Oxidative damages in tubular epithelial cells in IgA nephropathy: role of crosstalk between angiotensin II and aldosterone

Abstract

Background: Inhibition of the renin-angiotensin-aldosterone system (RAAS) slows down the progression of chronic renal diseases (CKD) including IgA nephropathy (IgAN). Herein, we studied the pathogenetic roles of aldosterone (Aldo) in IgAN.

Methods: Human mesangial cells (HMC) was activated with polymeric IgA (pIgA) from IgAN patients and the effects on the expression of RAAS components and TGF-β synthesis examined. To study the roles of RAAS in the glomerulotubular communication, proximal tubular epithelial cells (PTEC) was cultured with conditioned medium from pIgA-activated HMC with eplerenone or PD123319, the associated apoptotic event was measured by the generation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and reactive oxygen species (ROS).

Results: Polymeric IgA up-regulated the Aldo synthesis and aldosterone synthase expression by HMC. The release of TGF-β by HMC was up-regulated synergistically by AngII and Aldo and this was inhibited by incubation of HMC with losartan plus eplerenone. Cultured PTEC express the mineralocorticoid receptor, but not synthesizing aldosterone. Apoptosis, demonstrated by cleaved PARP expression and caspase 3 activity, was induced in PTEC activated by conditioned medium prepared from HMC cultured with pIgA from IgAN patients. This apoptotic event was associated with increased generation of NADPH oxidase and ROS. Pre-incubation of PTEC with PD123319 and eplerenone achieved complete inhibition of PTEC apoptosis.

Conclusions: Our data suggest that AngII and Aldo, released by pIgA activated HMC, served as mediators for inducing apoptosis of PTEC in glomerulo-tubular communications. Crosstalk between AngII and Aldo could participate in determining the tubular pathology of IgAN.

Figure 1

Increased expression of CYP11B2 in HMC cultured with pIgA. (A) There was increased expression of CYP11B2 mRNA and (B) CYP11B2 protein, but not 11β-HSD2, by HMC cultured with pIgA from patients (IgAN, n = 27) as compared to that of the controls (Control, n = 22). The results represent the mean ± SD. (C) Increased expression of CYP11B2 (arrow) in HMC was confirmed by immunofluorescence staining (magnification × 200).

Figure 2

Dose- and time-dependent CYP11B2 expression in HMC. (A) Significant up-regulation of CYP11B2 mRNA expression and (B) CYP11B2 protein synthesis by HMC exposed to pIgA from IgAN patients (IgAN), but not with pIgA from controls (Control). (C) HMC cultured with pIgA (0.5 mg/ml) from IgAN, but not with pIgA from controls, exhibited an time-dependent increase of CYP11B2 mRNA expression (from 6 h, peaked at 12 h) and (D) CYP11B2 protein synthesis (from 24 h peaked at 48 h). The results represent the mean ± SD from five individual experiments. * signifies p < 0.05 when compared with data from HMC cultured in plain medium or data from time zero.

Figure 3

Dose- and time-dependent Aldo release and CYP11B2 expression in HMC. (A) Significant increase in Aldo release by HMC exposed to increasing concentration of pIgA from IgAN patients. (B) HMC cultured with pIgA (0.5 mg/ml) from IgAN patients, exhibited time-related increase in Aldo release (from 12 h, peaked at 48 h). (C) AngII induced Aldo release (at concentration > 10^-12 M) and CYP11B2 protein synthesis (at concentration > 10^-11 M) from HMC. (D) AngII (10^-10 M) induced time-related increase of Aldo release and CYP11B2 protein synthesis in HMC. The results represent the mean ± SD from five individual experiments. * signifies p < 0.05 when compared with data from HMC cultured in plain medium or data from time zero.

Figure 4

Effect of Aldo or pIgA on TGF-β synthesis and RAS of HMC. (A) Aldo at concentration >10^-11 M increased the mRNA expression of angiotensinogen and ACE, and AngII release by HMC. (B) AngII or Aldo at concentration >10^-11 M increased TGF-β synthesis by HMC. AngII and Aldo showed synergistic effect on TGF-β synthesis by HMC. * signifies p < 0.05 when compared with data from control without Aldo or AngII. (C) Pre-incubation with losartan (100 mM) or eplerenone (10 μM) one hour before cultured with pIgA from IgAN patients partially reduced the pIgA-induced TGF-β synthesis by HMC. Combining losartan and eplerenone completely abolished the increased TGF-β synthesis by pIgA. The AngII effect on TGF-β synthesis was blocked by losartan and the Aldo effect blocked by eplerenone; with complete normalization of TGF-β synthesis with the presence of both blockers. ** and * signify p < 0.01 and p < 0.05 respectively when compared with data from HMC cultured with pIgA from controls (Control). ## and # signify p < 0.01 and p < 0.05 respectively when compared with data from HMC pre-incubated with both inhibitors. The results represent the mean ± SD from five individual experiments.

Figure 5

Expression of MR, 11β-HSD2 or CYP11B2 in cultured HMC, PTEC and kidney biopsies. (A) HMC expressed MR, 11β-HSD2 and CYP11B2 mRNAs and PTEC expressed only MR mRNA. Result was expressed as the mean fold change ± SD (related to the mean C_T value from positive control using purified kidney total mRNA) from five individual experiments. (B) Demonstration of MR expression (arrows) in cultured PTEC by immunofluorescence staining (magnification × 200). (C). Detection of MR, 11β-HSD2 and CYP11B2 in kidney biopsies from IgAN patients and controls. Immunoreactive MR was located in both the glomeruli and tubules. Signal from immunoreactive 11β-HSD2 and CYP11B2 was located in glomeruli but not tubules (magnification × 200). Compared to staining results in control biopsies, increased (D) glomerular CYP11B2 staining and (E) tubular MR staining in IgAN patients were semi-quantified using a five-point scale.

Figure 6

Effect of pIgA, IgA-HMC conditioned medium, AngII and Aldo on the expression of MR or AT2R. Growth arrested PTEC was cultured with (i) pIgA (IgA, 0.5 mg/ml) from IgAN patients; (ii) 4 fold diluted IgA-HMC medium prepared from controls or IgAN patients; (iii) Aldo (10^-10 M) or (iv) AngII (10^-10 M). Both the exogenous AngII and IgA-HMC medium prepared from IgAN patients significantly increased the expression of (A) MR mRNA, (B) MR protein, (C) AT2R mRNA and (D) AT2R protein as compared with PTEC cultured with plain medium control (** signifies p < 0.01). The results represent the mean ± SD from five individual experiments.

Figure 7

The effect of MR, AT1R or AT2R blockade on IgA-HMC medium or AngII induced MR or AT2R expression. PD123319 effectively abolished the up-regulation of MR and AT2R receptor mRNA (A) or protein expression (B & C) by IgA-HMC medium from IgAN patients (medium-IgA) or AngII. Neither losartan nor eplerenone altered these receptors expression in PTEC. * and ** signify p < 0.05 and p < 0.01 respectively when compared with data from PTEC cultured in plain medium. The results represent the mean ± SD from five individual experiments.

Figure 8

Time- and dose-related expression of cleaved PARP by PTEC. (A) Up-regulation of cleaved PARP expression by PTEC cultured with Aldo (10^-10 M), AngII (10^-10 M) or 4 fold diluted IgA-HMC medium prepared from IgAN patients (medium-IgAN). Similar up-regulation was not observed with IgA-HMC medium from controls (medium-Ctl) or pIgA alone (IgA). (B) Dose-dependent up-regulation of cleaved PARP expression by PTEC cultured with Aldo, AngII or IgA-HMC medium prepared from IgAN patients. * and ** signify p < 0.05 and p < 0.01 respectively when compared with data from time zero or from PTEC cultured in plain medium. The results represent the mean ± SD from five individual experiments.

Figure 9

Time- and dose-related assay of caspase 3 activity in PTEC. (A) Up-regulation of caspase 3 activity by PTEC cultured with Aldo (10^-10 M), AngII (10^-10 M) or 4 fold diluted IgA-HMC medium prepared from IgAN patients (medium-IgAN); Similar up-regulation was not observed with IgA-HMC medium from controls (medium-Ctl) or pIgA alone (IgA). (B) Dose-dependent up-regulation of caspase 3 activity by PTEC cultured with Aldo, AngII or IgA-HMC medium prepared from IgAN patients. * and ** signify p < 0.05 and p < 0.01 respectively when compared with data from time zero or from PTEC cultured in plain medium. The results represent the mean ± SD from five individual experiments.

Figure 10

ROS generation and NADPH oxidase activity by PTEC. (A) Up-regulation of ROS generation and (B) NADPH oxidase activity by PTEC cultured with Aldo (10^-10 M), AngII (10^-10 M) or 4 fold diluted IgA-HMC medium prepared from IgAN patients (IgAN); Similar up-regulation was not observed with IgA-HMC medium from controls (Ctl) or pIgA alone (IgA). ** signify p < 0.01 when compared with data from PTEC cultured in plain medium. The results represent the mean ± SD from five individual experiments.

Figure 11

The effect of MR, AT1R or AT2R blockade in ROS generation and caspase 3 activity by PTEC. (A) Individual use of MR, AT1R or AT2R antagonist failed to effectively inhibit the up-regulation of ROS generation and (B) caspase 3 activity by PTEC cultured with Aldo (10^-10 M), AngII (10^-10 M) or 4 fold diluted IgA-HMC medium prepared from IgAN patients (medium-IgAN). Abolishment of up-regulated ROS generation and caspase 3 activity by PTEC was only achieved by combining PD123319 and eplerenone. * and ** signify p < 0.05 and p < 0.01 respectively when compared with data from HMC cultured with plain medium control. ## and # signify p < 0.01 and p < 0.05 respectively when compared with data from HMC corresponding activator without pre-incubation of any receptor antagonist. The results represent the mean ± SD from five individual experiments.

Figure 12

Schema showing the possible role of RAAS components in the glomerulo-tubular communication operating the pathogenesis of IgAN. Polymeric IgA from IgAN patients is capable of enhancing AngII release or Aldo synthesis through increasing the expression of ACE/ANG or CYP11B2 in HMC. The increased synthesis of AngII and Aldo up-regulates the TGF-β synthesis via binding to their respective receptors: AT1R and MR. Aldo is able to further up-regulate the ACE/ANG expression and AngII release in HMC whereas AngII increases the Aldo release by HMC via increased expression of CYP11B2. This vicious cycle involving pIgA-induced AngII and Aldo in HMC amplifies the pIgA-induced HMC damages and initiates the downstream inflammatory cascade in PTEC through the glomerulo-tubular communication. AngII released by pIgA-activated HMC up-regulates the expression of AT2R and MR by PTEC. Binding of AngII and Aldo to their respective receptors on PTEC induces cellular apoptosis through increased NADPH activity and generation of ROS. Combined blockade of AT2R and MR can prevent pIgA-induced PTEC apoptosis through these glomerulo-tubular communications.