KCa3.1 mediates activation of fibroblasts in diabetic renal interstitial fibrosis

Abstract

Background: Fibroblast activation plays a critical role in diabetic nephropathy (DN). The Ca2+-activated K+ channel KCa3.1 mediates cellular proliferation of many cell types including fibroblasts. KCa3.1 has been reported to be a potential molecular target for pharmacological intervention in a diverse array of clinical conditions. However, the role of KCa3.1 in the activation of myofibroblasts in DN is unknown. These studies assessed the effect of KCa3.1 blockade on renal injury in experimental diabetes.

Methods: As TGF-β1 plays a central role in the activation of fibroblasts to myofibroblasts in renal interstitial fibrosis, human primary renal interstitial fibroblasts were incubated with TGF-β1+/- the selective inhibitor of KCa3.1, TRAM34, for 48 h. Two streptozotocin-induced diabetic mouse models were used in this study: wild-type KCa3.1+/+ and KCa3.1-/- mice, and secondly eNOS-/- mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Then, markers of fibroblast activation and fibrosis were determined.

Results: Blockade of KCa3.1 inhibited the upregulation of type I collagen, fibronectin, α-smooth muscle actin, vimentin and fibroblast-specific protein-1 in renal fibroblasts exposed to TGF-β1 and in kidneys from diabetic mice. TRAM34 reduced TGF-β1-induced phosphorylation of Smad2/3 and ERK1/2 but not P38 and JNK MAPK in interstitial fibroblasts.

Conclusions: These results suggest that blockade of KCa3.1 attenuates diabetic renal interstitial fibrogenesis through inhibiting activation of fibroblasts and phosphorylation of Smad2/3 and ERK1/2. Therefore, therapeutic interventions to prevent or ameliorate DN through targeted inhibition of KCa3.1 deserve further consideration.

Keywords: KCa3.1; diabetic nephropathy; fibroblast activation; renal interstitial fibrosis.

FIGURE 1:

KCa3.1 blocker TRAM34 inhibited ECM gene expression and the activation of renal fibroblasts induced by TGF-β1 in human renal interstitial fibroblasts. Human renal interstitial fibroblasts were treated with control, TGF-β1 (2 ng/mL) or TGF-β1 (2 ng/mL) combined with TRAM34 (2 μM) for 48 h. Real-time RT–PCR results showed that TRAM34 inhibited TGF-β1-induced type I collagen (A), type IV collagen (B) and fibronectin (C) mRNA expression. Western blot results showed that TRAM34 inhibited the induction of type IV collagen (D) and fibronectin (E) in TGF-β1 treated human renal interstitial fibroblasts. Quantitative RT–PCR demonstrated TRAM34 reversed TGF-β1-induced α-SMA (F), vimentin (G) and FSP-1 mRNA (H). Results are presented as means ± SEM. *P < 0.05 and **P < 0.01, n = 3.

FIGURE 2:

KCa3.1 blocker TRAM34 prevented TGF-β1-induced PAI-1, MMP2 and MMP9 expression in human renal interstitial fibroblasts. Human renal interstitial fibroblasts were treated with control, TGF-β1 (2 ng/mL) or TGF-β1 (2 ng/mL) combined with TRAM34 (2 μM) for 48 h. Quantitative RT–PCR and western blot results demonstrated TRAM34 suppressed TGF-β1-induced PAI-1 mRNA (A) and protein expression (B) as well as MMP2 (C) and MMP9 (D) mRNA expression in cultured human renal interstitial fibroblasts. (E) Zymographic analysis showed TRAM34 reversed TGF-β1-induced proteolytic activity of MMP2 and MMP9 in human renal interstitial fibroblasts. Results are presented as means ± SEM. *P < 0.05 and **P < 0.01, n = 3.

FIGURE 3:

KCa3.1 mediated TGF-β1-induced activation of renal cortical fibroblasts and fibrotic responses through Smad and ERK pathway but not P38 and JNK pathways. Human renal interstitial fibroblasts were treated with control, TGF-β1 (2 ng/mL) or TGF-β1 (2 ng/mL) combined with TRAM34 (2 μM) for 48 h. Western blot results showed that TRAM34 inhibited the TGF-β1-mediated increases in p-Smad2 (A), p-Smad3 (B) expression and p-ERK1/2 expression (C). However, TRAM34 had no effect on the P38 (D) and JNK (E) MAP kinase pathways. Results are presented as means ± SEM. *P < 0.05 and **P < 0.01, n = 3.

FIGURE 4:

Blockade of KCa3.1 improved renal injury in two STZ-induced diabetic models. Two STZ-induced diabetic mouse models are used in this study: wild-type KCa3.1+/+ and KCa3.1−/− mice, and secondly eNOS−/− mice treated with or without a selective inhibitor of KCa3.1 (TRAM34). Kidney function was assessed by measuring the 24-h urinary albumin excretion. The 24-h urinary albumin excretion is significantly reduced in both KCa3.1-deficient mice (A) and eNOS−/− mice treated with TRAM34 (B). Results are presented as means ± SEM. *P < 0.05 and **P < 0.01, n = 6–8.

FIGURE 5:

Blockade of KCa3.1 suppressed the overexpression of ECM in diabetic kidneys. Quantitative RT–PCR showed increased mRNA expression of type I collagen (A), and fibronectin (B) in the kidneys of KCa3.1+/+ diabetic mice compared with control mice but reduced in diabetic KCa3.1−/− kidneys (n = 8). Representative images (C) show picrosirius red and immunohistochemical staining of type I collagen and fibronectin in the renal cortex from control KCa3.1+/+ mice, KCa3.1+/+ diabetic mice and Kca3.1−/− diabetic mice (n = 8). Quantitative RT–PCR showed increased mRNA expression of type I collagen (G) and fibronectin (H) in the kidneys of diabetic eNOS−/− mice compared with control mice but reduced with TRAM34 treatment (DM+TRAM34) (n = 6). Representative images (I) show picrosirius red and immunohistochemical staining of type I collagen and fibronectin in the renal cortex from control mice, diabetic mice and diabetic mice treated with TRAM34. The degree of tubulointerstitial injury (D and J) and the quantitation of type I collagen (E and K) and fibronectin (F and L) were determined by computer-based morphometric analysis. Results are presented as mean ± SEM. *P < 0.05 and **P < 0.01. Original magnification: ×400.

FIGURE 5:

Blockade of KCa3.1 suppressed the overexpression of ECM in diabetic kidneys. Quantitative RT–PCR showed increased mRNA expression of type I collagen (A), and fibronectin (B) in the kidneys of KCa3.1+/+ diabetic mice compared with control mice but reduced in diabetic KCa3.1−/− kidneys (n = 8). Representative images (C) show picrosirius red and immunohistochemical staining of type I collagen and fibronectin in the renal cortex from control KCa3.1+/+ mice, KCa3.1+/+ diabetic mice and Kca3.1−/− diabetic mice (n = 8). Quantitative RT–PCR showed increased mRNA expression of type I collagen (G) and fibronectin (H) in the kidneys of diabetic eNOS−/− mice compared with control mice but reduced with TRAM34 treatment (DM+TRAM34) (n = 6). Representative images (I) show picrosirius red and immunohistochemical staining of type I collagen and fibronectin in the renal cortex from control mice, diabetic mice and diabetic mice treated with TRAM34. The degree of tubulointerstitial injury (D and J) and the quantitation of type I collagen (E and K) and fibronectin (F and L) were determined by computer-based morphometric analysis. Results are presented as mean ± SEM. *P < 0.05 and **P < 0.01. Original magnification: ×400.

FIGURE 6:

Blockade of KCa3.1 inhibited myofibroblast activation in kidneys of diabetic mice. Quantitative RT–PCR showed increased mRNA expression of α-SMA (A), vimentin (B) and FSP-1 (C) in the kidneys of diabetic KCa3.1+/+ mice compared with control mice but reduced in diabetic KCa3.1−/− kidneys (n = 8). (D) Immunohistochemical staining of α-SMA in the renal cortex from control KCa3.1+/+ mice, KCa3.1+/+ diabetic mice and Kca3.1−/− diabetic mice (n = 8). Quantitative RT–PCR showed increased mRNA expression of α-SMA (F), vimentin (G) and FSP-1 (H) in the kidneys of eNOS−/− diabetic mice compared with control mice but reduced in diabetic kidneys treated with TRAM34 (n = 6). (I) Immunohistochemical staining of α-SMA in the renal cortex from control mice, diabetic mice and diabetic mice treated with TRAM34 (n = 6). The quantitation of α-SMA expression in mice kidneys (E and J). Results are presented as mean ± SEM. *P < 0.05 and **P < 0.01. Original magnification: ×400.

FIGURE 7:

Blockade of KCa3.1 inhibited MMP2 and MMP9 expression in two STZ-induced diabetic models. Quantitative RT–PCR showed increased mRNA expression of MMP2 (A) and MMP9 (B) in the kidneys of diabetic KCa3.1+/+ mice compared with control mice but reduced in diabetic KCa3.1−/− kidneys (n = 8). Quantitative RT–PCR showed increased mRNA expression of MMP2 (C) and MMP9 (D) in the kidneys of eNOS−/− diabetic mice compared with control mice but reduced in diabetic kidneys treated with TRAM34 (n = 6). Results are presented as mean + SEM. *P < 0.05 and **P < 0.01.