Pediatr Surg Int (2014) 30:233–238 DOI 10.1007/s00383-013-3456-8
ORIGINAL ARTICLE
Effect of stem cells on renal recovery in rat model of partial unilateral upper ureteric obstruction N. Sugandhi • M. Srinivas • S. Agarwala D. K. Gupta • S. Sharma • A. Sinha • A. Dinda • S. Mohanty
•
Published online: 27 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Background Untreated obstructive uropathy produces irreversible renal damage and is an important cause of pediatric renal insufficiency. This study was designed to evaluate the effects of stem cell injection on morphological and pathological changes in the rat kidneys with partial unilateral upper ureteric obstruction (PUUUO). Methods Wistar rats (n = 30) were operated upon to create a PUUUO by the psoas hitch method and were randomized into Group I (control, n = 15) and Group II (stem cell, n = 15); at day 5, 10 and 15, a subgroup of rats (n = 5) from each group was killed and the kidneys harvested. Pathological and morphological changes in the harvested kidneys were studied and compared between the two groups. Results Morphologically, at day 15, Group II had significantly (p = 0.04) greater cortical thickness (0.48 ± 0.17 vs. 0.38 ± 0.09 mm). Histologically, at day 5, Group II had significantly (p = 0.032) lower peri-pelvic fibrosis. Group II group showed greater peri-pelvic inflammation as compared to Group I (p = 0.05). At day 10, lower grades of peri-pelvic fibrosis (p = 0.08), interstitial fibrosis (p = 0.037) and tubular atrophy (p = 0.05) were seen in the Group II. At day 15, Group II N. Sugandhi (&) M. Srinivas S. Agarwala D. K. Gupta S. Sharma A. Sinha Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India e-mail: [email protected] A. Dinda Department of Pathology, All India Institute of Medical Sciences, New Delhi, India S. Mohanty Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India
demonstrated significantly lower parenchymal loss (p = 0.037), glomerulosclerosis (p = 0.08), interstitial fibrosis (p = 0.08), tubular atrophy (p = 0.08) and peripelvic fibrosis (p = 0.08). Conclusions In a rat model of PUUUO, stem cell injection prevented detrimental changes in renal pathology and preserved renal parenchymal mass. Keywords Obstructive uropathy Kidney Stem cells Animal model Hydronephrosis
Introduction Obstructive uropathy (OU) is one of the leading causes of chronic renal insufficiency in children. It accounts for 23 % of pediatric renal insufficiency and 50 % of pediatric patients with end-stage renal disease (ESRD) undergoing renal transplantation [1, 2]. Current treatment options include maintenance dialysis and renal transplantation, both of which are less than ideal long-term alternatives in pediatric population, considering the shortage of donor organs, high cost, morbidity and reduced life expectancy. Therefore, the focus has shifted to regenerative cell-based therapeutics, where the inherent regenerative and reparative potential of kidneys can be stimulated for structural and functional recovery. Multiple independent studies have established stem cells to have therapeutic benefits in acute renal injury resulting from Doxorubicin, folic acid, radiation or ischemia [3–8]. The effect of stem cells on chronic renal injury due to obstructive nephropathy has not yet been explored. The goal of this pilot study was to establish a model of obstructive nephropathy in rats and elucidate the effect of stem cells on the kidneys damaged by chronic obstruction.
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Table 1 Division of rats into different groups (n = 30, each group = 5) Group I (control)
Group II (stem cell)
Killed at 5 days
IA
IIA
Killed at 10 days
IB
IIB
Killed at 15 days
IC
IIC
Materials and methods This experimental prospective interventional study was conducted after appropriate clearance from the institutional ethics committee [598/IAEC/11]. Thirty Wistar rats, 3–4 weeks old, were divided into two groups of 15 each. Group I (15 male rats) consisted of the control while Group II (15 female rats) formed the stem cell group. The rats were anasthetized using intraperitoneal ketamine anesthesia (50 mg/kg); laparotomy was done and the left ureter was partially obstructed at the upper end by hitching it to ipsilateral psoas muscle with silk 4-0 sutures (Ethicon, Johnson and Johnson Limited). Bone marrow was aspirated from the right tibia of Group I rats at the time of creation of upper ureteric obstruction using an 18-Gauze needle and 5 ml syringe. The bone marrow was pipetted repeatedly to make single cell suspension. Then mononuclear cells were separated by layering the single cell suspension over the density gradient media (Lymphoprep, Axis Shield) in 15 ml centrifuge tubes and centrifuged at 8009g for 25 min. The buffy layer at the interface was taken out in new 15 ml centrifuge tube and washed twice with Phosphate Buffer Saline (PBS) to remove the lymphoprep. A final volume of 2–4 ml of concentrated cell suspension was prepared in PBS. A small fraction of the cell suspension was used for cell counting and viability testing by trypan blue exclusion. Cell counting was performed using hemocytometer. 1 ml of this stem cell concentrate (1 million cells/1 ml) was injected into the median tail vein of Group II rats at the time of creation of the ureteric obstruction. Both the groups were further subdivided into three sub groups of five rats each (Subgroups A, B and C). The ureters were obstructed for 5 days in subgroup IA and IIA, 10 days in IB and IIB and 15 days in IC and IIC (Table 1). Affected kidneys were harvested at the end of the scheduled time. These kidneys were then subjected to morphometric and histopathological analysis. The kidneys were split longitudinally and photographed along with a scale, using a 12.1 megapixels 49 optical zoom digital camera kept vertically overhead at a distance of 25 cm and an angle of 15°. Various dimensions, viz., the renal height, pelvic dimensions (anterior–posterior, cranio-caudal and lateral) and renal cortical thickness at the upper and lower pole, were measured with a least count of 0.01 mm using
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Fig. 1 Morphometric measurements using I photomeasure
I-photo-measure software version 3.0 (DigiContractor Corp., USA) (Fig. 1). The slides were stained with hematoxylin and eosin and Masson’s trichrome stains. The distances were measured with Image Pro Plus software with a least count of 0.00001 lm (Media Cybernetics, UK). Each kidney was graded according to seven distinct parameters—Tubular Atrophy, Glomerulosclerosis, Interstitial Fibrosis, Peri-Pelvic fibrosis, Peri-Pelvic Inflammation, Parenchymal Inflammation and Parenchymal loss (Table 2) [9–11]. The data analysis was done using STATA software version 9.0 (Stata Corp LP, Texas, USA). The continuous data were compared using t test and dichotomous data were compared using Mann–Whitney U test. A p value of \0.05 was considered statistically significant.
Results The morphological and histological parameters were compared between the two groups (Tables 3, 4). The two groups were not significantly different with regard to the morphological parameters except for the pelvic volume and cortical thickness. At 15 days, the pelvic volume in Group I was 0.612 cm3 as compared to 0.318 cm3 in Group II with a p value of 0.008. The cortical thickness was significantly greater in Group II at 15 days (0.48 cm) as compared to that in Group I (0.38 cm) with a p value of 0.04 (Fig. 2a, b). Pathologically, there was a distinct difference in most of the parameters of renal injury between the two groups especially at day 10 and day 15. Tubular atrophy was significantly less in Group II at day 10 (p = 0.056) and day 15 (0.008). Interstitial fibrosis at day 10 (p = 0.037), day 15 (p = 0.008) and Glomerulosclerosis at day 15 (p = 0.032) were also notably less in Group II as compared
Pediatr Surg Int (2014) 30:233–238 Table 2 The grades of histological parameters Histological parameter
Grade
Changes
Peri-pelvic fibrosis
I
No abnormality
II
Mild perivascular fibrosis with muscle hypertrophy
III
Moderate perivascular fibrosis with muscle hypertrophy
IV
Severe perivascular fibrosis with muscle atrophy
Peri-pelvic inflammation
Tubular atrophy
Interstitial fibrosis
Glomerulosclerosis
Parenchymal inflammation
0
No or rare inflammatory cells
I
\15 inflammatory cells/hpf
II
15–30 inflammatory cells/hpf
III
[30 inflammatory cells/hpf
0
No abnormality
I
Tubular atrophy affecting \25 % of tubules
II
Tubular atrophy affecting 25–50 % of tubules
III
Tubular atrophy affecting [50 % of tubules
0 I
No fibrosis Mild interstitial fibrosis
II
Moderate interstitial fibrosis
III
Severe interstitial fibrosis
0
None
I
Glomerulosclerosis affecting \25 % of glomeruli
II
Glomerulosclerosis affecting 25–50 % of glomeruli
III
Glomerulosclerosis affecting 50 % of glomeruli
0
None
I
Inflammation affecting \25 % of parenchyma
II
Inflammation affecting 25–50 % of parenchyma Inflammation affecting [50 % of parenchyma
III Cortical loss
0
None
I
Mild decrease in cortical thickness
II
Moderate decrease in cortical thickness
III
Severe decrease in cortical thickness
to Group I (Fig. 3a, b). Peri-pelvic fibrosis as assessed by Mason’s Trichrome strain was appreciably less in Group II at all times of assessment with p value of 0.032 at day 5, 0.008 at day 10 and 0.008 at day 15. Importantly, parenchymal loss was significantly less in Group II at the end of 15 days with a p value of 0.037. The concentration of parenchymal mononuclear cells as graded by parenchymal inflammation was the only histological parameter higher in Group II as compared to Group I. Even though this
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difference was statistically significant only at day 10 (p = 0.032), even at day 5 and day 15 Group II demonstrated a trend towards higher parenchymal inflammation.
Discussion In this study, experimentally created PUUUO in rats replicated renal pathological changes resulting from OU in humans. Injection of stems cells in Group II restricted the severity of renal damage as assessed by various pathological parameters. Irreversible renal injury in the form of decreased cortical thickness and parenchymal loss was significantly lesser in Group II indicating protective effects of stem cells. Obstructive uropathy associated with congenital anomalies of the urinary tract accounts for 30–50 % of all endstage renal disease (ESRD) cases in children [1, 2]. The most common causes of obstructive uropathy in pediatric population are posterior urethral valves (PUV), neurogenic bladder and pelvi-ureteric junction obstruction (PUJO), especially those with bilateral PUJO or those with delayed diagnosis. Untreated obstruction impairs the growth and development of the kidney leading to renal dysplasia. Tubular atrophy, interstitial fibrosis and glomerulosclerosis are early changes and may be demonstrable even before the evidence of clinical hydronephrosis. Parenchymal and peripelvic inflammation and fibrosis are other common features. Unrelieved obstruction leads to progressive destruction and fibrosis finally resulting in irreversible loss of renal parenchyma, evident as decrease in cortical thickness. Histopathological changes vary with degree and duration of obstruction in various studies [9–11]. These changes may be transient in early stages, but the structural and functional disruption of the kidney becomes irreversible with prolonged obstruction. Various studies have documented these histological changes in the kidney in obstructive nephropathy [9–11]. Congenital obstructive lesions of the urinary tract can initiate renal damage in utero. Consequently many of such kidneys show irreversible injury or dysplasia at birth and such children show symptoms of chronic renal failure very early in life. The current options available for the treatment of pediatric ESRD are limited and far from optimal. Even with recent advances, renal transplantation is not possible in infants and toddlers. The shortage of donor kidneys remains a universal problem. Moreover, the life expectancy of a child after renal transplant is far less than the desired normal life-span due to the necessity of taking long-term immunosuppressants and risk of graft rejection, opportunistic infections and secondary malignancies. Furthermore, there is a growing recognition that the disease state arising from renal failure is the result of more than just the absence of toxin clearance and volume regulation
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Fig. 2 a, b Cortical thickness at day 15 in Group I and II, respectively, showing visibly thicker cortex in Group II
Table 3 Difference in the renal morphological parameters between Group I and II. 5 days (IA vs. IIA)
10 days (IB vs. IIB)
15 days (IC vs. IIC)
Renal height (cm)
1.66 vs. 1.56 (p = 0.47)
1.76 vs. 1.74 (p = 0.84)
1.92 vs. 1.84 (p = 0.84)
Renal breadth (cm)
1.00 vs. 0.94 (p = 0.63)
1.08 vs. 0.92 (p = 0.19)
1.28 vs. 1.26 (p = 0.74)
Pelvic cranio-caudal diameter (cm)
0.90 vs. 0.84 (p = 0.37)
0.98 vs. 0.82 (p = 0.03)
1.16 vs. 0.88 (p = 0.008)
Pelvic AP diameter (cm)
0.52 vs. 0.40 (p = 0.14)
0.60 vs. 0.60 (p = 1.00)
0.68 vs. 0.64 (p = 0.48)
Pelvic lateral diameter (cm)
0.72 vs. 0.66 (p = 0.37)
0.76 vs. 0.72 (p = 0.48)
0.78 vs. 0.68 (p = 0.09)
Pelvic volume (cm3)
0.336 vs. 0.244 (p = 0.33)
0.448 vs. 0.347 (p = 0.13)
0.612 vs. 0.381 (p = 0.008)
Cortical thickness (cm)
0.38 vs. 0.36 (p = 0.66)
0.40 vs. 0.46 (p = 0.17)
0.38 vs. 0.48 (p = 0.04)
Bold values highlight the significant differences
Table 4 Difference in the renal histopathological changes between Group I and II. 5 days (IA vs. IIA)
10 days (IB vs. IIB)
15 days (IC vs. IIC)
Interstitial fibrosis
p = 0.151
p = 0.037
p = 0.008
Tubular atrophy
p = 0.22
p = 0.056
p = 0.008
Glomerulosclerosis
p = 0.32
p = 0.22
p = 0.032
Peri-pelvic fibrosis
p = 0.032
p = 0.008
p = 0.008
Peri-pelvic inflammation
p = 0.056 (Group II [ Group I)
p = 0.151
p = 0.222
Parenchymal inflammation
p = 0.095 (Group II [ Group I)
p = 0.032 (Group II > Group I)
p = 0.095 (Group II > Group I)
Parenchymal loss
p = 0.41
p = 0.222
p = 0.032
Bold values highlight the significant differences
that are dealt with by conventional dialysis therapy. The other metabolic renal functions including gluconeogenesis, ammoniagenesis, catabolism of peptide hormones and growth factors, production and regulation of cytokines are not addressed by dialysis. Thus, there is a need to investigate and explore potential cellular- or molecular-based regenerative approaches for the kidney, thereby reducing the morbidity, mortality and overall economic impact associated with the condition. A number of experimental animal models have been established to study the effect of obstruction on a
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developing kidney. These include partial or complete ureteral obstruction, unilateral or bilateral ureteral obstruction, and urethral obstruction in young rats, rabbits, opossum, sheep or guinea pigs [12, 13]. The renal development and physiology in rats in the early post-natal period closely resembles human fetal renal development and hence can be used to study the effects of obstruction on the kidney. The present study recruited 3-week-old Wistar rats in which the renal maturation continues for almost 3–4 weeks postnatally, and hence the present model is best suited to compare the obstructive uropathy in children.
Pediatr Surg Int (2014) 30:233–238
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Fig. 3 a, b Marked tubular atrophy, glomerulosclerosis and interstitial fibrosis in Group I (a) as compared to significantly less severe tubular atrophy, glomerulosclerosis and tubular atrophy in Group II (b)
Fig. 4 Mitotic figures in regenerating tubules indicate the reparative effect of stem cells in Group II
The ‘psoas hitch method’ is one of the popular methods for creating a partial ureteral obstruction and involves hitching the upper one-third or one-fourth ureter in the psoas muscle to create an acute angulation. This gives a reliable, constant, reproducible and reversible means of creating a partial upper ureteric obstruction, as validated in previous studies [14]. Stem cells are multipotent cells capable of self-renewal and differentiation. Various studies have demonstrated that renal repair after injury occurs due to multipotent stem cells that are derived either from dedifferentiation of the tubular cells or by contribution from circulating/bone marrow-derived mesenchymal stem cells which are spontaneously drawn to the site of injury, or even from both sources [3–7, 15–17]. Typically, the degree of structural contribution by indigenous stem cells in renal repair is believed to be in the range of 1–8 % [3, 15]. This percentage is found to be more if stem cells are reinforced by external supplementation [3, 4]. Here, we loaded external stem cells in the circulation of Group II rats through the
median tail vein. Various theories have been put forward to explain the mechanism of renal repair by stem cells. Stem cells can induce tubular and glomerular regeneration by differentiating into the tubular progenitor cells [4]. They may also act in a paracrine manner, secreting growth factors and cytokines which may aid regeneration [18]. The mechanical effects of ureteric obstruction were comparable in both groups. The renal height and width, anterior–posterior, lateral, cranio-caudal diameter of the pelvis and the pelvic volume showed a progressive increase with increasing duration of obstruction. The cortical thickness showed a decreasing trend with increasing duration of obstruction in both the groups due to parenchymal atrophy. However, the cortical thickness was significantly more in Group II at 15 days indicating less cortical atrophy. Thus, stem cells may provide protection against parenchymal loss in cases of prolonged obstruction. Both groups demonstrated histopathological evidence of renal damage due to obstruction in the form of glomerulosclerosis, tubular atrophy, interstitial and peri-pelvic fibrosis. These changes were significantly less severe in Group II. Remarkably, the difference in severity of these pathological parameters became evident only at day 10 and 15. This lag time probably reflects the time required for the stem cells to establish/dedifferentiate and start multiplying/ secreting growth factors. Interestingly the high-power views of the pathological sections showed distinct mitotic figures with regenerating tubular epithelium cells at day 10 and day 15 in Group II (Fig. 4). It can be inferred that the injected stem cells caused slowing of the pathological damage and induced regeneration of the tubules. Loss of renal parenchyma is the end result of prolonged outflow obstruction and consequent back pressure. At 15 days, Group II had significantly lesser parenchymal loss compared to Group I. It is possible that stem cells had either modified the humoral environment in such a way that cell destruction and apoptosis (leading to loss of
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parenchyma) is slowed down, or they differentiated into the renal progenitor cells and replenished the damaged tissue, thus maintaining the parenchymal thickness. Intriguingly, Group II showed a higher concentration of mononuclear cells (parenchymal inflammation) at all times. It was observed that most of the inflammatory cells in Group II were mononuclear even at day 5 and 10, whereas polymorphs predominated in Group I. It is probable that these mononuclear cells really represented the migrated and transdifferentiated stem cells, rather than actual inflammatory cells. The high percentage of inflammatory cell infiltration has also been shown in other studies. These studies contend that the stem cells modify the humoral environment in such a way so as to attract more leukocytes which may aid repair by secretion of growth factors and cytokines [18]. Overall, these results indicate that supplemental stem cells may have a role in slowing down and preventing renal damage in obstructive uropathy. This has potential clinical implications. The present study beacons to explore stem cells for renal recovery in children with poorly recovering kidneys after successful pyeloplasty and ESRD due to obstructive uropathy. Our study is encumbered by certain limitations. The presence of stem cells in the affected kidney was inferred indirectly by the decreased degree of pathological damage in Group II. Their presence can be directly established by molecular markers such as CD34 or tracking of the Y chromosome of injected male stem cells by fluorescent in situ hybridization (FISH) [19]. This is being done and the data would be incorporated into the present draft manuscript.
Conclusion In a rat model of unilateral partial upper ureteric obstruction, injection of allogenic stem cells decreases the severity of renal pathological injury and cortical loss.
References 1. Grandaliano G, Gesualdo L, Bartoli F et al (2000) MCP-1 and EGF renal expression and urine excretion in human congenital obstructive nephropathy. Kidney Int 58:182–192
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Pediatr Surg Int (2014) 30:233–238 2. Ruiz E, Ferraris J (2007) 25 years of live related renal transplantation in children: the Buenos Aires experience. Indian J Urol 23:443–445 3. Lin F, Cordes K, Li L et al (2003) Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemia-reperfusion injury in mice. J Am Soc Nephrol 14:1188–1199 4. Kale S, Karihaloo A, Clark PR et al (2003) Bone marrow stem cells contribute to repair of the ischemically injured renal tubule. J Clin Invest 112:42–49 5. Wong CY, Cheong SK, Mok PL, Leong CF (2008) Differentiation of human mesenchymal stem cells into mesangial cells in post-glomerular injury murine model. Pathology 40:52–57 6. Togel F, Cohen A, Zhang P et al (2009) Autologous and allogeneic marrow stromal cells are safe and effective for the treatment of acute kidney injury. Stem Cell Dev 18:475–485 7. Humphreys BD, Bonventre JD (2008) Mesenchymal stem cells in acute kidney injury. Annu Rev Med 59:311–325 8. Sharma S, Gupta DK, Kumar L et al (2003) Are therapeutic stem cells justified in bilateral multicystic kidney disease? A review of literature with insights into the embryology. J Clin Invest 112:1776–1784 9. Mizuno S, Matsumoto K, Nakamura T (2001) Hepatocyte growth factor suppresses interstitial fibrosis in a mouse model of obstructive nephropathy. Kidney Int 59:1304–1314 10. Elder JS, Stansbrey R, Dahms BB, Selzman AA (1995) Renal histological changes secondary to ureteropelvic junction obstruction. J Urol 154:719–722 11. Ping LZ, Craig AP, Seymour R (2000) Ureteropelvic junction obstruction: morphological and clinical studies. Pediatr Nephrol 14:820–826 12. Chevalier RL, Kaiser DL (1984) Chronic partial ureteral obstruction in the neonatal guinea pig I: influence of uninephrectomy on growth and hemodynamics. Pediatr Res 18:1266–1271 13. Kaneto H, Morrissey JJ, Klahr S (1993) Increased expression of TGF-beta 1 mRNA in the obstructed kidney of the rats with unilateral ureteral ligation. Kidney Int 44:313–321 14. Wen JG (2002) Partial unilateral ureteral obstruction in rats. Neurol Urodyn 21:231–250 15. Poulsom R, Forbes SJ, Hodivala-Dilke K et al (2001) Bone marrow contributes to renal parenchymal turnover and regeneration. J Pathol 195:229–235 16. Witzgall R, Brown D, Schwartz C, Bonventre JV (1994) Localization of proliferating cell nuclear antigen, vimentin, c-Fos and clusterin in the post-ischemic kidney. Evidence for a heterogenous genetic response among nephron segments and a large pool of mitotically active and dedifferentiated cells. J Clin Invest 93:2175–2188 17. Imgrund M, Grone E, Grone HJ, Kretzler M, Holzman L, Schlondorff D, Rothenpieler UW (1999) Re-expression of the developmental gene Pax-2 during experimental acute tubular necrosis in mice. Kidney Int 56:1423–1431 18. Hopkins C, Li J, Rae F, Little MF (2009) Stem cell options for kidney disease. J Pathol 217:265–281 19. James CB, Humes HD (2005) Stem Cell approaches for treatment of renal failure. Pharmacol Rev 57:299–313
ORIGINAL ARTICLE
Effect of stem cells on renal recovery in rat model of partial unilateral upper ureteric obstruction N. Sugandhi • M. Srinivas • S. Agarwala D. K. Gupta • S. Sharma • A. Sinha • A. Dinda • S. Mohanty
•
Published online: 27 December 2013 Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Background Untreated obstructive uropathy produces irreversible renal damage and is an important cause of pediatric renal insufficiency. This study was designed to evaluate the effects of stem cell injection on morphological and pathological changes in the rat kidneys with partial unilateral upper ureteric obstruction (PUUUO). Methods Wistar rats (n = 30) were operated upon to create a PUUUO by the psoas hitch method and were randomized into Group I (control, n = 15) and Group II (stem cell, n = 15); at day 5, 10 and 15, a subgroup of rats (n = 5) from each group was killed and the kidneys harvested. Pathological and morphological changes in the harvested kidneys were studied and compared between the two groups. Results Morphologically, at day 15, Group II had significantly (p = 0.04) greater cortical thickness (0.48 ± 0.17 vs. 0.38 ± 0.09 mm). Histologically, at day 5, Group II had significantly (p = 0.032) lower peri-pelvic fibrosis. Group II group showed greater peri-pelvic inflammation as compared to Group I (p = 0.05). At day 10, lower grades of peri-pelvic fibrosis (p = 0.08), interstitial fibrosis (p = 0.037) and tubular atrophy (p = 0.05) were seen in the Group II. At day 15, Group II N. Sugandhi (&) M. Srinivas S. Agarwala D. K. Gupta S. Sharma A. Sinha Department of Pediatric Surgery, All India Institute of Medical Sciences, New Delhi, India e-mail: [email protected] A. Dinda Department of Pathology, All India Institute of Medical Sciences, New Delhi, India S. Mohanty Stem Cell Facility, All India Institute of Medical Sciences, New Delhi, India
demonstrated significantly lower parenchymal loss (p = 0.037), glomerulosclerosis (p = 0.08), interstitial fibrosis (p = 0.08), tubular atrophy (p = 0.08) and peripelvic fibrosis (p = 0.08). Conclusions In a rat model of PUUUO, stem cell injection prevented detrimental changes in renal pathology and preserved renal parenchymal mass. Keywords Obstructive uropathy Kidney Stem cells Animal model Hydronephrosis
Introduction Obstructive uropathy (OU) is one of the leading causes of chronic renal insufficiency in children. It accounts for 23 % of pediatric renal insufficiency and 50 % of pediatric patients with end-stage renal disease (ESRD) undergoing renal transplantation [1, 2]. Current treatment options include maintenance dialysis and renal transplantation, both of which are less than ideal long-term alternatives in pediatric population, considering the shortage of donor organs, high cost, morbidity and reduced life expectancy. Therefore, the focus has shifted to regenerative cell-based therapeutics, where the inherent regenerative and reparative potential of kidneys can be stimulated for structural and functional recovery. Multiple independent studies have established stem cells to have therapeutic benefits in acute renal injury resulting from Doxorubicin, folic acid, radiation or ischemia [3–8]. The effect of stem cells on chronic renal injury due to obstructive nephropathy has not yet been explored. The goal of this pilot study was to establish a model of obstructive nephropathy in rats and elucidate the effect of stem cells on the kidneys damaged by chronic obstruction.
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Table 1 Division of rats into different groups (n = 30, each group = 5) Group I (control)
Group II (stem cell)
Killed at 5 days
IA
IIA
Killed at 10 days
IB
IIB
Killed at 15 days
IC
IIC
Materials and methods This experimental prospective interventional study was conducted after appropriate clearance from the institutional ethics committee [598/IAEC/11]. Thirty Wistar rats, 3–4 weeks old, were divided into two groups of 15 each. Group I (15 male rats) consisted of the control while Group II (15 female rats) formed the stem cell group. The rats were anasthetized using intraperitoneal ketamine anesthesia (50 mg/kg); laparotomy was done and the left ureter was partially obstructed at the upper end by hitching it to ipsilateral psoas muscle with silk 4-0 sutures (Ethicon, Johnson and Johnson Limited). Bone marrow was aspirated from the right tibia of Group I rats at the time of creation of upper ureteric obstruction using an 18-Gauze needle and 5 ml syringe. The bone marrow was pipetted repeatedly to make single cell suspension. Then mononuclear cells were separated by layering the single cell suspension over the density gradient media (Lymphoprep, Axis Shield) in 15 ml centrifuge tubes and centrifuged at 8009g for 25 min. The buffy layer at the interface was taken out in new 15 ml centrifuge tube and washed twice with Phosphate Buffer Saline (PBS) to remove the lymphoprep. A final volume of 2–4 ml of concentrated cell suspension was prepared in PBS. A small fraction of the cell suspension was used for cell counting and viability testing by trypan blue exclusion. Cell counting was performed using hemocytometer. 1 ml of this stem cell concentrate (1 million cells/1 ml) was injected into the median tail vein of Group II rats at the time of creation of the ureteric obstruction. Both the groups were further subdivided into three sub groups of five rats each (Subgroups A, B and C). The ureters were obstructed for 5 days in subgroup IA and IIA, 10 days in IB and IIB and 15 days in IC and IIC (Table 1). Affected kidneys were harvested at the end of the scheduled time. These kidneys were then subjected to morphometric and histopathological analysis. The kidneys were split longitudinally and photographed along with a scale, using a 12.1 megapixels 49 optical zoom digital camera kept vertically overhead at a distance of 25 cm and an angle of 15°. Various dimensions, viz., the renal height, pelvic dimensions (anterior–posterior, cranio-caudal and lateral) and renal cortical thickness at the upper and lower pole, were measured with a least count of 0.01 mm using
123
Fig. 1 Morphometric measurements using I photomeasure
I-photo-measure software version 3.0 (DigiContractor Corp., USA) (Fig. 1). The slides were stained with hematoxylin and eosin and Masson’s trichrome stains. The distances were measured with Image Pro Plus software with a least count of 0.00001 lm (Media Cybernetics, UK). Each kidney was graded according to seven distinct parameters—Tubular Atrophy, Glomerulosclerosis, Interstitial Fibrosis, Peri-Pelvic fibrosis, Peri-Pelvic Inflammation, Parenchymal Inflammation and Parenchymal loss (Table 2) [9–11]. The data analysis was done using STATA software version 9.0 (Stata Corp LP, Texas, USA). The continuous data were compared using t test and dichotomous data were compared using Mann–Whitney U test. A p value of \0.05 was considered statistically significant.
Results The morphological and histological parameters were compared between the two groups (Tables 3, 4). The two groups were not significantly different with regard to the morphological parameters except for the pelvic volume and cortical thickness. At 15 days, the pelvic volume in Group I was 0.612 cm3 as compared to 0.318 cm3 in Group II with a p value of 0.008. The cortical thickness was significantly greater in Group II at 15 days (0.48 cm) as compared to that in Group I (0.38 cm) with a p value of 0.04 (Fig. 2a, b). Pathologically, there was a distinct difference in most of the parameters of renal injury between the two groups especially at day 10 and day 15. Tubular atrophy was significantly less in Group II at day 10 (p = 0.056) and day 15 (0.008). Interstitial fibrosis at day 10 (p = 0.037), day 15 (p = 0.008) and Glomerulosclerosis at day 15 (p = 0.032) were also notably less in Group II as compared
Pediatr Surg Int (2014) 30:233–238 Table 2 The grades of histological parameters Histological parameter
Grade
Changes
Peri-pelvic fibrosis
I
No abnormality
II
Mild perivascular fibrosis with muscle hypertrophy
III
Moderate perivascular fibrosis with muscle hypertrophy
IV
Severe perivascular fibrosis with muscle atrophy
Peri-pelvic inflammation
Tubular atrophy
Interstitial fibrosis
Glomerulosclerosis
Parenchymal inflammation
0
No or rare inflammatory cells
I
\15 inflammatory cells/hpf
II
15–30 inflammatory cells/hpf
III
[30 inflammatory cells/hpf
0
No abnormality
I
Tubular atrophy affecting \25 % of tubules
II
Tubular atrophy affecting 25–50 % of tubules
III
Tubular atrophy affecting [50 % of tubules
0 I
No fibrosis Mild interstitial fibrosis
II
Moderate interstitial fibrosis
III
Severe interstitial fibrosis
0
None
I
Glomerulosclerosis affecting \25 % of glomeruli
II
Glomerulosclerosis affecting 25–50 % of glomeruli
III
Glomerulosclerosis affecting 50 % of glomeruli
0
None
I
Inflammation affecting \25 % of parenchyma
II
Inflammation affecting 25–50 % of parenchyma Inflammation affecting [50 % of parenchyma
III Cortical loss
0
None
I
Mild decrease in cortical thickness
II
Moderate decrease in cortical thickness
III
Severe decrease in cortical thickness
to Group I (Fig. 3a, b). Peri-pelvic fibrosis as assessed by Mason’s Trichrome strain was appreciably less in Group II at all times of assessment with p value of 0.032 at day 5, 0.008 at day 10 and 0.008 at day 15. Importantly, parenchymal loss was significantly less in Group II at the end of 15 days with a p value of 0.037. The concentration of parenchymal mononuclear cells as graded by parenchymal inflammation was the only histological parameter higher in Group II as compared to Group I. Even though this
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difference was statistically significant only at day 10 (p = 0.032), even at day 5 and day 15 Group II demonstrated a trend towards higher parenchymal inflammation.
Discussion In this study, experimentally created PUUUO in rats replicated renal pathological changes resulting from OU in humans. Injection of stems cells in Group II restricted the severity of renal damage as assessed by various pathological parameters. Irreversible renal injury in the form of decreased cortical thickness and parenchymal loss was significantly lesser in Group II indicating protective effects of stem cells. Obstructive uropathy associated with congenital anomalies of the urinary tract accounts for 30–50 % of all endstage renal disease (ESRD) cases in children [1, 2]. The most common causes of obstructive uropathy in pediatric population are posterior urethral valves (PUV), neurogenic bladder and pelvi-ureteric junction obstruction (PUJO), especially those with bilateral PUJO or those with delayed diagnosis. Untreated obstruction impairs the growth and development of the kidney leading to renal dysplasia. Tubular atrophy, interstitial fibrosis and glomerulosclerosis are early changes and may be demonstrable even before the evidence of clinical hydronephrosis. Parenchymal and peripelvic inflammation and fibrosis are other common features. Unrelieved obstruction leads to progressive destruction and fibrosis finally resulting in irreversible loss of renal parenchyma, evident as decrease in cortical thickness. Histopathological changes vary with degree and duration of obstruction in various studies [9–11]. These changes may be transient in early stages, but the structural and functional disruption of the kidney becomes irreversible with prolonged obstruction. Various studies have documented these histological changes in the kidney in obstructive nephropathy [9–11]. Congenital obstructive lesions of the urinary tract can initiate renal damage in utero. Consequently many of such kidneys show irreversible injury or dysplasia at birth and such children show symptoms of chronic renal failure very early in life. The current options available for the treatment of pediatric ESRD are limited and far from optimal. Even with recent advances, renal transplantation is not possible in infants and toddlers. The shortage of donor kidneys remains a universal problem. Moreover, the life expectancy of a child after renal transplant is far less than the desired normal life-span due to the necessity of taking long-term immunosuppressants and risk of graft rejection, opportunistic infections and secondary malignancies. Furthermore, there is a growing recognition that the disease state arising from renal failure is the result of more than just the absence of toxin clearance and volume regulation
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Fig. 2 a, b Cortical thickness at day 15 in Group I and II, respectively, showing visibly thicker cortex in Group II
Table 3 Difference in the renal morphological parameters between Group I and II. 5 days (IA vs. IIA)
10 days (IB vs. IIB)
15 days (IC vs. IIC)
Renal height (cm)
1.66 vs. 1.56 (p = 0.47)
1.76 vs. 1.74 (p = 0.84)
1.92 vs. 1.84 (p = 0.84)
Renal breadth (cm)
1.00 vs. 0.94 (p = 0.63)
1.08 vs. 0.92 (p = 0.19)
1.28 vs. 1.26 (p = 0.74)
Pelvic cranio-caudal diameter (cm)
0.90 vs. 0.84 (p = 0.37)
0.98 vs. 0.82 (p = 0.03)
1.16 vs. 0.88 (p = 0.008)
Pelvic AP diameter (cm)
0.52 vs. 0.40 (p = 0.14)
0.60 vs. 0.60 (p = 1.00)
0.68 vs. 0.64 (p = 0.48)
Pelvic lateral diameter (cm)
0.72 vs. 0.66 (p = 0.37)
0.76 vs. 0.72 (p = 0.48)
0.78 vs. 0.68 (p = 0.09)
Pelvic volume (cm3)
0.336 vs. 0.244 (p = 0.33)
0.448 vs. 0.347 (p = 0.13)
0.612 vs. 0.381 (p = 0.008)
Cortical thickness (cm)
0.38 vs. 0.36 (p = 0.66)
0.40 vs. 0.46 (p = 0.17)
0.38 vs. 0.48 (p = 0.04)
Bold values highlight the significant differences
Table 4 Difference in the renal histopathological changes between Group I and II. 5 days (IA vs. IIA)
10 days (IB vs. IIB)
15 days (IC vs. IIC)
Interstitial fibrosis
p = 0.151
p = 0.037
p = 0.008
Tubular atrophy
p = 0.22
p = 0.056
p = 0.008
Glomerulosclerosis
p = 0.32
p = 0.22
p = 0.032
Peri-pelvic fibrosis
p = 0.032
p = 0.008
p = 0.008
Peri-pelvic inflammation
p = 0.056 (Group II [ Group I)
p = 0.151
p = 0.222
Parenchymal inflammation
p = 0.095 (Group II [ Group I)
p = 0.032 (Group II > Group I)
p = 0.095 (Group II > Group I)
Parenchymal loss
p = 0.41
p = 0.222
p = 0.032
Bold values highlight the significant differences
that are dealt with by conventional dialysis therapy. The other metabolic renal functions including gluconeogenesis, ammoniagenesis, catabolism of peptide hormones and growth factors, production and regulation of cytokines are not addressed by dialysis. Thus, there is a need to investigate and explore potential cellular- or molecular-based regenerative approaches for the kidney, thereby reducing the morbidity, mortality and overall economic impact associated with the condition. A number of experimental animal models have been established to study the effect of obstruction on a
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developing kidney. These include partial or complete ureteral obstruction, unilateral or bilateral ureteral obstruction, and urethral obstruction in young rats, rabbits, opossum, sheep or guinea pigs [12, 13]. The renal development and physiology in rats in the early post-natal period closely resembles human fetal renal development and hence can be used to study the effects of obstruction on the kidney. The present study recruited 3-week-old Wistar rats in which the renal maturation continues for almost 3–4 weeks postnatally, and hence the present model is best suited to compare the obstructive uropathy in children.
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Fig. 3 a, b Marked tubular atrophy, glomerulosclerosis and interstitial fibrosis in Group I (a) as compared to significantly less severe tubular atrophy, glomerulosclerosis and tubular atrophy in Group II (b)
Fig. 4 Mitotic figures in regenerating tubules indicate the reparative effect of stem cells in Group II
The ‘psoas hitch method’ is one of the popular methods for creating a partial ureteral obstruction and involves hitching the upper one-third or one-fourth ureter in the psoas muscle to create an acute angulation. This gives a reliable, constant, reproducible and reversible means of creating a partial upper ureteric obstruction, as validated in previous studies [14]. Stem cells are multipotent cells capable of self-renewal and differentiation. Various studies have demonstrated that renal repair after injury occurs due to multipotent stem cells that are derived either from dedifferentiation of the tubular cells or by contribution from circulating/bone marrow-derived mesenchymal stem cells which are spontaneously drawn to the site of injury, or even from both sources [3–7, 15–17]. Typically, the degree of structural contribution by indigenous stem cells in renal repair is believed to be in the range of 1–8 % [3, 15]. This percentage is found to be more if stem cells are reinforced by external supplementation [3, 4]. Here, we loaded external stem cells in the circulation of Group II rats through the
median tail vein. Various theories have been put forward to explain the mechanism of renal repair by stem cells. Stem cells can induce tubular and glomerular regeneration by differentiating into the tubular progenitor cells [4]. They may also act in a paracrine manner, secreting growth factors and cytokines which may aid regeneration [18]. The mechanical effects of ureteric obstruction were comparable in both groups. The renal height and width, anterior–posterior, lateral, cranio-caudal diameter of the pelvis and the pelvic volume showed a progressive increase with increasing duration of obstruction. The cortical thickness showed a decreasing trend with increasing duration of obstruction in both the groups due to parenchymal atrophy. However, the cortical thickness was significantly more in Group II at 15 days indicating less cortical atrophy. Thus, stem cells may provide protection against parenchymal loss in cases of prolonged obstruction. Both groups demonstrated histopathological evidence of renal damage due to obstruction in the form of glomerulosclerosis, tubular atrophy, interstitial and peri-pelvic fibrosis. These changes were significantly less severe in Group II. Remarkably, the difference in severity of these pathological parameters became evident only at day 10 and 15. This lag time probably reflects the time required for the stem cells to establish/dedifferentiate and start multiplying/ secreting growth factors. Interestingly the high-power views of the pathological sections showed distinct mitotic figures with regenerating tubular epithelium cells at day 10 and day 15 in Group II (Fig. 4). It can be inferred that the injected stem cells caused slowing of the pathological damage and induced regeneration of the tubules. Loss of renal parenchyma is the end result of prolonged outflow obstruction and consequent back pressure. At 15 days, Group II had significantly lesser parenchymal loss compared to Group I. It is possible that stem cells had either modified the humoral environment in such a way that cell destruction and apoptosis (leading to loss of
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parenchyma) is slowed down, or they differentiated into the renal progenitor cells and replenished the damaged tissue, thus maintaining the parenchymal thickness. Intriguingly, Group II showed a higher concentration of mononuclear cells (parenchymal inflammation) at all times. It was observed that most of the inflammatory cells in Group II were mononuclear even at day 5 and 10, whereas polymorphs predominated in Group I. It is probable that these mononuclear cells really represented the migrated and transdifferentiated stem cells, rather than actual inflammatory cells. The high percentage of inflammatory cell infiltration has also been shown in other studies. These studies contend that the stem cells modify the humoral environment in such a way so as to attract more leukocytes which may aid repair by secretion of growth factors and cytokines [18]. Overall, these results indicate that supplemental stem cells may have a role in slowing down and preventing renal damage in obstructive uropathy. This has potential clinical implications. The present study beacons to explore stem cells for renal recovery in children with poorly recovering kidneys after successful pyeloplasty and ESRD due to obstructive uropathy. Our study is encumbered by certain limitations. The presence of stem cells in the affected kidney was inferred indirectly by the decreased degree of pathological damage in Group II. Their presence can be directly established by molecular markers such as CD34 or tracking of the Y chromosome of injected male stem cells by fluorescent in situ hybridization (FISH) [19]. This is being done and the data would be incorporated into the present draft manuscript.
Conclusion In a rat model of unilateral partial upper ureteric obstruction, injection of allogenic stem cells decreases the severity of renal pathological injury and cortical loss.
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