Effect Of Renal Proximal Tubular Epithelial Cells Injury Induced By Albumin Overload On Peritubular Capillaries And Its Mechanism

Continuous and massive proteinuria, the most important clinical manifestation of many forms of glomerulus diseases, not only is closely associated with glomerulus sclerosis, but also can induce inflammatory response and fibrosis of tubulointerstitial. It is one of the independent risk factors for progression of chronic renal disease. Albumin is the major component of proteinuria in glomerulus diseases. Many studies have shown that increased reabsorption of albumin can activate the renal tubular epithelial cell (RTEC) to generate a large amount of inflammatory mediators, cytokines, growth factors and extracellular matrix poteins, mediate apoptosis and transdifferentiation of RTEC, and lead to of renal interstitial fibrogenesis. In addition, renal tubular epithelial cell activation induced by proteinuria can activate or damage other cells, such as fibroblast, facilitating the renal interstital fibrogenesis synergistically.Peritubular capillaries, adjacent to RTEC, is the major constitution of renal interstitial as well, and plays important roles in metaining the normal physical function. Peritubular capillaries injury can lead to renal interstitial ischemia and hypoxia, and is regarded as the basis of renal interstitial fibrosis in the progression of chronic renal diseases. However, the relationship between the peritubular microvescular injury and RTEC activation induced by proteinura and its mechanism remains unclear.The results in our previous studies have shown that before the occurence of renal interstitial fibrosis in patients with nephritic syndrome, the proliferation activity of peritubular microvecular epithelial cells declined obviously and the peritubular capillaries density decreased, and the latter is negatively correlated with the amount of albumin endocytosis and the expression of its receptor protein cubilin in RTEC. Whether there is relationship between the increased reabsorption of albumin by RTEC and chronic renal interstitial damage needs to be clarified. Does cubilin play a important role in this process? There is evidence that intracellular reactive oxygen species (ROS) generation is remarkably increased after albumin overloaded by RETC, and ROS plays important roles in pathogenesis of vascular diseases by influencing the cellular proliferation activity, inducing apoptosis of endothelial cells through increasing vascular permeability, inflammatory cell infiltration. Whether upregulated intracellular reactive oxygen species (ROS) generation by increased readsorption of albumin in renal tubular epithelial cell is one of the major reasons for peritubular microvescular injury in chronic renal diseases remains unknown.In this study, we observed the effect of albumin on the expression of cubilin and production of ROS in HKC. The influence of HKC activation by albumin on proliferation and apoptosis of HUVEC was investigated by cell coculture technique. After inhibition albumin reabsorption by cubilin siRNA, ROS generation in HKC and its influence on apoptosis of HUVEC and branching angiogenesis were observed. After inhibition the production of ROS by rotenone which blocks complex I of mitochondrial electron transport chain, the protective effect on HUVEC injury was studied.Methods.1. Construction of cubilin siRNA expression vector and identification of its function SiRNA expression vectors pSUPER EGFP-C1 and C2 were designed, constructed and transfected into HKC. HKC were stimulated with human serum albumin (HSA), changes of cubilin mRNA expression and the inhibitory effect of reconstructive vectors on cubilin mRNA in HKC were assessed.2. Inhibitory effect of Cubilin siRNA expression vector on albumin endocytosis by HKCDose- and time- dependent effects of HKC absorpting TRITC-BSA and the inhibitory effect of reconstructive vectors were investigated by using laser cofocal microscopy . Vector with higher inhibitory effect was selected for further studies.3. Effect of cubilin siRNA expression vector on ROS generation in HKC with albumin overloadedFluorescence probe technique and spectrometry were applied for determination of superoxide anion O₂^·- in HKC and H₂O₂ in supernatant and the time- dependent and dose-dependent effect were observed. The inhibitory effect of cubilin siRNA expression vector and retenone were studied.4. Effect of albumin overload inducing RTEC injury on vascular endothelial cells The effects of HKC activation by albumin on angiogenesis and apoptosis of HUVEC were studied. After albumin activating HKC pretreated with cubilin siRNA expression vector and rotenone,the effect of HKC on angiogenesis and apoptosis of HUVEC were studied.Results1. Enzyme digestive assay and sequencing results showed that two pairs of 64bp sequence are consistent with the designed cubilin siRNA, suggesting that cubilin siRNA expression vector, pSUPER EGFP-C1 and C2, were constructed successfully. Quantitative PCR results showed that cubilin mRNA expression was upregulated in a dose-dependent manner after HKC was stimulated with albumin. Compared with the normal control group, cublin mRNA expression was significantly reduced in HKC transfected with pSUPER EGFP-C1 and C2(P<0.01), and the inhibitory efficiency was 66% and 37%, respectively. There was no significant changes in HKC transfected withpSUPER EGFP.2. Albumin endocytosis by HKC is upregulated and the effect is dose- and time-dependent. However, compared with the normal control group, endocytosis of albumin by HKC transfected with pSUPER EGFP-C1 and C2 is significantly reduced(P<0.01), and the effect is more remarkable for pSUPER EGFP-C1. No significant differences were observed for HKC transfected with pSUPER EGFP(P>0.05).3. Generation of O₂^·-and in HKC and H₂O₂ in culture supernatant were increased after stimulating with albumin at high concentrations, and dose- and duration-dependent effect were observed. Generation of O₂^·- in HKC and H₂O₂ in culture supernatant were suppressed in HKC transfected with cubilin siRNA expression vector and rotenone (P<0.05).4. Compared with the control group, when HKC were activated by HSA at high concentration, angiogenesis of HUVEC decreased and the apoptosis of HUVEC increased (P<0.05). Compared with the HSA stimulation group, angiogenesis of HUVEC increased and the apoptosis of HUVEC decreased in HKC-HUEVC coculture system after HKC were pretreated with cubilin siRNA expression vector and rotenone(P<0.05).Conclusions1. Increased albumin endocytosis by HKC is accompanied with upregulation of cubilin mRNA expression and ROS generation, which is closely related to the increased apoptosis and decreased angiogenesis of HUVEC in the coculture system. Cubilin siRNA can inhibit the cubilin mRNA expression and albumin endocytosis in HKC, decrease the production of ROS, and have protective effect on endothelial cells in the coculture system, suggesting that cubilin may mediate the albumin reabsorption overload by RTEC in renal diseases .2. Albumin endocytosis can stimulate the mitochondrial electron transport chain to produce large amount of ROS in HKC, and the latter is associated with peritubular capillaries injury in renal diseases. Rotenone can inhibit the generation of ROS induced by albumin overload and exert protective effect on peritubular capillaries.