Effects Of Antisense Cubilin RNA On Renal Tubular-interstitial Lesion In Rats With Adriamycin-induced Nephropathy

The chronic heavy proteinuria is an independent risk factor for chronic renal failure. The persistent heavy proteinuria can not only cause damage to renal tubular interstitium and induce transdifferentiation or apoptosis of RTECs,but also initiate renal interstitial inflammatory reaction and activate interstitial fibroblast by activating RTECss(RTECs) and finally lead to interstitial fibrosis.Peritubular capillary(PTC) is the major constitutive component of renal interstitium,and the peritubular micro-vascular endothelial cell is adjacent to renal tubule in histology.Recent studies have confirmed that loss of PTCs is an important cause for interstitium fibrosis of chronic nephropathy.However,it still remains unclear whether proteinuria can also activate RTECs to injure the peritubular micro-vascular net.Our previous study indicated that the high level albumin could activate RTECs to produce massive ROS for inhibiting the proliferation of endothelial cell in co-culture system,inducing apoptosis and inhibiting the formation of tubular structure in the endothelial cells.Moreover,in the patient with nephrotic syndrome,there could be seen significant decrease of proliferative activity of peritubular micro-vascular epithelial cells and significant reduction of the peritubular microvessel density,the latter of which was significantly negatively correlated with the trapped albumin in the cytoplasm of peritubular micro-vascular epithelial cells,suggesting some possible internal correlation of proteinuria activating RTECs with the renal interstitial micro-vascular injury of chronic nephropathy.As the major component of urine protein,albumin has direct damage to RTECs. Studies indicated that the over-loaded reuptake of albumin was the major pathway injuring RTECs.Cubilin is a receptor responsible for the uptake of albumin by RTECs and play a "switch" role in the reuptake of albumin physiologically.In the previous study,we found that cubilin might play an important role in mediating the activation of RTECs by albumin. In this study,the effect of antisense cubilin adenovirus vector on renal interstitial inflammation and peritubular micro-vascular injury of the rat with adriamycin-induced nephropathy will be further observed,so as to provide scientific evidences for elucidating the mechanism of proteinuria-associated renal injury and finding possible intervention measure.Methods.1.SD rats were randomized into five groups,ie,normal control group,adriamycin model group,vector control group,pAdEasy-CUB group and pAdEasy-ACUB group,with 18 rats in each group.Except for normal control group injected with same dose of physiological saline,the there groups were respectively given the injection of single dose (7.5 mg/kg) of adriamycin hydrochloride via caudal vein to reproduce the model of adriamycin-induced nephropathy.At day 3 following the establishment of model,vector control group,pAdEasy-CUB group and pAdEasy-ACUB group were given the renal arterial perfusion(1.5×10¹¹pfu/kg) of pAdEasy-1,pAdEasy-Cub and pAdEasy-Acub, respectively,with equal dose of physiological saline perfused in normal control group and model group.2.Five rats were selected randomly from each group,among which 24-hour urine was collected at days 0,7,14,21 and 28 to determine the level of 24-hour urine protein.Other five rats were selected randomly from each group,and were killed via decapitation at days 7,14 and 28,among which serum was collected from heart so as to determine total proteins, albumin,urea nitrogen and creatinine in serum;meanwhile,the left kidneys were remained, from which partial renal tissues were taken out for preparation of frozen section and another partial renal tissues were fixed in 10%formalin solution for future use.3.The expression of green fluorescent protein was observed under laser scanning confocal microscope to decide the transfection efficacy of adenovirus.PAS staining was performed according to conventional method;the expressions of cubilin and MCP-1 in RTECs were detected with immunohistochemical SP method,the expression of JG12 and renal ED-1 with immunohistochemical two-steps method,and the accumulation of albumin in RTECs with immunohistochemical direct staining.Interstitial injury score of renal tubule, micro-vessel density(MVD) and count of ED-1-positive cell were calculated according to the methods reported in references.Results1.Identification of RTECs with the transfection of sense and antisense cubilinsAt day 3 following adenovirus transfection,laser scanning confocal microscope showed strong expression of green fluorescent protein in vector control group, pAdEasy-CUB group and pAdEasy-ACUB group but only weak expression in normal control group and model group,indicating a successful transfection of adenovirus in renal tissue of the rat with adriamycin-induced nephropathy.2.Interferential action of antisense cubilin on urine protein,renal function and pathological change of the rat with adriamycin-induced nephropathyCompared with normal control group,the other four groups showed significant increase of 24-hour urine protein over time(P＜0.01) and progressive increase in serous Cr and BUN(P＜0.01).Compared with model group,pAdEasy-ACUB group had more urine protein(P＜0.01) but less serous Cr and BUN(P＜0.05).In model group,vector control group and pAdEasy-CUB group,under light microscope,partial tubular expansion and vacuole-like degeneration were found and hyaline cast formed by urine protein,enlarged interstitial edema,inflammatory cell diffusion or focal infiltration could be observed in local region;however,in pAdEasy-ACUB group,the above pathological changes were reduced markedly.3.Inhibitory action of antisense cubilin on the expression of cubilin in renal tissue and the pinocytosis of albuminCompared with normal control group,in model group,vector control group and pAdEasy-CUB group,there could be found higher expression of cubilin in RTECs(P＜0.01) and more albumin taken up in cytoplasm(P＜0.01).Correlative analysis indicated that the accumulation of albumin in RTECs was significantly positively correlated with the expression of cubilin(γ=0.780,P＜0.01).Compared with model group,pAdEasy-ACUB group had lower expression of cubilin in RTECs and less albumin taken up in cytoplasm (P＜0.01).4.Interferential action of antisense cubilin on renal interstitial inflammation of the rat with adriamycin-induced nephropathy Significant higher expression of MCP-1 in RTECs and more ED-1-positive cells in renal interstitium were found in model group,vector control group,pAdEasy-CUB group, compared with normal control group(P＜0.01).The expression of MCP-1 in RTECs and the number of ED-1-positive cells in renal interstitium were significantly positively correlated with the accumulation of albumin(γ1=0.775 andγ2=0.863,respectively,P＜0.01). Compared with model group,pAdEasy-ACUB group showed lower expression of MCP-1 in RTECs and less ED-1-positive cell infiltration(P＜0.01).5.Interferential action of antisense cubilin on micro-vascular pathological changes of the rat with adriamycin-induced nephropathyCompared with normal control group,peritubular micro-vessel count with positive JG-12 was significantly reduced and was negatively correlated with the albumin taken up by RTECs and the injury score of renal tubular interstitium(γ1=-0.728 andγ2=-0.851, respectively,P＜0.01) in model group and vector control group.Peritubular micro-vessel with positive JG-12 was significantly increased in pAdEasy-ACUB group,compared with model group(P＜0.01).Conclusions1.Antisense cubilin RNA can inhibit expression of cubilin and pinocytosis of albumin by RTECs of the rat with adriamycin-induced nephropathy.2.Antisense cubilin RNA can down-regulate expression of MCP- 1 in RTECs of the rat with adriamycin-induced nephropathy,lessen the inflammatory reaction in renal interstitium and protect peritubular micro-vascular net,as suggests that inhibiting expression of pinocytosis receptor cubilin can reduce the over-loaded pinocytosis of albumin induced by RTECs of the rat with adriamycin-induced nephropathy and has a certain protective effect on the proteinuria-associated injury of renal tubular interstitium.