The Protective Effect And Underlying Mechanism Of Endothelial Progenitor Cells On Renal Fibrosis

Renal interstitial fibrosis is the final common pathological way of chronic kidney disease progressing to end-stage renal disease. Once initiate it is difficult to reverse. However, there still are no effective treatment for it in clinical practice. Chronic ischemic and hypoxia induced by vascular rarefaction is the key factor in the initiation and progress of renal interstitial fibrosis. To ameliorate renal fibrosis by improving renal microvascular network repair, associated knowledge and investigations are limited. It is reported that EPCs as precursor cells of vascular endothelial cells, in pathological conditions, it can home to ischemia and hypoxia organs, promote vascular repair and angiogenesis. Thus, we investigated the role and possible mechanisms of EPCs in kidney fibrosis. Unilateral ureteral obstruction surgery was used in mice to establish the renal interstitial fibrosis animal model. We detected the role of EPCs and explored the underlying mechanism as follows. On the one hand, bone marrow-derived EPCs was isolated, cultured, identified and infused into UUO mice model. On the other hand, AMD3100, a small molecular inhibitor of CXCR4, was used to inhibite the homing of endogenous EPCs into injured kidney in vivo experiment. The results showed that renal morphological changes and renal fibrosis were significantly decreased by EPCs infusion, while it is remarkably worsening if the EPCs homing were inhibited (which was related to the increased T cell infiltration and up-regulated inflammation) by AMD3100. The protecting effect of EPCs derived from its suppression impact on pericyte-myofibroblast transition and G2-M arrest of tubular epithelial cells. Therefore, the EPCs and its paracrine substance may become a new strategy in clinical treatment of renal interstitial fibrosis.Part I Vascular Rarefaction in Chronic Renal FibrosisObjective Chronic renal interstitial fibrosis often associated with renal vascular rarefaction, this part of study was designed to investigate the occurrence and mechanism of microvascular loss in renal fibrosis.Methods Thirty-two male C57b1/6 mice were randomly divided into four groups: Control group (Normal), UUO-3day group, UUO-7day group and UUO-14day group. Perform sham surgery or Unilateral Ureteral Occlusion (UUO) surgery and sacrificed the mice at the corresponding time point. Western Blot and RT-PCR were used to measure the expressions of a-SMA and Fibronectin; immunohistochemistry, immunofluorescence, and RT-PCR were used to observe the changes of CD31, CD34 and PDGFR-? expressions. The percentage and number of CD31+and CD34+cells during the course of renal fibrosis were detected by Flow Cytometry (FCM). The co-positive area of CD31, CD34, PDGFR-? and a-SMA were measured by immunofluorescence.Results Along with the development of UUO surgery, renal interstitial fibrosis markers a-SMA and Fibronectin were remarkably increased, while the interstitial microvascular (CD34 and CD31) were significantly decreased. In this process, neo--vascular exhibited dysmaturity, and the co-positive area of PDGFR-?(marker of pericyte) and a-SMA (marker of myofibroblast) was increased, too.Conclusion The process of chronic renal fibrosis was along with microvascular rarefaction, which induced by increased pericyte-myofibroblast transition and then, neovascular lost the support of pericytes.Part II Endothelial Progenitor Cells Attenuated Renal Fibrosis by Inhibiting the Pericyte-Myofibroblast TransitionObjective Endothelial progenitor cells has been proved participate in vascular repair in many vascular and ischemic diseases, but its effect on renal fibrosis, a vascular regression and rarefaction related pathology, remains unknown. In this part of study, we explored the effectiveness and the underlying mechanism of EPCs infusion in mice unilateral ureteral obstruction model.Methods Bone-marrow derived EPCs was isolated, cultured, identified, and then infused into the early renal fibrosis (UUO-5day) mice by tail vein injection. Morphology changes after transplantation was detected by Periodic Acid-Schiff staining (PAS). Collagen deposition evaluation was performed by Masson-trichrome staining (Masson), Sirius red staining and immunohistochemistry of Collagen-IV. The expressions of myofibroblast marker a-SMA and the pericyte marker PDGFR-? were measured in both protein and mRNA levels, their co-localization was performed by IF. Vascular repair changes after EPCs infusion was evaluated, too.Results Our data showed that after trans-infusion of EPCs, the pathologic changes and collagen deposition was significantly reduced. The expressions of a-SMA and PDGFR-? were decreased in EPCs group, too. Co-localization of a-SMA and PDGFR-? was obviously less in EPCs group, while there were no evident changes of vascular repair. What's more, the tubular epithelial cells G2-M arrest was also ameliorated by EPCs infusion.Conclusion In conclusion, EPCs infusion can effectively attenuated the obstruction induced renal fibrosis, which may be related to its inhibition on pericyt-myofibroblast transition and tubular epithelial cells G2-M arrest.Part ? AMD3100 Worsens Renal Fibrosis through Regulation of Endothelial Progenitor Cells Homing and T-cell-Related InflammationObjective AMD3100 is a small molecule inhibitor of CXCR4, which is located in the cell membranes of EPCs and a variety of inflammatory cells and has been reported to reduce organ fibrosis in the lung, liver and myocardium. However, the effect of AMD3100 on renal fibrosis is still unknown. This study investigated the impact of AMD3100 on renal fibrosis.Methods C57bl/6 mice were subjected to unilateral ureteral obstruction surgery with or without AMD3100 administration. Tubular injury, collagen deposition and renal fibrosis were detected and analyzed by histological staining, immunocytochemistry and Western Blot. Bone marrow derived EPCs (CD45+, CD34+ and CD309+ cells) and capillary density (CD31+) were measured by flow cytometry and immuno--fluorescence. Inflammatory cells, chemotactic factors and T cell proliferation were characterized, too.Results We found that AMD3100 treatment did not alleviate renal fibrosis but, rather, increased tissue damage and renal fibrosis. AMD3100 administration did not improve bone marrow derived endothelial progenitor cells mobilization but, rather, inhibited the migration of bone marrow derived endothelial progenitor cells into the fibrotic kidney. Additionally, T cell infiltration was significantly increased in AMD3100 treated kidneys compared to un-treated kidneys.Conclusion Thus, treatment of UUO mice with AMD3100 led to a decrease of EPCs homing and an increase of T cell infiltration, suggesting that AMD3100 aggravated renal fibrosis.