GPR97 deficiency ameliorates renal interstitial fibrosis in mouse hypertensive nephropathy

Abstract

Hypertensive nephropathy (HTN) ranks as the second-leading cause of end-stage renal disease (ESRD). Accumulating evidence suggests that persistent hypertension injures tubular cells, leading to tubulointerstitial fibrosis (TIF), which is involved in the pathogenesis of HTN. G protein-coupled receptors (GPCRs) are implicated in many important pathological and physiological processes and act as important drug targets. In this study, we explored the intrarenal mechanisms underlying hypertension-associated TIF, and particularly, the potential role of GPR97, a member of the adhesion GPCR subfamily, in TIF. A deoxycorticosterone acetate (DOCA)/salt-induced hypertensive mouse model was used. We revealed a significantly upregulated expression of GPR97 in the kidneys, especially in renal tubules, of the hypertensive mice and 10 patients with biopsy-proven hypertensive kidney injury. GPR97^-/- mice showed markedly elevated blood pressure, which was comparable to that of wild-type mice following DOCA/salt treatment, but dramatically ameliorated renal injury and TIF. In NRK-52E cells, we demonstrated that knockdown of GPR97 suppressed the activation of TGF-β signaling by disturbing small GTPase RhoA-mediated cytoskeletal reorganization, thus inhibiting clathrin-mediated endocytosis of TGF-β receptors and subsequent Smad activation. Collectively, this study demonstrates that GPR97 contributes to hypertension-associated TIF at least in part by facilitating TGF-β signaling, suggesting that GPR97 is a pivotal intrarenal factor for TIF progression under hypertensive conditions, and therapeutic strategies targeting GPR97 may improve the outcomes of patients with HTN.

Keywords: GPR97; Smad; TGF-β; endocytosis; hypertensive nephropathy; tubulointerstitial fibrosis.

Fig. 1. The level of GPR97 was significantly upregulated in the kidneys of DOCA/salt-induced hypertensive mice.

a The mRNA levels of GPR97 in the kidneys of DOCA/salt-induced hypertensive mice. *P < 0.05 versus sham-operated WT mice (n = 6). b Western blot analysis of the relative protein levels of GPR97 in the kidneys of DOCA/salt-induced hypertensive mice. *P < 0.05 versus sham-operated WT mice (n = 6). c Representative images of immunohistochemical staining of GPR97 in the kidneys of DOCA/salt-induced hypertensive mice. (n = 6). d Representative immunofluorescent images showing the colocalization of GPR97 with AQP1, AQP3 and Calbindin-D28k in the kidneys of DOCA/salt-induced hypertensive mice. *P < 0.05 versus sham-operated WT mice (n = 6). e Western blot analysis of GPR97 expression in the different groups of NRK-52E cells. *P < 0.05 versus control cells (n = 6). f Representative photomicrographs of GPR97 immunohistochemical staining in human renal cortical tissue from normal subjects (n = 9) and patients with hypertensive nephropathy (n = 10). *P < 0.05 versus normal. g Correlation analysis between the renal staining intensity of GPR97 and SCr levels (μmol/L) in all subjects (whose SCr was available). Arrows indicate positive staining. Scale bar = 50 μm.

Fig. 2. GPR97 deficiency ameliorated kidney injury and tubulointerstitial fibrosis in DOCA/salt-induced hypertensive mice.

a The genotype of GPR97^−/− mice was confirmed by PCR and tail preparation. b qRT‒PCR analysis of GPR97 expression in the kidneys of WT mice and GPR97^−/− mice. *P < 0.05 versus WT mice (n = 6). c The urine albumin-to-creatinine ratio (UACR) in the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). d Representative images of PAS staining showing typical changes in renal structure. Arrows indicate injured tubules. e Masson’s trichrome staining of kidneys from the different groups of mice. f Western blot analysis of collagen-1 and fibronectin expression in the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). g Representative images of immunohistochemical staining of Collagen-1 in the kidneys in the different groups of mice. h Western blot analysis of E-cadherin expression in the kidneys in the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). i Representative images of immunohistochemical staining of E-cadherin in kidneys in the different groups of mice. j Western blot analysis of Slug and Snail expression in the kidneys in the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). k Representative images of immunohistochemical staining of Snail1 in kidneys in the different groups of mice. Arrows indicate positive staining. Scale bar = 50 μm. l Western blot analysis of α-SMA expression in the kidneys in the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). m Representative images of immunohistochemical staining of α-SMA in kidneys in the different groups of mice. Arrows indicate positive staining. Scale bar = 50 μm.

Fig. 3. GPR97 silencing attenuated the TGF-β1-induced fibrotic phenotype of NRK-52E cells.

a Western blot analysis confirmed the silencing efficiency in NRK-52E cells transfected with siRNA-GPR97. *P < 0.05 versus scramble siRNA-transfected cells (n = 6). b, c Western blot analysis of Collagen-1, Fibronectin, Slug, Snail1 and α-SMA expression in NRK-52E cells after the different treatments. *P < 0.05 versus PBS-treated cells transfected with scramble siRNA, ^#P < 0.05 versus TGF-β1-treated cells transfected with scramble siRNA (n = 6). d Representative immunofluorescent images showing the expression and location of E-cadherin in NRK-52E cells after the different treatments. NRK-52E cells were treated with 10 ng/mL TGF-β1 for 24 h. The arrow indicates the location of E-cadherin at cell junctions. *P < 0.05 versus PBS-treated cells transfected with scramble siRNA, ^#P < 0.05 versus TGF-β1-treated cells transfected with scramble siRNA (n = 6). Scale bar = 20 μm.

Fig. 4. GPR97 participated in the activation of TGF-β/Smad signaling in tubule epithelial cells.

a Western blot analysis of p-Smad2 and Smad2 levels in the kidneys from the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). b Representative images of immunohistochemical staining of Smad2 in kidneys from the different groups of mice. Arrows indicate positive staining. Scale bar = 50 μm. c Western blot analysis of p-Smad2 and Smad2 levels in NRK-52E cells after different treatments (NRK-52E cells were treated with 10 ng/mL TGF-β1 for 30 min). *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group (n = 6). d Western blot analysis of p-Smad3 and Smad3 levels in NRK-52E cells after different treatments. *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group (n = 6). e, f Representative immunofluorescent images showing the location of Smad2 and Smad4 in NRK-52E cells after different treatments. Scale bar = 20 μm. (NRK-52E cells were treated with 10 ng/mL TGF-β1 for 24 h).

Fig. 5. GPR97 silencing reduced clathrin-mediated TβRII endocytosis in TGF-β1-treated NRK-52E cells.

a–d Representative confocal microscopic images and summarized data showing the colocalization of TβRII with clathrin (CLTA), EEA1, caveolin-1 (Cav1), and Lamp1 in NRK-52E cells after different treatments (NRK-52E cells starved for 24 h then incubated with 10 ng/mL TGF-β1 for 30 min, summarized at least 15 cells). Scale bar = 5 μm. *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group (n = 6). e Western blot analysis of TβRII expression in NRK-52E cells after different treatments (NRK-52E cells were treated with 10 ng/mL TGF-β1 for 24 h). *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group (n = 6). f Representative images of immunohistochemical staining of TβRII in kidneys from the different groups of mice. Arrows indicate positive staining. Scale bar = 50 μm. g Western blot analysis of TβRII expression in the different groups of mice. *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6).

Fig. 6. GPR97 modulates the activity of RhoA, which participates in the activation of the TGF-β1 signaling pathway.

a Representative immunofluorescent images of F-actin staining in NRK-52E cells after different treatments (NRK-52E cells were treated with 10 ng/mL TGF-β1 for 24 h). Scale bar = 20 μm. b Representative images of immunohistochemical staining of RhoA in kidneys from the different groups of mice. Scale bar = 50 μm. c Western blot analysis of RhoA levels in the different groups of mice (n = 6). *P < 0.05 versus sham-operated WT mice, ^#P < 0.05 versus DOCA/salt-treated WT mice (n = 6). d RhoA-GTP and RhoA levels in NRK-52E cells after different treatments (n = 6). *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group. e Western blot analysis of p-Smad2 levels in NRK-52E cells after different treatments (n = 6). *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group. ^&P < 0.05 versus si-GPR97 and TGF-β1 treatment group. f, g Representative immunofluorescent images and Western blot analysis showing the relative protein levels of HIC-5 in NRK-52E cells after different treatments. Scale bar = 20 μm. (n = 6). *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group. h, i Representative immunofluorescent images and Western blot gel documents and summarized data showing the relative protein levels of YAP in NRK-52E cells after different treatments. Scale bar = 20 μm. (n = 6). *P < 0.05 versus scramble group, ^#P < 0.05 versus TGF-β1 treatment group. j, k Representative images of immunohistochemical staining of HIC-5 and YAP in kidneys from the different groups of mice. Scale bar = 50 μm. Arrows indicate positive staining.

Fig. 7. Schematic depicting GPR97 as a novel regulator of TGF-β signaling.

GPR97-mediated RhoA activation is as a key mechanism for TGF-β signaling activation by promoting the redistribution of F-actin, thereby enhancing clathrin-mediated endocytosis of TGF-β receptors and subsequent Smad activation.