Multipotent mesenchymal stem cells reduce interstitial fibrosis but do not delay progression of chronic kidney disease in collagen4A3-deficient mice
2006; Elsevier BV; Volume: 70; Issue: 1 Linguagem: Inglês
10.1038/sj.ki.5001521
ISSN1523-1755
AutoresVolha Ninichuk, Oliver Groß, Stephan Segerer, Reinhard Hoffmann, Ewa Radomska, Andrea Buchstaller, Ralf Huss, Nese Akis, Detlef Schlöndorff, Hans‐Joachim Anders,
Tópico(s)Renal and related cancers
ResumoMultipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the α3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice. Multipotent mesenchymal stem or stromal cells (MSC) have shown to improve outcome of acute renal injury models, but whether MSC can delay renal failure in chronic kidney disease is not known. We injected primary MSC or saline into mice that lack the α3-chain of type IV collagen (COL4A3), a model of chronic kidney disease with close similarities to human Alport disease. Weekly injections of MSC from week 6 to 10 of life prevented the loss of peritubular capillaries and reduced markers of renal fibrosis, that is, interstitial volume, numbers of smooth muscle actin-positive interstitial cells, and interstitial collagen deposits as compared to saline-injected COL4A3-deficient mice. However, renal function, that is, blood urea nitrogen, creatinine levels, proteinuria as well as survival of COL4A3-deficient mice were not affected by MSC injections. Although MSC were found to localize to kidneys of COL4A3-deficient mice after injection, differentiation into renal cells was not detected. However, MSC expressed growth factors, that is, vascular endothelial growth factor (VEGF) and bone morphogenetic protein-7 under basal culture conditions. In fact, VEGF mRNA levels were increased in kidneys of MSC-injected COL4A3-deficient mice and MSC supernatants enhance endothelial cell proliferation in vitro. Thus, weekly injections with MSC prevent loss of peritubular capillaries possibly owing to local production of growth factors rather than by differentiation into renal cells. The maintenance of interstitial vasculature is associated with less interstitial fibrosis but, is insufficient to delay renal failure and survival of COL4A3-deficient mice. Stem cells have been proposed as a new opportunity for the treatment of many diseases. In view of ethical concerns regarding the use of embryonic stem cells, adult stem cells, for example, bone marrow-derived and fibroblast-like plastic-adherent mesenchymal stem cells or their closely related counterpart, multipotent mesenchymal stromal cells (MSCs), may represent a valuable substitute.1.Korbling M. Estrov Z. Adult stem cells for tissue repair – a new therapeutic concept?.N Engl J Med. 2003; 349: 570-582Crossref PubMed Scopus (667) Google Scholar MSCs reside in the bone marrow's non-hematopoietic stromal compartment and can be isolated using selection owing to plastic adherence.2.Horwitz E. Le Blanc K. Dominici M. et al.Clarification of the nomenclature for MSC: the International Society for Cellular Therapy position statement.Cytotherapy. 2005; 7: 393-395Abstract Full Text Full Text PDF PubMed Scopus (1520) Google Scholar MSCs produce growth factors and cytokines that support hematopoiesis in vivo and in vitro, but MSC can also differentiate into a wide variety of cell lineages.3.Pittenger M.F. Mackay A.M. Beck S.C. et al.Multilineage potential of adult human mesenchymal stem cells.Science. 1999; 284: 143-147Crossref PubMed Scopus (17938) Google Scholar In rodents, injections with MSC have proven beneficial in either toxic or ischemic tubular necrosis,4.Herrera M.B. Bussolati B. Bruno S. et al.Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury.Int J Mol Med. 2004; 14: 1035-1041PubMed Google Scholar, 5.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. 2004; 15: 1794-1804Crossref PubMed Scopus (664) Google Scholar, 6.Togel F. Hu Z. Weiss K. et al.Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.Am J Physiol Renal Physiol. 2005; 289: F31-F42Crossref PubMed Scopus (1035) Google Scholar ischemic limb, or heart disease.7.Huss R. Heil M. Moosmann S. et al.Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL(+) multipotent adult progenitor cell clones from peripheral blood.J Vasc Res. 2004; 41: 422-431Crossref PubMed Scopus (25) Google Scholar, 8.Toma C. Pittenger M.F. Cahill K.S. et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation. 2002; 105: 93-98Crossref PubMed Scopus (1997) Google Scholar Less well-defined bone marrow-derived cells reconstitute the mesangium after mesangiolysis.9.Ito T. Suzuki A. Imai E. et al.Bone marrow is a reservoir of repopulating mesangial cells during glomerular remodeling.J Am Soc Nephrol. 2001; 12: 2625-2635PubMed Google Scholar However, the effects of MSC observed in acute and potentially reversible models of mesangial or tubular injury may not apply to chronic kidney disease.10.Rookmaaker M.B. Verhaar M.C. van Zonneveld A.J. et al.Progenitor cells in the kidney: biology and therapeutic perspectives.Kidney Int. 2004; 66: 518-522Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar In the fibrotic kidney, MSC could also promote fibrogenesis-associated progression of tubulointerstitial damage similar to what has been observed in obstructive nephropathy or the fibrotic lung.11.Iwano M. Plieth D. Danoff T.M. et al.Evidence that fibroblasts derive from epithelium during tissue fibrosis.J Clin Invest. 2002; 110: 341-350Crossref PubMed Scopus (1714) Google Scholar, 12.Phillips R.J. Burdick M.D. Hong K. et al.Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis.J Clin Invest. 2004; 114: 438-446Crossref PubMed Scopus (920) Google Scholar To date, the effect of MSCs on the progression of chronic kidney disease is unknown.13.Yokoo T. Sakurai K. Ohashi T. et al.Stem cell gene therapy for chronic renal failure.Curr Gene Ther. 2003; 3: 387-394Crossref PubMed Scopus (19) Google Scholar We addressed this question by preparing primary murine MSCs that were injected into mice deficient for the α3-chain of type IV collagen (COL4A3). These mice are characterized by an abnormal assembly of glomerular basement membranes similar to human Alport disease.14.Hudson B.G. Reeders S.T. Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar, 15.Noel L.H. Renal pathology and ultrastructural findings in Alport's syndrome.Renal Failure. 2000; 22: 751-758Crossref PubMed Scopus (17) Google Scholar COL4A3-deficient mice represent a model for chronic kidney disease associated with interstitial fibrosis and uremia-related death around 10 weeks of age.14.Hudson B.G. Reeders S.T. Tryggvason K. Type IV collagen: structure, gene organization, and role in human diseases. Molecular basis of Goodpasture and Alport syndromes and diffuse leiomyomatosis.J Biol Chem. 1993; 268: 26033-26036Abstract Full Text PDF PubMed Google Scholar, 16.Cosgrove D. Meehan D.T. Grunkemeyer J.A. et al.Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar We show that injected MSC localize to kidneys of COL4A3-deficient mice, but remain within peritubular capillaries and do not transdifferentiate into renal cells. MSCs prevent the loss of peritubular capillaries and interstitial fibrosis, which, however, is not associated with prolonged survival of COL4A3-deficient mice. In order to determine their intrarenal distribution MSCs were labeled with a red fluorescent dye and kidneys of 6- and 9.3-week-old COL4A3-deficient mice were obtained 3 days after intravenous injection. Labeled MSCs were found to locate to the renal cortex outside the tubular compartment, which was apparent by costaining renal epithelial cells for cytokeratin and laminin for which costaining with MSCs was not observed (Figure 1a and b). Staining renal endothelial cells with an MECA-32-specific antibody revealed that MSCs localized within peritubular capillaries (Figure 1c). MSCs neither colocalized with MECA-32 (Figure 1c) nor were MSCs detected in the glomeruli of COL4A3-deficient mice (not shown). Costaining with a Ki-67-specific antibody addressed the question of MSC proliferation in kidneys of COL4A3-deficient mice. The intrarenal MSC (10%) stained positive for Ki-67 (Figure 1d). Next, we intended to assess the fate of MSCs that distribute to peritubular capillaries. For this purpose, fluorescently labeled MSCs were injected at various intervals before being killing at 9.3 weeks of age. Two hours after injection, 11.0±2.1 MSCs were detected per high power field in kidneys of COL4A3-deficient mice (Figure 2). Three and 7 days after injection, the number of MSCs in peritubular capillaries had declined to 6.7±1.6 and 4.4±1.2 MSCs/high power field, respectively (Figure 2, n=6–8/group). MSCs injected into 6-week-old COL4A3-deficient mice were detected at very low numbers in the renal interstitium, indicating that MSC recruitment increases in more advanced stages of kidney disease.Figure 2Interstitial MSC after intravenous injection in COL4A3-deficient mice. Numbers of interstitial MSCs at various intervals after intravenous injection in kidneys of 9.3-week-old COL4A3-deficient mice (n=6–8 for each time point). Values represent means±s.e.m. *P<0.05 vs 2 h.View Large Image Figure ViewerDownload (PPT) We hypothesized that recruitment of injected MSCs to the kidney would modulate renal pathology of COL4A3-deficient mice. Thus, COL4A3-deficient mice were killed at the age of 9.3 weeks after 4 weekly injections of MSCs or saline for collection of renal tissue. Saline-injected COL4A3-deficient mice had diffuse glomerulosclerosis as compared to age-matched wild-type control mice (Figure 3, Table 1). Glomerulosclerosis was characterized by segmental or global increase of glomerular matrix, adhesions to Bowman's capsule, or crescent formation. Injections with MSC did not affect the percentage of either segmental or global glomerulosclerosis or the number of Mac2-positive glomerular macrophages in COL4A3-deficient mice (Table 1, Figure 3).Table 1Serum, urinary, and histological findings in collagen4A3-deficient miceWild type (n=6)Collagen4A3-/-+saline (n=10)Collagen4A3-/-+MSC (n=10)Renal function Blood urea nitrogen (mg/dl)32.3±5.2107.0±83.6*P<0.05 vs wild type.131.6±57.7*P<0.05 vs wild type. Serum creatinine (mg/dl)0.40±0.070.77±0.450.82±0.43*P<0.05 vs wild type.Glomerulosclerosis score 0 (no lesion in %)91±33±5*P<0.05 vs wild type.3±4*P<0.05 vs wild type. 1 (1–24%)9±113±215±7*P<0.05 vs wild type. 2 (25–49%)0±022±3*P<0.05 vs wild type.26±5*P<0.05 vs wild type. 3 (50–74%)0±029±5*P<0.05 vs wild type.29±5*P<0.05 vs wild type. 4 (75–100%)0±033±11*P<0.05 vs wild type.29±11*P<0.05 vs wild type.Cellular response (cells/glomeruli or high-power field) Glom. Mac2+0.1±0.11.2±0.7*P<0.05 vs wild type.1.1±0.6*P<0.05 vs wild type. Interst. Mac2+0.9±0.514.3±3.8*P<0.05 vs wild type.11.5±5.8*P<0.05 vs wild type. Smooth muscle actin+ (%/high-power field)0.6±0.68.2±3.1*P<0.05 vs wild type.2.9±1.4*P<0.05 vs wild type.*P<0.05 MSC vs saline.Peritubular capillaries (capillary cross sections/hpf) MECA-32+115.1±13.342.8±8.4*P<0.05 vs wild type.79.5±12.0*P<0.05 vs wild type.P<0.05 MSC vs saline.MSC, mesenchymal stromal cells; HPF, high-power field.Values represent mean±s.e.m.* P<0.05 vs wild type.# P<0.05 MSC vs saline. Open table in a new tab MSC, mesenchymal stromal cells; HPF, high-power field. Values represent mean±s.e.m. COL4A3-deficient mice showed diffuse interstitial fibrosis when compared to wild-type mice (Figure 4). This was evident from quantitative morphometry, as saline-injected COL4A3-deficient mice showed an increase of the respective indices for interstitial volume, interstitial collagen, and smooth muscle actin-positive area as compared to wild-type mice (Figure 4, Table 1). MSCs significantly reduced all these markers compared to vehicle-treated COL4A3-deficient mice (Figure 4). In saline-injected COL4A3-deficient mice interstitial fibrosis was associated with markedly reduced numbers of peritubular capillary cross-sections. MSC injection partially restored the density of peritubular capillaries as compared to wild-type mice (Table 1, Figure 3). Interstitial fibrosis in COL4A3-deficient mice was associated with an increased number of interstitial Mac2-positive macrophages when compared to saline-treated mice. MSC injection did not affect the numbers of interstitial Mac2-positive macrophages as compared to saline-treated COL4A3-deficient mice (Figure 3, Table 1). COL4A3-deficient mice had proteinuria consistent with their diffuse glomerulosclerotic lesions (Figure 5). Weekly injections with MSC from week 6 to 9 did not affect protein/creatinine ratios in urines compared to saline-treated mice at week 9 (Figure 5). No statistical significant differences were noted in blood urea nitrogen in saline- and MSC-treated COL4A3-deficient mice at week 9 (Table 1). Although serum creatinine levels determined by the Jaffe method can overestimate creatinine levels in mice,17.Meyer M.H. Meyer Jr., R.A. Gray R.W. Irwin R.L. Picric acid methods greatly overestimate serum creatinine in mice: more accurate results with high-performance liquid chromatography.Anal Biochem. 1985; 144: 285-290Crossref PubMed Scopus (75) Google Scholar no significant difference was detected in serum creatinine levels in saline- and MSC-treated COL4A3-deficient mice at week 9 (Table 1). These data show that MSCs can prevent loss of peritubular capillaries and interstitial fibrosis, but not glomerulosclerosis, proteinuria, and renal failure in COL4A3-deficient mice. Next, we studied the effects of MSC injection on survival of COL4A3-deficient mice treated with either MSC or saline. Saline-treated COL4A3-deficient mice showed a mean survival of 75 days (95% confidence interval 68–82 days), whereas weekly injections with MSC from week 6 to 9 had a mean survival of 84 days (95% confidence interval 76–93 days). Kaplan–Meier analysis using log-rank two-tailed testing revealed a P=0.16, indicating that MSC injections did not significantly affect survival of COL4A3-deficient mice (Figure 6). Mortality of COL4A3-deficient mice was likely to be related to uremic death as within the last week of life the physical activity of COL4A3-deficient mice continuously declined until death as noted in previous studies.16.Cosgrove D. Meehan D.T. Grunkemeyer J.A. et al.Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar, 18.Gross O. Beirowski B. Koepke M.L. et al.Preemptive ramipril therapy delays renal failure and reduces renal fibrosis in COL4A3-knockout mice with Alport syndrome.Kidney Int. 2003; 63: 438-446Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar Wild-type control mice remained healthy until the end of the study. As we did not observe MSC differentiation into renal cells in kidneys of COL4A3-deficient mice, paracrine secretion of mediators, for example, cytokines or growth factors, may represent another mechanism how MSC cells modulate local tissue injury. For example, small amounts of bone morphogenetic protein (BMP)-7 and vascular endothelial growth factor (VEGF) have been shown to have strong modulatory effects on the progression of renal injury.19.Kang D.H. Hughes J. Mazzali M. et al.Impaired angiogenesis in the remnant kidney model: II. Vascular endothelial growth factor administration reduces renal fibrosis and stabilizes renal function.J Am Soc Nephrol. 2001; 12: 1448-1457PubMed Google Scholar, 20.Kalluri R. Zeisberg M. Exploring the connection between chronic renal fibrosis and bone morphogenic protein-7.Histol Histopathol. 2003; 18: 217-224PubMed Google Scholar In fact, MSCs cultured for 24 h either with or without fetal calf serum expressed significant levels of growth factors, that is, VEGF and BMP-7 mRNA (Figure 7a). Next, we hypothesized that the microenvironment of a chronically injured kidney may modulate MSC gene expression and growth factor production. In order to address this issue, we examined the gene expression profile of MSCs cultured without serum using 430 Affymetrix gene array. MSCs were incubated with supernatants of cells prepared from kidneys of either 9.3-week-old healthy wild-type mice or diseased COL4A3-deficient mice (n=3). After 12 h of incubation, MSCs were harvested and total RNA was prepared for gene array analysis. The analysis intended to identify genes differentially expressed by MSCs exposed to either a nephritic or a non-nephritic cytokine environment. The correlation coefficient between the two culture conditions for the expression of each gene was greater than 0.975 (Figure 7b). Comparison of the gene expression profiles of MSCs exposed to healthy or diseased kidney supernatants showed that no genes were statistically significantly differentially expressed (Figure 8). Thus, 12 h exposure of MSCs to supernatants of kidney cells prepared from COL43A-deficient mice does not modulate the gene expression profile of MSC.Figure 8Expression of VEGF and BMP-7 in kidneys of COL4A3-deficient mice. Expression of mRNA was determined by real-time RT-PCR using total renal RNA from 10 mice in each group. mRNA levels for saline- and MSC-injected COL4A3-deficient mice are expressed as mean±s.e.m. of the ratio to respective glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression of each kidney. Ratios of saline-treated COL4A3-deficient mice are set as 1.View Large Image Figure ViewerDownload (PPT) In order to test whether kidneys of MSC-treated COL4A3-deficient mice show higher expression levels of the a forementioned growth factors, we performed real-time reverse transcription-polymerase chain reaction (RT-PCR) for mRNAs of VEGF and BMP-7 on total RNA isolates from COL4A3-deficient mice that had been injected with MSCs or saline 7 days before being killing. MSC-injected kidneys expressed twofold higher levels of VEGF mRNA compared to saline-injected COL4A3-deficient mice; however, this did not reach statistical significance (Figure 8). BMP-7 mRNA levels were comparable in both groups. These data suggest that MSCs prevent the loss of peritubular vasculature in association with increased VEGF levels in kidneys of COL4A3-deficient mice. The observation that MSC injection maintains the peritubular capillary network in COL4A3-deficient mice can either be explained by enhanced endothelial cell proliferation or reduced endothelial cell death. To address this question, we coincubated a murine endothelial cell line with supernatants of either MSC, cultured under starving conditions, or VEGF under serum-free conditions. MSCs and VEGFs both markedly increased endothelial cell proliferation (Figure 9). In addition, we asked whether MSC supernatants effects improve EC survival under serum-free culture conditions. Using identical culture conditions, endothelial cell death was determined by costaining for propidium iodine and annexin 5. MSC and tubular cell supernatants both did not affect the percentage of apoptotic or necrotic endothelial cells after 24 h of incubation (data not shown). These data suggest that soluble factors released by MSCs increase endothelial cell proliferation but do not prevent endothelial cells death under serum-free culture conditions. Injection of cultured MSCs can have beneficial effects on experimental models of acute kidney disease,4.Herrera M.B. Bussolati B. Bruno S. et al.Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury.Int J Mol Med. 2004; 14: 1035-1041PubMed Google Scholar, 5.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. 2004; 15: 1794-1804Crossref PubMed Scopus (664) Google Scholar, 6.Togel F. Hu Z. Weiss K. et al.Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.Am J Physiol Renal Physiol. 2005; 289: F31-F42Crossref PubMed Scopus (1035) Google Scholar but their effects on chronic kidney disease is unknown. We used COL4A3-deficient mice as a model of chronic kidney disease associated with interstitial fibrosis and renal failure. Morphometry revealed that MSC injections reduced interstitial volume in COL4A3-deficient mice. In adult COL4A3-deficient mice, the interstitial compartment consists of the peritubular vasculature, myofibroblasts, interstitial matrix, and immune cell infiltrates.16.Cosgrove D. Meehan D.T. Grunkemeyer J.A. et al.Collagen COL4A3 knockout: a mouse model for autosomal Alport syndrome.Genes Dev. 1996; 10: 2981-2992Crossref PubMed Scopus (296) Google Scholar Numbers of interstitial macrophages were comparable in both groups. By contrast, collagen deposits and the smooth muscle actin-positive area were reduced in the renal interstitium of MSC- vs saline-injected COL4A3-deficient mice. Thus, the reduced interstitial volume in MSC-injected COL4A3-deficient mice relates to a decreased number of interstitial myofibroblasts and collagen deposits. Renal fibroblast proliferation and matrix production are driven by renal ischemia.21.Thomas S.E. Anderson S. Gordon K.L. et al.Tubulointerstitial disease in aging: evidence for underlying peritubular capillary damage, a potential role for renal ischemia.J Am Soc Nephrol. 1998; 9: 231-242PubMed Google Scholar In fact, the numbers of peritubular capillaries increased with MSC injections as determined by immunostaining for the endothelial cell marker MECA32. MSC have shown to enhance neoangiogenesis and tissue repair in ischemic skeletal muscle,7.Huss R. Heil M. Moosmann S. et al.Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL(+) multipotent adult progenitor cell clones from peripheral blood.J Vasc Res. 2004; 41: 422-431Crossref PubMed Scopus (25) Google Scholar myocardium,8.Toma C. Pittenger M.F. Cahill K.S. et al.Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart.Circulation. 2002; 105: 93-98Crossref PubMed Scopus (1997) Google Scholar, 22.Tang Y.L. Zhao Q. Zhang Y.C. et al.Autologous mesenchymal stem cell transplantation induce VEGF and neovascularization in ischemic myocardium.Regul Peptide. 2004; 117: 3-10Crossref PubMed Scopus (355) Google Scholar and kidney.6.Togel F. Hu Z. Weiss K. et al.Administered mesenchymal stem cells protect against ischemic acute renal failure through differentiation-independent mechanisms.Am J Physiol Renal Physiol. 2005; 289: F31-F42Crossref PubMed Scopus (1035) Google Scholar Thus, the proangiogenic effects of injected MSC may prevent loss of peritubular capillaries and subsequent fibroblast proliferation and matrix production in the renal interstitium of COL4A3-deficient mice. Although the extent of interstitial fibrosis is considered to be a major factor of progression in chronic kidney disease, MSC-related reduction of interstitial fibrosis did not delay renal failure in COL4A3-deficient mice. These data are consistent with a recent study that found progression to uremic death of COL4A3-deficient mice to be independent of the extent of interstitial fibrosis.23.Cosgrove D. Rodgers K. Meehan D. et al.Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar In this study, blockade of tumor growth factor (TGF)-β reduced myofibroblast proliferation and interstitial matrix deposition, which did not result in prolonged survival of COL4A3-deficient mice. Obviously, in COL4A3-deficient mice, interstitial fibrosis is a marker of chronic kidney disease, but progressive loss of renal function is driven by other factors. For example, previous studies have identified integrin-α1β1, angiotensin II, vasopeptidase, and chemokine receptor CCR1-dependent interstitial macrophage recruitment as factors that contribute to the progression of kidney disease in COL4A3-deficient mice.18.Gross O. Beirowski B. Koepke M.L. et al.Preemptive ramipril therapy delays renal failure and reduces renal fibrosis in COL4A3-knockout mice with Alport syndrome.Kidney Int. 2003; 63: 438-446Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar, 23.Cosgrove D. Rodgers K. Meehan D. et al.Integrin alpha1beta1 and transforming growth factor-beta1 play distinct roles in alport glomerular pathogenesis and serve as dual targets for metabolic therapy.Am J Pathol. 2000; 157: 1649-1659Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 24.Ninichuk V. Gross O. Reichel C. et al.Delayed chemokine receptor 1 blockade prolongs survival in collagen 4A3-deficient mice with Alport disease.J Am Soc Nephrol. 2005; 16: 977-985Crossref PubMed Scopus (77) Google Scholar, 25.Gross O. Koepke M.L. Beirowski B. et al.Nephroprotection by antifibrotic and anti-inflammatory effects of the vasopeptidase inhibitor AVE7688.Kidney Int. 2005; 68: 456-463Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar Given the low number of MSCs recruiting to the renal interstitium of 6-week-old COL4A3-deficient mice, it is rather unlikely that an earlier onset of MSC injections may have shown different results. Thus, our data argue for a limited role of MSC injections in chronic kidney disease. The MSCs we used in our experiments have the capacity to differentiate into multiple cell lineages, for example, skeletal muscle cells, and hematopoietic cells.7.Huss R. Heil M. Moosmann S. et al.Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL(+) multipotent adult progenitor cell clones from peripheral blood.J Vasc Res. 2004; 41: 422-431Crossref PubMed Scopus (25) Google Scholar, 26.Lange C. Kaltz C. Thalmeier K. et al.Hematopoietic reconstitution of syngeneic mice with a peripheral blood-derived, monoclonal CD34-, Sca-1+, Thy-1(low), c-kit+ stem cell line.J Hematother Stem Cell Res. 1999; 8: 335-342Crossref PubMed Scopus (43) Google Scholar However, when MSCs were injected into mice after ligation of the femoral artery neoangiogenesis was markedly enhanced in the absence of MSC differentiation into for example, endothelial cells.7.Huss R. Heil M. Moosmann S. et al.Improved arteriogenesis with simultaneous skeletal muscle repair in ischemic tissue by SCL(+) multipotent adult progenitor cell clones from peripheral blood.J Vasc Res. 2004; 41: 422-431Crossref PubMed Scopus (25) Google Scholar Data from two different models of acute tubular necrosis suggested that injected MSCs contribute to tubular regeneration by differentiation into tubular epithelial cells.4.Herrera M.B. Bussolati B. Bruno S. et al.Mesenchymal stem cells contribute to the renal repair of acute tubular epithelial injury.Int J Mol Med. 2004; 14: 1035-1041PubMed Google Scholar, 5.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. 2004; 15: 1794-1804Crossref PubMed Scopus (664) Google Scholar Our results show that MSCs do not differentiate into renal cells in kidneys of COL4A3-deficient mice. MSCs were found at considerable numbers within peritubular capillaries, but not in the interstitial, tubular, or glomerular compartment. The latter may relate to the lack of chemokine receptor CCR5 in MSC, which is required for the recruitment of leukocytes to glomeruli and transendothelial diapedesis.27.Luttichaux I.V. Notohamiprodjo M. Wechselberger A. et al.Human adult CD34 - progenitor cells functionally express the chemokine receptors CCR1, CCR4, CCR7, CXCR5, and CCR10 but not CXCR4.Stem Cells Dev. 2005; 14: 329-336Crossref PubMed Scopus (164) Google Scholar In fact, differentiation into tubular epithelial cells or interstitial cells would require that MSC migrate from the intravascular into the tubular or interstitial compartment of the kidney. However, MSCs remained up to 7 days within peritubular c
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