Klotho protein supplementation reduces blood pressure and renal hypertrophy in db/db mice, a model of type 2 diabetes

Abstract

Aims: Klotho interacts with various membrane proteins, such as receptors for transforming growth factor (TGF)-β and insulin-like growth factor (IGF), to alter their function. Renal expression of klotho is diminished in diabetes. The present study examined whether exogenous klotho protein supplementation ameliorates kidney injury and renin-angiotensin system (RAS) in db/db mice.

Methods: We investigated the effects of klotho supplementation on diabetic kidney injury and RAS. Recombinant human klotho protein (10 μg/kg/d) was administered to db/db mice daily.

Results: Klotho protein supplementation reduced kidney weight, systolic blood pressure (SBP), albuminuria, glomerular filtration rate, and 8-epi-prostaglandin F2α excretion without affecting body weight. Although klotho supplementation did not alter glycated albumin, it reduced renal angiotensin II levels associated with reduced renal expression of angiotensinogen. Klotho supplementation improved renal expression of superoxide dismutase (SOD), and endogenous renal expression of klotho. Klotho supplementation reduced the levels of hypoxia-inducible factor, phosphorylated Akt, and phosphorylated mTOR and decreased the renal expression of TGF-β, tumour necrosis factor (TNF), and fibronectin.

Conclusions: These data indicate that klotho supplementation reduces blood pressure and albuminuria along with ameliorating renal RAS activation in db/db mice. Furthermore, these results suggest that klotho inhibits IGF signalling, induces SOD expression to reduce oxidative stress, and suppresses Akt-mTOR signalling to inhibit abnormal kidney growth. Collectively, the results suggest that klotho inhibits TGF-β and TNF signalling, resulting in a decline in renal fibrosis.

Keywords: epithelial-mesenchymal transition; insulin-like growth factor; mTOR; superoxide dismutase; transforming growth factor; tumour necrosis factor.

FIGURE 1

Impact of exogenous klotho protein supplementation on renal expressions of TGF-β (A), collagen I (B), fibronectin (C), and E-cadherin (D), Smad3 distribution (E), and interstitial fibrosis (F) in db/db mice (db). The * indicates statistically significant differences between the two groups (n = 10 for each)

FIGURE 2

Influences of exogenous klotho protein supplementation on phosphorylation of Akt (A, 56 kDa), mTOR (B, 289 kDa), and p70-S6k (C, 70 kDa), and phosphorylated mTOR staining (D) in db/db mice (db). The * indicates statistically significant differences between the two groups (n = 10 for each). db + k depicts db/db mice with klotho supplementation

FIGURE 3

Effects of exogenous klotho protein supplementation on aortic (A) and renal (B) expressions of superoxide dismutase (SOD), renal abundance of hypoxia-inducible factor-1α (C, 110 kDa, HIF-1α), renal expression of tumour necrosis factor-α (D, TNF-α), plasma concentration of TNF-α (E), and phosphorylation of Iκβ (F, 36 kDa) in db/db mice (db). β-actin was observed at 42 kDa. The * indicates statistically significant differences between the two groups (n = 10 for each). db + k depicts db/db mice with klotho supplementation

FIGURE 4

Summary of in vitro studies in HK-2 cells. Hydrogen peroxide induced angiotensinogen expression (A) and klotho suppressed this response (For time: F = 36, df = 1, P < 0.005; for klotho treatment: F = 14, df = 1, P < 0.001; for interaction: F = 5, df = 1, P < 0.05; for error: df = 20). An interaction between time and klotho treatment may relate to transcytosis of klotho protein by proximal tubular cells. Similarly, hydrogen peroxide induced the expression of tumour necrosis factor-α (B, TNF-alpha), and klotho inhibited this (For time: F = 85, df = 1, P < 0.001; for klotho treatment: F = 8, df = 1, P < 0.05; for interaction: F = 13, df = 1, P < 0.01; for error: df = 20). Insulin-like growth factor repressed expression of superoxide dismutase (C, SOD), and klotho opposed this response (For time: F = 96, df = 1, P < 0.001; for klotho treatment: F = 6, df = 1, P < 0.05; for interaction: F = 9, df = 1, P < 0.01; for error: df = 20). Blue and grey bars depict control and klotho-treated groups respectively. The * indicates statistically significant differences between the two groups