitis pigmentosa, hypertension
To identify novel genes coding for pharmaceutically relevant disease targets, Lexicon Pharmaceuticals evaluated more than 4650 knockout mouse lines in a high-throughput mutagenesis and phenotyping program.124,125 Coincidentally, many disease phenotypes were discovered during this process,100 and some of these have proven useful in elucidating fundamental processes in biology or in determining the underlying pathogenesis of heritable diseases.69,75,76,79,102,104–111 Not surprising, a large proportion of pathologic phenotypes were linked to inactivation of genes involved in the regulation of complicated and tightly controlled processes (eg, embryologic development and immunity). Many other pathologic phenotypes resulted from abnormal development of structurally complex tissues/organs (eg, eyes, teeth, sperm).2,86 Over time, it also became clear that numerous pathologic phenotypes in knockout mice were associated with the inactivation of genes involved in the biogenesis and/or function of the extremely complex multifunctional organelles known as cilia.22 Recognition that polycystin 1, polycystin 2, and nephrocystin proteins could all be localized to the primary cilium/
basal body/centrosome indicated the involvement of dysfunctional primary cilia in the pathogenesis of polycystic kidney disease (PKD) and nephronophthisis (NPHP).62,68 Presently, mutations in more than 30 cilia-related genes have been linked to cystic kidney diseases.40 Most likely due to the nearly ubiquitous distribution of cilia and their involvement in several critical developmental
1 Department of Pathology, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA 2 Department of Ophthalmology, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA 3 Department of Molecular Genetics, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA 4 Department of Metabolism, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA
Corresponding Author: Peter Vogel, Department of Pathology, Lexicon Pharmaceuticals Inc., 262 Danny Thomas Place, MS 250, Memphis, TN 38105-3678, USA. Email: [email protected]
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pathways, defective ciliary proteins have been linked to a large number of diseases collectively called ciliopathies (reviewed by Yildiz et al120). Multiorgan involvement is common with ciliopathies, and the most frequently observed extrarenal lesions in NPHP include retinopathies (15%) and cerebellar ataxia (10%–15%).37 The diagnosis applied to most patients showing both NPHP and retinal degeneration is Senior-Løken syndrome (SLSN).50,88 The recognition that the mutated genes in patients with SLSN coded for proteins that were localized to the cilia/centrosome led to the classification of SLSN as a ciliopathy.35,55,62,64,65,82 Transmembrane protein 218 (TMEM218) is a small 115 amino acid protein of unknown function. TMEM218 is evolutionarily well conserved, with 81% amino acid identity (92% similarity) between mouse and humans. The location of the TMEM218 gene in humans (11q24.2) does not map to any known disease locus, and we did not investigate any possible associations between Tmem218 and human disease in this study. To the best of our knowledge, the only previous mentions of TMEM218 in the scientific literature are that its expression is downregulated in testicular feminized mice60 and upregulated in confluent cultures of NIH3T3 fibroblasts.46 We did not investigate the molecular functions of TMEM218 or any of the underlying mechanisms involved in the pathogenesis of progressive NPHP and retinal degeneration in Tmem218–/– mice. Nevertheless, the lesions that develop in the kidneys and eyes of TMEM218-null mice (Tmem218Gt(OST40451)Lex; abbreviated as Tmem218–/– in this report) indicate that TMEM218 plays an important role in maintaining normal structure and function in the kidney and retina of mice. Furthermore, the concurrent development of progressive cystic kidney disease and retinal degeneration suggests that TMEM218 is involved in ciliary biogenesis or function. The Tmem218–/– mouse should provide a useful model for investigating the role of TMEM218 in normal ciliary functions and developmental processes, as well as in elucidating molecular mechanisms involved in the pathogenesis and progression of cilia-associated disease in both the kidney and the retina. In addition, the Tmem218–/– mouse might be used in the development and testing of novel therapeutic approaches to the prevention and treatment of the renal and retinal ciliopathies seen in SLSN.
Materials and Methods Mouse Production For the sake of brevity, the symbol for mice homozygous for this gene-trapped allele—Tmem218Gt(OST40451)Lex—has been abbreviated as Tmem218–/– in this report. The mutant mice used in this study were produced by Lexicon Pharmaceuticals Inc. (The Woodlands, TX) but are now distributed through Taconic Farms Inc. (New York City, NY). The methods used for gene trapping in embryonic stem cells, identification of trapped genes using OmniBank sequence tags (OSTs), characterization of retroviral gene trap vector insertion site, and reverse transcription polymerase chain reaction (PCR) analysis
of knockout and wild-type transcripts are published.122 Gene trapping was performed with strain 129S5SvEvBrd-derived embryonic stem cells obtained from the OmniBank library.123 OST40451 was selected from the OmniBank library based on a BLAST search of all available sequence tags using the Tmem218 genomic interval as a query. The mutation in this clone was confirmed to be intergenic according to inverse genomic PCR, as previously described.31 Briefly, oligonucleotide primers complementary to the gene trap vector were used to amplify the vector insertion site from clone OST40451, which was then compared to mouse genome sequence assemblies to localize the insertion within intron 1 of the Tmem218 gene (Fig. 1a). The gene-trapped embryonic stem cell clone was microinjected into C57BL/6-Tyrc–Brd(albino) blastocysts to generate chimeric animals that were bred to C57BL/6-Tyrc–Brd(albino) females, and the resulting heterozygous offspring were interbred to produce homozygous gene-deficient mice. The knockout F2 mice used in phenotyping studies were produced by intercrossing the F1 heterozygous knockout (–/þ) offspring of chimeric founder parents and were therefore of mixed C57BL and 129 genetic background. Using the albino variant of C57BL/6 mice (C57BL/6-Tyrc–Brd) permits simple visual recognition of chimeric offspring, because they have dark eyes and patches of dark hair that derive from stem cells from the agouti 129S5/SvEvBrd. Genotypes of offspring were determined by quantitative PCR as previously described.31 Briefly, DNA isolated from tail biopsy samples was assayed by quantitative PCR for the neo gene, which is present in VICTR24, the gene-trapping vector used to generate the mutation described in this study. Gene disruption in vivo was confirmed by a direct analysis of gene expression based on reverse transcription PCR. RNA was extracted from kidney and spleen of wild-type and homozygous mutant mice via a bead homogenizer and RNAzol (Ambion, Austin, TX) according to manufacturer’s instructions. Reverse transcription was performed with SuperScript II (Invitrogen, Carlsbad, CA) and random hexamer primers according to the manufacturer’s instructions. PCR amplification was performed with oligonucleotide primers complementary to exons flanking the insertion site (Fig. 1b). The primers used were F: 50 -GGAACCTGAGAGCCGGGTGAGGA-30 and R: 50 - CAGAGCTCCGAGGAAGACAAAGA-30 , are complementary to Tmem218 exons 1 and 3, respectively, and amplify a product of 196 nucleotides. Reverse transcription PCR analysis based on actb primers (50 -GGCTGGCCGGGACCTGACGGACTACCTCAT-30 and 50 - GCCTAGAAGCACTTGCGGTGCACGATGGAG-30 ) complementary to the mouse beta actin gene (accession M12481) with an expected product size of 591 bp was performed in the same reaction as an internal amplification control. In all studies reported here, mutant mice were compared directly with their wild-type littermates used as negative controls.
Mouse Husbandry Mice were housed in microisolator cages within a barrier facility at 24 C on a fixed 12-hour light and 12-hour dark cycle and
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Veterinary Pathology 52(3) prepulse inhibition of the acoustic startle response, tail suspension, marble burying, and context trace conditioning), fundoscopy and retinal angiography exams, blood pressure and heart rate measurements, serum chemistries, insulin levels, glucose tolerance testing, urinalysis, quantitative magnetic resonance, dual-energy x-ray absorptiometry scans, computerized axial tomography scans, micro–computed tomography scans, fertility testing, skin fibroblast proliferation assays, and pathology. Immunology assays included hematology, peripheral blood FACS analysis, acute phase response, and ovalbumin challenge.
Histopathology Figure 1. Generation of Tmem218Gt(OST40451)Lex mice. (a) Retroviral gene trap vector VICTR24 was used to produce OmniBank clone OST40451 (accession CG498846.1), which contains an insertion within intron 1 of the Tmem218 gene. This mutation would be expected to interrupt endogenous transcription within the 50 UTR, preventing transcription and translation of the protein. Open boxes denote untranslated exons; filled boxes denote coding exons. LTR, viral long terminal repeat; SA, splice acceptor sequence; bGEO, fusion of the beta-galactosidase and the neomycin phosphotransferase genes; pA, polyadenylation sequence; Pgk, phosphoglycerate kinase 1 promoter; Btk-SD, Bruton’s tyrosine kinase splice donor sequence. (b) Reverse transcription polymerase chain reaction expression analysis of Tmem218 transcript. Endogenous Tmem218 transcript was detected in the kidney and spleen of wildtype (þ/þ) mice. No endogenous Tmem218 transcript was detected in homozygous (–/–) tissues. The primers used were F: 50 -GGAACCTGAGAGCCGGGTGAGGA-30 and R: 50 -CAGAGCTCCGAGGAAGACAAAGA-30 , which are complementary to Tmem218 exons 1 and 3, respectively, and amplify a product of 196 nucleotides. Reverse transcription polymerase chain reaction analysis using actb primers (50 GGCTGGCCGGGACCTGACGGACTACCTCAT-30 and 50 -GCCTAGAAGCACTTGCGGTGCACGATGGAG-30 ) complementary to the mouse beta actin gene (accession M12481) with an expected product size of 591 bp was performed in the same reaction as an internal amplification control. M, molecular weight marker.
were provided ad libitum acidified water and Purina rodent chow (5001, Purina, St Louis, MO). Procedures involving animals were conducted in conformance with Lexicon Pharmaceuticals’ Institutional Animal Care and Use Committee guidelines, which are in compliance with state and federal laws and the standards outlined in the National Research Council’s Guide for the Care and Use of Laboratory Animals (2011). Quarterly sentinel surveillance showed no evidence of pathogenic rodent viruses, Mycoplasma sp, or Helicobacter sp in the Lexicon Pharmaceuticals source colonies.
Phenotype Screening Wild-type and homozygous-null mice were subjected to a comprehensive battery of phenotype screening exams as previously described.9,122 These screening assays included behavioral tests (eg, circadian rhythm, open field, inverted screen,
Age-matched knockout and wild-type control mice (2 male and 2 female of each genotype) were euthanized at 7 and 14 weeks of age and fixed by cardiac perfusion with 10% neutral buffered formalin. Lungs were infused with formalin via the trachea, and all tissues were immersed in 10% neutral buffered formalin for an additional 48 hours, except for the eyes, which were removed and fixed by immersion in Davidson’s fixative (Poly Scientific, NY) overnight at room temperature. All tissues (to include heart, skeletal muscle, tongue, lungs, trachea, thyroid gland, liver, kidney, adrenal gland, salivary glands, lymph nodes, white adipose, brown adipose, aorta, thymus, spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder, skin, brain, eyes, nose, teeth, ear, bone, bone marrow, testes, epididymis, prostate gland, seminal vesicles, vas deferens, uterus, ovaries) were embedded in paraffin, sectioned at 4 mm, mounted on positively charged glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA), and stained with hematoxylin and eosin for histopathologic examination. A full set of tissues from a pair (1 male and 1 female) of 14-week-old heterozygote mice was similarly collected and examined. Additional histopathologic evaluations were conducted on kidneys and eyes only from at least 2 age-matched knockout mice and 1 control mouse collected at several other time points (kidneys at 9, 14, 17, 29, and 58 weeks of age; eyes at 4, 8, 12, and 42 weeks of age). Kidney sections were stained with Masson’s trichrome and periodic acid–Schiff to highlight interstitial fibrosis and basement membrane changes respectively.
b-Galactosidase (LacZ) Staining The b-galactosidase reporter gene (b-geo) was under transcriptional control of the Tmem218 regulatory elements, allowing localization of the expression pattern of Tmem218 in tissues of homozygous or heterozygous mutant mice by X-gal staining. b-Galactosidase reporter gene activity was assessed in situ as previously described.111 Briefly, 2 heterozygous mice and 2 homozygous mice were anesthetized and perfused sequentially with b-Gal fixative (0.2% glutaraldehyde, 1.5% paraformaldehyde, 2 mM MgCl2, 5 mM EGTA [ethylene glycol tetraacetic acid], 100 mM sodium phosphate [pH 7.3]), followed by 2 ml of b-Gal rinse (0.2% Nonidet-P40, 0.1% sodium deoxycholate, 2 mM MgCl2,100 mM sodium phosphate), and finally 10 ml of
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b-Gal stain (5 mM K3Fe(CN)6 [potassium ferricyanide], 5 mM K4Fe(CN)6 [potassium ferrocyanide], 1 mg of 5-bromo-4chloro-3-indolyl-D galactopyranoside [X-Gal; dissolved in dimethylformamide] per ml, 0.2% Nonidet-P40, 0.1% sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate [pH 7.3]). Tissues were postfixed in b-Gal fix, rinsed in the b-Gal rinse, and then incubated in b-Gal stain solution for 48 hours. After 3 additional washes in b-Gal rinse, the tissues were postfixed in Bouin’s fixative before dehydration and embedding in paraffin. Tissues examined included heart, skeletal muscle, trachea, lung, thyroid gland, parathyroid gland, liver, kidney, adrenal gland, salivary glands, lymph nodes, thymus, spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder, skin, brain, eyes, nose, teeth, ear, bone marrow, testes, epididymis, prostate gland, seminal vesicles, vas deferens, uterus, and ovaries. Sections were cut at 4 mm and counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA).
Blood Pressure Measurements Systolic blood pressures were measured with a noninvasive tail cuff method on a Visitech BP-2000 blood pressure analysis system (Visitech Systems, Apex, NC). Systolic pressure and heart rate were recorded in conscious mice (8 homozygotes, 8 wild type, and 8 heterozygotes at 10 weeks of age) 10 times each day for 4 days, and all readings were averaged for each mouse.
Clinical Chemistry and Renal Function Serum and urine chemistry parameters were measured with a Cobas Integra 400 autoanalyzer (Roche Diagnostics, Indianapolis, IN). For urine studies, 24-hour urine samples were collected for 3 consecutive days from mice individually housed in metabolic cages (product MTB-0311; Nalgene, Rochester, NY) and acclimatized for 3 days prior to the start of urine collection.
Electroretinography Electroretinograms (ERGs) were recorded from wild-type (n ¼ 5), heterozygous (n ¼ 5), and homozygous (n ¼ 5) mice at 2 and 6 months of age. All procedures were carried out under dim red light (>650 nm). ERGs from both eyes were recorded simultaneously with the UTAS-E 3000 Visual Electrodiagnostic System (LKC Technologies, Gaithersburg, MD). After an overnight dark-adaptation period, the pupil was dilated with 0.1% atropine/phenylephrine (Alcon Laboratories Inc., Fort Worth, TX), and 0.5% proparacaine (Alcon) was applied for topical anesthesia. Mice were deeply anesthetized with (per milliliter) ketamine (7.5 mg), xylazine (0.38 mg), and acepromazine (0.074 mg), delivered at 10 ml/kg body weight. Silver electrodes were placed on the corneal surface with a drop of methylcellulose. A reference electrode was placed subcutaneously on the head, and a ground
electrode was placed in the right hind leg. The mice were placed in a Ganzfeld illumination dome. Full-field scotopic ERGs of both eyes were elicited simultaneously with 10-ms light flashes. For a- and b-wave ERGs, 5 recordings per flash intensity (0.006, 0.04, and 24 cds/m2) were averaged. Mixed rod-cone (scotopic)–driven responses to light flashes were recorded, with the intervals between flashes increasing from 10 to 30 seconds, with an increasing flash intensity range (0.0094–40.41 cd sec/mm2).
Results Phenotype Screening At the time of genotyping (3 weeks of age), the number of surviving knockout mice (188 wild type, 331 heterozygote, 145 homozygote) was slightly below expected Mendelian ratios (w2 ¼ 5.58, P ¼ .0614). Although young Tmem218–/– mice were indistinguishable from wild-type littermates upon macroscopic examination and showed normal growth rates (length and weight) between 2 and 16 weeks, they tended to have lower than normal body weight at 19 weeks (Table 1). At 17 weeks, Tmem218–/– mice also showed trends toward higher serum creatinine and blood urea nitrogen compared to Tmem218þ/– and Tmem218þ/þlittermates, suggesting the presence of impaired kidney function; these findings were accompanied by modest but significant increases in serum chloride, calcium, and alkaline phosphatase (Table 2). Although 24-hour urine volume and osmolality were not significantly different in Tmem218–/– mice, correlations between serum creatinine and 24-hour urine volume (r ¼ 0.62, P < .05) and between serum creatinine and urine osmolality (r ¼ –0.71, P < .05) in Tmem218–/– mice suggest that urinary concentrating ability became compromised after the onset of renal failure. At 14 weeks, Tmem218–/– mice also showed a trend toward higher systolic blood pressure compared to Tmem218þ/þ littermates; this finding, confirmed by studying additional older mice (Table 3), was more pronounced in males and correlated with serum creatinine (r ¼ 0.76, P < .01). Overall, the data suggest that the elevated systolic blood pressure was likely secondary to renal failure. Interestingly, fundoscopy and retinal angiography exams were normal in baseline screens performed at 8 weeks of age. In other phenotyping assays, Tmem218–/– mice showed normal results in behavioral tests (eg, circadian rhythm, open field, inverted screen, prepulse inhibition of the acoustic startle response, tail suspension, marble burying, and context trace averse conditioning). In screening assays, insulin levels, glucose tolerance testing, urinalysis, quantitative magnetic resonance, dual-energy x-ray absorptiometry scans, computerized axial tomography scans, micro–computed tomography scans, and skin fibroblast proliferation assays were also within normal limits. Immunologic assays included hematology, peripheral blood FACS analysis, acute phase response, and ovalbumin challenge, and standard hematology workups were unremarkable.
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Table 1. Body Weight and Urine Measurements: 19-Week-Old Mice. Micea Metabolic Cages
WT
P
LacZ Staining Results
Body weight, g Males Females 24-hour urine Volume, ml Creatinine, mg Osmolality, mOsm/kg H2O
KO
in any other tissue of homozygous mice. Similarly, no lesions were noted in any tissue in a single pair of heterozygote mice examined at 14 weeks of age.
38 + 2 (4)
25 + 5 (8)
25 + 2 (4)
22 + 9 (8)
1.3 + 0.9 (8) 2.2 + 1.7 (16) 413 + 116 (8) 338 + 89 (16) 2208 + 759 (8) 1877 + 1354 (16)
KO < WT, P < .01 ns ns ns ns
Abbreviations: KO, knockout; ns, not significant; WT, wild type. a Mean + SEM (n).
Pathology Findings Situs inversus was not observed in any knockout mice, and both males and females were fertile. The key histologic findings in Tmem218–/– mice were in the kidneys and eyes. Tmem218–/– mice had variable numbers of cysts at all ages examined, but the kidneys were generally of normal or slightly reduced size compared to wild-type mice (Figs. 2–4). A few cases displayed sporadic or scattered cysts in the cortex (Fig. 3), but severe cyst formation was present in most mice of the same age (Fig. 4). Histologically, the lesions were characterized by tubular atrophy and tubulointerstitial inflammatory cell infiltrates (Fig. 5), with interstitial fibrosis (Fig. 6) and disruption, thickening, and splitting of tubular basement membranes (Fig. 7). Although renal cyst formation could be extensive in young mice, evaluation of kidney sections taken at various time points demonstrated that cortex, inner medulla, and outer medulla were becoming progressively cystic and atrophic (not shown). Diffuse tubulointerstitial cell infiltration with interstitial fibrosis and deposition of collagen throughout the kidney, along with tubular basement membrane disintegration with tubular atrophy and tubular cysts, plus scattered glomerular cysts, were present diffusely at 14 weeks of age. Lesions in the eyes of Tmem218–/– mice were characterized by slowly progressive retinal degeneration. At 9 weeks, routine histologic sections from wild-type (Fig. 8) and homozygous retinas (Fig. 9) were indistinguishable, with similar thickness and cellularity of the outer and inner nuclear layers as well as the retinal ganglion cell layer. However, by 14 weeks of age, subtle photoreceptor loss, as evidenced by a thinning of the outer nuclear layers, became evident, and by 4 months, there was diffuse moderate thinning of the outer nuclear layer in Tmem218–/– mice that often became pronounced by 29 weeks (Figs. 10, 11). Degeneration of the photoreceptor cells was demonstrated by diffuse thinning of the outer nuclear layer and photoreceptor inner and outer segments (OSs). In contrast, the inner retina of these animals appeared to be intact, with apparently normal inner nuclear and ganglion cell layers. No notable lesions were detected
Tmem218 gene promoter expression was analyzed in Tmem218–/– mice by using b-galactosidase enzyme histochemistry. In Tmem218–/– mice, specific X-gal staining representative of TMEM218 expression was observed in several cell types: These included renal epithelium (Figs. 12, 13), retina (Fig. 14), and a variety of ciliated/flagellated cells (respiratory epithelium, Fig. 15; ependymal and choroid plexus, Fig. 16); vas deferens, epididymis, and spermatids, Fig. 17). In addition, several different endocrine cells (pancreatic islets, pituitary gland, adrenal medulla, parathyroid gland, C-cells of thyroid gland) were stained positively (data not shown). Although the intensity of staining was generally lower in heterozygote mice, the positive tissues and cell types were identical (data not shown).
ERG Results The gradual and progressive loss of photoreceptors was accompanied by a functional deficit, as demonstrated by ERG results at 2 and 6 months of age (Figs. 18–21). At 2 months of age, Tmem218–/– mice had statistically significant reductions in both a-wave (Fig. 18) and b-wave (Fig. 20) ERG responses at only the highest (24 cd.s/m2) of the light exposures tested (0.006, 0.04, and 24 cd.s/m2). By 6 months of age, the progressive loss of vision in Tmem218–/– mice was demonstrated by statistically significant reductions in a-wave ERG responses (Fig. 19) at all 3 light intensities tested (0.006, 0.04, and 24 cd.s/m2) as well as reductions in b-wave responses (Fig. 21), although these differences were statistically significant only at 24 cd.s/m2).
Discussion The 2 striking pathologic phenotypes that we observed in Tmem218–/– mice included PKD culminating in renal failure and progressive retinal degeneration resulting in blindness. Retinopathies are the most common extrarenal lesions associated with NPHP, being present in 10% to 15% of human patients.50,88 Other relatively common extrarenal findings with NPHP in humans that were not observed in Tmem218–/– mice include cerebellar vermis hypoplasia (Joubert’s syndrome [JBTS]) in 10% to 15% of cases and hepatic fibrosis in 5%.37,83 Other syndromes that may have concurrent NPHP include congenital ocular motor apraxia type Cogan, cognitive impairment, phalangeal cone-shaped epiphyses in MainzerSaldino syndrome, Bardet-Biedl syndrome, Ellis-van Creveld syndrome, Jeune asphyxiating thoracic dystrophy, Alstro¨m syndrome, and Meckel-Gruber syndrome (MKS).1,32,33,83,92 Given the low penetrance of many of these phenotypes and the small number of mice and limited time points that we
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Table 2. Serum Chemistries: 17–18 Weeks. Micea P WT Glucoseb Sodiumc Chloridec Potassiumc Calciumb Phosphorusb Blood urea nitrogenb Creatinineb Cholesterolb Triglyceridesb ALTd Total bilirubinb Alkaline phosphatased
186 149 111 5.5 9.5 6.1 19 0.12 133 138 40 0.33 83
HET
+ 30 (27) + 2 (27) + 1 (27) + 0.7 (27) + 0.4 (27) + 0.9 (27) + 4 (27) + 0.03 (27) + 47 (27) + 68 (27) + 36 (27) + 0.12 (27) + 38 (27)
186 + 150 + 112 + 5.6 + 9.6 + 6.0 + 20 + 0.12 + 143 + 130 + 34 + 0.29 + 78 +
KO
33 (24) 3 (24) 2 (24) 0.6 (24) 0.2 (24) 1.0 (24) 3 (24) 0.03 (24) 44 (24) 50 (24) 16 (24) 0.10 (24) 21 (24)
186 + 151 + 114 + 5.6 + 10.1 + 6.3 + 45 + 0.26 + 139 + 106 + 27 + 0.25 + 117 +
40 (28) 3 (28) 3 (28) 0.6 (28) 0.6 (28) 1.2 (28) 35 (28) 0.21 (28) 30 (28) 38 (28) 6 (28) 0.10 (28) 44 (28)
ns ns KO > HET þ WT, P < .001 ns KO > HET þ WT, P < .001 ns KO > HET þ WT, P < .001 KO > HET þ WT, P < .001 ns ns ns KO < WT, P < .05 KO > HET þ WT, P < .01
Abbreviations: HET, heterozygote; KO, knockout; ns, not significant; WT, wild type. a Mean + SEM (n). b mg/dl. c mmol/liter. d U/liter.
Motile Cilia: Primary Ciliary Dyskinesia
Table 3. Systolic Blood Pressure, mm Hg. Micea Weeks 14–20 Males Females Combined 23–25 Males Females Combined
WT
KO
P
108 + 7 (10) 117 + 14 (6) KO > WT, P < .05 109 + 10 (10) 109 + 9 (6) ns 108 + 7 (20) 114 + 13 (12) KO > WT, P < .001 113 + 12 (15) 137 + 27 (7) KO > WT, P < .01 123 + 18 (8) 138 + 23 (10) ns 117 + 15 (23) 138 + 24 (17) KO > WT, P < .01
Abbreviations: KO, knockout; ns, not significant; WT, wild type. a Mean + SEM (n).
examined, it is possible that some of these lesions could be linked to TMEM218 deficiency in mice in the future. In our experience, the appearance of very similar phenotypes (phenocopies) in either mice or humans with mutations in different genes often indicates that the proteins encoded by these mutant genes involve different steps in a common metabolic pathway or affect the same process or structure.86,106–109 It is often instructive to consider what is known about the underlying pathogenic mechanisms in phenocopies (whether in humans or in animal models) when attempting to understand the pathogenesis of lesions in a novel line of knockout mice. We believe that the simultaneous development of NPHP and retinal degeneration in Tmem218–/– mice is highly suggestive of an underlying defect in the structure/function of the sensory primary cilium of renal epithelium and the connecting cilium of retinal photoreceptors, respectively. Due to the highly conserved nature of most known ciliary genes, we believe that it is possible that TMEM218 will be linked to human ciliopathies in the future.
Cilia are highly conserved structurally complex organelles composed of 9 microtubule doublets (called the axoneme) that emanate from basal bodies, which are the microtubule-organizing centers of the cell. There are 2 types of cilia: motile cilia that are necessary for locomotion and fluid movement and primary (immotile) cilia that serve mainly sensory functions. Either motile cilia or immotile primary cilia are present on most cell types, and as a result of this wide distribution, it is possible for defective cilia to affect a very wide range of tissues and organ systems. In fact, mutations in genes whose products have been localized to the cilium–centrosome complex have in recent years been linked to a wide range of monogenic developmental and degenerative disorders now recognized as ciliopathies.33 Dysfunctional motile cilia and flagella were first recognized as being the cause of a group of disorders (usually autosomal recessive) now classified as primary ciliary dyskinesias (PCD).3 Normally, motile flagella and cilia in the upper and lower respiratory tract, middle ear, reproductive tract, and brain create fluid flow over cell surfaces.93 Respiratory tract cilia help clear respiratory secretions in the airways, and when mucociliary clearance is impaired in PCD,25 patients can develop chronic otitis media, rhinitis, sinusitis, and recurrent infections of the lower respiratory tract.29,54 Impaired ependymal flow also appears to contribute to the development of hydrocephalus in some humans13 and to the randomization of left/right body asymmetry caused by dysfunctional cilia in the embryonic node during early development. Laterality defects (situs inversus) occur in approximately 50% of PCD patients.3 Similarly, the deletion or mutation of genes linked to motile cilia in mice have been linked to the development of chronic upper respiratory tract infections, hydrocephalus, infertility, and/or laterality defects.105,108,109
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Figure 2. Normal kidney; 14-week-old Tmem218þ/þ mouse. Normal-sized kidney. HE. Figure 3. Polycystic kidney, 14-week-old Tmem218–/– mouse. Scattered renal cysts are evident in both the cortex and the medulla of normal-sized kidney. HE. Figure 4. Nephronophthisis, 14-weekold Tmem218–/– mouse. There is diffuse cyst formation in an essentially normal-sized kidney. HE. Figure 5. Nephronophthisis, 14-week-old Tmem218–/– mouse. Renal cysts are accompanied by tubular atrophy and tubulointerstitial inflammatory cell infiltrates and with interstitial fibrosis. HE. Figure 6. Nephronophthisis, 14-week-old Tmem218–/– mouse. Extensive interstitial fibrosis is associated with cystic lesions. Masson’s trichrome stain. Figure 7. Nephronophthisis, 14-week-old Tmem218–/– mouse. There is widespread disruption, thickening, and splitting of tubular basement membranes. Periodic acid–Schiff. Figure 8. Retina, 9-week-old Tmem218þ/þ. Normal thickness of all retinal layers. HE. Figure 9. Retina, 9-week-old Tmem218–/– mouse. All retinal layers were indistinguishable from wild-type retinas in terms of thickness and cellularity of the outer and inner nuclear layers as well as the retinal ganglion cell layer. HE. Figure 10. Retina, 29-week-old Tmem218þ/þ. Normal thickness of mouse retina. Note that external nuclear layer is normally thicker than inner nuclear layer. HE. Figure 11. Retinopathy, 29-week-old Tmem218–/– mouse. There is pronounced thinning of the retina due to diffuse thinning of the outer nuclear layer and photoreceptor inner and Downloaded from vet.sagepub.com at QUEENS UNIV LIBRARIES on October 4, 2015
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Primary/Immotile Cilia: Renal Ciliopathies For many decades after its discovery, it was thought that the immotile primary cilium was a purely vestigial organelle. However, in recent years, many crucial roles for primary cilia in development and physiology have been reported.12 In contrast to the well-defined and easily understood pathogenic mechanisms responsible for the lesions associated with dysfunctional motile cilia, defects in immotile primary cilia, which have a range of different sensory functions,10 can be associated with a remarkably broad spectrum of pleiotropic phenotypes. These include PKD, NPHP, retinitis pigmentosa, anosmia, ataxia, cardiac defects, liver fibrosis, and obesity, among many others.33 Of these phenotypes, the PKDs were the first to be linked definitively to defects in primary immotile cilia, and many genes have since been linked to the development of cystic kidney diseases (reviewed in Loftus and Ong49). The most common and clinically significant type of PKD in humans is inherited as an adult-onset autosomal dominant trait (ADPKD). Other less common hereditary renal cystic diseases include autosomal recessive PKDs and adult-onset autosomal dominant medullary cystic kidney disease.115 In addition, there are the genetically heterogeneous renal medullary cystic diseases that constitute the NPHP group of renal diseases. In humans, NPHP includes a group of autosomal recessive cystic kidney diseases that together compose the most common genetic cause of end-stage renal disease in children and young adults.36 Depending on time of onset of end-stage renal disease, NPHP in humans may be classified as adolescent, juvenile, or infantile,80 but all variants of human NPHP share a triad of characteristic histologic findings, which include corticomedullary renal cysts, disruption of tubular basement membrane, and tubulointerstitial nephropathy.40,112,127 In contrast to the massive enlargement of polycystic kidneys in the forms of PKD resulting from disordered orientation and proliferation of renal tubular epithelium, the kidneys are not significantly enlarged in NPHP.11 Because kidney size is usually normal or slightly reduced in NPHP, the renal cysts appear to develop as a result of loss of normal tissue.127 This is in contrast to PKD, in which cysts are disseminated throughout the organ, causing gross enlargement of the kidneys.34,36 There is an increasing number of genes implicated in NPHP, which most often appears as an autosomal recessive disease due to homozygous single-gene mutations/deletions or compound heterozygous mutations occurring in a single NPHP gene. However, oligogenicity, where allelic variants at multiple loci contribute to disease, has also been documented for NPHP and a wide spectrum of clinical variants with any mutant gene (or genes) is possible.37 At this time, mutations in at least 13 distinct and unrelated causative genes
(Table 4) have been identified in human NPHP.49,78 These genes include NPHP1, Inversin/NPHP2, NPHP3, NPHP4, IQCB1/ NPHP5, CEP290/NPHP6, GLIS2/NPHP7, RPGRIP1L/NPHP8, NEK8/NPHP9, SDCAAG8/NPHP10, TMEM67/NPHP11, TTC21B/NPHP12, and WDR19/NPHP13.4,15,40,117 The proteins encoded by these genes are generally referred to as nephrocystins, and with the exception of NPHP-like 1 gene (NPHPL1), which encodes a mitochondrial enzyme (X-prolyl aminopeptidase 3),61 they have all been localized to the cilium/centrosome/basal body complex. This led to the conclusion that NPHP should be considered a ciliopathy.34,83,92 Many of these NPHP proteins form interacting protein complexes that copurify in distinct subcellular ciliary locations. For example, NPHP 1, 4, and 8 are present in the transition zone of primary cilia and regulate apical junction formation, whereas NPHP 5 and 6 are located at the centrosomes and regulate ciliogenesis.81 The MKS proteins MKS1 and MKS6 regulate hedgehog signaling, and the NPHP2/INVS and JBTS3/AHI1 proteins act as molecular bridges between different modules.81 The many molecular links between proteins involved in the development of NPHP, JBTS, and MKS help explain the overlapping clinical presentations seen in these diseases.26,39,91,114 However, since the known NPHP genes account for about only 30% of NPHP in humans,67 it can be expected that many more causative genes will be identified in the future. For example, whole-exome resequencing was recently used to identify mutations in MRE11, ZNF423, and CEP164 that result in NPHP in humans, and knockdown of cep164 in zebrafish was shown to produce a NPHP phenotype.14 The characteristic progressive renal lesions in Tmem218 –/– mice are very similar to those seen in human NPHP. Grossly, the kidneys of Tmem218 –/– mice are of normal or even mildly reduced size, which is typical of NPHP in humans. In addition, Tmem218 –/– kidneys display the classic histologic lesions of cyst formation, tubulointerstitial cell infiltrates with interstitial fibrosis, and tubular basement membrane disruption that are characteristic of human NPHP. These lesions were present diffusely within the renal medulla and cortex in most Tmem218 –/– mice, although some milder cases showed only sporadic cysts that were restricted to the corticomedullary junctional area. The precise pathogenetic mechanisms responsible for cyst formation in PKD and NPHP have not been determined, but it is known that mutations in NPHP genes disrupt signaling mechanisms involving the noncanonical Wnt and Sonic Hedgehog signaling pathways, which in turn lead to alterations in planar cell polarity and cell cycle control.32 In PKD, abnormal proliferation in tubular epithelial cells plays a crucial role in cyst development and/or growth, but dysregulation of
Figure 11. (continued) outer segments. HE. Figure 12. Kidney; Tmem218–/– mouse LacZ staining. Positive Tmem218 gene promoter expression is detected in renal tubular epithelium and glomeruli. Figure 13. Kidney; Tmem218–/– mouse LacZ staining. At higher magnification, it is evident that Tmem218 gene promoter expression is most common in renal tubular epithelial cells lining cystic tubules. Figure 14. Eye; Tmem218–/– mouse LacZ staining. The diffuse positive Tmem218 gene promoter expression in the retina is most intense in the inner segment, inner plexiform layer, and ganglion cell layer. Figure 15. Nose; Tmem218–/– mouse LacZ staining. Ciliated respiratory epithelium is strongly positive for Tmem218 gene promoter. Figure 16. Choroid plexus and ependyma; Tmem218–/– mouse LacZ staining: Ciliated ependymal cells and the choroid plexus epithelium are both strongly positive for Tmem218 gene promoter. Figure 17. Testis; Tmem218–/– mouse LacZ staining: Testis. Spermatids are strongly positive for Tmem218 gene promoter. Downloaded from vet.sagepub.com at QUEENS UNIV LIBRARIES on October 4, 2015
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Figure 18. Electroretinograms: a-wave. At 2 months of age, Tmem218–/– mice had statistically significant reductions in a-wave response at only the highest (24 cd.s/m2) of the light exposures tested (0.006, 0.04, and 24 cd.s/m2). ERG, electroretinogram; HET, heterozygous; HOM, homozygous; WT, wild type. Figure 19. Electroretinograms: a-wave. By 6 months of age, the progressive loss of vision is demonstrated by statistically significant reductions in a-wave electroretinogram responses at all 3 light intensities tested (0.006, 0.04, and 24 cd.s/m2). Figure 20. Electroretinograms: b-wave. 2 months of age: There are statistically significant reductions in b-wave response at only the highest (24 cd.s/m2) of the light exposures tested (0.006, 0.04, and 24 cd.s/m2). Figure 21. Electroretinograms: b-wave. 6 months of age: Reductions in b-wave responses were seen at all three light intensities tested, however, these differences were statistically significant only at 24 cd.s/m2).
apoptosis also appears to contribute to the progression of PKD, juvenile NPHP, and other dysplastic renal disease associated with cyst formation in humans and animal models (reviewed in Goilav30). Although apoptosis is present in both cystic and noncystic tubular cells, most available evidence suggests that the proliferative response predominates in PKD, with the hallmark lesion of kidney enlargement most likely a result of disordered and increased proliferation of renal tubular epithelium (reviewed in Goilav30). We previously found similar evidence of increased epithelial proliferation in another mouse model that developed early-onset PKD and retinopathy.87 In marked contrast, apoptosis of renal tubular epithelium was relatively common in Tmem218–/– mice, suggesting that the primary
cilia-induced abnormalities in cell cycling may favor apoptosis over proliferation in NPHP and that increased apoptosis may contribute to the loss of renal tubules that eventually results in small- to normal-sized cystic kidneys typical of NPHP.
Primary/Immotile Cilia: Retinal Ciliopathies The progressive retinal degeneration (determined by histology and electroretinography) that develops along with cystic renal disease in Tmem218–/– mice provides additional supporting evidence that TMEM218 has a role in ciliary biogenesis or function. NPHP frequently presents with extrarenal manifestations, and the involvement of multiple organ systems may be
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GLIS2 (GLIS family zinc finger 2)
RPGRIP1 L (RP GTPase regulator- < 10 interacting 1 like protein) (NPHP8)
NEK8 (NIMA [never in mitosis gene a]–related kinase 8)
NPHP7
NPHP8
NPHP9
0
100 (SLSN7)85
0
0
100 (SLSN6)84
Abbreviations: NPHP, nephronophthisis; SLSN, Senior-Løken syndrome.
NPHP12 TTC21B (tetratricopeptide 0 repeat domain 21B) NPHP13 WDR19 (WD repeat domain 19)
Nephronophthisis and Retinal Degeneration in Tmem218–/– Mice: A Novel Mouse Model for Senior-Løken Syndrome?
Veterinary Pathology 2015, Vol. 52(3) 580-595 ª The Author(s) 2014 Reprints and permission: sagepub.com/journalsPermissions.nav DOI: 10.1177/0300985814547392 vet.sagepub.com
P. Vogel1, C. M. Gelfman2, T. Issa2, B. J. Payne1, G. M. Hansen3, R. W. Read1, C. Jones2, M. R. Pitcher2, Z.-M. Ding4, C. M. DaCosta4, M. K. Shadoan4, R. B. Vance1, and D. R. Powell4
Abstract Mice deficient in TMEM218 (Tmem218–/–) were generated as part of an effort to identify and validate pharmaceutically tractable targets for drug development through large-scale phenotypic screening of knockout mice. Routine diagnostics, expression analysis, histopathology, and electroretinogram analyses completed on Tmem218–/– mice identified a previously unknown role for TMEM218 in the development and function of the kidney and eye. The major observed phenotypes in Tmem218–/– mice were progressive cystic kidney disease and retinal degeneration. The renal lesions were characterized by diffuse renal cyst development with tubulointerstitial nephropathy and disruption of tubular basement membranes in essentially normal-sized kidneys. The retinal lesions were characterized by slow-onset loss of photoreceptors, which resulted in reduced electroretinogram responses. These renal and retinal lesions are most similar to those associated with nephronophthisis (NPHP) and retinitis pigmentosa in humans. At least 10% of NPHP cases present with extrarenal conditions, which most often include retinal degeneration. Senior-Løken syndrome is characterized by the concurrent development of autosomal recessive NPHP and retinitis pigmentosa. Since mutations in the known NPHP genes collectively account for only about 30% of NPHP cases, it is possible that TMEM218 could be involved in the development of similar ciliopathies in humans. In reviewing all other reported mouse models of NPHP, we suggest that Tmem218–/– mice could provide a useful model for elucidating the pathogenesis of cilia-associated disease in both the kidney and the retina, as well as in developing and testing novel therapeutic strategies for Senior-Løken syndrome. Keywords Senior-Løken, nephronophthisis, cystic kidney disease, retina, retinal degeneration, retinitis pigmentosa, hypertension
To identify novel genes coding for pharmaceutically relevant disease targets, Lexicon Pharmaceuticals evaluated more than 4650 knockout mouse lines in a high-throughput mutagenesis and phenotyping program.124,125 Coincidentally, many disease phenotypes were discovered during this process,100 and some of these have proven useful in elucidating fundamental processes in biology or in determining the underlying pathogenesis of heritable diseases.69,75,76,79,102,104–111 Not surprising, a large proportion of pathologic phenotypes were linked to inactivation of genes involved in the regulation of complicated and tightly controlled processes (eg, embryologic development and immunity). Many other pathologic phenotypes resulted from abnormal development of structurally complex tissues/organs (eg, eyes, teeth, sperm).2,86 Over time, it also became clear that numerous pathologic phenotypes in knockout mice were associated with the inactivation of genes involved in the biogenesis and/or function of the extremely complex multifunctional organelles known as cilia.22 Recognition that polycystin 1, polycystin 2, and nephrocystin proteins could all be localized to the primary cilium/
basal body/centrosome indicated the involvement of dysfunctional primary cilia in the pathogenesis of polycystic kidney disease (PKD) and nephronophthisis (NPHP).62,68 Presently, mutations in more than 30 cilia-related genes have been linked to cystic kidney diseases.40 Most likely due to the nearly ubiquitous distribution of cilia and their involvement in several critical developmental
1 Department of Pathology, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA 2 Department of Ophthalmology, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA 3 Department of Molecular Genetics, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA 4 Department of Metabolism, Lexicon Pharmaceuticals Inc., The Woodlands, TX, USA
Corresponding Author: Peter Vogel, Department of Pathology, Lexicon Pharmaceuticals Inc., 262 Danny Thomas Place, MS 250, Memphis, TN 38105-3678, USA. Email: [email protected]
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pathways, defective ciliary proteins have been linked to a large number of diseases collectively called ciliopathies (reviewed by Yildiz et al120). Multiorgan involvement is common with ciliopathies, and the most frequently observed extrarenal lesions in NPHP include retinopathies (15%) and cerebellar ataxia (10%–15%).37 The diagnosis applied to most patients showing both NPHP and retinal degeneration is Senior-Løken syndrome (SLSN).50,88 The recognition that the mutated genes in patients with SLSN coded for proteins that were localized to the cilia/centrosome led to the classification of SLSN as a ciliopathy.35,55,62,64,65,82 Transmembrane protein 218 (TMEM218) is a small 115 amino acid protein of unknown function. TMEM218 is evolutionarily well conserved, with 81% amino acid identity (92% similarity) between mouse and humans. The location of the TMEM218 gene in humans (11q24.2) does not map to any known disease locus, and we did not investigate any possible associations between Tmem218 and human disease in this study. To the best of our knowledge, the only previous mentions of TMEM218 in the scientific literature are that its expression is downregulated in testicular feminized mice60 and upregulated in confluent cultures of NIH3T3 fibroblasts.46 We did not investigate the molecular functions of TMEM218 or any of the underlying mechanisms involved in the pathogenesis of progressive NPHP and retinal degeneration in Tmem218–/– mice. Nevertheless, the lesions that develop in the kidneys and eyes of TMEM218-null mice (Tmem218Gt(OST40451)Lex; abbreviated as Tmem218–/– in this report) indicate that TMEM218 plays an important role in maintaining normal structure and function in the kidney and retina of mice. Furthermore, the concurrent development of progressive cystic kidney disease and retinal degeneration suggests that TMEM218 is involved in ciliary biogenesis or function. The Tmem218–/– mouse should provide a useful model for investigating the role of TMEM218 in normal ciliary functions and developmental processes, as well as in elucidating molecular mechanisms involved in the pathogenesis and progression of cilia-associated disease in both the kidney and the retina. In addition, the Tmem218–/– mouse might be used in the development and testing of novel therapeutic approaches to the prevention and treatment of the renal and retinal ciliopathies seen in SLSN.
Materials and Methods Mouse Production For the sake of brevity, the symbol for mice homozygous for this gene-trapped allele—Tmem218Gt(OST40451)Lex—has been abbreviated as Tmem218–/– in this report. The mutant mice used in this study were produced by Lexicon Pharmaceuticals Inc. (The Woodlands, TX) but are now distributed through Taconic Farms Inc. (New York City, NY). The methods used for gene trapping in embryonic stem cells, identification of trapped genes using OmniBank sequence tags (OSTs), characterization of retroviral gene trap vector insertion site, and reverse transcription polymerase chain reaction (PCR) analysis
of knockout and wild-type transcripts are published.122 Gene trapping was performed with strain 129S5SvEvBrd-derived embryonic stem cells obtained from the OmniBank library.123 OST40451 was selected from the OmniBank library based on a BLAST search of all available sequence tags using the Tmem218 genomic interval as a query. The mutation in this clone was confirmed to be intergenic according to inverse genomic PCR, as previously described.31 Briefly, oligonucleotide primers complementary to the gene trap vector were used to amplify the vector insertion site from clone OST40451, which was then compared to mouse genome sequence assemblies to localize the insertion within intron 1 of the Tmem218 gene (Fig. 1a). The gene-trapped embryonic stem cell clone was microinjected into C57BL/6-Tyrc–Brd(albino) blastocysts to generate chimeric animals that were bred to C57BL/6-Tyrc–Brd(albino) females, and the resulting heterozygous offspring were interbred to produce homozygous gene-deficient mice. The knockout F2 mice used in phenotyping studies were produced by intercrossing the F1 heterozygous knockout (–/þ) offspring of chimeric founder parents and were therefore of mixed C57BL and 129 genetic background. Using the albino variant of C57BL/6 mice (C57BL/6-Tyrc–Brd) permits simple visual recognition of chimeric offspring, because they have dark eyes and patches of dark hair that derive from stem cells from the agouti 129S5/SvEvBrd. Genotypes of offspring were determined by quantitative PCR as previously described.31 Briefly, DNA isolated from tail biopsy samples was assayed by quantitative PCR for the neo gene, which is present in VICTR24, the gene-trapping vector used to generate the mutation described in this study. Gene disruption in vivo was confirmed by a direct analysis of gene expression based on reverse transcription PCR. RNA was extracted from kidney and spleen of wild-type and homozygous mutant mice via a bead homogenizer and RNAzol (Ambion, Austin, TX) according to manufacturer’s instructions. Reverse transcription was performed with SuperScript II (Invitrogen, Carlsbad, CA) and random hexamer primers according to the manufacturer’s instructions. PCR amplification was performed with oligonucleotide primers complementary to exons flanking the insertion site (Fig. 1b). The primers used were F: 50 -GGAACCTGAGAGCCGGGTGAGGA-30 and R: 50 - CAGAGCTCCGAGGAAGACAAAGA-30 , are complementary to Tmem218 exons 1 and 3, respectively, and amplify a product of 196 nucleotides. Reverse transcription PCR analysis based on actb primers (50 -GGCTGGCCGGGACCTGACGGACTACCTCAT-30 and 50 - GCCTAGAAGCACTTGCGGTGCACGATGGAG-30 ) complementary to the mouse beta actin gene (accession M12481) with an expected product size of 591 bp was performed in the same reaction as an internal amplification control. In all studies reported here, mutant mice were compared directly with their wild-type littermates used as negative controls.
Mouse Husbandry Mice were housed in microisolator cages within a barrier facility at 24 C on a fixed 12-hour light and 12-hour dark cycle and
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Veterinary Pathology 52(3) prepulse inhibition of the acoustic startle response, tail suspension, marble burying, and context trace conditioning), fundoscopy and retinal angiography exams, blood pressure and heart rate measurements, serum chemistries, insulin levels, glucose tolerance testing, urinalysis, quantitative magnetic resonance, dual-energy x-ray absorptiometry scans, computerized axial tomography scans, micro–computed tomography scans, fertility testing, skin fibroblast proliferation assays, and pathology. Immunology assays included hematology, peripheral blood FACS analysis, acute phase response, and ovalbumin challenge.
Histopathology Figure 1. Generation of Tmem218Gt(OST40451)Lex mice. (a) Retroviral gene trap vector VICTR24 was used to produce OmniBank clone OST40451 (accession CG498846.1), which contains an insertion within intron 1 of the Tmem218 gene. This mutation would be expected to interrupt endogenous transcription within the 50 UTR, preventing transcription and translation of the protein. Open boxes denote untranslated exons; filled boxes denote coding exons. LTR, viral long terminal repeat; SA, splice acceptor sequence; bGEO, fusion of the beta-galactosidase and the neomycin phosphotransferase genes; pA, polyadenylation sequence; Pgk, phosphoglycerate kinase 1 promoter; Btk-SD, Bruton’s tyrosine kinase splice donor sequence. (b) Reverse transcription polymerase chain reaction expression analysis of Tmem218 transcript. Endogenous Tmem218 transcript was detected in the kidney and spleen of wildtype (þ/þ) mice. No endogenous Tmem218 transcript was detected in homozygous (–/–) tissues. The primers used were F: 50 -GGAACCTGAGAGCCGGGTGAGGA-30 and R: 50 -CAGAGCTCCGAGGAAGACAAAGA-30 , which are complementary to Tmem218 exons 1 and 3, respectively, and amplify a product of 196 nucleotides. Reverse transcription polymerase chain reaction analysis using actb primers (50 GGCTGGCCGGGACCTGACGGACTACCTCAT-30 and 50 -GCCTAGAAGCACTTGCGGTGCACGATGGAG-30 ) complementary to the mouse beta actin gene (accession M12481) with an expected product size of 591 bp was performed in the same reaction as an internal amplification control. M, molecular weight marker.
were provided ad libitum acidified water and Purina rodent chow (5001, Purina, St Louis, MO). Procedures involving animals were conducted in conformance with Lexicon Pharmaceuticals’ Institutional Animal Care and Use Committee guidelines, which are in compliance with state and federal laws and the standards outlined in the National Research Council’s Guide for the Care and Use of Laboratory Animals (2011). Quarterly sentinel surveillance showed no evidence of pathogenic rodent viruses, Mycoplasma sp, or Helicobacter sp in the Lexicon Pharmaceuticals source colonies.
Phenotype Screening Wild-type and homozygous-null mice were subjected to a comprehensive battery of phenotype screening exams as previously described.9,122 These screening assays included behavioral tests (eg, circadian rhythm, open field, inverted screen,
Age-matched knockout and wild-type control mice (2 male and 2 female of each genotype) were euthanized at 7 and 14 weeks of age and fixed by cardiac perfusion with 10% neutral buffered formalin. Lungs were infused with formalin via the trachea, and all tissues were immersed in 10% neutral buffered formalin for an additional 48 hours, except for the eyes, which were removed and fixed by immersion in Davidson’s fixative (Poly Scientific, NY) overnight at room temperature. All tissues (to include heart, skeletal muscle, tongue, lungs, trachea, thyroid gland, liver, kidney, adrenal gland, salivary glands, lymph nodes, white adipose, brown adipose, aorta, thymus, spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder, skin, brain, eyes, nose, teeth, ear, bone, bone marrow, testes, epididymis, prostate gland, seminal vesicles, vas deferens, uterus, ovaries) were embedded in paraffin, sectioned at 4 mm, mounted on positively charged glass slides (Superfrost Plus, Fisher Scientific, Pittsburgh, PA), and stained with hematoxylin and eosin for histopathologic examination. A full set of tissues from a pair (1 male and 1 female) of 14-week-old heterozygote mice was similarly collected and examined. Additional histopathologic evaluations were conducted on kidneys and eyes only from at least 2 age-matched knockout mice and 1 control mouse collected at several other time points (kidneys at 9, 14, 17, 29, and 58 weeks of age; eyes at 4, 8, 12, and 42 weeks of age). Kidney sections were stained with Masson’s trichrome and periodic acid–Schiff to highlight interstitial fibrosis and basement membrane changes respectively.
b-Galactosidase (LacZ) Staining The b-galactosidase reporter gene (b-geo) was under transcriptional control of the Tmem218 regulatory elements, allowing localization of the expression pattern of Tmem218 in tissues of homozygous or heterozygous mutant mice by X-gal staining. b-Galactosidase reporter gene activity was assessed in situ as previously described.111 Briefly, 2 heterozygous mice and 2 homozygous mice were anesthetized and perfused sequentially with b-Gal fixative (0.2% glutaraldehyde, 1.5% paraformaldehyde, 2 mM MgCl2, 5 mM EGTA [ethylene glycol tetraacetic acid], 100 mM sodium phosphate [pH 7.3]), followed by 2 ml of b-Gal rinse (0.2% Nonidet-P40, 0.1% sodium deoxycholate, 2 mM MgCl2,100 mM sodium phosphate), and finally 10 ml of
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b-Gal stain (5 mM K3Fe(CN)6 [potassium ferricyanide], 5 mM K4Fe(CN)6 [potassium ferrocyanide], 1 mg of 5-bromo-4chloro-3-indolyl-D galactopyranoside [X-Gal; dissolved in dimethylformamide] per ml, 0.2% Nonidet-P40, 0.1% sodium deoxycholate, 2 mM MgCl2, 100 mM sodium phosphate [pH 7.3]). Tissues were postfixed in b-Gal fix, rinsed in the b-Gal rinse, and then incubated in b-Gal stain solution for 48 hours. After 3 additional washes in b-Gal rinse, the tissues were postfixed in Bouin’s fixative before dehydration and embedding in paraffin. Tissues examined included heart, skeletal muscle, trachea, lung, thyroid gland, parathyroid gland, liver, kidney, adrenal gland, salivary glands, lymph nodes, thymus, spleen, pancreas, stomach, duodenum, jejunum, ileum, cecum, colon, urinary bladder, skin, brain, eyes, nose, teeth, ear, bone marrow, testes, epididymis, prostate gland, seminal vesicles, vas deferens, uterus, and ovaries. Sections were cut at 4 mm and counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA).
Blood Pressure Measurements Systolic blood pressures were measured with a noninvasive tail cuff method on a Visitech BP-2000 blood pressure analysis system (Visitech Systems, Apex, NC). Systolic pressure and heart rate were recorded in conscious mice (8 homozygotes, 8 wild type, and 8 heterozygotes at 10 weeks of age) 10 times each day for 4 days, and all readings were averaged for each mouse.
Clinical Chemistry and Renal Function Serum and urine chemistry parameters were measured with a Cobas Integra 400 autoanalyzer (Roche Diagnostics, Indianapolis, IN). For urine studies, 24-hour urine samples were collected for 3 consecutive days from mice individually housed in metabolic cages (product MTB-0311; Nalgene, Rochester, NY) and acclimatized for 3 days prior to the start of urine collection.
Electroretinography Electroretinograms (ERGs) were recorded from wild-type (n ¼ 5), heterozygous (n ¼ 5), and homozygous (n ¼ 5) mice at 2 and 6 months of age. All procedures were carried out under dim red light (>650 nm). ERGs from both eyes were recorded simultaneously with the UTAS-E 3000 Visual Electrodiagnostic System (LKC Technologies, Gaithersburg, MD). After an overnight dark-adaptation period, the pupil was dilated with 0.1% atropine/phenylephrine (Alcon Laboratories Inc., Fort Worth, TX), and 0.5% proparacaine (Alcon) was applied for topical anesthesia. Mice were deeply anesthetized with (per milliliter) ketamine (7.5 mg), xylazine (0.38 mg), and acepromazine (0.074 mg), delivered at 10 ml/kg body weight. Silver electrodes were placed on the corneal surface with a drop of methylcellulose. A reference electrode was placed subcutaneously on the head, and a ground
electrode was placed in the right hind leg. The mice were placed in a Ganzfeld illumination dome. Full-field scotopic ERGs of both eyes were elicited simultaneously with 10-ms light flashes. For a- and b-wave ERGs, 5 recordings per flash intensity (0.006, 0.04, and 24 cds/m2) were averaged. Mixed rod-cone (scotopic)–driven responses to light flashes were recorded, with the intervals between flashes increasing from 10 to 30 seconds, with an increasing flash intensity range (0.0094–40.41 cd sec/mm2).
Results Phenotype Screening At the time of genotyping (3 weeks of age), the number of surviving knockout mice (188 wild type, 331 heterozygote, 145 homozygote) was slightly below expected Mendelian ratios (w2 ¼ 5.58, P ¼ .0614). Although young Tmem218–/– mice were indistinguishable from wild-type littermates upon macroscopic examination and showed normal growth rates (length and weight) between 2 and 16 weeks, they tended to have lower than normal body weight at 19 weeks (Table 1). At 17 weeks, Tmem218–/– mice also showed trends toward higher serum creatinine and blood urea nitrogen compared to Tmem218þ/– and Tmem218þ/þlittermates, suggesting the presence of impaired kidney function; these findings were accompanied by modest but significant increases in serum chloride, calcium, and alkaline phosphatase (Table 2). Although 24-hour urine volume and osmolality were not significantly different in Tmem218–/– mice, correlations between serum creatinine and 24-hour urine volume (r ¼ 0.62, P < .05) and between serum creatinine and urine osmolality (r ¼ –0.71, P < .05) in Tmem218–/– mice suggest that urinary concentrating ability became compromised after the onset of renal failure. At 14 weeks, Tmem218–/– mice also showed a trend toward higher systolic blood pressure compared to Tmem218þ/þ littermates; this finding, confirmed by studying additional older mice (Table 3), was more pronounced in males and correlated with serum creatinine (r ¼ 0.76, P < .01). Overall, the data suggest that the elevated systolic blood pressure was likely secondary to renal failure. Interestingly, fundoscopy and retinal angiography exams were normal in baseline screens performed at 8 weeks of age. In other phenotyping assays, Tmem218–/– mice showed normal results in behavioral tests (eg, circadian rhythm, open field, inverted screen, prepulse inhibition of the acoustic startle response, tail suspension, marble burying, and context trace averse conditioning). In screening assays, insulin levels, glucose tolerance testing, urinalysis, quantitative magnetic resonance, dual-energy x-ray absorptiometry scans, computerized axial tomography scans, micro–computed tomography scans, and skin fibroblast proliferation assays were also within normal limits. Immunologic assays included hematology, peripheral blood FACS analysis, acute phase response, and ovalbumin challenge, and standard hematology workups were unremarkable.
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Table 1. Body Weight and Urine Measurements: 19-Week-Old Mice. Micea Metabolic Cages
WT
P
LacZ Staining Results
Body weight, g Males Females 24-hour urine Volume, ml Creatinine, mg Osmolality, mOsm/kg H2O
KO
in any other tissue of homozygous mice. Similarly, no lesions were noted in any tissue in a single pair of heterozygote mice examined at 14 weeks of age.
38 + 2 (4)
25 + 5 (8)
25 + 2 (4)
22 + 9 (8)
1.3 + 0.9 (8) 2.2 + 1.7 (16) 413 + 116 (8) 338 + 89 (16) 2208 + 759 (8) 1877 + 1354 (16)
KO < WT, P < .01 ns ns ns ns
Abbreviations: KO, knockout; ns, not significant; WT, wild type. a Mean + SEM (n).
Pathology Findings Situs inversus was not observed in any knockout mice, and both males and females were fertile. The key histologic findings in Tmem218–/– mice were in the kidneys and eyes. Tmem218–/– mice had variable numbers of cysts at all ages examined, but the kidneys were generally of normal or slightly reduced size compared to wild-type mice (Figs. 2–4). A few cases displayed sporadic or scattered cysts in the cortex (Fig. 3), but severe cyst formation was present in most mice of the same age (Fig. 4). Histologically, the lesions were characterized by tubular atrophy and tubulointerstitial inflammatory cell infiltrates (Fig. 5), with interstitial fibrosis (Fig. 6) and disruption, thickening, and splitting of tubular basement membranes (Fig. 7). Although renal cyst formation could be extensive in young mice, evaluation of kidney sections taken at various time points demonstrated that cortex, inner medulla, and outer medulla were becoming progressively cystic and atrophic (not shown). Diffuse tubulointerstitial cell infiltration with interstitial fibrosis and deposition of collagen throughout the kidney, along with tubular basement membrane disintegration with tubular atrophy and tubular cysts, plus scattered glomerular cysts, were present diffusely at 14 weeks of age. Lesions in the eyes of Tmem218–/– mice were characterized by slowly progressive retinal degeneration. At 9 weeks, routine histologic sections from wild-type (Fig. 8) and homozygous retinas (Fig. 9) were indistinguishable, with similar thickness and cellularity of the outer and inner nuclear layers as well as the retinal ganglion cell layer. However, by 14 weeks of age, subtle photoreceptor loss, as evidenced by a thinning of the outer nuclear layers, became evident, and by 4 months, there was diffuse moderate thinning of the outer nuclear layer in Tmem218–/– mice that often became pronounced by 29 weeks (Figs. 10, 11). Degeneration of the photoreceptor cells was demonstrated by diffuse thinning of the outer nuclear layer and photoreceptor inner and outer segments (OSs). In contrast, the inner retina of these animals appeared to be intact, with apparently normal inner nuclear and ganglion cell layers. No notable lesions were detected
Tmem218 gene promoter expression was analyzed in Tmem218–/– mice by using b-galactosidase enzyme histochemistry. In Tmem218–/– mice, specific X-gal staining representative of TMEM218 expression was observed in several cell types: These included renal epithelium (Figs. 12, 13), retina (Fig. 14), and a variety of ciliated/flagellated cells (respiratory epithelium, Fig. 15; ependymal and choroid plexus, Fig. 16); vas deferens, epididymis, and spermatids, Fig. 17). In addition, several different endocrine cells (pancreatic islets, pituitary gland, adrenal medulla, parathyroid gland, C-cells of thyroid gland) were stained positively (data not shown). Although the intensity of staining was generally lower in heterozygote mice, the positive tissues and cell types were identical (data not shown).
ERG Results The gradual and progressive loss of photoreceptors was accompanied by a functional deficit, as demonstrated by ERG results at 2 and 6 months of age (Figs. 18–21). At 2 months of age, Tmem218–/– mice had statistically significant reductions in both a-wave (Fig. 18) and b-wave (Fig. 20) ERG responses at only the highest (24 cd.s/m2) of the light exposures tested (0.006, 0.04, and 24 cd.s/m2). By 6 months of age, the progressive loss of vision in Tmem218–/– mice was demonstrated by statistically significant reductions in a-wave ERG responses (Fig. 19) at all 3 light intensities tested (0.006, 0.04, and 24 cd.s/m2) as well as reductions in b-wave responses (Fig. 21), although these differences were statistically significant only at 24 cd.s/m2).
Discussion The 2 striking pathologic phenotypes that we observed in Tmem218–/– mice included PKD culminating in renal failure and progressive retinal degeneration resulting in blindness. Retinopathies are the most common extrarenal lesions associated with NPHP, being present in 10% to 15% of human patients.50,88 Other relatively common extrarenal findings with NPHP in humans that were not observed in Tmem218–/– mice include cerebellar vermis hypoplasia (Joubert’s syndrome [JBTS]) in 10% to 15% of cases and hepatic fibrosis in 5%.37,83 Other syndromes that may have concurrent NPHP include congenital ocular motor apraxia type Cogan, cognitive impairment, phalangeal cone-shaped epiphyses in MainzerSaldino syndrome, Bardet-Biedl syndrome, Ellis-van Creveld syndrome, Jeune asphyxiating thoracic dystrophy, Alstro¨m syndrome, and Meckel-Gruber syndrome (MKS).1,32,33,83,92 Given the low penetrance of many of these phenotypes and the small number of mice and limited time points that we
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Table 2. Serum Chemistries: 17–18 Weeks. Micea P WT Glucoseb Sodiumc Chloridec Potassiumc Calciumb Phosphorusb Blood urea nitrogenb Creatinineb Cholesterolb Triglyceridesb ALTd Total bilirubinb Alkaline phosphatased
186 149 111 5.5 9.5 6.1 19 0.12 133 138 40 0.33 83
HET
+ 30 (27) + 2 (27) + 1 (27) + 0.7 (27) + 0.4 (27) + 0.9 (27) + 4 (27) + 0.03 (27) + 47 (27) + 68 (27) + 36 (27) + 0.12 (27) + 38 (27)
186 + 150 + 112 + 5.6 + 9.6 + 6.0 + 20 + 0.12 + 143 + 130 + 34 + 0.29 + 78 +
KO
33 (24) 3 (24) 2 (24) 0.6 (24) 0.2 (24) 1.0 (24) 3 (24) 0.03 (24) 44 (24) 50 (24) 16 (24) 0.10 (24) 21 (24)
186 + 151 + 114 + 5.6 + 10.1 + 6.3 + 45 + 0.26 + 139 + 106 + 27 + 0.25 + 117 +
40 (28) 3 (28) 3 (28) 0.6 (28) 0.6 (28) 1.2 (28) 35 (28) 0.21 (28) 30 (28) 38 (28) 6 (28) 0.10 (28) 44 (28)
ns ns KO > HET þ WT, P < .001 ns KO > HET þ WT, P < .001 ns KO > HET þ WT, P < .001 KO > HET þ WT, P < .001 ns ns ns KO < WT, P < .05 KO > HET þ WT, P < .01
Abbreviations: HET, heterozygote; KO, knockout; ns, not significant; WT, wild type. a Mean + SEM (n). b mg/dl. c mmol/liter. d U/liter.
Motile Cilia: Primary Ciliary Dyskinesia
Table 3. Systolic Blood Pressure, mm Hg. Micea Weeks 14–20 Males Females Combined 23–25 Males Females Combined
WT
KO
P
108 + 7 (10) 117 + 14 (6) KO > WT, P < .05 109 + 10 (10) 109 + 9 (6) ns 108 + 7 (20) 114 + 13 (12) KO > WT, P < .001 113 + 12 (15) 137 + 27 (7) KO > WT, P < .01 123 + 18 (8) 138 + 23 (10) ns 117 + 15 (23) 138 + 24 (17) KO > WT, P < .01
Abbreviations: KO, knockout; ns, not significant; WT, wild type. a Mean + SEM (n).
examined, it is possible that some of these lesions could be linked to TMEM218 deficiency in mice in the future. In our experience, the appearance of very similar phenotypes (phenocopies) in either mice or humans with mutations in different genes often indicates that the proteins encoded by these mutant genes involve different steps in a common metabolic pathway or affect the same process or structure.86,106–109 It is often instructive to consider what is known about the underlying pathogenic mechanisms in phenocopies (whether in humans or in animal models) when attempting to understand the pathogenesis of lesions in a novel line of knockout mice. We believe that the simultaneous development of NPHP and retinal degeneration in Tmem218–/– mice is highly suggestive of an underlying defect in the structure/function of the sensory primary cilium of renal epithelium and the connecting cilium of retinal photoreceptors, respectively. Due to the highly conserved nature of most known ciliary genes, we believe that it is possible that TMEM218 will be linked to human ciliopathies in the future.
Cilia are highly conserved structurally complex organelles composed of 9 microtubule doublets (called the axoneme) that emanate from basal bodies, which are the microtubule-organizing centers of the cell. There are 2 types of cilia: motile cilia that are necessary for locomotion and fluid movement and primary (immotile) cilia that serve mainly sensory functions. Either motile cilia or immotile primary cilia are present on most cell types, and as a result of this wide distribution, it is possible for defective cilia to affect a very wide range of tissues and organ systems. In fact, mutations in genes whose products have been localized to the cilium–centrosome complex have in recent years been linked to a wide range of monogenic developmental and degenerative disorders now recognized as ciliopathies.33 Dysfunctional motile cilia and flagella were first recognized as being the cause of a group of disorders (usually autosomal recessive) now classified as primary ciliary dyskinesias (PCD).3 Normally, motile flagella and cilia in the upper and lower respiratory tract, middle ear, reproductive tract, and brain create fluid flow over cell surfaces.93 Respiratory tract cilia help clear respiratory secretions in the airways, and when mucociliary clearance is impaired in PCD,25 patients can develop chronic otitis media, rhinitis, sinusitis, and recurrent infections of the lower respiratory tract.29,54 Impaired ependymal flow also appears to contribute to the development of hydrocephalus in some humans13 and to the randomization of left/right body asymmetry caused by dysfunctional cilia in the embryonic node during early development. Laterality defects (situs inversus) occur in approximately 50% of PCD patients.3 Similarly, the deletion or mutation of genes linked to motile cilia in mice have been linked to the development of chronic upper respiratory tract infections, hydrocephalus, infertility, and/or laterality defects.105,108,109
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Figure 2. Normal kidney; 14-week-old Tmem218þ/þ mouse. Normal-sized kidney. HE. Figure 3. Polycystic kidney, 14-week-old Tmem218–/– mouse. Scattered renal cysts are evident in both the cortex and the medulla of normal-sized kidney. HE. Figure 4. Nephronophthisis, 14-weekold Tmem218–/– mouse. There is diffuse cyst formation in an essentially normal-sized kidney. HE. Figure 5. Nephronophthisis, 14-week-old Tmem218–/– mouse. Renal cysts are accompanied by tubular atrophy and tubulointerstitial inflammatory cell infiltrates and with interstitial fibrosis. HE. Figure 6. Nephronophthisis, 14-week-old Tmem218–/– mouse. Extensive interstitial fibrosis is associated with cystic lesions. Masson’s trichrome stain. Figure 7. Nephronophthisis, 14-week-old Tmem218–/– mouse. There is widespread disruption, thickening, and splitting of tubular basement membranes. Periodic acid–Schiff. Figure 8. Retina, 9-week-old Tmem218þ/þ. Normal thickness of all retinal layers. HE. Figure 9. Retina, 9-week-old Tmem218–/– mouse. All retinal layers were indistinguishable from wild-type retinas in terms of thickness and cellularity of the outer and inner nuclear layers as well as the retinal ganglion cell layer. HE. Figure 10. Retina, 29-week-old Tmem218þ/þ. Normal thickness of mouse retina. Note that external nuclear layer is normally thicker than inner nuclear layer. HE. Figure 11. Retinopathy, 29-week-old Tmem218–/– mouse. There is pronounced thinning of the retina due to diffuse thinning of the outer nuclear layer and photoreceptor inner and Downloaded from vet.sagepub.com at QUEENS UNIV LIBRARIES on October 4, 2015
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Primary/Immotile Cilia: Renal Ciliopathies For many decades after its discovery, it was thought that the immotile primary cilium was a purely vestigial organelle. However, in recent years, many crucial roles for primary cilia in development and physiology have been reported.12 In contrast to the well-defined and easily understood pathogenic mechanisms responsible for the lesions associated with dysfunctional motile cilia, defects in immotile primary cilia, which have a range of different sensory functions,10 can be associated with a remarkably broad spectrum of pleiotropic phenotypes. These include PKD, NPHP, retinitis pigmentosa, anosmia, ataxia, cardiac defects, liver fibrosis, and obesity, among many others.33 Of these phenotypes, the PKDs were the first to be linked definitively to defects in primary immotile cilia, and many genes have since been linked to the development of cystic kidney diseases (reviewed in Loftus and Ong49). The most common and clinically significant type of PKD in humans is inherited as an adult-onset autosomal dominant trait (ADPKD). Other less common hereditary renal cystic diseases include autosomal recessive PKDs and adult-onset autosomal dominant medullary cystic kidney disease.115 In addition, there are the genetically heterogeneous renal medullary cystic diseases that constitute the NPHP group of renal diseases. In humans, NPHP includes a group of autosomal recessive cystic kidney diseases that together compose the most common genetic cause of end-stage renal disease in children and young adults.36 Depending on time of onset of end-stage renal disease, NPHP in humans may be classified as adolescent, juvenile, or infantile,80 but all variants of human NPHP share a triad of characteristic histologic findings, which include corticomedullary renal cysts, disruption of tubular basement membrane, and tubulointerstitial nephropathy.40,112,127 In contrast to the massive enlargement of polycystic kidneys in the forms of PKD resulting from disordered orientation and proliferation of renal tubular epithelium, the kidneys are not significantly enlarged in NPHP.11 Because kidney size is usually normal or slightly reduced in NPHP, the renal cysts appear to develop as a result of loss of normal tissue.127 This is in contrast to PKD, in which cysts are disseminated throughout the organ, causing gross enlargement of the kidneys.34,36 There is an increasing number of genes implicated in NPHP, which most often appears as an autosomal recessive disease due to homozygous single-gene mutations/deletions or compound heterozygous mutations occurring in a single NPHP gene. However, oligogenicity, where allelic variants at multiple loci contribute to disease, has also been documented for NPHP and a wide spectrum of clinical variants with any mutant gene (or genes) is possible.37 At this time, mutations in at least 13 distinct and unrelated causative genes
(Table 4) have been identified in human NPHP.49,78 These genes include NPHP1, Inversin/NPHP2, NPHP3, NPHP4, IQCB1/ NPHP5, CEP290/NPHP6, GLIS2/NPHP7, RPGRIP1L/NPHP8, NEK8/NPHP9, SDCAAG8/NPHP10, TMEM67/NPHP11, TTC21B/NPHP12, and WDR19/NPHP13.4,15,40,117 The proteins encoded by these genes are generally referred to as nephrocystins, and with the exception of NPHP-like 1 gene (NPHPL1), which encodes a mitochondrial enzyme (X-prolyl aminopeptidase 3),61 they have all been localized to the cilium/centrosome/basal body complex. This led to the conclusion that NPHP should be considered a ciliopathy.34,83,92 Many of these NPHP proteins form interacting protein complexes that copurify in distinct subcellular ciliary locations. For example, NPHP 1, 4, and 8 are present in the transition zone of primary cilia and regulate apical junction formation, whereas NPHP 5 and 6 are located at the centrosomes and regulate ciliogenesis.81 The MKS proteins MKS1 and MKS6 regulate hedgehog signaling, and the NPHP2/INVS and JBTS3/AHI1 proteins act as molecular bridges between different modules.81 The many molecular links between proteins involved in the development of NPHP, JBTS, and MKS help explain the overlapping clinical presentations seen in these diseases.26,39,91,114 However, since the known NPHP genes account for about only 30% of NPHP in humans,67 it can be expected that many more causative genes will be identified in the future. For example, whole-exome resequencing was recently used to identify mutations in MRE11, ZNF423, and CEP164 that result in NPHP in humans, and knockdown of cep164 in zebrafish was shown to produce a NPHP phenotype.14 The characteristic progressive renal lesions in Tmem218 –/– mice are very similar to those seen in human NPHP. Grossly, the kidneys of Tmem218 –/– mice are of normal or even mildly reduced size, which is typical of NPHP in humans. In addition, Tmem218 –/– kidneys display the classic histologic lesions of cyst formation, tubulointerstitial cell infiltrates with interstitial fibrosis, and tubular basement membrane disruption that are characteristic of human NPHP. These lesions were present diffusely within the renal medulla and cortex in most Tmem218 –/– mice, although some milder cases showed only sporadic cysts that were restricted to the corticomedullary junctional area. The precise pathogenetic mechanisms responsible for cyst formation in PKD and NPHP have not been determined, but it is known that mutations in NPHP genes disrupt signaling mechanisms involving the noncanonical Wnt and Sonic Hedgehog signaling pathways, which in turn lead to alterations in planar cell polarity and cell cycle control.32 In PKD, abnormal proliferation in tubular epithelial cells plays a crucial role in cyst development and/or growth, but dysregulation of
Figure 11. (continued) outer segments. HE. Figure 12. Kidney; Tmem218–/– mouse LacZ staining. Positive Tmem218 gene promoter expression is detected in renal tubular epithelium and glomeruli. Figure 13. Kidney; Tmem218–/– mouse LacZ staining. At higher magnification, it is evident that Tmem218 gene promoter expression is most common in renal tubular epithelial cells lining cystic tubules. Figure 14. Eye; Tmem218–/– mouse LacZ staining. The diffuse positive Tmem218 gene promoter expression in the retina is most intense in the inner segment, inner plexiform layer, and ganglion cell layer. Figure 15. Nose; Tmem218–/– mouse LacZ staining. Ciliated respiratory epithelium is strongly positive for Tmem218 gene promoter. Figure 16. Choroid plexus and ependyma; Tmem218–/– mouse LacZ staining: Ciliated ependymal cells and the choroid plexus epithelium are both strongly positive for Tmem218 gene promoter. Figure 17. Testis; Tmem218–/– mouse LacZ staining: Testis. Spermatids are strongly positive for Tmem218 gene promoter. Downloaded from vet.sagepub.com at QUEENS UNIV LIBRARIES on October 4, 2015
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Figure 18. Electroretinograms: a-wave. At 2 months of age, Tmem218–/– mice had statistically significant reductions in a-wave response at only the highest (24 cd.s/m2) of the light exposures tested (0.006, 0.04, and 24 cd.s/m2). ERG, electroretinogram; HET, heterozygous; HOM, homozygous; WT, wild type. Figure 19. Electroretinograms: a-wave. By 6 months of age, the progressive loss of vision is demonstrated by statistically significant reductions in a-wave electroretinogram responses at all 3 light intensities tested (0.006, 0.04, and 24 cd.s/m2). Figure 20. Electroretinograms: b-wave. 2 months of age: There are statistically significant reductions in b-wave response at only the highest (24 cd.s/m2) of the light exposures tested (0.006, 0.04, and 24 cd.s/m2). Figure 21. Electroretinograms: b-wave. 6 months of age: Reductions in b-wave responses were seen at all three light intensities tested, however, these differences were statistically significant only at 24 cd.s/m2).
apoptosis also appears to contribute to the progression of PKD, juvenile NPHP, and other dysplastic renal disease associated with cyst formation in humans and animal models (reviewed in Goilav30). Although apoptosis is present in both cystic and noncystic tubular cells, most available evidence suggests that the proliferative response predominates in PKD, with the hallmark lesion of kidney enlargement most likely a result of disordered and increased proliferation of renal tubular epithelium (reviewed in Goilav30). We previously found similar evidence of increased epithelial proliferation in another mouse model that developed early-onset PKD and retinopathy.87 In marked contrast, apoptosis of renal tubular epithelium was relatively common in Tmem218–/– mice, suggesting that the primary
cilia-induced abnormalities in cell cycling may favor apoptosis over proliferation in NPHP and that increased apoptosis may contribute to the loss of renal tubules that eventually results in small- to normal-sized cystic kidneys typical of NPHP.
Primary/Immotile Cilia: Retinal Ciliopathies The progressive retinal degeneration (determined by histology and electroretinography) that develops along with cystic renal disease in Tmem218–/– mice provides additional supporting evidence that TMEM218 has a role in ciliary biogenesis or function. NPHP frequently presents with extrarenal manifestations, and the involvement of multiple organ systems may be
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GLIS2 (GLIS family zinc finger 2)
RPGRIP1 L (RP GTPase regulator- < 10 interacting 1 like protein) (NPHP8)
NEK8 (NIMA [never in mitosis gene a]–related kinase 8)
NPHP7
NPHP8
NPHP9
0
100 (SLSN7)85
0
0
100 (SLSN6)84
Abbreviations: NPHP, nephronophthisis; SLSN, Senior-Løken syndrome.
NPHP12 TTC21B (tetratricopeptide 0 repeat domain 21B) NPHP13 WDR19 (WD repeat domain 19)
