Activation of TGR5 Increases Urine Concentration by Inducing AQP2 and AQP3 Expression in Renal Medullary Collecting Ducts.

Introduction: G protein-coupled bile acid receptor (TGR5), the first G protein-coupled receptor for bile acids identified, is capable of activating a variety of intracellular signaling pathways after interacting with bile acids. TGR5 plays an important role in multiple physiological processes and is considered to be a potential target for the treatment of various metabolic diseases, including type 2 diabetes. Evidence has emerged that genetic deletion of TGR5 results in an increase in basal urine output, suggesting that it may play a critical role in renal water and salt reabsorption. The present study aims to elucidate the effect and mechanism of TGR5 activation on urine concentration. Methods: Mice were treated with TGR5 agonists (LCA and INT-777) for 3 days. The 24-h urine of mice was collected and analyzed for urine biochemical parameters. The mRNA expressions were detected by real-time PCR, and the protein expressions were detected by western blot. Immunohistochemistry and immunofluorescence were performed to examine the cellular location of proteins. The cultured primary medullary collecting duct cells were pretreated with H89 (a PKA inhibitor) for 1 h, followed by 12-h treatment of LCA and INT-777. Luciferase reporter assays were used to detect the effect of CREB on the gene transcription of AQPs. Gel electrophoretic mobility shift assays were used to analyze DNA-protein interactions. Results: Treatment of mice with the TGR5 agonist LCA and INT-777 markedly reduced urine output and increased urine osmolality, accompanied by a marked increase in AQP2 and AQP3 protein expression and membrane translocation. In cultured primary medullary collecting duct cells, LCA and INT-777 dose-dependently upregulated AQP2 and AQP3 expression in a cAMP/PKA-dependent manner. Mechanistically, both AQP2 and AQP3 gene promoter contains a putative CREB-binding site, which can be bound and activated by CREB as assessed by both gene promoter-driven luciferase and gel shift assays. Conclusion: Collectively, our findings demonstrate that activation of TGR5 can promote urine concentration by upregulation of AQP2 and AQP3 expression in renal collecting ducts. TGR5 may represent an attractive target for the treatment of patients with urine concentration defect.

Expression and localization of HSD17B13 along mouse urinary tract.

17ß-Hydroxysteroid dehydrogenase-13 (HSD17B13), a newly identified lipid droplet-associated protein, plays an important role in the development of nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Emerging evidence demonstrates that NASH is an independent risk factor for chronic kidney disease, which is frequently accompanied by renal lipid accumulation. In addition, the HSD17B13 rs72613567 variant is associated with lower levels of albuminuria in patients with biopsy-proven NAFLD. At present, the role of HSD17B13 in lipid accumulation in the kidney is unclear. This study utilized bioinformatic and immunostaining approaches to examine the expression and localization of HSD17B13 along the mouse urinary tract. We found that HSD17B13 is constitutively expressed in the kidney, ureter, and urinary bladder. Our findings reveal for the first time, to our knowledge, the precise localization of HSD17B13 in the mouse urinary system, providing a basis for further studying the pathogenesis of HSD17B13 in various renal and urological diseases.NEW & NOTEWORTHY HSD17B13, a lipid droplet-associated protein, is crucial in nonalcoholic fatty liver disease (NAFLD) development. NAFLD also independently raises chronic kidney disease (CKD) risk, often with renal lipid buildup. However, HSD17B13's role in CKD-related lipid accumulation is unclear. This study makes the first effort to examine HSD17B13 expression and localization along the urinary system, providing a basis for exploring its physiological and pathophysiological roles in the kidney and urinary tract.

The NF-κB/FXR/TonEBP pathway protects renal medullary interstitial cells against hypertonic stress.

Farnesoid X receptor (FXR), a ligand-activated transcription factor, plays an important role in maintaining water homeostasis by up-regulating aquaporin 2 (AQP2) expression in renal medullary collecting ducts; however, its role in the survival of renal medullary interstitial cells (RMICs) under hypertonic conditions remains unclear. We cultured primary mouse RMICs and found that the FXR was expressed constitutively in RMICs, and that its expression was significantly up-regulated at both mRNA and protein levels by hypertonic stress. Using luciferase and ChIP assays, we found a potential binding site of nuclear factor kappa-B (NF-κB) located in the FXR gene promoter which can be bound and activated by NF-κB. Moreover, hypertonic stress-induced cell death in RMICs was significantly attenuated by FXR activation but worsened by FXR inhibition. Furthermore, FXR increased the expression and nuclear translocation of hypertonicity-induced tonicity-responsive enhance-binding protein (TonEBP), the expressions of its downstream target gene sodium myo-inositol transporter (SMIT), and heat shock protein 70 (HSP70). The present study demonstrates that the NF-κB/FXR/TonEBP pathway protects RMICs against hypertonic stress.

Pregnane X receptor (PXR) deficiency protects against spinal cord injury by activating NRF2/HO-1 pathway.

INTRODUCTION: As a devastating neurological disease, spinal cord injury (SCI) results in severe tissue loss and neurological dysfunction. Pregnane X receptor (PXR) is a ligand-activated nuclear receptor with a major regulatory role in xenobiotic and endobiotic metabolism and recently has been implicated in the central nervous system. In the present study, we aimed to investigate the role and mechanism of PXR in SCI. METHODS: The clip-compressive SCI model was performed in male wild-type C57BL/6 (PXR+/+ ) and PXR-knockout (PXR-/- ) mice. The N2a H2 O2 -induced injury model mimicked the pathological process of SCI in vitro. Pregnenolone 16α-carbonitrile (PCN), a mouse-specific PXR agonist, was used to activate PXR in vivo and in vitro. The siRNA was applied to knock down the PXR expression in vitro. Transcriptome sequencing analysis was performed to discover the relevant mechanism, and the NRF2 inhibitor ML385 was used to validate the involvement of PXR in influencing the NRF2/HO-1 pathway in the SCI process. RESULTS: The expression of PXR decreased after SCI and reached a minimum on the third day. In vivo, PXR knockout significantly improved the motor function of mice after SCI, meanwhile, inhibited apoptosis, inflammation, and oxidative stress induced by SCI. On the contrary, activation of PXR by PCN negatively influenced the recovery of SCI. Mechanistically, transcriptome sequencing analysis revealed that PXR activation downregulated the mRNA level of heme oxygenase-1 (HO-1) after SCI. We further verified that PXR deficiency activated the NRF2/HO-1 pathway and PXR activation inhibited this pathway in vitro. CONCLUSION: PXR is involved in the recovery of motor function after SCI by regulating NRF2/HO-1 pathway.

Pregnane X receptor activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/ß-catenin signaling.

Renal fibrosis is a common pathological feature of chronic kidney disease (CKD) with various etiologies, which seriously affects the structure and function of the kidney. Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily and plays a critical role in regulating the genes related to xenobiotic and endobiotic metabolism in mammals. Previous studies show that PXR is expressed in the kidney and has protective effect against acute kidney injury (AKI). In this study, we investigated the role of PXR in CKD. Adenine diet-induced CKD (AD) model was established in wild-type and PXR humanized (hPXR) mice, respectively, which were treated with pregnenolone-16α-carbonitrile (PCN, 50 mg/kg, twice a week for 4 weeks) or rifampicin (RIF, 10 mg·kg-1·d-1, for 4 weeks). We showed that both PCN and RIF, which activated mouse and human PXR, respectively, improved renal function and attenuated renal fibrosis in the two types of AD mice. In addition, PCN treatment also alleviated renal fibrosis in unilateral ureter obstruction (UUO) mice. On the contrary, PXR gene deficiency exacerbated renal dysfunction and fibrosis in both adenine- and UUO-induced CKD mice. We found that PCN treatment suppressed the expression of the profibrotic Wnt7a and ß-catenin in AD mice and in cultured mouse renal tubular epithelial cells treated with TGFß1 in vitro. We demonstrated that PXR was colocalized and interacted with p53 in the nuclei of tubular epithelial cells. Overexpression of p53 increased the expression of Wnt7a, ß-catenin and its downstream gene fibronectin. We further revealed that p53 bound to the promoter of Wnt7a gene to increase its transcription and ß-catenin activation, leading to increased expression of the downstream profibrotic genes, which was inhibited by PXR. Taken together, PXR activation alleviates renal fibrosis in mice via interacting with p53 and inhibiting the Wnt7a/ß-catenin signaling pathway.

Estrogen receptor ß attenuates renal fibrosis by suppressing the transcriptional activity of Smad3.

Renal fibrosis (RF) is a common pathway leading to chronic kidney disease (CKD), which lacks effective treatment. While estrogen receptor beta (ERß) is known to be present in the kidney, its role in RF remains unclear. The present study aimed to investigate the role and underlying mechanism of ERß during RF progression in patients and animal models with CKD. We found that ERß was highly expressed in the proximal tubular epithelial cells (PTECs) in healthy kidneys but its expression was largely lost in patients with immunoglobin A nephropathy (IgAN) and in mice with unilateral ureter obstruction (UUO) and subtotal nephrectomy (5/6Nx). ERß deficiency markedly exacerbated, whereas ERß activation by WAY200070 and DPN attenuated RF in both UUO and 5/6Nx mouse models, suggesting a protective role of ERß in RF. In addition, ERß activation inhibited TGF-ß1/Smad3 signaling, while loss of renal ERß was associated with overactivation of the TGF-ß1/Smad3 pathway. Furthermore, deletion or pharmacological inhibition of Smad3 prevented the loss of ERß and RF. Mechanistically, activation of ERß competitively inhibited the association of Smad3 with the Smad-binding element, thereby downregulating the transcription of the fibrosis-related genes without altering Smad3 phosphorylation in vivo and in vitro. In conclusion, ERß exerts a renoprotective role in CKD by blocking the Smad3 signaling pathway. Thus, ERß may represent as a promising therapeutic agent for RF.

Neutral amino acid transporter SLC38A2 protects renal medulla from hyperosmolarity-induced ferroptosis.

Hyperosmolarity of the renal medulla is essential for urine concentration and water homeostasis. However, how renal medullary collecting duct (MCD) cells survive and function under harsh hyperosmotic stress remains unclear. Using RNA-Seq, we identified SLC38A2 as a novel osmoresponsive neutral amino acid transporter in MCD cells. Hyperosmotic stress-induced cell death in MCD cells occurred mainly via ferroptosis, and it was significantly attenuated by SLC38A2 overexpression but worsened by Slc38a2-gene deletion or silencing. Mechanistic studies revealed that the osmoprotective effect of SLC38A2 is dependent on the activation of mTORC1. Moreover, an in vivo study demonstrated that Slc38a2-knockout mice exhibited significantly increased medullary ferroptosis following water restriction. Collectively, these findings reveal that Slc38a2 is an important osmoresponsive gene in the renal medulla and provide novel insights into the critical role of SLC38A2 in protecting MCD cells from hyperosmolarity-induced ferroptosis via the mTORC1 signalling pathway.