Glomerulotubular interaction in IgA nephropathy induces oxidative damages in tubular epithelial cells: role of crosstalk between angiotensin II and aldosterone

Abstract

INTRODUCTION AND AIMS: Glomerulotubular crosstalk network is pivotal in inducing tubulointerstitial damage in IgA nephropathy (IgAN). We had previously shown that polymeric IgA1 (pIgA1) from IgAN patients activated the renin-angiotensin system (RAS) in mesangial cells and up-regulated the inflammatory cascade in proximal tubular epithelial cells (PTEC). We further examined the role of aldosterone (Aldo) in tubular injury induced through glomerulotubular crosstalk in IgAN. METHODS: Expression of the major components of renin-angiotensin-aldosterone system (RAAS), which regulate the synthesis and action of Aldo, including mineralocorticoid receptor (MR), 11β-hydroxysteroid dehydrogenase type II (11β-HSD2) and aldosterone synthase (CYP11B2), was examined by real-time quantitative PCR (qPCR) and immunofluorescence staining. Polymeric IgA were purified from IgAN patients (n=35) and normal subjects (n=32) using affinity and size exclusion chromatography. Apoptosis in PTEC was measured by cleaved PARP expression (western blot) and caspase 3 activity (fluorometric kit). Expression of NADPH oxidase was determined by qPCR and western blot. Generation of the intracellular reactive oxidative species (ROS) was determined the 2′7′-dichlorofluorescein (DCF) assay. RESULTS: PTEC expresses only the MR, but not 11β-HSD2 and CYP11B2, suggesting the lack of aldosterone synthesis by PTEC. Apoptosis, measured by cleaved PARP expression and caspase 3 activity, was induced in PTEC activated by conditioned medium prepared from mesangial cells (HMC) cultured with pIgA1 from IgAN patients but not from normal subjects. Exogenous angiotensin II (AngII) and Aldo, but not purified pIgA1 protein, induced cleaved PARP expression and caspase 3 activity in dose- and time-dependent manner in PTEC. Significant increased expression of NADPH oxidase and ROS formation were also detected in PTEC cultured with AngII, Aldo or conditioned media prepared from IgAN patients. Pre-incubation of PTEC with AngII receptor subtype-II (AT2R) antagonist (PD123319) or MR antagonist (eplererone), but not AT1R antagonist (losartan), partially suppressed the pIgA1-conditioned media induced ROS generation (25%) and caspase 3 activity (< 35%). Combined blockade with PD123319 and eplererone achieved more than 95% inhibition of ROS generation and apoptosis. Interestingly, AngII and pIgA1 conditioned media, but not Aldo, up-regulated the expression of AT2R and MR in PTEC. Pre-incubated PTEC with PD123319, but not losartan or eplerone, abrogated these up-regulated AT2R and MR expression. CONCLUSIONS: Our in vitro data suggest that AngII and aldosterone, released by pIgA1 activated HMC, served as the mediators for glomerulotubular crosstalk in inducing apoptosis of PTEC through generation of ROS species. In addition, crosstalk between AngII and aldosterone could play a role in pIgA1 induced PTEC apoptosis.