Role of mineralocorticoid receptor/Rho/Rho-kinase pathway in obesity-related renal injury

Abstract

Objective: We examined whether aldosterone/Rho/Rho-kinase pathway contributed to obesity-associated nephropathy.

Subjects: C57BL/6J mice were fed a high fat or low fat diet, and mice on a high fat diet were treated with a mineralocorticoid receptor antagonist, eplerenone.

Results: The mice on a high fat diet not only developed obesity, but also manifested renal histological changes, including glomerular hypercellularity and increased mesangial matrix, which paralleled the increase in albuminuria. Furthermore, enhanced Rho-kinase activity was noted in kidneys from high fat diet-fed mice, as well as increased expressions of inflammatory chemokines. All of these changes were attenuated by eplerenone. In high fat diet-fed mice, mineralocorticoid receptor protein levels in the nuclear fraction and SGK1, an effector of aldosterone, were upregulated in kidneys, although serum aldosterone levels were unaltered. Furthermore, aldosterone and 3β-hydroxysteroid dehydrogenase in renal tissues were upregulated in high fat diet-fed mice. Finally, in cultured mesangial cells, stimulation with aldosterone enhanced Rho-kinase activity, and pre-incubation with eplerenone prevented the aldosterone-induced activation of Rho kinase.

Conclusion: Excess fat intake causes obesity and renal injury in C57BL/6J mice, and these changes are mediated by an enhanced mineralocorticoid receptor/Rho/Rho-kinase pathway and inflammatory process. Mineralocorticoid receptor activation in the kidney tissue and the subsequent Rho-kinase stimulation are likely to participate in the development of obesity-associated nephropathy without elevation in serum aldosterone levels.

Figure 1

Effects of eplerenone on animal phenotype. (a–c) Systolic BP, mean BP and heart rate were neither unaltered by HFD nor was reduced by eplerenone. (d) Albuminuria was markedly increased in mice on HFD compared with those on LFD. Eplerenone reduced albuminuria. (e, f) Body weights and kidney weights of mice on HFD were markedly greater than those of mice on LFD. The diet-induced obesity was ameliorated by the treatment with eplerenone. Data were expressed as mean±s.e.m. Cr, creatinine. ^*P<0.05, ^**P<0.01 vs mice on LFD; ^†P<0.05 vs untreated mice on HFD.

Figure 2

The effects of eplerenone on the HFD-induced renal damages, and renal expression of inflammatory chemokines. (a) Histology of F4/80-stained kidney section from mice on LFD, HFD and HFD with eplerenone. Magnification, × 400. Compared with mice on LFD, untreated mice showed marked glomerular hypercellularity and enlarged glomerular size. Eplerenone-treated mice showed near-normal glomerular histology. (b) Histology of Masson's modified trichrome-stained kidney section from mice on LFD, HFD and HFD with eplerenone. HFD showed no significant fibrotic changes. Alternatively, the stain-negative round spots are increased in tubules in obese mice and were reduced by eplerenone. (c) Glomeruli were markedly enlarged in mice on HFD, which change was reduced by eplerenone. (d) Glomerular cellularity was assessed by the number of nuclei per glomerular cross-section (GCS) in 50 hilar glomeruli per animal. (e) Macrophages were markedly infiltrated in the renal tissue of mice on HFD, which was improved by the treatment with eplerenone. (f) The stain-negative round spots are increased in obese mice and were reduced by eplerenone. HFD-fed mice showed increases in renal expressions of MCP-1 (g, h), TNF-α (i, j) and PDGF-B (k), all of which were attenuated by eplerenone. Data were expressed as the ratio of mRNA levels of MCP-1 (h), TNF-α (j) and PDGF-B (k) to that of β-actin in arbitrary units (a.u.), relative to controls assigned as a value of 1. Data were expressed as mean±s.e.m. ^**P<0.01 vs C57BL mice on LFD; ^†P<0.05 vs untreated mice on HFD.

Figure 3

MR and SGK1 expression and aldosterone synthesis enzyme expression in kidneys from HFD-fed mice. (a) The protein in the nuclear fraction representing the MR was increased in HFD, suggesting that MR was enhanced with obesity. (b) HFD-fed mice showed increases in renal expressions of SGK1, which was attenuated by eplerenone. Data were expressed as the ratio of mRNA level of SGK1 to that of β-actin in arbitrary units (a.u.), relative to controls assigned as a value of 1. (c, left) Serum aldosterone levels did not differ between mice on LFD and those on HFD. (c, right) Tissue aldosterone in the kidneys was markedly increased. (d) HFD-fed mice showed increases in renal expressions of 3β-HSD, when compared with mice on LFD. Other aldosterone-synthesizing enzyme CYP21 hydroxylase (e), CYP11B1 (f) or B2 (g) was unchanged in mice on HFD. Data were expressed as the ratio of mRNA levels of 3β-HSD (d), CYP21 hydroxylase (e), CYP11B1 (f) and CYP11B2 (g) to that of β-actin in arbitrary units (a.u.), relative to controls assigned as a value of 1. Data were expressed as mean±s.e.m. ^*P<0.05, ^**P<0.01 vs mice on LFD; ^†P<0.05 vs untreated mice on HFD.

Figure 4

(Upper) Rho-kinase activity in the kidney of mice on HFD and the effects of eplerenone on the Rho-kinase activation. (a) Phosphorylation of MYPT was significantly increased in mice on HFD. The activation of Rho kinase was completely blocked by eplerenone treatment. (b, c) p42/44 or p38 in the kidney was unchanged in mice on HFD. Data were expressed as mean±s.e.m. ^*P<0.05 vs mice on LFD; ^†P<0.05 vs untreated mice on HFD. (Lower) Effects of aldosterone on Rho/Rho-kinase activity and effects of insulin on MR signaling pathway in primary mesangial cells. The stimulation with aldosterone increased Rho-kinase activity in a dose- (d, n=4) and a time-dependent manner (e, n=4). (d) Mesangial cells were incubated with various concentration of aldosterone for 1 h and the activation of Rho kinase was assayed by immunoblotting. Densitometric analysis of immunoblots is shown as values normalized by the expression levels of total MYPT1. ^*P<0.05 vs quiescent cells. (e) Stimulation with aldosterone (1 nmol l⁻¹) significantly increased the level of phospho-MYPT1 at 90 min and 3 h. ^**P<0.01, ^*P<0.05 vs time 0. (f) Pre-incubation with eplerenone (10 μmol l⁻¹) attenuated the aldosterone-induced increase in MYPT-1 phosphorylation in a dose-dependent manner. ^**P<0.01 vs quiescent; ^††P<0.01, ^†P<0.05 vs aldosterone stimulation without pre-treatment. (g) mRNA levels of SGK1 with high insulin treatment. ^*P<0.05 vs 1 nM, 10 nM of insulin. Results are presented as mean±s.e.m.

Figure 5

Effects of fasudil on animal phenotype. (a–c) Systolic BP, mean BP and heart rate were unaltered by fasudil. (d) Increased albuminuria in mice on HFD was reduced by fasudil. (e, f) The diet-induced obesity and enlarged kidneys were ameliorated by the treatment with fasudil. Data were expressed as mean±s.e.m. Cr, creatinine. ^†P<0.05 vs untreated mice on HFD.

Figure 6

Effects of fasudil on renal morphological changes and renal expression of inflammatory chemokines. (a) Histology of F4/80-stained kidney section from mice on HFD and HFD with fasudil. (b) Periodic acid-Schiff's-stained kidney section. (c) Masson's modified trichrome-stained kidney section. (d, e) The HFD-induced changes in glomerular size and cellularity were nearly completely abolished by the treatment with fasudil. (f) Macrophages were markedly infiltrated in the renal tissue of mice on HFD, which was improved by the treatment with fasudil. (g) The stain-negative round spots are increased in obese mice and were reduced by fasudil. HFD-fed mice showed increases in renal expressions of MCP-1 (h, i), TNF-α (j, k) and PDGF-B (l), all of which were attenuated by fasudil. Data were expressed as the ratio of mRNA levels of MCP-1 (i), TNF-α (k) and PDGF-B (l) to that of β-actin in arbitrary units (a.u.), relative to controls assigned as a value of 1. Data were expressed as mean±s.e.m. ^†P<0.05 vs untreated C57BL/6J mice with HFD.