C1q/tumor necrosis factor-related protein-3 improves renal fibrosis via inhibiting notch signaling pathways

C1q/tumor necrosis factor-related protein-3 (CTRP3) has been extensively reported as an important role involved in antifibrosis, antiapoptosis, and anti-inflammation. However, the role of CTRP3 involved in renal fibrosis remains unclear. Our current study explored the role of CTRP3 in renal fibrosis and its underlying mechanisms by using serums and renal biopsy specimens from renal fibrosis patients and control subjects, rats models with the surgery of unilateral ureteral obstruction (UUO) and human renal proximal tubular epithelial cells (HRPTEpiCs). We found that circulating levels of CTRP3 had no significant difference between renal fibrosis patients and healthy subjects; however, renal CTRP3 expression was markedly downregulated in the fibrotic region with an abundant expression of collagen-I. In UUO rat models, circulating levels of CTRP3 have not changed with the prolonged obstruction of the kidney; renal CTRP3 expression was decreased with the severity of renal fibrosis; adenovirus-mediated CTRP3 treatment inhibited renal interstitial fibrosis. In vitro experiments revealed that CTRP3 attenuates TGF-β1 induced tubular epithelial cells fibrotic changes; CTRP3 knockdown facilitates the expression of fibrotic markers in TGF-β1-induced HRPTEpiCs; recombinant CTRP3 or adenovirus-mediated CTRP3 overexpression significantly inhibited the Notch signaling pathway-associated factors, and knockdown of CTRP3 increased TGF-β1-mediated activation of the Notch signaling pathways. Collectively, our current study found that CTRP3 could improve renal fibrosis, to some extent, through inhibiting the Notch pathway.     

Up-regulation of the human-specific CHRFAM7A gene protects against renal fibrosis in mice with obstructive nephropathy

Renal fibrosis is a major factor in the progression of chronic kidney diseases. Obstructive nephropathy is a common cause of renal fibrosis, which is also accompanied by inflammation. To explore the effect of human-specific CHRFAM7A expression, an inflammation-related gene, on renal fibrosis during obstructive nephropathy, we studied CHRFAM7A transgenic mice and wild type mice that underwent unilateral ureteral obstruction (UUO) injury. Transgenic overexpression of CHRFAM7A gene inhibited UUO-induced renal fibrosis, which was demonstrated by decreased fibrotic gene expression and collagen deposition. Furthermore, kidneys from transgenic mice had reduced TGF-β1 and Smad2/3 expression following UUO compared with those from wild type mice with UUO. In addition, the overexpression of CHRFAM7A decreased release of inflammatory cytokines in the kidneys of UUO-injured mice. In vitro, the overexpression of CHRFAM7A inhibited TGF-β1-induced increase in expression of fibrosis-related genes in human renal tubular epithelial cells (HK-2 cells). Additionally, up-regulated expression of CHRFAM7A in HK-2 cells decreased TGF-β1-induced epithelial-mesenchymal transition (EMT) and inhibited activation f TGF-β1/Smad2/3 signalling pathways. Collectively, our findings demonstrate that overexpression of the human-specific CHRFAM7A gene can reduce UUO-induced renal fibrosis by inhibiting TGF-β1/Smad2/3 signalling pathway to reduce inflammatory reactions and EMT of renal tubular epithelial cells.     

Suppression of LMCD1 ameliorates renal fibrosis by blocking the activation of ERK pathway

Tubulointerstitial fibrosis is a common pathway of chronic kidney disease (CKD) and is closely related to the progression of CKD. LMCD1, acting as an intermediary, has been reported to play a role in cardiac fibrosis. However, its role in renal fibrosis is yet to be deciphered. Based on the GEO database, we found the expression of LMCD1 is increased in kidney tissues of CKD patients and in human proximal tubular epithelial (HK-2) cells treated with transforming growth factor-β1 (TGF-β1), suggesting that LMCD1 may be involved in tubulointerstitial fibrosis. Herein, we investigated the role of LMCD1 in mice with unilateral ureteral obstruction (UUO) and in TGF-β1-stimulated HK-2 cells. In the UUO model, the expression of LMCD1 was upregulated. UUO-induced renal histopathological changes were mitigated by knockdown of LMCD1. LMCD1 silence alleviated renal interstitial fibrosis in UUO mice by decreasing the expression of TGF-β1, fibronectin, collagen I, and collagen III. LMCD1 deficiency suppressed cell apoptosis in kidney to prevent UUO-triggered renal injury. Furthermore, LMCD1 deficiency blocked the activation of ERK signaling in UUO mice. In vitro, LMCD1 was upregulated in HK-2 cells after TGF-β1 stimulation. LMCD1 silence abrogated TGF-β1-mediated upregulation of fibrotic genes. Treatment of HK-2 cells with ERK-specific inhibitor SCH772984 and agonist TPA validated LMCD1 exerted its function via activating ERK signaling. Together, our findings suggest that inhibition of LMCD1 protects against renal interstitial fibrosis by impeding ERK activation.     

WNT1-inducible signaling protein-1 mediates TGF-β1-induced renal fibrosis in tubular epithelial cells and unilateral ureteral obstruction mouse models via autophagy

Renal fibrosis is a common pathway for the progression of all chronic kidney diseases to end-stage kidney disease. Studies show that WNT1-inducible signaling pathway protein-1 (WISP-1) is involved in the fibrosis of various organs. The aim of the study was to explore the functional role and potential mechanism of WISP-1 in renal fibrosis. We observed that overexpression of WISP-1 in rat tubular epithelial cells (TECs) enhanced transforming growth factor-β1 (TGF-β1)-induced production of fibrotic markers, including collagen I (Col I), fibronectin (FN) and TGF-β1, while inhibition of WISP-1 suppressed such production. In vivo, the messenger RNA and protein levels of Col I, FN, and α-smooth muscle actin were significantly inhibited after anti-WISP-1 antibody treatment for 7 days in unilateral ureteral obstruction mouse models. Moreover, blockade of WISP-1 by anti-WISP-1 antibody significantly reduced autophagy-related markers, including anti-microtubule-associated protein-1 light chain 3 (LC3) and beclin 1, while increasing sequestosome 1. In addition, overexpression of WISP-1 in TECs increased autophagy as evidenced by greater numbers of GFP-LC3 puncta and increased expression of LC3 and beclin 1 in response to TGF-β1. In contrast, knockdown of WISP-1 by small interfering RNA decreased the number of GFP-LC3 puncta and the expression of LC3 and beclin 1 in TGF-β1-treated TECs. Collectively, these data suggest that WISP-1, as a profibrotic protein, may mediate renal fibrosis by inducing autophagy in both obstructive nephropathy and TGF-β1-treated TECs. WISP-1 may serve as an effective therapeutic target for the treatment of renal fibrosis.     

PTEN improve renal fibrosis in vitro and in vivo through inhibiting FAK/AKT signaling pathway

Renal fibrosis, the ultimate common pathway of progressive nephropathy, is characterized by excess accumulation and deposition of extracellular matrix (ECM) within the renal interstitium and glomeruli, finally resulting in end-stage kidney failure. TGFβ1 is not only abnormally increased during fibrosis but also involved in ECM induction and accumulation. Based on the bioinformative analyses, phosphatase and tensin homolog deleted on chromosome ten (PTEN) and focal adhesion kinase (FAK) signaling pathway might be involved in TGFβ1 functions on renal fibrosis development. In the present study, fibrosis was induced in HK-2 cells using TGFβ1 and PTEN expression was significantly suppressed by 24 or 48 hours TGFβ1 treatment. PTEN overexpression in HK-2 cells improved TGFβ1-induced fibrosis within α-SMA and E-cadherin. According to the KEGG signaling pathway annotation analyses on microarray profiles (GSE23338 and GSE20247) and immunoblotting validation, FAK signaling might be involved in PTEN functions in TGFβ1-induced fibrosis. PTEN overexpression significantly inhibited TGFβ1- or unilateral ureteral obstruction (UUO)-induced FAK signaling pathway activation both in vitro and in vivo; more importantly, PTEN silence enhanced TGFβ1- or UUO-induced fibrosis, while FAK inhibitor PF567721 significantly reversed the effects of PTEN silence, indicating that PTEN exerted its effects on TGFβ1- and UUO-induced fibrotic development in vitro and in vivo via inhibiting FAK signaling pathway. In summary, these findings indicate that PTEN could improve cellular fibrotic changes and renal fibrosis via inhibiting FAK/AKT signaling pathway. Restoring PTEN expression to target FAK/AKT signaling pathway might be a potent strategy for renal fibrosis treatment.     

LncRNA ENST00000453774.1 contributes to oxidative stress defense dependent on autophagy mediation to reduce extracellular matrix and alleviate renal fibrosis

Although long noncoding RNA (LncRNA) are important players in the initiation and progression of many pathological processes, the role of LncRNAENST00000453774.1 (LncRNA 74.1) in renal fibrosis still remains unclear. Lentivirus mediated LncRNA 74.1 overexpressing HK2 cells and overexpression mice models were constructed. HK2 cells induced by transforming growth factor-β (TGF-β) in vitro, and the mice UUO model in vivo were used to simulate renal fibrosis. The expression of LncRNA 74.1 was significantly downregulated in the TGF-β-induced HK-2 cell fibrosis and clinical renal fibrosis specimens. LncRNA 74.1 overexpression obviously attenuated renal fibrosis in vitro and unilateral ureteral obstruction-induced renal fibrosis in vivo. LncRNA 74.1 promoted reactive oxygen species defense by activating prosurvival autophagy then decreased ECM-related proteins fibronectin and collagen I involved in renal fibrosis. We also found that Nrf2-keap1 signaling played important roles in the remission of ECM mediated by LncRNA 74.1. This study indicates that LncRNA 74.1 downregulation would contribute to renal fibrosis and its overexpression might represent a novel anti-fibrotic treatment in renal diseases.     

Nicotinamide reduces renal interstitial fibrosis by suppressing tubular injury and inflammation

Renal interstitial fibrosis is a common pathological feature in progressive kidney diseases currently lacking effective treatment. Nicotinamide (NAM), a member of water‐soluble vitamin B family, was recently suggested to have a therapeutic potential for acute kidney injury (AKI) in mice and humans. The effect of NAM on chronic kidney pathologies, including renal fibrosis, is unknown. Here we have tested the effects of NAM on renal interstitial fibrosis using in vivo and in vitro models. In vivo, unilateral urethral obstruction (UUO) induced renal interstitial fibrosis as indicated Masson trichrome staining and expression of pro‐fibrotic proteins, which was inhibited by NAM. In UUO, NAM suppressed tubular atrophy and apoptosis. In addition, NAM suppressed UUO‐associated T cell and macrophage infiltration and induction of pro‐inflammatory cytokines, such as TNF‐α and IL‐1β. In cultured mouse proximal tubule cells, NAM blocked TGF–β‐induced expression of fibrotic proteins, while it marginally suppressed the morphological changes induced by TGF‐β. NAM also suppressed the expression of pro‐inflammatory cytokines (eg MCP‐1 and IL‐1β) during TGF‐β treatment of these cells. Collectively, the results demonstrate an anti‐fibrotic effect of NAM in kidneys, which may involve the suppression of tubular injury and inflammation.     

Reduced Klotho expression level in kidney aggravates renal interstitial fibrosis

Renal expression of the klotho gene is markedly suppressed in chronic kidney disease (CKD). Since renal fibrosis is the final common pathology of CKD, we tested whether decreased Klotho expression is a cause and/or a result of renal fibrosis in mice and cultured renal cell lines. We induced renal fibrosis by unilateral ureteral obstruction (UUO) in mice with reduced Klotho expression (kl/+ mice) and compared them with wild-type mice. The UUO kidneys from kl/+ mice expressed significantly higher levels of fibrosis markers such as α-smooth muscle actin (α-SMA), fibronectin, and transforming growth factor-β(1) (TGF-β(1)) than those from wild-type mice. In addition, in cultured renal fibroblast cells (NRK49F), the levels of α-SMA and PAI1 expression were significantly suppressed by addition of recombinant Klotho protein to the medium. The similar effects were observed by a TGF-β(1) receptor inhibitor (ALK5 inhibitor). These observations suggest that low renal Klotho expression enhances TGF-β(1) activity and is a cause of renal fibrosis. On the other hand, TGF-β(1) reduced Klotho expression in renal cultured epithelial cells (inner medullary collecting duct and human renal proximal tubular epithelium), suggesting that low renal Klotho expression is a result of renal fibrosis. Taken together, renal fibrosis can trigger a deterioration spiral of Klotho expression, which may be involved in the pathophysiology of CKD progression.     

Autophagy promotes intracellular degradation of type I collagen induced by transforming growth factor (TGF)-β1

Autophagy is a highly conserved cellular process regulating turnover of cytoplasmic proteins via a lysosome-dependent pathway. Here we show that kidneys from mice deficient in autophagic protein Beclin 1 exhibited profibrotic phenotype, with increased collagen deposition. Reduced Beclin 1 expression, through genetic disruption of beclin 1 or knockdown by specific siRNA in primary mouse mesangial cells (MMC), resulted in increased protein levels of type I collagen (Col-I). Inhibition of autolysosomal protein degradation by bafilomycin A(1) also increased Col-I protein levels and colocalization of Col-I with LC3, an autophagy marker, or LAMP-1, a lysosome marker, whereas treatment with TFP, an inducer of autophagy, resulted in decreased Col-I protein levels induced by TGF-β1, without alterations in Col-I α1 mRNA. Heterozygous deletion of beclin 1 increased accumulation of aggregated Col-I under nonstimulated conditions, and stimulation with TGF-β1 further increased aggregated Col-I. These data indicate that Col-I and aggregated, insoluble procollagen I undergo intracellular degradation via autophagy. A cytoprotective role of autophagy is implicated in kidney injury, and we demonstrate that low-dose carbon monoxide, shown to exert cytoprotection against renal fibrosis, induces autophagy to suppress accumulation of Col-I induced by TGF-β1. We also show that TGF-β1 induces autophagy in MMC via TAK1-MKK3-p38 signaling pathway. The dual functions of TGF-β1, as both an inducer of Col-I synthesis and an inducer of autophagy and Col-I degradation, underscore the multifunctional nature of TGF-β1. Our findings suggest a novel role of autophagy as a cytoprotective mechanism to negatively regulate and prevent excess collagen accumulation in the kidney.     

Poricoic acid A activates AMPK to attenuate fibroblast activation and abnormal extracellular matrix remodelling in renal fibrosis

BackgroundIn chronic kidney disease, although fibrosis prevention is beneficial, few interventions are available that specifically target fibrogenesis. Poricoic acid A (PAA) isolated from Poria cocos exhibits anti-fibrotic effects in the kidney, however the underlying mechanisms remain obscure.PurposeWe isolated PAA and investigated its effects and the underlying mechanisms in renal fibrosis.Study designUnilateral ureteral obstruction (UUO) and 5/6 nephrectomy (Nx) animal models and TGF-β1-induced renal fibroblasts (NRK-49F) were used to investigate the anti-fibrotic activity of PAA and its underlying mechanisms.MethodsWestern blots, qRT-PCR, immunofluorescence staining, co-immunoprecipitation and molecular docking methods were used. Knock-down and knock-in of adenosine monophosphate-activated protein kinase (AMPK) in the UUO model and cultured NRK-49F cells were employed to verify the mechanisms of action of PAA.ResultsPAA improved renal function and alleviated fibrosis by stimulating AMPK and inhibiting Smad3 specifically in Nx and UUO models. Reduced AMPK activity was associated with Smad3 induction, fibroblast activation, and the accumulation and aberrant remodelling of extracellular matrix (ECM) in human renal puncture samples and cultured NRK-49F cells. PAA stimulated AMPK activity and decreased fibrosis in a dose-dependent manner, thus showing that AMPK was essential for PAA to exert its anti-fibrotic effects. AMPK deficiency reduced the anti-fibrotic effects of PAA, while AMPK overexpression enhanced its effect.ConclusionPAA activated AMPK and further inhibited Smad3 specifically to suppress fibrosis by preventing aberrant ECM accumulation and remodelling and facilitating the deactivation of fibroblasts.     

The regulatory peptide apelin: a novel inhibitor of renal interstitial fibrosis

Epithelial–mesenchymal transition (EMT) of tubular epithelial cells is a key event in renal interstitial fibrosis and the progression of chronic kidney disease (CKD). Apelin is a regulatory peptide involved in the regulation of normal renal hemodynamics and tubular functions, but its role in renal fibrosis remains unknown. In this study, we examined the inhibitory effects of apelin on transforming growth factor-β1 (TGF-β1)-induced EMT in HK-2 cells, and evaluated its therapeutic efficacy in mice with complete unilateral ureteral obstruction (UUO). In vitro, apelin inhibited TGF-β1-mediated upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin. Increased levels of phosphorylated Smad-2/3 and decreased levels of Smad7 in TGF-β1-stimulated cells were reversed by apelin co-treatment. In the UUO model, administration of apelin significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin and laminin expression, and markedly suppressed expression of α-SMA, TGF-β1 and its type I receptor, as well as interstitial matrix components. Interestingly, in UUO mice, there was a reduction in the plasma level of apelin, which was compensated by upregulation of APJ expression in the injured kidney. Exogenous supplementation of apelin normalized the level of plasmatic apelin and renal APJ. In conclusion, our study provides the first evidence that apelin is able to ameliorate renal interstitial fibrosis by suppression of tubular EMT through a Smad-dependent mechanism. The apelinergic system itself may promote some compensatory response in the renal fibrotic process. These results suggest that apelin has potential renoprotective effects and may be an effective agent for retarding CKD progression.     

Identification of a microRNA signature in renal fibrosis: role of miR-21

Renal fibrosis is a final stage of many forms of kidney disease and leads to impairment of kidney function. The molecular pathogenesis of renal fibrosis is currently not well-understood. microRNAs (miRNAs) are important players in initiation and progression of many pathologic processes including diabetes, cancer, and cardiovascular disease. However, the role of miRNAs in kidney injury and repair is not well-characterized. In the present study, we found a unique miRNA signature associated with unilateral ureteral obstruction (UUO)-induced renal fibrosis. We found altered expression in UUO kidneys of miRNAs that have been shown to be responsive to stimulation by transforming growth factor (TGF)-β1 or TNF-α. Among these miRNAs, miR-21 demonstrated the greatest increase in UUO kidneys. The enhanced expression of miR-21 was located mainly in distal tubular epithelial cells. miR-21 expression was upregulated in response to treatment with TGF-β1 or TNF-α in human renal tubular epithelial cells in vitro. Furthermore, we found that blocking miR-21 in vivo attenuated UUO-induced renal fibrosis, presumably through diminishing the expression of profibrotic proteins and reducing infiltration of inflammatory macrophages in UUO kidneys. Our data suggest that targeting specific miRNAs could be a novel therapeutic approach to treat renal fibrosis.     

Blocking IL-17A promotes the resolution of pulmonary inflammation and fibrosis via TGF-beta1-dependent and -independent mechanisms

Pulmonary fibrosis is the pathologic basis for a variety of incurable human chronic lung diseases. IL-17A, a glycoprotein secreted from IL-17-producing cells, has recently been shown to be a proinflammatory cytokine involved in chronic inflammation and autoimmune disease. In this study, we report that IL-17A increased the synthesis and secretion of collagen and promoted the epithelial-mesenchymal transition in alveolar epithelial cells in a TGF-β1-dependent manner. Using in vivo fibrotic models, we found IL-17A expression to be elevated and IL-17A-associated signaling pathways to be activated in fibrotic lung tissues. Neutralization of IL-17A in vivo promoted the resolution of bleomycin-induced acute inflammation, attenuated pulmonary fibrosis, and increased survival. Additionally, IL-17A antagonism inhibited silica-induced chronic inflammation and pulmonary fibrosis. Targeting IL-17A resulted in a shift of the suppressive immune response in fibrotic lung tissue toward a Th1-type immune response, and it effectively induced autophagy, which promoted the autophagic degradation of collagen and autophagy-associated cell death. Moreover, IL-17A was found to attenuate the starvation-induced autophagy, and autophagy modulators regulated collagen degradation in the alveolar epithelial cells in a TGF-β1-independent manner. Administration of 3-methylamphetamine, an autophagy inhibitor, reversed the therapeutic efficacy of IL-17A antagonism in pulmonary fibrosis. Our studies indicate that IL-17A participates in the development and progression of pulmonary fibrosis in both TGF-β1-dependent and -independent manners and that the components of the IL-17A signaling pathway are potential therapeutic targets for the treatment of fibroproliferative lung diseases.     

Validation of highly selective sphingosine kinase 2 inhibitors SLM6031434 and HWG-35D as effective anti-fibrotic treatment options in a mouse model of tubulointerstitial fibrosis

Renal fibrosis is characterized by chronic inflammation and excessive accumulation of extracellular matrix and progressively leads to functional insufficiency and even total loss of kidney function. In this study we investigated the anti-fibrotic potential of two highly selective and potent SK2 inhibitors, SLM6031434 and HWG-35D, in unilateral ureter obstruction (UUO), a model for progressive renal fibrosis, in mice. In both cases, treatment with SLM6031434 or HWG-35D resulted in an attenuated fibrotic response to UUO in comparison to vehicle-treated mice as demonstrated by reduced collagen accumulation and a decreased expression of collagen-1 (Col1), fibronectin-1 (FN-1), connective tissue growth factor (CTGF), and α-smooth muscle actin (α-SMA). Similar to our previous study in Sphk2^−/− mice, we found an increased protein expression of Smad7, a negative regulator of the pro-fibrotic TGFβ/Smad signalling cascade, accompanied by a strong accumulation of sphingosine in SK2 inhibitor-treated kidneys. Treatment of primary renal fibroblasts with SLM6031434 or HWG-35D dose-dependently increased Smad7 expression and ameliorated the expression of Col1, FN-1 and CTGF.In summary, these data prove the anti-fibrotic potential of SK2 inhibition in a mouse model of renal fibrosis, thereby validating SK2 as pharmacological target for the treatment of fibrosis in chronic kidney disease.     

Transplantation of endothelial progenitor cells alleviates renal interstitial fibrosis in a mouse model of unilateral ureteral obstruction

AimsThe present study investigated whether transplantation of bone marrow-derived endothelial progenitor cells (BM-EPCs) in renal capillary network improves renal interstitial fibrosis in unilateral ureteral obstruction (UUO) model in mice.Main methodsEx vivo generated, characterized, and cultivated mice BM-EPCs were identified by their vasculogenic properties in vitro. BM-EPCs were labelled with carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) before transplantation. The animal models of UUO were used. Histological changes in renal tubular interstitium were observed with HE and Masson staining. The protein levels of vascular endothelial growth factor(VEGF), hypoxia inducible factor-1α (HIF-1α) and connective tissue growth factor (CTGF) were analyzed by western blotting and immunohistochemistry. Transforming growth factor-β1 (TGF-β1) was detected by immunohistochemistry. Peritubular capillary (PTC) density was determined by CD31 immunostaining.Key findingsTransplanted BM-EPCs were successfully incorporated into the capillary network in the obstructed kidney in vivo. UUO induced a significant decrease in VEGF levels and PTC density in the kidney tissue, which was accompanied by a significant increase in HIF-1α, CTGF and TGF-β1. Transplantation of BM-EPCs increased PTC density, VEGF expression and alleviated the development of renal interstitial fibrosis in UUO mice. No significant pathological changes were found in control mice.SignificanceThe reduction of PTC density and up-regulation of HIF-1α are the important mechanisms of interstitial fibrosis in UUO mice. BM-EPCs transplantation may increase the number of capillary density and alleviate the development of renal fibrosis in obstructive nephropathy in mice.     

Autophagy in FOXD1 stroma-derived cells regulates renal fibrosis through TGF-β and NLRP3 inflammasome pathway

Renal fibrosis is the final common pathway of various renal injuries and it leads to chronic kidney disease. Recent studies reported that FOXD1-lineage pericyte plays a critical role in tubulointerstitial fibrosis (TIF). However the regulatory mechanisms remain unclear. Autophagy is a cellular process of degradation of damaged cytoplasmic components that regulates cell death and proliferation. To investigate the role of autophagy in FOXD1-lineage pericytes on renal TIF, we generated the FOXD1-lineage stromal cell-specific Atg7 deletion (Atg7^△FOXD1) mice. FOXD1-lineage stromal cell-specific Atg7 deletion enhanced renal TIF through Smad-dependent transforming growth factor (TGF)-β signaling after unilateral ureteral obstruction (UUO). FOXD1-lineage stromal cell-specific Atg7 deletion increased the accumulation of interstitial myofibroblasts and enhanced the differentiation of pericytes into myofibroblasts after UUO. Peritubular capillary rarefaction was accelerated in Atg7^△FOXD1 mice after UUO. Atg7^△FOXD1 mice increased the accumulation of SQSTM1/p62-positive aggregates in the obstructed kidney and resulted in increased expression of NLRP3 inflammasome, interleukin (IL) 1-β and caspase-1 signaling pathway, which enhanced apoptosis of interstitial cells after UUO. In summary, our data showed that autophagy in FOXD1-lineage stromal cells plays a protective role in renal TIF through regulating the Smad4 dependent TGF-β an NLRP3 inflammasome signaling pathway.     

Response gene to complement 32 is essential for fibroblast activation in renal fibrosis

Response gene to complement 32 (RGC-32) is a downstream target of transforming growth factor-β (TGF-β). TGF-β is known to play a pathogenic role in renal fibrosis. In this study, we investigated RGC-32 function in renal fibrosis following unilateral ureteral obstruction (UUO) in mice, a model of progressive tubulointerstitial fibrosis. RGC-32 is normally expressed only in blood vessels of mouse kidney. However, UUO induces RGC-32 expression in renal interstitial cells at the early stage of kidney injury, suggesting that RGC-32 is involved in interstitial fibroblast activation. Indeed, expression of smooth muscle α-actin (α-SMA), an indicator of fibroblast activation, is limited to the interstitial cells at the early stage, and became apparent later in both interstitial and tubular cells. RGC-32 knockdown by shRNA significantly inhibits UUO-induced renal structural damage, α-SMA expression and collagen deposition, suggesting that RGC-32 is essential for the onset of renal interstitial fibrosis. In vitro studies indicate that RGC-32 mediates TGF-β-induced fibroblast activation. Mechanistically, RGC-32 interacts with Smad3 and enhances Smad3 binding to the Smad binding element in α-SMA promoter as demonstrated by DNA affinity assay. In the chromatin setting, Smad3, but not Smad2, binds to α-SMA promoter in fibroblasts. RGC-32 appears to be essential for Smad3 interaction with the promoters of fibroblast activation-related genes in vivo. Functionally, RGC-32 is crucial for Smad3-mediated α-SMA promoter activity. Taken together, we identify RGC-32 as a novel fibrogenic factor contributing to the pathogenesis of renal fibrosis through fibroblast activation.