Klotho Ameliorates Kidney Injury and Fibrosis and Normalizes Blood Pressure by Targeting the Renin-Angiotensin System

Abstract

Loss of Klotho and activation of the renin-angiotensin system (RAS) are common pathological findings in chronic kidney diseases. However, whether these two events are intricately connected is poorly understood. We hypothesized that Klotho might protect kidneys by targeted inhibition of RAS activation in diseased kidneys. To test this hypothesis, mouse models of remnant kidney, as well as adriamycin nephropathy and unilateral ureteral obstruction, were utilized. At 6 weeks after 5/6 nephrectomy, kidney injury was evident, characterized by elevated albuminuria and serum creatinine levels, and excessive deposition of interstitial matrix proteins. These lesions were accompanied by loss of renal Klotho expression, up-regulation of RAS components, and development of hypertension. In vivo expression of exogenous Klotho through hydrodynamic-based gene delivery abolished the induction of multiple RAS proteins, including angiotensinogen, renin, angiotensin-converting enzyme, and angiotensin II type 1 receptor, and normalized blood pressure. Klotho also inhibited β-catenin activation and ameliorated renal fibrotic lesions. Similar results were obtained in mouse models of adriamycin and obstructive nephropathy. In cultured kidney tubular epithelial cells, Klotho dose-dependently blocked Wnt1-triggered RAS activation. Taken together, these results demonstrate that Klotho exerts its renal protection by targeted inhibition of RAS, a pathogenic pathway known to play a key role in the evolution and progression of hypertension and chronic kidney disorders.

Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Repression of Klotho is associated with renal renin-angiotensin system (RAS) activation in a mouse remnant kidney model. A: Western blot analyses of renal expression of Klotho and RAS components in sham control and 5/6 nephrectomized mice (5/6NX). Kidney lysates from sham control and 5/6NX mice at 6 weeks as indicated were immunoblotted with antibodies against Klotho, AGT, renin, AT1, and actin, respectively. B–E: Graphic presentation of the relative abundance of Klotho (B), AGT (C), renin (D) and AT1 (E) in two groups as indicated. F: Representative micrographs show Klotho, AGT, renin, ACE, and AT1 protein expression in two groups. Paraffin-embedded kidney sections were stained with different antibodies. Arrows indicate positive staining. ∗P < 0.05 versus sham controls. n = 5 to 6 (B–E). Scale bar = 50 μm (F).

Figure 2

Expression of β-catenin and its target genes in the mouse remnant kidney model. A: Representative Western blot analyses show β-catenin and its target proteins in kidney lysates. Whole kidneys were homogenized and blotted with antibodies against β-catenin and its targets plasminogen activator inhibitor-1 (PAI-1), Snail1, and matrix metalloproteinase (MMP)-7. Actin was used as a loading control. B–E: Quantitative analyses demonstrate the overexpression of β-catenin (B), PAI-1 (C), Snail1 (D), and MMP-7 (E) in 5/6NX mice. F: Representative micrographs show β-catenin and FSP1 and α-SMA expression in two groups as indicated. Kidney sections were stained with different antibodies against β-catenin, FSP1, and α-SMA. Arrows indicate positive staining. G–I: Western blot analyses show the expression of fibronectin and α-SMA in different groups as indicated. Western blot (G) and graphical presentation of fibronectin (H) and α-SMA (I). n = 5 to 6 (B–E, H, and I). ∗P < 0.05 versus sham controls. Scale bar = 50 μm (F).

Figure 3

Expression of secreted Klotho in vivo ameliorates renal injury in mouse model of remnant kidney. A: Experimental design. Green arrows indicate the injection of pV5-sKlotho plasmid; black arrowheads, the surgical resection of left kidney and nephrectomy of right kidney. B: Urinary albumin levels in mice at 6 weeks after 5/6NX. Urinary albumin was expressed as mg/mg creatinine. C: Klotho reduces serum creatinine levels in 5/6NX mice. Serum creatinine was assessed at 6 weeks after 5/6NX. D–F: Representative images show that expression of Klotho attenuates glomerular lesions and interstitial fibrosis as indicated by periodic acid-Schiff (PAS) and Masson's trichrome staining (D) and quantitative data of glomerular lesions (E) and interstitial fibrosis score (F) in different groups. Paraffin sections were used for periodic acid-Schiff and Masson's trichrome staining. Black arrow indicates positive staining. ∗P < 0.05 versus sham controls; †P < 0.05 versus 5/6NX alone. Scale bar = 50 μm (D). 2/3NX, two thirds of left kidney by surgical resection; 5/6NX, 5/6 nephrectomized mice injected with pcDNA3; 5/6NX+K, 5/6 nephrectomized mice injected with pV5-sKlotho; UNX, intact nephrectomy of right kidney.

Figure 4

Expression of secreted Klotho in vivo abolishes renin-angiotensin system (RAS) induction in mouse remnant kidney model. A: Representative micrographs show Klotho and RAS components in different groups as indicated. Kidney sections were stained with different antibodies against Klotho, AGT, renin, ACE, and AT1. Arrows indicate positive staining. B: Representative Western blot analyses show membranous and secreted Klotho in kidney lysates. Renal endogenous membranous Klotho (mKlotho) and secreted Klotho (sKlotho) were indicated. Quantitative analyses demonstrate the levels of renal mKlotho (C) and sKlotho (D) at 6 weeks in 5/6NX mice (7 days after last injection of pV5-sKlotho plasmid). E: Representative Western blot analyses reveal that Klotho abolishes RAS induction in 5/6NX mice. Kidney lysates from different groups as indicated were immunoblotted with antibodies against AGT, renin, ACE, AT1, and actin, respectively. Numbers (1 to 5) represent different animals in a given group. F–I: Graphic presentations of the relative abundances of AGT (F), renin (G), ACE (H), and AT1 (I) in different groups as indicated. ∗P < 0.05 versus 5/6NX alone. Scale bar = 50 μm (A). 5/6NX, 5/6 nephrectomized mice injected with pcDNA3; 5/6NX+K, 5/6 nephrectomized mice injected with pV5-sKlotho.

Figure 5

Expression of secreted Klotho in vivo normalizes blood pressure in 5/6NX mice. A: Tail-cuff blood pressure measurements show that delivery of pV5-sKlotho plasmid normalizes systolic blood pressure (SBP) in mice at 6 weeks after 5/6NX. B: Exogenous sKlotho normalizes mean arterial blood pressure (MABP) at 6 weeks after 5/6NX. Solid blocks, dots, and triangles represent different animals in a given group. n = 6 to 8 (each group). ∗P < 0.05. 5/6NX, 5/6 nephrectomized mice injected with pcDNA3; 5/6NX+K, 5/6 nephrectomized mice injected with pV5-sKlotho.

Figure 6

Klotho represses various fibrogenic genes in the mouse remnant kidney model. A: Representative micrographs show that Klotho inhibits TGF-β1 and α-SMA expression and reduces collagen I and fibronectin deposition in the kidneys after 5/6NX. Arrows indicate positive staining. B–D: Western blot analyses of renal expression of fibronectin and α-SMA in 5/6NX and 5/6NX injected with pV5-sKlotho groups. Numbers (1 to 5) represent different animals in a given group. Graphic representations of fibronectin (C) and α-SMA (D) expression in different groups as indicated. ∗P < 0.05 versus 5/6NX alone. Scale bar = 50 μm (A). 5/6NX, 5/6 nephrectomized mice injected with pcDNA3; 5/6NX+K, 5/6 nephrectomized mice injected with pV5-sKlotho.

Figure 7

Klotho inhibits renal expression of β-catenin and its target genes in the mouse remnant kidney model. A: Representative micrographs show β-catenin, FSP-1, MMP-7, and PAI-1 expression in different groups. Paraffin sections were stained with different antibodies. Arrows indicate positive staining. B: Western blot analyses of renal expression of β-catenin and its target genes in different groups. Numbers (1 to 5) represent different animals in a given group. Graphic representations of β-catenin (C) and Snail1 (D) expression in different groups as indicated. ∗P < 0.05 versus 5/6NX alone. Scale bar = 50 μm (A). 5/6NX, 5/6 nephrectomized mice injected with pcDNA3; 5/6NX+K, 5/6 nephrectomized mice injected with pV5-sKlotho.

Figure 8

Expression of Klotho inhibits renin-angiotensin system (RAS) activation in adriamycin (ADR) nephropathy. A–C: Representative micrographs show renal expression and localization of RAS components in different groups. Paraffin-embedded or frozen kidney sections were stained with AGT, renin, ACE, and AT1 antibodies. Arrows indicate positive staining. B: Western blot analyses show AT1 expression in different groups as indicated. Numbers (1 to 4) represent different animals in a given group. C: Graphic presentation shows that expression of Klotho inhibits AT1 expression in ADR nephropathy. Relative protein levels over the controls (fold induction) are reported. n = 5 to 6 (C). ∗P < 0.05. Scale bar = 50 μm (A). ADR, ADR mice injected with pcDNA3; ADR+K, ADR mice injected with pV5-sKlotho.

Figure 9

Expression of sKlotho inhibits renin-angiotensin system (RAS) activation in obstructive nephropathy. A: Representative micrographs show renal expression and localization of RAS components in different groups. Kidney sections were stained with AGT, renin, ACE, and AT1 antibodies. Arrows indicate positive staining. B: Western blot analyses show that exogenous sKlotho inhibits renin and AT1 expression in different groups as indicated. Numbers (1 to 3) represent different animals in a given group. C: Graphic presentation show expression of sKlotho inhibits renin (white bars) and AT1 (black bars) expression in unilateral ureteral obstruction (UUO) kidneys. Relative protein levels over the controls (fold-induction) are reported. n = 5 to 6 (C). ∗P < 0.05 versus sham controls; †P < 0.05 versus UUO. Scale bar = 50 μm (A). UUO, UUO mice injected with pcDNA3; UUO/K-1, UUO mice injected with pV5-sKlotho 1 day before surgery; UUO/K+3, UUO mice injected with pV5-sKlotho 3 days after surgery.

Figure 10

Klotho inhibits Wnt1-mediated renin-angiotensin system (RAS) induction in vitro. A: Klotho dose-dependently inhibits Wnt1-mediated induction of RAS components, such as AGT, renin, ACE, and AT1. HKC-8 cells were transfected with Wnt1 expression plasmid (pHA-Wnt1) and Klotho expression vector (pV5-mKlotho) as indicated. Cells lysates were then blotted with antibodies against different RAS components. B–D: Quantitative data show the expression of AGT, ACE, renin, and AT1 in different groups as indicated. ∗P < 0.05 versus pcDNA3 alone; †P < 0.05 versus pHA-Wnt1 alone.