Protective effect of TRPV1 against renal fibrosis via inhibition of TGF-β/Smad signaling in DOCA-salt hypertension

Abstract

To investigate the effects of the transient receptor potential vanilloid type 1 (TRPV1) channel on renal extracellular matrix (ECM) protein expression including collagen deposition and the transforming growth factor β (TGF-β)/Smad signaling pathway during salt-dependent hypertension, wild-type (WT) and TRPV1-null (TRPV1⁻/⁻) mutant mice were uninephrectomized and given deoxycorticosterone acetate (DOCA)-salt for 4 wks. TRPV1 gene ablation exaggerated DOCA-salt-induced impairment of renal function as evidenced by increased albumin excretion (μg/24 h) compared with WT mice (83.7 ± 7.1 versus 28.3 ± 4.8, P < 0.05), but had no apparent effect on mean arterial pressure (mmHg) as determined by radiotelemetry (141 ± 4 versus 138 ± 3, P > 0.05). Morphological analysis showed that DOCA-salt-induced glomerulosclerosis, tubular injury and macrophage infiltration (cells/mm²) were increased in TRPV1⁻/⁻ compared with WT mice (0.74 ± 0.08 versus 0.34 ± 0.04; 3.14 ± 0.26 versus 2.00 ± 0.31; 68 ± 5 versus 40 ± 4, P < 0.05). Immunostaining studies showed that DOCA-salt treatment decreased nephrin but increased collagen type I and IV as well as phosphorylated Smad2/3 staining in kidneys of TRPV1⁻/⁻ compared with WT mice. Hydroxyproline assay and Western blot showed that DOCA-salt treatment increased collagen content (μg/mg dry tissue) and fibronectin protein expression (%β-actin arbitrary units) in the kidney of TRPV1⁻/⁻ compared with WT mice (26.7 ± 2.7 versus 17.4 ± 1.8; 0.93 ± 0.07 versus 0.65 ± 0.08, P < 0.05). Acceleration of renal ECM protein deposition in DOCA-salt-treated TRPV1⁻/⁻ mice was accompanied by increased TGF-β1, as well as phosphorylation of Smad2/3 protein expression (%β-actin arbitrary units) compared with DOCA-salt-treated WT mice (0.61 ± 0.07 versus 0.32 ± 0.05; 0.57 ± 0.07 versus 0.25 ± 0.05; 0.71 ± 0.08 versus 0.40 ± 0.06, P < 0.05). These results show that exaggerated renal functional and structural injuries are accompanied by increased production of ECM protein and activation of the TGF-β/Smad2/3 signaling pathway. These data suggest that activation of TRPV1 attenuates the progression of renal fibrosis possibly via suppression of the TGF-β and its downstream regulatory signaling pathway.

Figure 1

Time course responses of mean arterial pressure (A) and heart rate (B) to deoxycorticosterone acetate (DOCA)-salt treatment for 4 wks in WT and TRPV1^−/− mice. Mean arterial pressure and heart rate were determined by radiotelemetry and averaged in 24 h. Values are mean ± SE (n = 6).

Figure 2

Changes in renal morphology in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. Representative PAS-stained (A) or Masson trichrome–stained (B) kidney sections showing glomerulosclerosis or tubulointerstitial injury in control WT and TRPV1^−/− mice (a and b), and WT and TRPV1^−/− mice (c and d) treated with DOCA-salt. Magnification, 400× (A) or 200× (B).

Figure 3

Changes in renal cortical immunostaining of F4/80-positive cells (monocytes/macrophages) in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. (A) Representative immunostained kidney sections showing the immunostaining of F4/80-positive cells (monocytes/macrophages in red) in control WT and TRPV1^−/− mice (a and b), and WT and TRPV1^−/− mice (c and d) treated with DOCA-salt. Magnification, 400×. (B) Bar graph shows the changes in renal cortical F4/80-positive cells (monocytes/macrophages) in WT and TRPV1^−/− mice with or without DOCA-salt treatment. Values are mean ± SE (n = 7 to 8). *P < 0.05 compared to control WT or TRPV1^−/− mice; ^†P < 0.05 compared to DOCA-WT mice.

Figure 4

Changes in renal glomerular immunostaining of nephrin in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. (A) Representative immunostained kidney sections showing the immunostaining of nephrin in brown in control WT and TRPV1^−/− mice (a and b), and WT and TRPV1^−/− mice (c and d) treated with DOCA-salt. Magnification, 400×. (B) Bar graph shows the changes in renal glomerular immunostaining of nephrin in WT and TRPV1^−/− mice with or without DOCA-salt treatment. Values are mean ± SE (n = 7 to 8). *P < 0.05 compared to control WT or TRPV1^−/− mice; ^†P < 0.05 compared to DOCA-WT mice.

Figure 5

Changes in renal cortical immunolocalization of collagen type I and type IV and collagen levels in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. (A, B) Representative immunostained kidney sections showing the immunostaining of collagen type I and type IV (in red) in control WT and TRPV1^−/− mice (a and b), and WT and TRPV1^−/− mice (c and d) treated with DOCA-salt. Arrows point to collagen type I-positive staining (A) or collagen type IV-positive staining (B). Magnification, 400×. (C) Bar graph shows the changes in renal collagen levels in WT and TRPV1^−/− mice with or without DOCA-salt treatment. Values are mean ± SE (n = 7 to 8). *P < 0.05 compared to control WT or TRPV1^−/− mice; ^†P < 0.05 compared to DOCA-WT mice.

Figure 6

Changes in renal fibronectin protein expression in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. (A) Representative Western blot of renal fibronectin in WT and TRPV1^−/− mice with or without DOCA-salt treatment. (B) Bar graph shows the relative optical density values for renal fibronectin in WT and TRPV1^−/− mice with or without DOCA-salt treatment. Results were expressed as ratio of fibronectin to corresponding β-actin. Values are mean ± SE (n = 4 to 5). *P < 0.05 compared to control WT or TRPV1^−/− mice; ^†P < 0.05 compared to DOCA-WT mice.

Figure 7

Changes in renal TGF-β1 protein expression in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. (A) Representative Western blot of renal TGF-β1 in WT and TRPV1^−/− mice with or without DOCA-salt treatment. (B) Bar graph shows the relative optical density values for renal TGF-β1 in WT and TRPV1^−/− mice with or without DOCA-salt treatment. Results were expressed as ratio of TGF-β1 to corresponding β-actin. Values are mean ± SE (n = 4 to 5). *P < 0.05 compared to control WT or TRPV1^−/− mice; ^†P < 0.05 compared to DOCA-WT mice.

Figure 8

Changes in renal cortical immunostaining of phosphorylated Smad2/3-positive cells in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. Representative immunostained kidney sections showing the immunostaining of phosphorylated Smad2/3 in brown in control WT and TRPV1^−/− mice (a and b), and WT and TRPV1^−/− mice (c and d) treated with DOCA-salt. Arrows point to phosphorylated Smad2/3-positive cells. Magnification, 400×.

Figure 9

Changes in renal phosphorylated Smad2 (pSmad2) and phosphorylated Smad3 (pSmad3) protein expression in WT and TRPV1^−/− mice after 4 wks of deoxycorticosterone acetate (DOCA)-salt treatment. (A, C) Representative Western blot of renal pSmad2 (A) and pSmad3 (C) in WT and TRPV1^−/− mice with or without DOCA-salt treatment. (B, D) Bar graphs show the relative optical density values for renal pSmad2 (B) and pSmad3 (D) in WT and TRPV1^−/− mice with or without DOCA-salt treatment. Results were expressed as ratio of pSmad2 and pSmad3 to corresponding β-actin. Values are mean ± SE (n = 4 to 5). *P < 0.05 compared to control WT or TRPV1^−/− mice; ^†P < 0.05 compared to DOCA-WT mice.