Effect Of Endothelial Progenitor Cells Transplantation On Reconstituting Peritubular Capillary Of Unilateral Ureteral Occlusion Rats

Background and objectiveChronic kidney disease(CKD) is an important problem of world public health and irreversibly progresses into end stage renal failure(ESRF). At present the main treatment of ESRF is dependent on dialysis or kidney transplantation, but there are high mortality and a lot of complications in dialysis patients and transplantation is also limited for absence of donors and expensive immunodepressants. So it is one of the most important research fields in chronic kidney disease to find more effective ways to repair impairment or inhibit the progression.Recently, close pathologic analysis shows that functional impairment of the kidney is better correlated with the degree of tubulointerstitial damage than with that of glomerular injury, and this finding in turn has led to the broad recognition that the final common pathway of renal failure operates principally in the tubulointerstitium. Furthermore, rarefaction of peritubular capillaries(PTC) and chronic hypoxia is crucial for the progression of tubulointerstitial injury. And the former is the major factor of hypoxia. Another research suggests that either vascular injury or vascular factors are the most likely triggers for pericyte migration and differentiation into myofibroblasts, which serve to refocus fibrosis research to injury of the vasculature rather than injury to the epithelium. So it is important to improve peritubular capillaries density and hypoxia for regressing and inhibiting progression of CKD.Neovascularization in adults is often considered to result exclusively from the proliferation, migration and remodeling of preexisting endothelial cells, a process referred to as angiogenesis. Vasculogenesis, the formation of new blood vessels from endothelial progenitor cells, was thought to occur only during embryonic life. The discovery of adult endothelial progenitor cells (EPCs) has major implications for angiogenic therapy (Asahara et al., 1997)in adult,and EPCs derive from the bone marrow and contribute to the formation of new blood vessels in adults. EPCs differ from circulating endothelial cells that randomly enter the circulation as a result of vascular injury because they develop strong proliferative potential when cultured in medium containing specific growth factors. When these expanded cells are injected intravenously or locally into mice with hind-limb ischemia and myocardial infarction, they form new vessels and restore blood flow sufficiently to save the limb and improve cardiac function. EPCs also secrete angiogenic factors,i.e. VEGF, bFGF, and so on. Therefore, EPCs can reconstitute capillaries similar to embryonic vasculogenesis, which is significantly different from simple angiogenic factor.CKD patients have reduced numbers of circulating CD34+ EPCs. Furthermore, EPCs from patients with CKD displayed functional impairments, i.e. hampered adherence, reduced endothelial outgrowth potential and reduced antithrombogenic function. These impairments are already observed at Stage 1 of CKD and become more apparent when CKD progresses. Bone marrow-derived endothelial progenitor cells can repair glomerular capillary in habu snake venom-induced glomerulonephritis, and restore renal function in chronic experimental renovascular disease. There is limited research about reconstitution of peritubular capillaries by ECPs. So we design the present study that we attempt to demonstrate if EPCs participate in reconstituting of peritubular capillaries in unilateral ureteral obstruction rats. For this purpose, we first evaluated the effects of conditioned medium from renal tubular epithelial cells under hypoxic on the proliferation, migration and secretion of rat bone marrow-derived EPCs ex vivo. Then in vivo we investigated the homing of EPCs in obstructive kidneys of UUO rats and reconstitution of peritubular capillaries and possible mechanisms after transplantation.MethodIn part one, mononuclear cells(MNCs) in rat bone marrow were isolated by density gradient centrifugation and were cultured according to previously described techniques. Rat bone marrow-derived EPCs were characterized as adherent cells double positive for acLDL-DiI-uptake and FITC-lectin binding by direct fluorescent staining under confocal microscopy and further documented by demonstrating the expression of cd133, cd34 and VEGFR-2 by fluorescent microscope. Renal tubular epithelial cells(RTEC) were cultured primarily with routine tissue block adhering wall method. And conditioned medium (CM) was harvested from renal tubular epithelial cells cultivated for 48 hours in hypoxia or normoxia. Rats bone marrow EPCs were divided into 2 groups: control group, cultured with CM from normoxia(5%Fetal Calf Serum, FCS); CM group, cultured with CM from hypoxia(5%FCS). Each group cells were collected at 12, 24, 48 and 96 hours. EPCs proliferation and migration were evaluated by MTT assay and transwell method. The protein and mRNA expression of SDF-1, VEGF, angiogenin(Ang-1) and CXCR4 were assessed by western bloting and RT-PCR method.In part two, we established a tubularinterstitial fibrosis model of rat kidney with unilateral ureteral obstruction method and EPCs were expanded in vitro and marked with 5-bromodeoxyuridine(BrdU) and then transplanted by direct renal artery injection. Sixty health male SD rats were divided into 4 groups.1. Sham group were operated by sham surgery, and then rats were injected 1ml PBS by renal artery.2. Transplantation control group were operated by the same surgery as sham group and then rats were injected EPCs by renal artery..3. UUO group were operated by ligation of left ureter, and then were injected 1ml PBS by left renal artery.4. Transplantation group were operated by ligation of left ureter, and then were injected EPCs by left renal artery.Five rats of each group were killed on the 7th, 14th, and 21st day. The blood, urine and renal tissue aliquot were collected. At each time point, items were detected as follows:①changes of renal weight/body weight, urine protein in 24 hours, serum alanine aminotransferase, serum aspartate aminotransferase, serum urea nitrogen and serum creatinine.②histopathological changes in kidney slices and severity by interstitial lesion scoring by HE, PAS, and Masson methods.③Peritubular capillaries density was evaluated by expression of immunohistochemistry JG12.④The expression of proliferating cell nuclear antigen(PCNA), SDF-1,α-SMA and HIF-1αwere detected by immunohistochemistry method. The protein expression of SDF-1, VEGF, angiogenin(Ang-1) and HIF-1αwere assessed by western blot method.⑤Localization and semiquantitative of transplantation cells were detected by fluorescent microscope. resultsPart oneAfter 7~10 days in endothelial progenitor cells selection medium, bone marrow mononuclear cells turned into spindle-shaped, endothelial cell-like cells. Most of them showed uptake of ac-LDL and lectin binding, and they were characterized further by demonstrating the expression of CD133, CD34 and VEGFR2. By tissue block adhering wall method renal tubular epithelial cells were cultivated and expression of keratinose was positive by fluorescent microscope. Expression of VEGF and SDF-1 was increased in RTEC in hypoxic condition, and data in MTT assay showed that the activity of proliferation and migration of EPCs were improved by CM. The results in RT-PCR and western blot showed that up-regulation of CXCR4, VEGF and Ang-1 mRNA and VEGF, Ang-1 Protein in EPCs was evident in 24~48h.Part twoThe rat UUO model was successfully established. In sham group, all renal tubules and the interstitium were intact, and no significantly infiltrating cells were observed. On the 7th day, dilation of tubules in UUO group began to appear in some regions, with mononuclear cell infiltration of the interstitium, mild edema, and fibrosis. Also during this period, there was a significant increase in the ligated kidney/body weight ratio. On the 14th day, tubular deformation, including dilation and atrophy, became more severe. Inflammatory cells infiltration became conspicuous, reaching a maximum in both the cortex and the medulla on the 21st day. Intense interstitial fibrosis was also observed, mainly around the affected tubules.Hepatic function,renal function and urine protein in 24 hours did not vary among transplantation group and UUO group, sham group except for temporary abnormity of renal function on day 7th in UUO group and transplantation group. In transplantation group peritubular capillary index on the 14th and 21st day in transplantation was significantly lower than that in Sham group, and higher than that in UUO group. The interstitial fibrosis and tubular injury in transplantation group was lighter than that in UUO group. Compared with sham group, expression of alpha-SMA was significantly higher in UUO group on the 14th and 21st(P<0.05), but that in transplantation group was lower than that in UUO group(p<0.05) respectively on day 14th and 21st. We found the BrdU and JG12 double positive cells in peritubular capillaries in transplantation group. On day 7th we could detect a lot of BrdU-positive cells, and gradually decreased, but significantly higher that transplantation control group(P<0.05). Compared with sham group, expression of Ang-1 and VEGF increased on 7th day(P<0.05), but quickly decreased, and in transplantation group the increase was higher and longer(P<0.05). Compared with sham group, expression of SDF-1 in UUO and transplantation groups was up-regulated on day 7th and 14th(P<0.05). And that in transplantation group significantly increased, and higher than that in UUO group respectively on day 7th, 14th and 21st(P<0.05). There was trace amount of expression of HIF in sham group. But in UUO group that was significantly increased (P<0.05), and on the 21st reached at peak. And expression in transplantation group was lower than that in UUO group respectively on day 7th, 14th and 21st(P<0.05). At last, we checked expression of interstitial PCNA, and we found it was very low in sham group, but in UUO group the expression of PCNA significantly up-regulated respectively on day 7th, 14th and 21st. After tansplantation, expression of intersticial PCNA was significantly down-regulated.Conclusion1. Rat bone marrow EPCs and renal tubular epithelial cells were successfully cultured in our experiment.2. Hypoxia increased the expression of VEGF and SDF-1 in renal tubular epithelial cells.3. Conditioned medium in hypoxic renal tubular cells improved proliferation, migration and expression of VEGF, Ang-1 and CXCR4 of EPCs, which may partly be dependent on SDF-1 and VEGF.4. EPCs transplantation improved peritubular capillaries density and interstitial fibrosis of the ligated kidney.5. Expression of SDF-1 in ligated kidney contribute to homing of EPCs, which reconstituted peritubular capillaies.6. Up-regulation expression of VEGF, Ang-1 in ligated kidney EPCs suggests that EPCs also participated in peritubular capillaries reconstitution by autocrine and paracrine.