Mechanisms Of MiRNA And AQP2 In Zhen-wu-tang Treatment On Adriamycin-induced Nephrotic Syndrome Rats

ObjectivesNephrotic syndrome (NS) is an intractable and multiple disease in children, making it the second leading cause of kidney disease in China The disease is seen mostly in boys and the incidence rate is as high as 2～10/100000. The global pandemic of NS is progressing at an alarming rate and severely diminishes the quality of life for millions. Despite the changing face of NS, treatment options and resources remain woefully. The first line of treatment uses old but important approaches to reduce kidney tissue damage hormone therapy. But hormone therapy would have recrudesce, hormonal resistance and other side effects for long-term treatment. Hence, there is a critical need to search for alternative medicine with higher safety and efficacy to treat NS.Zhen-wu-tang (ZWT), a classic prescription in Febrile Disease, is widely used to treat chronic kidney diseases. The effect of ZWT on curing NS was confirmed both in clinic and experiment researches. AQP2 is a key target for diuretic traditional Chinese medicines and plays a key role in maintaining body water homeostasis and tubular function. Dysfunction of AQP2 is one of the most important factors contributing to NS pathogenesis. Further, it reported that miRNAs have fundamental roles in regulation of kidney homeostasis, and the water sodium metabolism in kidney. Our research was to explore the possible mechanism underlying the curing of ZWT on NS by regulation of miRNAs and AQP2 on Adriamycin-induced NS rat model.Methods1. The protect effect of ZWT on ADR-induced nephrotic syndrome rat modelThe NS rats model was established with a single tail-intravenous injection of Adriamycin (ADR) (6.0 mg/kg). One week of after modeling, repeated doses of ZWT (16.8 and 8.4 g/kg/d) and Prednisone (5 mg/kg/d, reference drug) were administrated orally once daily, for 4 weeks, to these rats.24h urine protein excretion, serum levels of total protein (TG), albumin (ALB), total cholesterol (TC), triglycerides (TG), blood urea nitrogen (BUN) and serum creatinine (Scr) were assessed through biochemical parameters. Plasma SOD and MDA levels were tested according to instructions of assay kits, and the assay of interleukin-4 and interleukin-8 levels using ELISA kits.2. Effect of ZWT on edema and AQP2 expression in ADR-induced NS rat modelThe levels of potassium, sodium, and chloride in blood and urine were measured with autobiochemical analyzer. And serum levels of AVP and ALD were detected with ELISA kits according to instructions of assay kits. AQP2 expression and distribution in kidney were detected using qRT-PCR, westernblot and immunocytochemistry tests, respectively.3. miRNA microarray analysisTotal RNA was harvested using TRIzoland miRNAeasy mini kit.And post RNA quantity measurement using the NanoDrop 1000, samples were labeled using the miRCURYTM Hy3TM Power labeling kit and hybridized on the miRCURYTM LNA Array (v.18.0). The slides were scanned using the Axon GenePix 4000B microarray scanner. Expressed data were normalized using the Median normalization and differentially expressed miRNAs were identified using fold-change filtering (Fold change> 2.0). Target genes and transcription factor of miRNAs were identified using bioinformatics methods. And then the candidate genes were carried for GO and Pathway analysis.4. Determination of miRNA targeting AQP2 based on the treatment of ZWT on NSTargetScan (Release 6.2:June 2012), Microcosm Targets (Version 5.0), and Miranda (Release:August 2010) were used to predict the miRNAs targeting AQP2. Candidate miRNAs were obtained from the intersection of the three prediction methods. We selected those miRNA that appeared in both approaches, the candidate miRNAs and differentially expressed miRNAs were identified by miRNA microarray analysis, as the target miRNAs. Reverse-transcriptase (RT)-PCR was performed to confirm the expression of target miRNAs. And miRNA transfection in mouse cortical collecting duct cells was conducted to identify the effect of miRNA on AQP2. Further, after increasing or decreasing miRNAs expression by using lipofectamine, we performed cellular growth experiments and flow cytometry analysis to examine the influence of miRNAs on cell growth and proliferation. To further investigate the direct effect of miR-92b on AQP2, wild-type (WT) and mutant 3’-UTRs of AQP2 were cloned into the luciferase reporter and co-transfected with miR-92b mimic into M1 cells.Results1. The proctect effect of ZWT on Adriamycin-induced nephrotic syndrome rat model(1) In our study, we demonstrated that a single injection of ADR at 6.0 mg/kg increased 24 h uric protein excretion 2 weeks after the injection (5.13± 1.76;14.97±7.81, P<0.01). ZWT decreased urine protein excretion, the serum levels of of TC, TG, BUN and Scr significantly in diseased rats. A decrease in plasma levels of TP and ALB were also recorded in nephropathic rats. Pathological results showed an improved pathological state and recovered glomerular structure in ZWT treatment groups.(2) The biochemical results showed that ZWT decreased renal IL-8 level but increased renal IL-4 level. Furthermore, ZWT reduced the level of MDA (lipid peroxidation marker) and increased SOD activity.2. Effect of ZWT on edema and AQP2 expression in Adriamycin-induced NS rat model(1) ADR-induced NS rats presented with obvious edema-associated pathologies, manifested by marked ascites, increased kidney index, decreased urine volume and swollen kidney ZWT treatment ameliorated ascites, reduced the ratio of absolute kidney weight (K.W) to total body weight (BW), and reduced tubular injury induced by ADR injection. Further, ZWT promoted sodium excretion in NS rats and has no effect on potassium and chloride excretion. Elisa results showed that ZWT reduced the serum level of AVP and ALD.(2) In the NS model group, AQP2 mRNA and protein expression was reduced 2.58-fold and 1.88-fold compared with the normal control group, respectively. In rats treated with ZWT, AQP2 mRNA and protein expression significantly increased and was comparable with that in the normal control group. These results were confirmed by immunoreactivity using kidney sections. However, the residual AQP2 in ADR-induced NS rats was chiefly distributed in the apical region of the collecting ducts cells as compared to normal control rats, suggesting that the intracellular trafficking mechanism of AQP2 is normal3. miRNA microarray analysisThere are 123 miRNAs differentially expressed in normal control group and NS model group. Among these miRNAs,48 miRNAs were up-regulated (Model vs control) and 75 miRNA were down-regulated. Compared with the microarray data conducted by Qing-chun Li in PAN-induced nephropathy,24 miRNAs coincide exactly with the present study. Bioinformatics methods showed that miRNAs are associated with renal sodium water metabolism, immunoregulation, interstitial fibrosis and autophagy in NS.4. Determination of miRNA targeting AQP2 based and the intervention of ZWT(1) Bioinformatics search was conducted in 3 databases, which yielded 6 miRNAs against AQP2. Among these, only miR-92b and miR-340-3p were differentially expressed in the miRNA microarray analysis. Then RT-PCR was used to confirm the expression of miR-92b and miR-340-3p. Consistent with the miRNA microarray analysis, the expression of miR-92b was decreased 5-fold in the NS model rats. In the miR-92b mimic transfected cells, decrease in AQP2 expression was detected. Likewise, the mRNA and protein levels of AQP2 were significantly increased due to the introduction of miR-92b inhibitor. These findings suggest that miR-92b functions as a modulator of AQP2.(2) Fluorescent activity of the reporter was significantly decreased in the cells transfected with WT 3’-UTR, while that in cells transfected with mutant AQP23’-UTR was not changed, indicating that the miR-92b effect was abolished by site-directed mutagenesis. These results indicated that miR-92b could directly target AQP2. Functional studies showed that miR-92b could decrease cell viability and induce apoptosis in collecting duct cell. ZWT treatment could down-regulated miRNA-92b expression, thus protecting cortical collecting duct cells from injury.Conclusion1. ZWT could ameliorate the proteinuria, low serum albumin, hypercholesterolemia and loss of kidney function of NS rats. These data provides direct evidence for ZWT treating NS, at least in part, by modulating the balance between inflammatory and anti-inflammatory response, enhancing antioxidant capacity and the elimination capacity of ROS.2. The protective effect of ZWT on NS edema in Adriamycin-induced NS rats was demonstrated. The possible mechanism might involve the diuretic and natriuretic effect of ZWT, and the decreased of serum ALD by ZWT treatment.3. There is an obvious differential expression profile of miRNA between ADR-induced NS rats and normal control rats. Understanding the regulation mechanism of miRNAs is of great significance to the pathogenesis of the NS.4. AQP2 mRNA and protein expression significantly decreased in NS rats companied with an unchanged distribution in the apical region of collecting duct cell and an increased serum AVP. It indicates that some other mechanisms may involve in the regulation of AQP2 expression.5. This study revealed that miR-92b is involved in the pathogenesis of NS, at least in part by targeting AQP2, thus correcting water and sodium disturbances as well as reducing renal tubular lesions. And miR-92b may also participate in the pathogenesis of NS through other regulation pathways.In summary, the mechanism underlying the effect of ZWT in treating NS might involve:(1) ZWT inhibits inflammatory infiltration in kidney, improved inflammation injury and attenuated oxidative damage; (2) ZWT ameliorates NS edema and regulate sodium and water metabolism; (3) Treatment with ZWT could up-regulated AQP2 expression, thus maintaining the structure and function of renal tubular; (4) ZWT affects miR-92b function in the development of NS.