| Hypertension is a major public health problem worldwide with high morbidity and mortality,and it is also considered as a major risk factor for cardiovascular diseases(such as stroke,myocardial infarction,and heart failure).Renal fibrosis is a major pathogenic factor in end-stage renal disease caused by hypertension.Although numerous studies have demonstrated that oxidative stress,renin-aldosterone-angiotensin system(RAAS)activation,chronic inflammation and vasoactive substances are closely related to renal fibrosis induced by hypertensive kidney injury,the pathogenesis remains largely unknown Moreover,current clinical therapies to treat and reverse renal fibrosis are very limited Therefore,to further explore the pathogenesis of renal fibrosis induced by hypertensive kidney injury is of great significance for finding a new effective target and the diagnosis and treatment of hypertensive nephropathy.Recent studies have shown that G-protein coupled receptors(GPCR)are closely related to renal fibrosis.GPCR is one of the largest and most diversified protein families in the genome.GPCR regulate a variety of important physiological functions.As its abundant expression on cell membrane and clear binding sites,they have become common drug targets,accounting for about 35%of all FDA-approved drugs.However,the function of most GPCR remains unclear.GPR97 belongs to the orphan-adherent GPCR family and is currently found to be highly expressed on heart,kidney,and bone marrow,as well as the surface of some immune cells.Current research indicates that GPR97 plays an important role in the regulation of immune cells and inflammatory responses.Our previous research found that GPR97 aggravated acute kidney injury through Sema3 A signaling pathway.The role of GPR97 in renal fibrosis induced by hypertensive kidney injury remains unclear.Therefore,further elucidating the role of GPR97 in the pathogenesis of renal fibrosis induced by hypertensive kidney injury and clarifying its ligands have very important clinical significance for finding potential effective therapeutic targets for hypertensive nephropathyObjective1.To determine the expression patterns of GPR97 in hypertensive renal fibrosis,and to explore whether GPR97 is involved in the pathogenesis of renal fibrosis induced by hypertensive kidney injury2.To explore the role of GPR97 in renal fibrosis induced by hypertensive kidney injury 3.To investigate the molecular mechanism of GPR97 in renal fibrosis induced by hypertensive kidney injuryMethodsPart 1:The expression of GPR97 in two independent hypertension models1.1 Establishing 2 hypertension modelsDOCA/salt-induced hypertension model:12-week male WT C57BL/6J mice were selected.After recovering from unilateral nephrectomy for one week,continuous 21-day release DOC A pellets were implanted subcutaneously.Afterwards,the drinking water was switched to 1%saline.AngⅡ-induced hypertension model:12-week male WT mice were chosen.AngⅡ-induced hypertensive mice were established by infusing AngⅡ at a dose of 1500 ng/kg/min using osmotic minipumps subcutaneously for 28 days.1.2 The expression of GPR97 in the kidney from hypertensive miceWestern blot(WB)was used to detect the protein levels of GPR97 in kidneys of DOCA/salt or Angll induced hypertensive mice.IHC assay was used to further determine the expression of GPR97 in the kidneys of DOCA/salt or AngⅡ induced hypertensive mice.1.3 The levels of GPR97 in different kinds of renal tubules Immunofluorescence(IF)was used to confirm the expression of GPR97 in different kinds of renal tubules.1.4 The levels of GPR97 in NRK-52E in response to AngⅡ and TGF-β which were closely related to the etiology of hypertensionIn vitro,WB was performed to detect the expression of GPR97 under AngⅡ and TGF-βtreatment in NRK-52E.Part 2:The role of GPR97 in DOCA/salt-induced hypertensive renalinjury2.1 Validation of GPR97 knockout miceGenotyping of mice was performed by DNA extracted from tail using PCR.The knockout efficiency of GPR97 in mice was also evaluated by gene chip..2.2 Effects of GPR97 on blood pressureSystolic blood pressure,diastolic blood pressure,mean blood pressure were measured using tail-cuff in different groups of mice.2.3 Role of GPR97 on renal functionThe kidney weight-to-body weight ratio,proteinuria,and PAS staining were performed to observe the changes of renal function in different groups of mice.2.4 Effects of GPR97 on renal fibrosis caused by hypertensionMasson staining was used to observe the pathological characteristics of renal fibrosis in mice of each group.WB and IHC were used to detect the expression of fibrosis-related proteins Collagen-1,Fibronectin,Slug,and E-cadherin in the kidneys of mice from different groups.2.5 Effects of GPR97 on fibrosis-related proteins in NRK-52E cells in vitroGene silencing of GPR97 in NRK-52E cells was performed by transfecting small interfering RNA(siRNA).WB method was used to observe the expression of fibrosis related proteins Collagen-1,Collagen-3,Fibronectin,and MMP-14 in NRK-52E cells under different treatmentsPart 3:The mechanisms that GPR97 affected DOCA/salt-induced hypertensive renal fibrosis3.1 Effects of GPR97 on fibrosis-related Smad2 signalingWB was used to detect the expression of p-Smad2 in the kidneys of mice in different treatment groups.IF and WB were used to verify the results in NRK-52E cells in vitro.3.2 Effects of GPR97 on Hippo/YAP signalingThe expression level of YAP,a key molecule of Hippo signaling pathway in the kidneys of mice in each group was detected by WB,and the expression of YAP under different treatment in NRK-52E were further detected by IF and WB.ResultsPart 1:The expression patterns of GPR97 in the kidneys from 2 independent hypertension models1.1 The levels of GPR97 were significantly increased in kidney from DOCA/salt-induced hypertensionWB results showed that compared with the sham group,the protein level of GPR97 was significantly increased in the kidneys from DOCA/salt-induced hypertensive mice.The change was not significant in the kidneys from AngⅡ-induced hypertensive mice.At the same time,the results of immunohistochemistry are basically consistent with the results of WB.1.2 GPR97 was abundantly expressed in the proximal tubules of mouse kidney GPR97 was co-localized with three renal tubule markers by IF,and GPR97 was found to be abundantly expressed in the proximal and distal tubules of the mouse kidney.1.3 The levels of GPR97 was increased in NRK-52E under TGFβ treatment WB results showed that the protein level of GPR97 in NRK-52E was significantly increased after TGF-β stimulation,but the protein level of GPR97 was not significantly changed under AngⅡ treatment.Part 2:The role of GPR97 in DOCA/salt-induced hypertensive renal injury2.1 Validation of GPR97 knockout miceAgarose gel electrophoresis was performed on the DNA extracted from mouse tails,and mice with successful GPR97 knockout were identified and selected for subsequent experiments.Similarly,the gene chip results confirmed that GPR97 knockout is highlyefficient.2.2 Deletion of GPR97 had no effect on blood pressure in mice The tail-cuff method was used to measure the systolic blood pressure,diastolic blood pressure,and average blood pressure of mice in different treatment groups.It was found that the deletion of GPR97 had no significant effect on blood pressure in mice.2.3 GPR97 deficiency improved renal function in hypertensive miceThe results of renal weight-to-weight ratio and proteinuria showed that GPR97 knockout could improve renal function in DOCA/salt-induced hypertensive mice.PAS staining showed that knockdown of GPR97 could improve DOCA/salt-induced mesangial expansion.2.4 Knockout of GPR97 could alleviate renal fibrosis caused by hypertensionMasson staining showed that the absence of GPR97 could significantly improve tubulointerstitial fibrosis in DOC/salt-induced hypertensive mice.WB and IHC showed that knockout of GPR97 could obviously restore the abnormal expression of fibrosis-related proteins in the kidneys of mice caused by hypertension,further indicating that the knockout of GPR97 could relieve the renal fibrosis caused by hypertension.2.5 Silencing GPR97 improved TGF-β-induced abnormal expression of fibrosis-related proteins in NRK-52E cellsWB experiments confirmed that gene silencing of GPR97 in NRK-52E cells was achieved by siRNA transfection.It was further found that silencing GPR97 could significantly restore the abnormal expression of fibrosis-related proteins Collagen-1,Collagen-3,Fibronectin and MMP-14 in NRK-52E cells caused by TGF-βPart 3:The mechanisms that GPR97 affected DOCA/salt-induced hypertensive renal fibrosis3.1 GPR97 positively regulated Smad2 signalingThe results of WB experiments showed that the loss of GPR97 significantly reduced the increased level of p-Smad2 in the kidney caused by hypertension.Consistent results were found in NRK-52E cells in vitro by IF and WB3.2 GPR9 7 positively regulated Hippo/YAP signalingWB results showed that the knockout of GPR97 could significantly decreased the level of YAP,a key molecule of Hippo pathway in mouse kidney caused by hypertension.At the same time,IF and WB experiments also confirmed this results in vitro.Conclusion and innovation1.Our study confirmed for the first time that GPR97 expression was significantly increased in the kidney of DOCA/salt-induced hypertensive mice.Further research showed that knockout GPR97 could improve renal fibrosis in DOCA/salt-induced hypertensive mice.2.This study demonstrated that knockout of GPR97 could inhibit the expression of proteins associated with renal fibrosis and retard the development of renal fibrosis under the pathological conditions of hypertension.Further,Hippo/YAP signaling pathway had been proved to be one of the key pathways for GPR97 to regulate renal fibrosis induced by hypertensive kidney injuryfor the first time.This study provided an important experimental and theoretical basis for further exploring the biological functions of GPR97 and finding new therapeutic targets for hypertensive nephropathy. |
