Aldosterone-induced Podocyte Injury And Its Underlying Mechanisms

Partâ… Aldosterone modulates the production of matrix metalloproteinases-2and -9 via TGF-β₁-signaling pathway in podocytes.Objectives To assess the effect of aldosterone (ALD) on the production of matrixmetalloproteinases-2 (MMP-2), matrix metalloproteinases-9 (MMP-9) and collagenâ…£inculture supernatants of podocytes and possible molecular mechanisms involved in theinfluence of aldosterone on the synthesis and degradation of extracellular matrix (ECM)produced by podocytes.Methods (1) To evaluate the time and dose effect of aldosterone on the catabolicenzymes and related components of ECM, podocytes were treated with aldosterone at theconcentration of 10^-11M, 10^-9M, 10^-7M respectively. Conditioned media was harvestedat 24, 48 and 72 hours. The enzymatic activities of MMP-2 and MMP-9 were assayed bygelatin zymography. Collagen IVα5 chain and TGF-β₁ proteins released into culturesupernatants were assessed by Western blot and enzyme-linked immunosorbent assay(ELISA) analysis. The adhesion rates of podocytes were monitored by flow cytometry.(2) To determine whether the effect of aldosterone was mediated by mineralocorticoidreceptor (MR), podocytes were pretreated with aldosterone receptorantagonist-spironolactone (SPI) before stimulated with ALD. The above-mentionedindexes were examined under the same conditions. (3) To evaluate whether TGF-βsignaling pathway participated in the ALD-induced podocytes injury, the inhibitor ofTGF-β₁ receptor was applied before addition of ALD. The changes of enzymaticactivities of MMP-2 and MMP-9, the expression of collagenⅣα5 chain protein and theadhesion rates of podocytes were also tested. Results (1) Compared with the control group, aldosterone increased the activity ofMMP-2 and MMP-9 in culture supernatants of podocytes with a dose- andtime-dependent manner (P＜0.05). Opposite to the increases in MMPs activity,aldosterone decreased the level of collagenⅣα5 chain protein in culture supernatants(P＜0.05). Meanwhile, the expression of TGF-β₁ also increased (P＜0.05). (2)Spironolactone completely abolished the above-mentioned effects of aldosterone(P＜0.05). (3) Blockage of TGF-β₁ signaling pathway with SB431542 partly preventedthe aldosterone-induced upregulation of MMP-2 and MMP-9 as well as thedownregulation of the collagenⅣα5 chain protein and the adhesion rates of podocytes(P＜0.05). (4) Correlation analysis was performed between the expression of MMP-2,MMP-9, TGF-β₁ and collagenⅣα5 chain in culture supernatants of podocytes. Theenzymatic activities of MMP-2 and MMP-9 have significant negative correlation with theexpression of collagenⅣα5 chain protein (P＜0.05). There was a significant positivecorrelation between the enzymatic activities of MMP-2, MMP-9 and the expression ofTGF-β₁ (P＜0.05).Conclusions Aldosterone increased the activities of MMP-2 and MMP-9 butdecreased the expression of collagenⅣα5 chain and the adhesion rate of podocytespossibly via mechanisms involving TGF-β₁ signaling pathway. Such alterations maycontribute to glomerular podocytes injury associated with the GBM abnormality causedby the imbalance between matrix synthesis and degradation. Partâ…¡Aldosterone induces podocytes apoptosis: the role of reactive oxygenspeciesObjectives To investigate the effect of aldosterone and its receptor antagonist on theproduction of reactive oxygen species (ROS) and apoptosis in podocytes, and access theeffect of anti-oxidant on ALD induced podocytes apoptosis.Methods (1) To evaluate the effect of aldosterone and its receptor antagonist onpodocytes apoptosis and ROS production, podocytes were divided into control group,ALD group, SPI group and SPI+ALD group. The apoptosis rate of podocytes wasmonitored by flow cytometry. The level of ROS production and the expression ofNephrin were assayed by fluorescent spectrophotometry and indirectimmunofluorescence technology. The mRNA and protein expression levels of Bax andBcl-2 in podocytes were detected by reverse transcription polymerase chain reaction(RT-PCR) and Western blot methods. (2) To confirm whether ROS pathway participatedin the ALD-induced podocytes apoptosis, the antioxidant N-acetylcysteine (NAC) wasapplied before the addition of ALD. The apoptosis rate and the expression of Nephrin inpodocytes were accessed by flow cytometry and indirect immunofluorescencetechnology. The expression levels of Bax and Bcl-2 mRNA and protein in podocyteswere examined by RT-PCR and Western blot methods.Results (1) Compared with the control group, aldosterone increased the apoptosis rateand ROS production in podocytes (P＜0.05). Meanwhile, the expression of Nephrindecreased (P＜0.05). Aldosterone increased the expression of Bax mRNA and protein butdecreased the expression of Bcl-2 mRNA and protein (P＜0.05). Spironolactone completely abolished the above-mentioned effects of aldosterone (P＜0.05). (3) Theapplication of anti-oxidant prevented the aldosterone-induced upregulation of apoptosisrate as well as the downregulation of the expression of Nephrin in podocytes (P＜0.05).Treatment with anti-oxidant also decreased the expression of Bax mRNA and protein butincreased and the expression of Bcl-2 mRNA and protein (P＜0.05).Conclusions Aldosterone increased the expression of pro-apoptotic factor (Bax)mRNA and protein but decreased the expression of anti-apoptotic factor (Bcl-2) mRNAand protein to induce the apoptosis of podocytes possibly via the mechanisms involvingROS production. Partâ…¢Preventive and therapeutic effects of eplerenone on adriamycininduced nephropathy in ratsObjectives To investigate the preventive effects of eplerenone on adriamycin inducedrenal injury.Methods (1) 36 healthy male Sprague-Dawley rats were randomly divided into controlgroup, adriamycin-induced nephrotic syndrome (NS) group and eplerenone-treated group(100mg.kg^-1.d^-1). NS was induced by a single dose of ADR injected into the tail vein ofconscious rats. Rats in eplerenone-treated group were administered with a daily dose ofeplerenone by gastric gavage at 24 hours before surgery until being sacrificed.Correspondingly, rats in NS group or control group were daily administered with thesame amount of water. The experiment was terminated at the 28th day after operation. (2)Biochemical parameters and blood pressure (BP) measurement: On the day 28, aftermeasurement of systolic BP and collection of 24h proteinuria, animals were decapitated.Blood was taken from abdominal aorta of all rats. Serum potassium, sodium andcreatinine were measured on the 28th day after injection of adriamycin. (3) The leftkidney was rapidly removed and processed for light microscopic and transmissionelectron microscopic evaluation of renal histology. Fixed sections of the left kidney werecut and stained with hematoxylin-eosin (HE) and periodic acid-schiff's (PAS) reaction.Coded slides were examined using light microscopy and transmission electronmicroscopy to determine the degree of podocytes injury and renal pathological change.(4) Immunohistochemistry and Western blot methods were performed to examine theexpression of TGF-β₁ and desmin in renal cortex. Results (1) Biochemical parameters and BP measurement: There were no significantchanges in the level of serum potassium, sodium, creatinine concentrations and BP valuesamong the three groups. After 28 days of adriamycin injection, the 24h proteinuriaexcretion was significantly higher than that of control group (P＜0.01), but eplerenonereduced the 24h proteinuria excretion compared with NS group (P＜0.05). (2) Histologicalstudies: Twenty-eight days after adriamycin injection, HE and PAS staining displayedmild mesangial cells and martrix proliferation in some glomerulus. Diffuse deformationand confluence of foot processes in podocytes were also observed by transmissionelectron microscopy in NS group. By contrast, rats in eplerenone-treated group exhibitedobvious attenuation of these morphological lesions. (3) Immunohistochemical studies:The expression of glomerular TGF-β₁ and desmin of NS group was markedlyup-regulated in contrast to the control group (P＜0.01), whereas the expression ofglomerular TGF-β₁ and desmin in eplerenone-treated group was significantly less thanthat of NS group (P＜0.05). (4) Western blot: In comparison with the control group, theexpression of TGF-β₁ and desmin in kidney cortex of NS group was significantlyincreased (P＜0.01), however, the expression of TGF-β₁ and desmin protein ineplerenone-treated group was significantly less than that of NS group (P＜0.05).Conclusions Eplerenone ameliorated the proteinuria and the development of renalpathologic alteration in adriamycin induced nephropathy presumably via the inhibition ofcytokine release and restore the morphology of podocytes independent of its BP-lowingeffect.