Mesenchymal stem cells transplantation mildly ameliorates experimental diabetic nephropathy in rats

Diabetic nephropathy is one of the devastating complications in patients with diabetes. It has been considered that about 25%–40% of patients with type 1 or type 2 diabetes develop nephropathy within 20–25 years of the onset of diabetes.¹ Therefore, it is known to be the leading cause of end-stage renal disease and the most frequent cause of mortality in patients with diabetes.²

The major clinical treatment for diabetic nephropathy targets hyperglycemia and hypertension. UKPDS (1987–1991) has shown that with tight as compared with the less-tight control of blood pressure, there were relative risk reductions of 37% for microvascular disease.³ However, in the UKPDS post-trial, the benefits of previously improved blood pressure control were not sustained when between-group differences in blood pressure were lost. And good blood pressure control must be continued if the benefits are to be maintained.⁴ Intensive glucose control in the ADVANCE study has yielded a 21% relative reduction in nephropathy.⁵ But the ACCORD trial ended its intensive therapy early, after 3.5 years, because of a significant increase in deaths in the intensive-therapy group.⁶ Therefore, the need for alternative treatment strategies is compelling.

Theoretically, cell-based therapies may have the advantage of acting through multiple mechanisms in disorders with highly complex pathophysiology, such as diabetic nephropathy, while pharmacologic interventions often target only a single aspect of a disease. In the pathophysiology of diabetic nephropathy, roles of advanced glycation end products (AGEs), protein kinase C (PKC), rennin-angiotensin system (RAS) and growth factors such as transforming growth factor β (TGF-β) have been identified.⁷ Due to this complexity, it is thought that diabetic nephropathy may be an ideal target for a cell therapeutic approach.

Mesenchymal stem cells (MSCs) are a bone marrow-derived population which contains multipotent progenitors that can differentiate into multiple cellular lineages such as osteoblasts, chondrocytes, adipocytes^8-10 and also nervous cells.^11,12 MSCs are autologous, abundant, and relatively easy to harvest; they do not require immune suppression and they are associated with less ethical concern than are fetal or neonatal heart cells. For these reasons, adult MSCs could have a great therapeutic potential.

MSCs are attractive candidates for renal repair, because nephrons are of mesenchymal origin and stromal cells are of crucial importance for signaling, leading to differentiation of both nephrons and collecting ducts.¹³ Indeed, MSCs have been used in experimental acute renal failure, which could lower renal injury, accelerate tubular proliferation and improve renal function.^14-17 Chimeric rats carrying enhanced green fluorescence protein bone marrow cells responded to anti-Thy1 antibody-induced mesangiolysis with a dramatic repopulation of the mesangium, mainly of nonhematopoietic lineages.¹⁸ Furthermore, female mice recipients of male bone marrow grafts carried the Y chromosome in up to 7.9% of cortical tubular epithelial cells, indicating that bone marrow cells participated in the normal tubular turnover.¹⁹ Nevertheless, studies about MSCs on chronic kidney disease, particularly diabetic kidney disease, are largely lacking. The aim of the present study was to explore whether MSCs transplantation could attenuate renal change in rats with experimental diabetes, which could offer an alternative therapeutic strategy for diabetic nephropathy.

Cells and culture
Male Sprague-Dawley (SD) rats weighed 110–120 g were sacrificed by cervical dislocation and femora and tibiae were isolated. After dissection of attached muscle and connective tissue from the bones, the marrow was extruded by clipping of the epiphysial ends of the bones and flushing using a 26-gauge needle with 2 ml of Dulbecco′s modified Eagle′s medium-low glucose (DMEM-LG; Gibco, USA), supplemented with 10% fetal bovine serum (FBS, Hyclone, USA), L-glutamine (0.3 g/L) and kanamycin (100 mg/L) (MSC medium). Cells were filtered through 70 μm mesh (Beckton & Dickinson, San Jose, CA, USA) and incubated in 75 cm² primary culture flasks at 37°C in a humidified atmosphere consisting of 95% air and 5% CO₂. The first medium change was after 24 hours and twice a week thereafter. When these primary MSCs reached 80%–90% of confluence, they were detached with trypsin/EDTA (Sigma, USA), washed and resuspended at a concentration of 2×10⁴–5×10⁴ into new flasks. At second passage (P2 MSCs) when reaching 80% of confluence, the cells were collected and counted, and viability was assessed by trypan blue assay and flow-cytometric analysis. P2 MSCs were used for all of the experiments in this study.

Flow cytometry
After trypsin/EDTA treatment, MSCs were washed and resuspended in phosphate-buffered saline (PBS, Zhongshan Goldbridge, China) in aliquots of 2×10⁵ cells. CD45-PE (Biolegned, USA), CD29-FITC (Biolegend, USA), CD90-FITC (Serotec, UK) and CD34-PE-γ (Santa Cruz, USA) were added at a concentration of 2.5 μg/ml, 2.5 μg/ml, 5.0 μg/ml and 10.0 μg/ml, respectively. The samples were incubated with gentle shaking at room temperature for 20 minutes. The cells were pelleted, washed twice with PBS, and analyzed by flow cytometry.

In vitro differentiation assays
Bone marrow-derived cells obtained by plastic adhesion, as described above, were studied to verify their mesenchymal potential to differentiate toward osteoblasts and adipocytes. Osteogenic differentiation of MSCs was tested following the protocol of Pittenger et al.⁹ In brief, MSCs were expanded in complete medium containing 10% FBS until subconfluence after the second passage. Complete medium then was replaced by medium that was composed of DMEM-LG, 10% FBS, 100 nmol/L dexamethasone, 50 μmol/L ascorbic acid-2-phosphate, and 10 mmol/L-glycerophosphate (all from Sigma- Aldrich, Taufkirchen, Germany). After 3 weeks, cells were fixed with 4% paraformaldehyde in PBS for 15–30 minutes at room temperature followed by Alizarin Red S staining to observe calcium deposition. Adipogenic differentiation of MSCs from SD rats was tested following the protocol of Pittenger et al.⁹ After 15 days, Oil Red O staining was performed to assay the accumulation of lipid droplets.

BrdU labeling for in vivo tracking of cells
To identify cells derived from bone marrow, 5-bromo-2′-deoxyuridine (BrdU, 3 μg/ml), a thymidine analog and marker of newly synthesized DNA, was added to the medium 3 days before transplantation.²⁰ For immunostaining, the plastic-adherent cells were subcultured in chambered slides and >90% of plastic-adherent cells were BrdU immunoreactive.

Animal care
All animals received human care in compliance with the “Principles of Laboratory Animal Care”, formulated by the National Society for Medical Research, and “Guide for the Care and Use of Laboratory Animals” prepared by the institute of Laboratory Animal Research and published by the National Institutes of Health (NIH Pub. No. 86-23, revised 1985). The protocol was approved by the local animal study committee.

Diabetes was induced by a single injection of streptozo- tocin (STZ, Sigma) administered to male Sprague- Dawley (SD) rats (200–250 g). Briefly, the male rats (n=70) received a single intraperitoneal injection of STZ (60 mg/kg), dissolved immediately before administration in freshly prepared 0.1 mol/L citrate buffer (pH 4.5). Diabetes was defined as a random blood glucose reading of >16.7 mmol/L at 3 continuous days after 72 hours of STZ injection. Sixty-four rats met the criteria for diabetes. Four weeks after STZ, when the kidneys have recovered from the acute mild nephrotoxic effects of STZ,²¹ all animals were assigned to 4 groups: (1) MSC treated group with daily CsA gavage (the MSCA group, n=16); (2) MSC treated group (the MSC group, n=16); (3) CsA treatd group (the CsA group, n=16); (4) diabetes control group (the DC group, n=16). In MSC treated groups, 2×10⁶ labeled MSCs per animal in 0.2 ml serum free medium (SFM) were given via the left cardiac ventricle. Control diabetic animals were treated identically but infused with 0.2 ml SFM instead of cells. CsA, dissolved in olive oil, was administered at a dosage of 5 mg/kg by daily gavage, while control groups were administered with equal volume of olive oil. Eight rats were as normal controls. All groups were kept under identical conditions and had access to food and drinking water ad libitum.

Metabolic data
At 1, 2, 4, and 8 weeks after therapeutic intervention, random blood glucose was measured by Roche glucometer (Roche, Germany) through tail vein. And rats were placed in metabolic cages for collection of 24-hour urine specimens. Serum creatinine (SCr, mg/dl) and urine creatinine levels were measured using enzymatic methods. Urinary levels of albumin were determined by the enzyme-linked immunosorbent assay (ELISA) method according to the kit manufacturer's instructions (ADL, USA). Creatinine clearance rate (Ccr, ml/min) was calculated as urine creatinine (mg/dl) × urine volume (ml/min)/serum creatinine (mg/dl). Although the progression of human diabetic nephropathy is strongly associated with hypertension, the blood pressure changes seen in STZ induced diabetic rodent models is usually mild unless the strain being used is spontaneously hypertensive.²² In initial experiments, we tested blood pressure of some rats. No difference was discovered between diabetic rats and control rats (data not shown).

Kidney histology
At every time point, four rats of each group were weighted before they were killed. Then kidneys were removed, measured and weighed for morphological analysis. Renal mass index (RMI) was determined as the ratio of kidney weight/body mass. For pathohistological observation, all samples were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4 μm sections and subjected to periodic acid-Schiff (PAS) staining.

Immunohistochemical analysis
After deparaffinizing, kidney sections were sequentially incubated with anti-BrdU monoclonal antibodies and secondary antibody (anti-mouse Ig-alkoline phosphatase). 3′, 3-diaminobenzidine (DAB) was then used as a chromogen for BrdU light microscopy. All procedures were followed by 5-bromo-2′-deoxy-uridine Labeling and Detection Kit II (Roche, Germany). Proliferating cell nuclear antigen (PCNA) was detected to investigate whether labeled MSCs proliferated in recipient rats. For PCNA detection, a monoclonal mouse anti-rat PC10 antibody was used at dilution 1:400 (DakoCytomation, Denmark) and PBS was used for negative control.

Statistic analysis
Data are expressed as means ± standard deviation (SD). Statistical analyses were carried out using SAS 8.1. Two-way analysis of variance (ANOVA) (treatment vs time) or one-way ANOVA was used to compare differences in outcome variables among 4 groups. A result was considered statistically significant if P <0.05.

Validation of MSC culture
MSCs were generated by standard procedures and grown for at least two passages in culture. Contaminating hematopoietic cells were depleted during passage 1 and MSCs were morphologically defined by a fibroblast-like appearance (Figure 1). Flow cytometric analysis of passage 2 cells confirmed that cells were negative for CD34 (hematopoietic cell marker, 2.8% positive) and CD45 (leukocyte marker, 2.9% positive) and that cells were positive for CD29 (β1-integrin, 92.1% positive) and CD90 (Thy-1, 95.9% positive). Before use, each batch of MSC was further characterized by confirming their specific ability to undergo osteogenic and adipogenic differentiation (Figure 2). Only cells that met these criteria were used in subsequent experiments.

Blood glucose after MSC transplantation
Random blood glucose was assessed at time points up to 8 weeks after the injection of MSCs. Figure 3 shows that as early as 3 days, there was a significant decrease in blood glucose in the MSCA group. Over the course of the experiment, there was a significant difference in blood glucose among 4 groups (F=13.12, P <0.001, two-way ANOVA). And MSCA group showed a significant decrease in blood glucose compared to MSC group, CsA group and DC group (27.28±3.22, 28.75±1.86, 29.16±2.72, 29.80±2.70, P <0.05, respectively, two-way ANOVA). MSC group did not show a significant difference compared to DC group, though blood glucose was lower in MSC group (28.75±1.86 vs 29.80±2.70, P >0.05, two-way ANOVA). At day 3, blood glucose in MSCA group decreased from (28.2±3.0) mmol/L pretreatment to (25.9±3.6) mmol/L, significantly lower than (30.3±2.2) mmol/L in diabetes control group (P <0.05, one-way ANOVA). The blood glucose remained low throughout the experiment until week 8 when there was no significant difference between MSCA group and diabetes control group (29.4±3.9 vs 29.6±1.8, P >0.05, one-way ANOVA).

Urine albumin/creatinine ratio (Alb/Cr) after MSC transplantation
Alb/Cr was used to correct the value of urine albumin. Before MSCs transplantation, there were no significant differences in Alb/Cr among 4 groups (F=1.15, P=0.37, one-way ANOVA). In Figure 4, there was a significant difference in Alb/Cr among 4 groups (F=20.92, P <0.0001, two-way ANOVA). The Alb/Cr in both MSCA group and MSC group were significantly lower than CsA group and DC group over the course of the experiment (0.74±0.21, 0.84±0.17, 0.96±0.16, 1.02±0.21, respectively, P <0.05, two-way ANOVA). And the Alb/Cr in MSCA group showed a significant decrease compared to MSC group (0.74±0.21 vs 0.84±0.17, P <0.05, two-way ANOVA). Still, there was no significant difference in Alb/Cr at week 8 among 4 groups (F=0.75, P >0.05, one-way ANOVA).

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Figure 4. Urine albumin/creatinine ratio (Alb/Cr) after MSC transplantation. ^*P <0.05 vs DC group.

Creatinine clearance rate after MSC transplantation
No significant difference was found in endogenous Ccramong MSCA group, MSC group, CsA group and DC group over the whole experiment (0.70±0.23, 0.72±0.13, 0.76±0.35, 0.85±0.27, respectively, F=1.36, P=0.27, two-way ANOVA).

RMI after MSCs transplantation
RMI (total kidney weight/body mass) was significantly different among MSCA group, MSC group, CsA group and DC group over the experiment (5.66±0.41, 6.15±0.60, 6.15±0.50, 6.37±0.63, respectively, F=4.85, P=0.005, two-way ANOVA). There was a significant difference in kidney hypertrophy between the MSCA group and DC group (5.66±0.41 vs 6.37±0.63, P <0.05, two-way ANOVA).

Kidney histology
Histological examinations of the kidneys by light microscope are shown in Figure 5. In the diabetes control group, Figure 5E showed a glomerulus with glomerulosclerosis, diminution of capillary lumen, predominance of dense hyaline matrix and peripheral capillaries of thick stiff wall. The proximal tubules did not have a brush border and did not contain lysosomes or endocytic vacuoles. Treatment with MSC + CsA significantly ameliorated these diabetes-induced alterations in the kidney.

MSC in vivo homing and engraftment
For in vivo tracking of BrdU-marked MSCs, immunostaining for BrdU was performed in the heart, liver, spleen, pancreas, lung and kidney of recipient rats. BrdU positive cells were detected in the heart (into which the MSCs were infused), pancreas and kidney of recipient rats, while no positive cells were found in other organs. In the pancreas, BrdU positive cells were mainly located in the interstitium. And BrdU-marked MSC were mainly located in renal interstitium instead of glomerulus and renal tubles (Figure 6). However, no proliferating cells were found where the labeled MSCs were detected when PCNA immunostaining was performed (data not shown).

In this study, we established that adherent, bone marrow-derived, spindle-shaped cells expressed mesenchymal cell phenotype (CD29⁺, CD90⁺), rather than CD34 (hemato- poietic cell marker) and CD45 (leukocyte marker). Next, by in vitro differentiation, we further demonstrate that the cultured cells exhibit two key features of MSC, namely adipogenesis and osteoblastogenesis.

After introcardiac injection of MSCs, the present study provides the clear evidence that MSCs afford mild protection in rats with experimental diabetes by decreasing random blood glucose, Alb/Cr and RMI, especially at the early phase of the treatment (3 days to 2 weeks). In CsA group, there was no significant decrease in either blood glucose or Alb/Cr compared to diabetes control group. We assess that it is the MSCs that drop the blood glucose and urine Alb/Cr, and CsA strengthens the effects of MSCs, for MSCA group seems to get a more decrease of blood glucose and Alb/Cr compared to MSC group. Early diabetic kidney disease usually experience kidney hypertrophy, and treatment with MSCs ameliorates the RMI in our experiment. In the present study, Ccr was used as a surrogate for glomerular filtration rate (GFR) to assess the renal function. We failed to find any significant difference in Ccr between MSC treatment groups and diabetes control group. Maybe it is at the early phase of diabetic nephropathy, so there is no obvious renal dysfunction up to the end of the experiment.

To explore whether MSCs proliferated in vivo, PCNA immunostaining was performed. However, we failed to find PCNA positive cells where BrdU-labeled cells located. In addition, immunostaining of renal sections for BrdU revealed either brightly positive cells or no staining at all. This indicates that the majority of our MSC did not proliferate in the kidney and therefore not dilute the BrdU content of their DNA during nuclear division. Recent studies revealed that MSC might mediate the renoprotective effects via paracrine mechanisms. In one study, administered MSCs protected against ischemic acute renal failure by downregulating proinflammatory cytokines interleukin (IL)-1β, tumor necrosis factor (TNF)-α, interferon (IFN)-γ, as well as iNOS, and upregulating anti-inflammatory and organ-protective IL-10, as well as basic fibroblast growth factor, transforming growth factor-α and Bcl-2.¹⁴ Insulin-like growth factor-1 (IGF-1) is also discovered to sustain stem cell-mediated renal repair, since either blocking the growth factor′s function or knocking down IGF-1 expression in MSC by small interfering-RNA resulted in a significant decrease in proximal tubular epithelial cells proliferation and increased apoptosis in a cisplatin-induced acute kidney injury model.²³ In one study, conditioned media from cultured stromal cells induced migration and proliferation of kidney-derived epithelial cells in vitro and diminished tubular cell apoptosis, increased survival, and limited renal injury of mice injected with cisplatin, indicating that identification of the stromal cell-derived protective factors may provide new therapeutic options to explore in humans with acute kidney injury.²⁴ Therefore, in our experiment, MSC might act more likely through paracrine mechanisms. Further studies are needed to explore the exact mechanisms that underlie MSCs′ renal protective efforts.

The tracking of administered MSC in vivo is critical to the interpretation of MSC therapy. It has been argued that genetic markers like enhanced green fluorescence protein (eGFP) are superior to nongenetic tracking methods. However, the utility of eGFP as a cell marker is also limited because the kidney possesses intensive autofluorescence, which makes it difficult to detect eGFP-positive MSC unless confocal microscopy is used.¹⁴β-Gal, another cell marker, has been shown to yield false positive results when pH is not strictly controlled during histological processing.²⁵ In the present study, we utilized BrdU as the cell marker, a synthetic analog of thymidine, which is incorporated into cells in S-phase. Studies have shown that BrdU did not inhibit MSCs′ “stemness” and their responsiveness to injury.²⁶Immunohistochemical detection of BrdU positive cells indicated that there are BrdU positive cells in the kidney and pancreas of recipient rats, rather than organs, such as liver, lung and spleen, confirming the fact that MSCs are capable of specific migration to a site of injury (present study). And intracardiac infusion instead of intravenous infusion of the cells probably decreased trapping of the cells in the capillary beds of the lung. In the kidney, BrdU-marked MSCs mainly located in renal interstitium instead of glomerulus and renal tubles, which was in line with Bi et al.²⁴ We suppose that MSCs may act through paracrine mechanisms rather than direct transdifferentiation.^14-16,24

In vitro, MSCs have been shown to suppress the function of a broad range of immune cells, including T cells, B cells, NK cells and antigen-presenting cells.²⁷ However, in vivo, some studies discovered that allogeneic MSC transplantation increased graft rejection.^28,29 To inhibit the possible immunologic rejection of MSC, CsA was utilized. Signs of chronic nephrotoxicity were observed at doses of CsA >10 mg/kg administered for at least 2 weeks.³⁰ Therefore, for our experiment, we chose a dose of 5 mg/kg CsA to minimize possible nephrotoxicity, which was also demonstrated to be able to inhibit inflammation.³⁰ In the present study, compared to MSC group, MSC treated group with daily CsA gavage got a lower blood glucose, Alb/Cr and RMI, especially at day 3 and week 1. We suppose that MSC might have mild immunogeneic effects, which could be ameliorated by low dose of CsA.

We notice that at the end of the experiment, all indexes, including blood glucose, Alb/Cr and whole kidney hypertrophy, rebounded to the pretreatment level even with MSC treatment. We consider that it might be the limited long term effects of MSC. Since most studies concentrated on the short term effects of MSC,^14-16there are only limited data on the role of MSC in models of chronic kidney disease. Weekly injections of MSC in a murine model of Alports disease prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, but were not associated with an improvement in creatinine, proteinuria and animal survival.³¹ It is still unclear whether renal protection revealed in the present study is due to the indirect effects of blood glucose lowering or MSCs′ direct effects on the kidney. In the experiment, we noticed that some rats showed improved Alb/Cr and RMI, though the blood glucose was still high. We believe that MSCs might have direct effects on the diabetic kidney. In a recent study, ex vivo pretreatment with melatonin improves survival, proangiogenic/ mitogenic activity, and efficiency of MSCs injected into ischemic kidney.³² Pretreatment of MSC could enhance the effects of MSC, which serves us a new aspect for MSC therapy.

In summary, we explore the effects of MSC on diabetic nephropathy. MSC could mildly ameliorate diabetic nephropathy by decreasing blood glucose, Alb/Cr and RMI.

1. Remuzzi G, Schieppati A, Ruggenenti P. Clinical practice. Nephropathy in patients with type 2 diabetes. N Engl J Med 2002; 346: 1145-1151.

2. Maisonneuve P, Agodoa L, Gellert R, Stewart JH, Buccianti G, Lowenfels AB, et al. Distribution of primary renal diseases leading to end-stage renal failure in the United States, Europe, and Australia/New Zealand: results from an international comparative study. Am J Kidney Dis 2000; 35: 157-165.

3. Group UKPDS. Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. BMJ 1998; 317: 703-713.

4. Holman RR, Paul SK, Bethel MA, Neil HA, Matthews DR. Long-term follow-up after tight control of blood pressure in type 2 diabetes. N Engl J Med 2008; 359: 1565-1576.

5. Patel A, MacMahon S, Chalmers J, Neal B, Billot L, Woodward M, et al. Intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. N Engl J Med 2008; 358: 2560-2572.

6. Gerstein HC, Miller ME, Byington RP, Goff DC Jr, Bigger JT, Buse JB, et al. Effects of intensive glucose lowering in type 2 diabetes. N Engl J Med 2008; 358: 2545-2559.

7. Yamagishi S, Fukami K, Ueda S, Okuda S. Molecular mechanism of diabetic nephropathy and its therapeutic intervention. Current Drug Targets 2007; 8: 952-959.

8. Prockop DJ. Marrow stromal cells as stem cells for non-hematopoietic tissues. Science 1997; 276: 71-74.

9. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999; 284: 143-147.

10. Digirolamo CM, Stokes D, Colter D, Phinney DG, Class R, Prockop DJ. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with greatest potential to propagate and differentiate. Br J Haematol 1999; 107: 275-281.

11. Sanchez-Ramoz J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, et al. Adult bone marrow stromal cells differentiate into neuronal cells in vitro. Exp Neurol 2000; 164: 247-256.

12. Brazelton TR, Rossi FM, Keshet GI, Blau HM. From marrow to brain: expression of neuronal phenotype in adult mice. Science 2000; 290: 1775-1779.

13. Anglani F, Forino M, Del Prete D, Tosetto E, Torregrossa R, D'Angelo A. In search of adult renal stem cells. J Cell Mol Med 2004; 8: 474-487.

14. Togel F, Hu Z, Weiss K, Isaac J, Lange C, Westenfelder C. Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation- independent mechanisms. Am J Physiol Renal Physiol 2005; 289: 31-42.

15. Lange C, Togel F, Ittrich H, Clayton F, Nolte-Ernsting C, Zander AR, et al. Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int 2005; 68: 1613-1617.

16. Kunter U, Rong S, Djuric Z, Boor P, Müller-Newen G, Yu D, et al. Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis. J Am Soc Nephrol 2006; 17: 2202-2212.

17. Morigi M, Imberti B, Zoja C, Corna D, Tomasoni S, Abbate M, et al. Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol 2004; 15: 1794-1804.

18. Ito T, Suzuki A, Imai E, Okabe M, Hori M. Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling. J Am Soc Nephrol 2001; 12: 2625-2635.

19. Poulsom R, Forbes SJ, Hodivala-Dilke K, Ryan E, Wyles S, Navaratnarasah S, et al. Bone marrow contributes to renal parenchymal turnover and regeneration. J Pathol 2001; 195: 229-235.

20. Gratzner HG. Monoclonal antibody to 5-bromo- and 5-iododeoxyuridine: a new reagent for detection of DNA replication. Science 1982; 218: 474-475.

21. Kraynak AR, Storer RD, Jensen RD, Kloss MW, Soper KA, Clair JH, et al. Extent and persistence of streptozotocin-induced DNA damage and cell proliferation in rat kidney as determined by in vivo alkaline elution and BrdUrd labeling assays. Toxicol Appl Pharmacol 1995; 135: 279-286.

22. Tesch GH, Allen TJ. Rodent models of streptozotocin-induced diabetic nephropathy. Nephrology (Carlton) 2007; 12: 261-266.

23. Imberti B, Morigi M, Tomasoni S, Rota C, Corna D, Longaretti L, et al. Insulin-like growth factor-1 sustains stem cell mediated renal repair. J Am Soc Nephrol 2007; 18: 2921-2928.

24. Bi B, Schmitt R, Israilova M, Nishio H, Cantley LG. Stromal cells protect against acute tubular injury via an endocrine effect. J Am Soc Nephrol 2007; 18: 2486-2496.

25. DuBose TD Jr, Warnock DG, Mehta RL, Bonventre JV, Hammerman MR, Molitoris BA, et al. Acute renal failure in the 21st century: recommendations for management and outcomes assessment. Am J Kidney Dis 1997; 29: 793-799.

26. Oliver JA, Maarouf O, Cheema FH, Martens TP, Al-Awqati Q. The renal papilla is a niche for adult kidney stem cells. J Clin Invest 2004; 114: 795-804.

27. Stagg J. Immune regulation by mesenchymal stem cells: two sides to the coin. Tissue Antigens 2007; 69: 1-9.

28. Nauta AJ, Westerhuis G, Kruisselbrink AB, Lurvink EG, Willemze R, Fibbe WE. Donor-derived mesenchymal stem cells are immunogenic in an allogeneic host and stimulate donor graft rejection in a nonmyeloablative setting. Blood 2006; 108: 2114-2120.

29. Eliopoulos N, Stagg J, Lejeune L, Pommey S, Galipeau J. Allogeneic marrow stromal cells are immune rejected by MHC class I-and class II-mismatched recipient mice. Blood 2005; 106: 4057-4065.

30. Savikko J, Kallio EA, Taskinen E, von Willebrand E. The effect of acute rejection and cyclosporin A-treatment on induction of platelet-derived growth factor and its receptors during the development of chronic rat renal allograft rejection. Transplantation 2002; 73: 506-511.

31. Ninichuk V, Gross O, Segerer S, Hoffmann R, Radomska E, Buchstaller A, et al. Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice. Kidney Int 2006; 70: 121-129.

32. Mias C, Trouche E, Seguelas MH, Calcagno F, Dignat-George F, Sabatier F, et al. Ex vivo pretreatment with melatonin improves survival, proangiogenic/mitogenic activity, and efficiency of mesenchymal stem cells injected into ischemic kidney. Stem Cells 2008; 26: 1749-1757.