- Email: [email protected]
Mesenchymal Stem Cells Delivered at the Subcapsule of the Kidney Ameliorate Renal Disease in the Rat Remnant Kidney Model
Mesenchymal Stem Cells Delivered at the Subcapsule of the Kidney Ameliorate Renal Disease in the Rat Remnant Kidney Model R.C. Cavaglieri, D. Martini, M.C. Sogayar, and I.L. Noronha ABSTRACT Stem cells (SC) are potential therapeutic tools in the treatment of chronic renal diseases. Number and engraftment of SC in the injured sites are important for possible differentiation into renal cells and paracrine effect. The aim of this study was to analyze the effect of subcapsular injection of mesenchymal stem cells (MSC) in the 5/6 nephrectomy model (5/6 Nx). MSC obtained from Wistar rats were isolated by their capacity to adhere to plastic surfaces, characterized by flow cytometry, and analyzed by their differentiation potential into osteoblasts. MSC (2 ⫻ 105) were injected into the subcapsule of the remnant kidney of male Wistar rats, and were followed for 15 or 30 days. 5/6 Nx rats showed significant hypertension at 15 and 30 days, which was reduced by MSC at 30 days. Increased albuminuria and serum creatinine at 15 and 30 days in 5/6 Nx rats were also reduced by subcapsular injection of MSC. We also observed a significant reduction of glomerulosclerosis index 30 days after injection of MSC. 4 – 6 diamidino-2-phenylindole dihydrochloride (DAPI)-stained MSC showed a migration of these cells into renal parenchyma 5, 15, and 30 days after subcapsular injection. In conclusion, our data demonstrated that subcapsular injection of MSC in 5/6 Nx rats is associated with renoprotective effects. These results suggest that locally implanted MSC in the kidney allow a large number of cells to migrate into the injured sites and demonstrate that subcapsular injection represent an effective route for MSC delivery.
C
HRONIC kidney diseases feature progressive loss of function, and culminate with total organ failure. One of the most important challenges in nephrology is to reduce the incidence of end-stage kidney disease, by blocking or at least slowing down the progression of the disease. Current therapeutic strategies aiming at preventing the progression of renal diseases have not been shown to be very effective. In this context, the study of alternative approaches, such as stem cell (SC) therapy, is of central relevance. Mesenchymal stem cells (MSC) are multipotent cells that reside in the bone marrow and other tissues, and are capable of differentiating in vitro and in vivo into different cell types, such as chondrocytes, osteocytes, and adipocytes. They can be isolated from bone marrow based on their ability to adhere to plastic surfaces and to adopt a fibroblast-like morphology, termed colony-forming unitfibroblasts. In addition, MSC express specific cell surface markers, such as CD29, CD44, CD73, CD90, and CD105, but not CD31 (differentiated endothelial cells), CD34 (hematopoietic stem cells), or CD45 (leukocyte common antigen).1
© 2009 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 947–951 (2009)
Previous studies have shown that SC have the potential to enhance recovery from acute tubular injury2,3 and repair glomerular diseases.4 –7 The mechanisms involved in renoprotection and renal repair associated with SC are not clear, but they could be due to differentiation of SC into organspecific renal cell types or protection mediated by paracrine/ endocrine actions of these cells. In this context, the number and the engraftment of SC in the injured compartments may have an important role, and, therefore, intravascular From the Laboratory of Cellular and Molecular Nephrology (R.C., I.L.N.), Faculty of Medicine, University of São Paulo, and Nephrology Clinic (D.M., I.L.N.), Beneficencia Portuguesa Hospital, São Paulo, Brazil; and NUCEL (M.C.S.), Cell and Molecular Therapy Center, University of São Paulo, São Paulo, Brazil. Supported by CNPq (Brazilian Council for Scientific and Technologic Development) number 552644/2005-6 and by FAPESP (Foundation for Research Support) number 2006/56628-4. Address reprint requests to Irene L. Noronha, MD, PhD, Laboratory of Molecular and Cellular Nephrology University of São Paulo, Av. Dr. Arnaldo, 455 - 4° andar - Lab 4304, CEP: 01246-903 São Paulo/SP - Brazil. E-mail: [email protected] 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.01.072 947
948
injection of extrinsic SC, which allows only a rapid transit passage through the kidney, may not be the most adequate method to deliver SC in the target organ. An alternative delivery technique, evaluated in the present study, consists of administering SC at the subcapsule of the kidney. MATERIALS AND METHODS MSC Isolation and Expansion Briefly, bone marrow from femur and tibia of male Wistar rats (180 –220 g, obtained from local colony) were flushed out and cultivated with Dulbecco’s modified Eagle’s medium–low glucose (DMEM-low) supplemented with 10% fetal bovine serum. MSC were isolated using their characteristic to adhere to plastic culture dishes. After 2 weeks of culture, adherent cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and kept on ice until the moment of the infusion. For the present study, cells were used between passage 1 and 4.
Phenotype Identification Using Flow Cytometry MSC were harvested, washed in PBS, fixed with 4% paraformaldehyde, and labeled with isothiocyanate (FITC)-conjugated antibodies against CD31 and CD90, phycoerythrin (PE)-conjugated antibodies against CD34 and CD44, Pe-cy5.5-conjugated antibody against CD45, and FITC- or PE-conjugated nonspecific immunoglobulin (Ig)G (Caltag Laboratories, Carlsbad, Calif, United States). Flow cytometry was carried out before each inoculation, using a FACScalibur cytometer equipped with 488 nm argon laser (Becton Dickinson, San Diego, Calif, United States) with CellQuest software. At least 10,000 events were collected.
Osteogenic Differentiation MSC were cultivated in specific osteogenic induction medium (DMEM-L, 10% fetal bovine serum, 10 mmol/L -glycerophosphate, 100 nmol/L dexamethasone, and 50 g/mL ascorbate-2phosphate (Sigma-Aldrich, Saint Louis, Mo., United States) for 3 weeks. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and calcium phosphate deposition with alizarin red solution, as previously described.8
Fig 1. Characterization of MSC. Expression of cell surface markers was measured using flow cytometry. MSC express CD44 and CD90, but not CD31, CD34, or CD45.
CAVAGLIERI, MARTINI, AND NORONHA
Experimental 5/6 Ablation Model and MSC Infusion The 5/6 ablation model was performed as previously described.9 For the infusion of MSC, recipient rats were anesthetized and subjected to dorsal incision on the left side. A 5-mm incision was made in the capsule of the left kidney. A sterile plastic capillary was placed under the kidney capsule and 2 ⫻ 105 MSC (in 10 L sterile PBS) were injected with the help of a micro-injector. After MSC infusion, the kidney capsule was cauterized with an electric scalpel, and the dorsal incision was sutured. Injection of MSC or PBS in the subcapsule of the kidney was performed on the same day as the 5/6 nephrectomy (5/6 Nx). Male Wistar rats (n ⫽ 40) were divided into 4 groups: shamoperated rats (sham), sham-operated rats receiving MSC (sham ⫹ MSC), rats subjected to 5/6 Nx, and Nx rats receiving MSC (Nx ⫹ MSC). Half of the animals of each group were sacrificed at 15 days and 30 days. One day before humane killing, systolic blood pressure was measured using manometry and rats were placed in metabolic cages to collect 24-hour urine. Rats were anesthetized with a cocktail of Ketamine ⫹ Xilazine injected intraperitoneally and blood samples were collected and kidneys were prepared for histology. All experimental procedures were conducted in accordance with our institutional guidelines and were approved by the institutional ethics committee (CAPPesq 856/06).
DAPI Staining for In Vivo Detection of MSC Before inoculation, SC were labeled with the nuclear stain 4-6 diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich). Briefly, DAPI was added to the culture medium for 1 hour when the cells were 70% confluent. Then, the cells were harvested using trypsinization and prepared for inoculation into the rat renal subcapsule. Kidneys were harvested 1, 5, 15, and 30 days after inoculation. Frozen kidney specimens were cut and fixed in acetone. DAPI fluorescence was analyzed using a fluorescence microscope.
Renal Histology The kidneys were in situ perfused, excised, and 2 midcoronal sections were embedded in paraffin for simple histological analysis. Sections 2–3 m thick were stained with periodic acid-Schiff reaction and the Masson trichrome technique.
949
0.15 ⫾ 0.02 0.44 ⫾ 0.14 0.77 ⫾ 0.08* 0.90 ⫾ 0.05 0.3 ⫾ 0.3 1.3 ⫾ 1.1 22.0 ⫾ 6.1* † 5.4 ⫾ 2.4 0.2 ⫾ 0.2 0.3 ⫾ 0.3 13 ⫾ 3.0* 10 ⫾ 2.1 0.3 ⫾ 0.1 0.3 ⫾ 0.1 1.2 ⫾ 0.3* † 0.5 ⫾ 0.2 0.2 ⫾ 0.01 0.4 ⫾ 0.1 1.1 ⫾ 0.1* † 0.6 ⫾ 0.03 2.3 ⫾ 0.1 0.5 ⫾ 0.1 116.1 ⫾ 34.5* † 17.6 ⫾ 4.3 0.8 ⫾ 0.2 0.4 ⫾ 0.1 12.1 ⫾ 6.1* † 4.7 ⫾ 2.1 10 ⫾ 0.1 6 ⫾ 0.2 96 ⫾ 2* † 23 ⫾ 5 13 ⫾ 1 9⫾1 28 ⫾ 8* 29 ⫾ 12
30 d 15 d 30 d
GS (%) 15 d 30 d
Screat (mg/dL)
15 d 30 d
Ualb (mg/24 h)
15 d
Abbreviations: BP, blood pressure; Uprot, proteinuria; Ualb, albuminuria; Screat, serum creatinine; GS, glomerulosclerosis. Note: Data are presented as mean ⫾ SEM. *P ⬍ .05 vs sham. † P ⬍ .05 vs Nx.
When compared with sham-operated rats, nephrectomized rats showed increased glomerulosclerosis. Sclerotic glomerular lesions were evident at 15 days, and were more severe at 30 days (Fig 3), indicating the progressive nature of the lesions. Nx rats receiving subcapsular injection of MSC showed a significantly lower degree of glomerulosclerosis at
122 ⫾ 1 124 ⫾ 2 192 ⫾ 3* † 145 ⫾ 7
Histology
124 ⫾ 5 120 ⫾ 2 150 ⫾ 9* † 120 ⫾ 3
MSC detection in the kidney after inoculation into the renal subcapsule was possible by DAPI staining. Figure 2A shows that 24 hours after inoculation, MSC are present in the subcapsular region and start to infiltrate the cortex. Five and 15 days after injection, DAPI-positive cells populated the renal cortex and migrate toward the medulla (Fig 2B and 2C). Thirty days after injection, DAPI-positive cells were still localized in the cortical region, but the intensity of fluorescence was lower than at 15 days.
Sham Sham ⫹ MSC Nx Nx ⫹ MSC
In Vivo Detection of MSC After Inoculation
30 d
As compared with sham-operated animals, 5/6 Nx rats displayed significant elevated levels of tail-cuff blood pressure, proteinuria, albuminuria, and serum creatinine, already detected 15 days after the induction of the model, and markedly pronounced at 30 days (Table 1). Subcapsular administration of MSC significantly reduced blood pressure, proteinuria, albuminuria, and serum creatinine levels in 5/6 Nx rats at 30 days. It is interesting to note that these renoprotective effects of MSC infusion were already detected at 15 days. Injection of MSC had no effect on these parameters in sham-operated rats.
Uprot (mg/24 h)
MSC Infusion Ameliorated Renal Function in the 5/6 Ablation Model
15 d
After 3 weeks of culture in osteogenic induction medium, MSC acquired an osteoblast-like morphology. Osteogenic differentiation was confirmed by measuring alkaline phosphatase activity and calcium phosphate deposition.
30 d
Osteogenic Differentiation of MSC
BP (mm Hg)
MSC isolated from femur and tibia bone marrow formed colonies of fibroblast-like cells after about 12–20 days of culture, and grew into a homogeneous population of cells, with a mainly fusiform morphology. Analysis of cell surface markers using flow cytometry showed that MSC were positive for CD44 (61 ⫾ 8%) and CD90 (85 ⫾ 4%), and mostly negative for CD31 (2.5 ⫾ 0.5%), CD34 (4 ⫾ 1%), and CD45 (19 ⫾ 4%; Fig 1.).
15 d
RESULTS MSC Culture and Characterization
Table 1. Effects of the Inoculation of MSC in Renal Function and Histological Parameters in the 5/6 Nx Model After 15 and 30 Days
Data are presented as mean ⫾ SEM, and analyzed by analysis of variance (ANOVA) using Bonferroni post- test analysis (GraphPad Prism Software, La Jolla, Calif). P ⬍ .05 was considered significant.
Interstitial Fibrosis (%)
Statistical Analysis
0.20 ⫾ 0.06 0.38 ⫾ 0.06 1.42 ⫾ 0.11* 1.15 ⫾ 0.07*
MESENCHYMAL STEM CELLS
950
CAVAGLIERI, MARTINI, AND NORONHA
Fig 2. Localization of DAPI-labelled MSC delivered at the subcapsule of the kidney, 1 day after inoculation (A). Trafficking of these cells into the renal cortex can be clearly observed 5 days after inoculation, and also infiltrating glomeruli (B). Fifteen days after inoculation DAPI-labelled MSC can still be detected in the renal parenchyma, reaching the renal medulla (C).
30 days, but not at 15 days, when compared with Nx rats. A modest increase in interstitial expansion was observed in the Nx at 15 days, whereas at 30 days interstitial expansion was significantly increased when compared with the sham group. Injection of MSC tended to decrease interstitial fibrosis at 30 days in the Nx group, although it did not reach statistical significance. DISCUSSION
In this study, we have shown that MSC isolated from rat bone marrow and injected into the subcapsule of remnant kidney rats with the 5/6 Nx experimental model of chronic progressive nephropathy significantly ameliorated renal function 30 days after nephrectomy, however, it was already detected at 15 days. Although no unique surface marker can be used for specific characterization of MSC, these cells can be identified by combined expression of cell surface markers and exclusion of others. In our experiments, using flow cytometry, adherent bone marrow– derived cells have been shown to express CD44 and CD90, but very low expression of CD34, CD45, and CD31. The MSC isolated from rat bone marrow retained their differentiation potential in culture. Our results demonstrated that, under favorable conditions and appropriate stimulus, MSC isolated from Wistar rats could differentiate into osteoblasts. The cells adopted an osteoblast-like morphology, expressed alkaline phosphatase activity, and showed calcium phosphate deposition. Together, these findings suggested that the cells used in our experiment were MSC.
The results of the present work demonstrated that renal subcapsular administration of MSC significantly promoted renoprotective effects in the 5/6 Nx experimental model of chronic kidney disease, by reducing blood pressure, proteinuria, albuminuria, and serum creatinine levels. It is interesting to note that infusion of MSC significantly reduced blood pressure in the diseased animals, demonstrating a protective hemodynamic effect. In the Thy1.1-induced glomerulonephritis model, infusion of MSC in the renal artery failed to improve hypertension.6 These discrepant results may be due to the different nature of kidney injuries in the 2 models. An alternative explanation is the different route of MSC delivery, which may account for differences in the number of stem cells homing in the kidney. Our study also showed that MSC delivered at the subcapsule of the kidney led to a significant reduction of proteinuria, albuminuria, and serum creatinine levels in Nx rats already at 15 days and, remarkably, 30 days after 5/6 Nx. The effect of MSC on the development of interstitial fibrosis was not so remarkable but injection of MSC promoted a significant reduction of glomerulosclerosis in Nx rats after 30 days. Our results are in agreement with previous reports using SC in a model of Thy1.1 aggravated with nephrectomy4,10 and also in the ablation model.7 The possible beneficial effects of SC have not been clearly established but they may rely on their ability to differentiate into renal cell types and repopulate regions that have been damaged during injury. However, the detection of SC at sites of injury has been reported as a rare phenome-
Fig 3. Renal histology of rat kidney tissue. Sham, normal glomerulus (A); Nx, glomerulosclerosis lesions (B); Nx ⫹ MSC, amelioration of glomerulosclerotic lesions in animal receiving MSC (C).
MESENCHYMAL STEM CELLS
non.2,11–13 Alternatively, the renoprotective effect of SC can be due to the local production and secretion of hormones or growth factors (paracrine effects), which seems to be a more reliable mechanism. Independent of the predominant mechanism of renoprotection induced by the inoculation of SC, it seems reasonable to hypothesize that the delivery of SC at the subcapsular region allows a larger number of cells to home in and interact for a longer time with the injured site, which could be important for these cells to play their role. Injection of SC in the circulation, either intravenous or intraarterial, provide only a transient passage of the cells and this may negatively affect the homing of these cells to the site of injury. Our study showed that subcapsular delivery of MSC allowed a great amount of cells to remain in the kidney, which may be of crucial relevance for efficient homing of these cells. The present study also documented a curious trafficking of MSC into the renal parenchyma, observed already 5 days after inoculation and detected until 30 days, when the immunofluorescence signal weakened. In conclusion, our study demonstrated a renoprotective effect of the administration of MSC in the rat remnant kidney model. In addition, the inoculation of MSC at the subcapsule of the kidney may represent an effective alternative route for MSC delivery in the kidney. ACKNOWLEDGMENTS The authors thank Laila Casado and Fernanda Cobucci for their skillful technical assistance and gratefully acknowledge Dr Denis Feliers for his excellent help in reviewing the manuscript.
REFERENCES 1. Meirelles LD, Chagastelles PC, Nardi NB: Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 119:2204, 2006
951 2. Morigi M, Imberti B, Zoja C, et al: Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol 15:1794, 2004 3. Lange C, Toegel F, Ittrich H, et al: Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int 68:1613, 2005 4. Li B, Morioka T, Uchiyama M, et al: Bone marrow cell infusion ameliorates progressive glomerulosclerosis in an experimental rat model. Kidney Int 69:323, 2006 5. Sugimoto H, Mundel TM, Sund M, et al: Bone-marrowderived stem cells repair basement membrane collagen defects and reverse. Proc Natl Acad Sci USA 103:7321, 2006 6. Kunter U, Rong S, Djuric Z, et al: Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis. J Am Soc Nephrol 17:2202, 2006 7. Caldas HC, Fernandes IMM, Gerbi F, et al: Effect of whole bone marrow cell infusion in the progression of experimental chronic renal failure. Transplant Proc 40:853, 2008 8. Pittenger MF, Mackay AM, Beck SC, et al: Multilineage potential of adult human mesenchymal stem cells. Science 284:143, 1999 9. Fujihara CK, Noronha IL, Malheiros DMA, et al: Combined mycophenolate mofetil and losartan therapy arrest established injury in the remnant kidney. J Am Soc Nephrol 11:283, 2000 10. Kunter U, Rong S, Boor P, et al: Mesenchymal stem cells prevent progressive experimental renal failure but maldifferentiate into glomerular adipocytes. J Am Soc Nephrol 18:1754, 2007 11. Lin F, Cordes K, Li L, et al: Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemiareperfusion injury in mice. J Am Soc Nephrol 14:1188, 2003 12. Kale S, Karihaloo A, Clark PR, et al: Bone marrow stem cells contribute to repair of the ischemically injured renal tubule. J Clin Invest 112:42, 2003 13. Tögel F, Weiss K, Yangl Y, et al: Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury. Am J Physiol Renal Physiol 292:F1626, 2007
C
HRONIC kidney diseases feature progressive loss of function, and culminate with total organ failure. One of the most important challenges in nephrology is to reduce the incidence of end-stage kidney disease, by blocking or at least slowing down the progression of the disease. Current therapeutic strategies aiming at preventing the progression of renal diseases have not been shown to be very effective. In this context, the study of alternative approaches, such as stem cell (SC) therapy, is of central relevance. Mesenchymal stem cells (MSC) are multipotent cells that reside in the bone marrow and other tissues, and are capable of differentiating in vitro and in vivo into different cell types, such as chondrocytes, osteocytes, and adipocytes. They can be isolated from bone marrow based on their ability to adhere to plastic surfaces and to adopt a fibroblast-like morphology, termed colony-forming unitfibroblasts. In addition, MSC express specific cell surface markers, such as CD29, CD44, CD73, CD90, and CD105, but not CD31 (differentiated endothelial cells), CD34 (hematopoietic stem cells), or CD45 (leukocyte common antigen).1
© 2009 Published by Elsevier Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 41, 947–951 (2009)
Previous studies have shown that SC have the potential to enhance recovery from acute tubular injury2,3 and repair glomerular diseases.4 –7 The mechanisms involved in renoprotection and renal repair associated with SC are not clear, but they could be due to differentiation of SC into organspecific renal cell types or protection mediated by paracrine/ endocrine actions of these cells. In this context, the number and the engraftment of SC in the injured compartments may have an important role, and, therefore, intravascular From the Laboratory of Cellular and Molecular Nephrology (R.C., I.L.N.), Faculty of Medicine, University of São Paulo, and Nephrology Clinic (D.M., I.L.N.), Beneficencia Portuguesa Hospital, São Paulo, Brazil; and NUCEL (M.C.S.), Cell and Molecular Therapy Center, University of São Paulo, São Paulo, Brazil. Supported by CNPq (Brazilian Council for Scientific and Technologic Development) number 552644/2005-6 and by FAPESP (Foundation for Research Support) number 2006/56628-4. Address reprint requests to Irene L. Noronha, MD, PhD, Laboratory of Molecular and Cellular Nephrology University of São Paulo, Av. Dr. Arnaldo, 455 - 4° andar - Lab 4304, CEP: 01246-903 São Paulo/SP - Brazil. E-mail: [email protected] 0041-1345/09/$–see front matter doi:10.1016/j.transproceed.2009.01.072 947
948
injection of extrinsic SC, which allows only a rapid transit passage through the kidney, may not be the most adequate method to deliver SC in the target organ. An alternative delivery technique, evaluated in the present study, consists of administering SC at the subcapsule of the kidney. MATERIALS AND METHODS MSC Isolation and Expansion Briefly, bone marrow from femur and tibia of male Wistar rats (180 –220 g, obtained from local colony) were flushed out and cultivated with Dulbecco’s modified Eagle’s medium–low glucose (DMEM-low) supplemented with 10% fetal bovine serum. MSC were isolated using their characteristic to adhere to plastic culture dishes. After 2 weeks of culture, adherent cells were harvested by trypsinization, washed with phosphate-buffered saline (PBS), and kept on ice until the moment of the infusion. For the present study, cells were used between passage 1 and 4.
Phenotype Identification Using Flow Cytometry MSC were harvested, washed in PBS, fixed with 4% paraformaldehyde, and labeled with isothiocyanate (FITC)-conjugated antibodies against CD31 and CD90, phycoerythrin (PE)-conjugated antibodies against CD34 and CD44, Pe-cy5.5-conjugated antibody against CD45, and FITC- or PE-conjugated nonspecific immunoglobulin (Ig)G (Caltag Laboratories, Carlsbad, Calif, United States). Flow cytometry was carried out before each inoculation, using a FACScalibur cytometer equipped with 488 nm argon laser (Becton Dickinson, San Diego, Calif, United States) with CellQuest software. At least 10,000 events were collected.
Osteogenic Differentiation MSC were cultivated in specific osteogenic induction medium (DMEM-L, 10% fetal bovine serum, 10 mmol/L -glycerophosphate, 100 nmol/L dexamethasone, and 50 g/mL ascorbate-2phosphate (Sigma-Aldrich, Saint Louis, Mo., United States) for 3 weeks. Osteogenic differentiation was assessed by measuring alkaline phosphatase activity and calcium phosphate deposition with alizarin red solution, as previously described.8
Fig 1. Characterization of MSC. Expression of cell surface markers was measured using flow cytometry. MSC express CD44 and CD90, but not CD31, CD34, or CD45.
CAVAGLIERI, MARTINI, AND NORONHA
Experimental 5/6 Ablation Model and MSC Infusion The 5/6 ablation model was performed as previously described.9 For the infusion of MSC, recipient rats were anesthetized and subjected to dorsal incision on the left side. A 5-mm incision was made in the capsule of the left kidney. A sterile plastic capillary was placed under the kidney capsule and 2 ⫻ 105 MSC (in 10 L sterile PBS) were injected with the help of a micro-injector. After MSC infusion, the kidney capsule was cauterized with an electric scalpel, and the dorsal incision was sutured. Injection of MSC or PBS in the subcapsule of the kidney was performed on the same day as the 5/6 nephrectomy (5/6 Nx). Male Wistar rats (n ⫽ 40) were divided into 4 groups: shamoperated rats (sham), sham-operated rats receiving MSC (sham ⫹ MSC), rats subjected to 5/6 Nx, and Nx rats receiving MSC (Nx ⫹ MSC). Half of the animals of each group were sacrificed at 15 days and 30 days. One day before humane killing, systolic blood pressure was measured using manometry and rats were placed in metabolic cages to collect 24-hour urine. Rats were anesthetized with a cocktail of Ketamine ⫹ Xilazine injected intraperitoneally and blood samples were collected and kidneys were prepared for histology. All experimental procedures were conducted in accordance with our institutional guidelines and were approved by the institutional ethics committee (CAPPesq 856/06).
DAPI Staining for In Vivo Detection of MSC Before inoculation, SC were labeled with the nuclear stain 4-6 diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich). Briefly, DAPI was added to the culture medium for 1 hour when the cells were 70% confluent. Then, the cells were harvested using trypsinization and prepared for inoculation into the rat renal subcapsule. Kidneys were harvested 1, 5, 15, and 30 days after inoculation. Frozen kidney specimens were cut and fixed in acetone. DAPI fluorescence was analyzed using a fluorescence microscope.
Renal Histology The kidneys were in situ perfused, excised, and 2 midcoronal sections were embedded in paraffin for simple histological analysis. Sections 2–3 m thick were stained with periodic acid-Schiff reaction and the Masson trichrome technique.
949
0.15 ⫾ 0.02 0.44 ⫾ 0.14 0.77 ⫾ 0.08* 0.90 ⫾ 0.05 0.3 ⫾ 0.3 1.3 ⫾ 1.1 22.0 ⫾ 6.1* † 5.4 ⫾ 2.4 0.2 ⫾ 0.2 0.3 ⫾ 0.3 13 ⫾ 3.0* 10 ⫾ 2.1 0.3 ⫾ 0.1 0.3 ⫾ 0.1 1.2 ⫾ 0.3* † 0.5 ⫾ 0.2 0.2 ⫾ 0.01 0.4 ⫾ 0.1 1.1 ⫾ 0.1* † 0.6 ⫾ 0.03 2.3 ⫾ 0.1 0.5 ⫾ 0.1 116.1 ⫾ 34.5* † 17.6 ⫾ 4.3 0.8 ⫾ 0.2 0.4 ⫾ 0.1 12.1 ⫾ 6.1* † 4.7 ⫾ 2.1 10 ⫾ 0.1 6 ⫾ 0.2 96 ⫾ 2* † 23 ⫾ 5 13 ⫾ 1 9⫾1 28 ⫾ 8* 29 ⫾ 12
30 d 15 d 30 d
GS (%) 15 d 30 d
Screat (mg/dL)
15 d 30 d
Ualb (mg/24 h)
15 d
Abbreviations: BP, blood pressure; Uprot, proteinuria; Ualb, albuminuria; Screat, serum creatinine; GS, glomerulosclerosis. Note: Data are presented as mean ⫾ SEM. *P ⬍ .05 vs sham. † P ⬍ .05 vs Nx.
When compared with sham-operated rats, nephrectomized rats showed increased glomerulosclerosis. Sclerotic glomerular lesions were evident at 15 days, and were more severe at 30 days (Fig 3), indicating the progressive nature of the lesions. Nx rats receiving subcapsular injection of MSC showed a significantly lower degree of glomerulosclerosis at
122 ⫾ 1 124 ⫾ 2 192 ⫾ 3* † 145 ⫾ 7
Histology
124 ⫾ 5 120 ⫾ 2 150 ⫾ 9* † 120 ⫾ 3
MSC detection in the kidney after inoculation into the renal subcapsule was possible by DAPI staining. Figure 2A shows that 24 hours after inoculation, MSC are present in the subcapsular region and start to infiltrate the cortex. Five and 15 days after injection, DAPI-positive cells populated the renal cortex and migrate toward the medulla (Fig 2B and 2C). Thirty days after injection, DAPI-positive cells were still localized in the cortical region, but the intensity of fluorescence was lower than at 15 days.
Sham Sham ⫹ MSC Nx Nx ⫹ MSC
In Vivo Detection of MSC After Inoculation
30 d
As compared with sham-operated animals, 5/6 Nx rats displayed significant elevated levels of tail-cuff blood pressure, proteinuria, albuminuria, and serum creatinine, already detected 15 days after the induction of the model, and markedly pronounced at 30 days (Table 1). Subcapsular administration of MSC significantly reduced blood pressure, proteinuria, albuminuria, and serum creatinine levels in 5/6 Nx rats at 30 days. It is interesting to note that these renoprotective effects of MSC infusion were already detected at 15 days. Injection of MSC had no effect on these parameters in sham-operated rats.
Uprot (mg/24 h)
MSC Infusion Ameliorated Renal Function in the 5/6 Ablation Model
15 d
After 3 weeks of culture in osteogenic induction medium, MSC acquired an osteoblast-like morphology. Osteogenic differentiation was confirmed by measuring alkaline phosphatase activity and calcium phosphate deposition.
30 d
Osteogenic Differentiation of MSC
BP (mm Hg)
MSC isolated from femur and tibia bone marrow formed colonies of fibroblast-like cells after about 12–20 days of culture, and grew into a homogeneous population of cells, with a mainly fusiform morphology. Analysis of cell surface markers using flow cytometry showed that MSC were positive for CD44 (61 ⫾ 8%) and CD90 (85 ⫾ 4%), and mostly negative for CD31 (2.5 ⫾ 0.5%), CD34 (4 ⫾ 1%), and CD45 (19 ⫾ 4%; Fig 1.).
15 d
RESULTS MSC Culture and Characterization
Table 1. Effects of the Inoculation of MSC in Renal Function and Histological Parameters in the 5/6 Nx Model After 15 and 30 Days
Data are presented as mean ⫾ SEM, and analyzed by analysis of variance (ANOVA) using Bonferroni post- test analysis (GraphPad Prism Software, La Jolla, Calif). P ⬍ .05 was considered significant.
Interstitial Fibrosis (%)
Statistical Analysis
0.20 ⫾ 0.06 0.38 ⫾ 0.06 1.42 ⫾ 0.11* 1.15 ⫾ 0.07*
MESENCHYMAL STEM CELLS
950
CAVAGLIERI, MARTINI, AND NORONHA
Fig 2. Localization of DAPI-labelled MSC delivered at the subcapsule of the kidney, 1 day after inoculation (A). Trafficking of these cells into the renal cortex can be clearly observed 5 days after inoculation, and also infiltrating glomeruli (B). Fifteen days after inoculation DAPI-labelled MSC can still be detected in the renal parenchyma, reaching the renal medulla (C).
30 days, but not at 15 days, when compared with Nx rats. A modest increase in interstitial expansion was observed in the Nx at 15 days, whereas at 30 days interstitial expansion was significantly increased when compared with the sham group. Injection of MSC tended to decrease interstitial fibrosis at 30 days in the Nx group, although it did not reach statistical significance. DISCUSSION
In this study, we have shown that MSC isolated from rat bone marrow and injected into the subcapsule of remnant kidney rats with the 5/6 Nx experimental model of chronic progressive nephropathy significantly ameliorated renal function 30 days after nephrectomy, however, it was already detected at 15 days. Although no unique surface marker can be used for specific characterization of MSC, these cells can be identified by combined expression of cell surface markers and exclusion of others. In our experiments, using flow cytometry, adherent bone marrow– derived cells have been shown to express CD44 and CD90, but very low expression of CD34, CD45, and CD31. The MSC isolated from rat bone marrow retained their differentiation potential in culture. Our results demonstrated that, under favorable conditions and appropriate stimulus, MSC isolated from Wistar rats could differentiate into osteoblasts. The cells adopted an osteoblast-like morphology, expressed alkaline phosphatase activity, and showed calcium phosphate deposition. Together, these findings suggested that the cells used in our experiment were MSC.
The results of the present work demonstrated that renal subcapsular administration of MSC significantly promoted renoprotective effects in the 5/6 Nx experimental model of chronic kidney disease, by reducing blood pressure, proteinuria, albuminuria, and serum creatinine levels. It is interesting to note that infusion of MSC significantly reduced blood pressure in the diseased animals, demonstrating a protective hemodynamic effect. In the Thy1.1-induced glomerulonephritis model, infusion of MSC in the renal artery failed to improve hypertension.6 These discrepant results may be due to the different nature of kidney injuries in the 2 models. An alternative explanation is the different route of MSC delivery, which may account for differences in the number of stem cells homing in the kidney. Our study also showed that MSC delivered at the subcapsule of the kidney led to a significant reduction of proteinuria, albuminuria, and serum creatinine levels in Nx rats already at 15 days and, remarkably, 30 days after 5/6 Nx. The effect of MSC on the development of interstitial fibrosis was not so remarkable but injection of MSC promoted a significant reduction of glomerulosclerosis in Nx rats after 30 days. Our results are in agreement with previous reports using SC in a model of Thy1.1 aggravated with nephrectomy4,10 and also in the ablation model.7 The possible beneficial effects of SC have not been clearly established but they may rely on their ability to differentiate into renal cell types and repopulate regions that have been damaged during injury. However, the detection of SC at sites of injury has been reported as a rare phenome-
Fig 3. Renal histology of rat kidney tissue. Sham, normal glomerulus (A); Nx, glomerulosclerosis lesions (B); Nx ⫹ MSC, amelioration of glomerulosclerotic lesions in animal receiving MSC (C).
MESENCHYMAL STEM CELLS
non.2,11–13 Alternatively, the renoprotective effect of SC can be due to the local production and secretion of hormones or growth factors (paracrine effects), which seems to be a more reliable mechanism. Independent of the predominant mechanism of renoprotection induced by the inoculation of SC, it seems reasonable to hypothesize that the delivery of SC at the subcapsular region allows a larger number of cells to home in and interact for a longer time with the injured site, which could be important for these cells to play their role. Injection of SC in the circulation, either intravenous or intraarterial, provide only a transient passage of the cells and this may negatively affect the homing of these cells to the site of injury. Our study showed that subcapsular delivery of MSC allowed a great amount of cells to remain in the kidney, which may be of crucial relevance for efficient homing of these cells. The present study also documented a curious trafficking of MSC into the renal parenchyma, observed already 5 days after inoculation and detected until 30 days, when the immunofluorescence signal weakened. In conclusion, our study demonstrated a renoprotective effect of the administration of MSC in the rat remnant kidney model. In addition, the inoculation of MSC at the subcapsule of the kidney may represent an effective alternative route for MSC delivery in the kidney. ACKNOWLEDGMENTS The authors thank Laila Casado and Fernanda Cobucci for their skillful technical assistance and gratefully acknowledge Dr Denis Feliers for his excellent help in reviewing the manuscript.
REFERENCES 1. Meirelles LD, Chagastelles PC, Nardi NB: Mesenchymal stem cells reside in virtually all post-natal organs and tissues. J Cell Sci 119:2204, 2006
951 2. Morigi M, Imberti B, Zoja C, et al: Mesenchymal stem cells are renotropic, helping to repair the kidney and improve function in acute renal failure. J Am Soc Nephrol 15:1794, 2004 3. Lange C, Toegel F, Ittrich H, et al: Administered mesenchymal stem cells enhance recovery from ischemia/reperfusion-induced acute renal failure in rats. Kidney Int 68:1613, 2005 4. Li B, Morioka T, Uchiyama M, et al: Bone marrow cell infusion ameliorates progressive glomerulosclerosis in an experimental rat model. Kidney Int 69:323, 2006 5. Sugimoto H, Mundel TM, Sund M, et al: Bone-marrowderived stem cells repair basement membrane collagen defects and reverse. Proc Natl Acad Sci USA 103:7321, 2006 6. Kunter U, Rong S, Djuric Z, et al: Transplanted mesenchymal stem cells accelerate glomerular healing in experimental glomerulonephritis. J Am Soc Nephrol 17:2202, 2006 7. Caldas HC, Fernandes IMM, Gerbi F, et al: Effect of whole bone marrow cell infusion in the progression of experimental chronic renal failure. Transplant Proc 40:853, 2008 8. Pittenger MF, Mackay AM, Beck SC, et al: Multilineage potential of adult human mesenchymal stem cells. Science 284:143, 1999 9. Fujihara CK, Noronha IL, Malheiros DMA, et al: Combined mycophenolate mofetil and losartan therapy arrest established injury in the remnant kidney. J Am Soc Nephrol 11:283, 2000 10. Kunter U, Rong S, Boor P, et al: Mesenchymal stem cells prevent progressive experimental renal failure but maldifferentiate into glomerular adipocytes. J Am Soc Nephrol 18:1754, 2007 11. Lin F, Cordes K, Li L, et al: Hematopoietic stem cells contribute to the regeneration of renal tubules after renal ischemiareperfusion injury in mice. J Am Soc Nephrol 14:1188, 2003 12. Kale S, Karihaloo A, Clark PR, et al: Bone marrow stem cells contribute to repair of the ischemically injured renal tubule. J Clin Invest 112:42, 2003 13. Tögel F, Weiss K, Yangl Y, et al: Vasculotropic, paracrine actions of infused mesenchymal stem cells are important to the recovery from acute kidney injury. Am J Physiol Renal Physiol 292:F1626, 2007