Role In Tubulointerstitial Fibrosis Research Articles

A PDE1 inhibitor, vinpocetine, ameliorates epithelial-mesenchymal transition and renal fibrosis in adenine-induced chronic kidney injury in rats by targeting the DNMT1/Klotho/β-catenin/Snail 1 and MMP-7 pathways.

Tubulointerstitial fibrosis (TIF) is present with chronic kidney disease (CKD). Vinpocetine (Vinpo) is used for treating cerebrovascular deficits, exhibiting some kidney-beneficial effects; however, its role in TIF is uncertain. So, the aim of this study was to investigate its potential impact on adenine-induced fibrotic CKD and explore the underlying mechanistic aspects. Eighteen male Wistar rats were categorized into three groups (n = 6 each). Group I was kept as controls and given saline; group II received adenine (300 mg/kg, twice weekly, i.p.) for induction of the CKD model; and group III was administered Vinpo (20 mg/kg/d, orally) concurrently with adenine. All treatments were administered for 4 weeks. Vinpo revealed an improvement in renal function and an alleviation of inflammation triggered by adenine via diminishing serum tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6) levels. Further, Vinpo repressed the epithelial-mesenchymal transition (EMT) with preserved E-cadherin mRNA expression and lowered gene and immune expression of fibronectin and vimentin, respectively, besides attenuating the elevated G2/M arrest-related molecules (renal Ki67 protein contents and p21 gene expression). Renal pathological alterations caused by adenine were attenuated upon Vinpo administration. Interestingly, Vinpo suppressed abnormal renal β-catenin immunoreactivity, Snail 1, and MMP-7 gene expression while simultaneously restored Klotho protein expression by downregulating DNA methyltransferase 1 enzyme (DNMT1) protein expression in the kidney. These data indicated that Vinpo effectively mitigated EMT and G2/M arrest-induced renal fibrosis in adenine-induced CKD rats by targeting DNMT1-associated Klotho suppression, subsequently inhibiting β-catenin and its fibrotic downstream genes.

#2006 CircMTND5 participated in tubulointerstitial fibrosis progression of lupus nephritis by binding with A1CF

Abstract Background and Aims lupus nephritis (LN) is the most common and severe complication of systemic lupus erythematosus (SLE). CircRNA MTND5 participated in the pathogenesis of LN by sponging miR6812. However, it remains unclear what RNA binding role of circMTND5 play in LN. We aim to investigate whether circMTND5 can interact with RBP in the development and progression in LN, and to clarify the corresponding mechanisms. Method A1CF, a RNA binding protein, E-cadherin, α-SMA, TGF-β, and Fibronectin were analyzed by immunohistochemistry on paraffin-embedded section of LN patients and NC kidney tissues. The above parameters and circMTND5 were also examined in Fcgr2b-/- spontaneous LN mice kidneys by Western blotting and qPCR. In human kidney (HK) -2 cells, the binding of circMTND5 to A1CF protein was examined by RNA immunoprecipitation (RIP) assay. After knockdown in HK-2 cells and overexpression in hTGF-β stimulated HK-2 cells, we examined the all above indices. Results the expression of A1CF was down-regulated in the kidneys of LN patients and was localized in tubular epithelial cells. E-cadherin was down-regulated while α-SMA was up-regulated. The expression of circMTND5 was down-regulated in kidneys of Fcgr2b-/- spontaneous LN mice. In HK-2 cells, the binding of circMTND5 to A1CF was confirmed by RIP. A1CF and E-cadherin were down-regulated, while α-SMA, TGF-β and FN were up-regulated in HK-2 cells with knockdown of circMTND5. The overexpression of circMTND5 reverted the changes of the those above parametered induced by hTGF-β in both mRNA and proteins levels. Conclusion The expression level of circMTND5 in renal tissue of LN mice was downregulated. CircMTND5 might play an important role in tubulointerstitial fibrosis of lupus nephritis by binding with A1CF protein.

#2104 Neutrophil extracellular traps have a pivotal role in tubulointerstitial fibrosis

Abstract Background and Aims Tubulointerstitial fibrosis (TIF) is a typical pathological change in chronic kidney disease (CKD). However, the mechanisms underlying tubulointerstitial fibrosis are still to be fully elucidated. Neutrophil extracellular traps (NETs) participate in the pathophysiological processes of various kidney diseases, but their role in CKD-TIF has not been reported. Here, we investigated the role of NETs in tubulointerstitial fibrosis. Method Immunofluorescence was employed to assess the levels and localization of NETs in the renal tissues of CKD patients. The GSE66494 human CKD microarray dataset was explored to analyze the changes of PAD4 levels. Three CKD mice models, including 14d unilateral ureteral obstruction (UUO), 28d ischemia/reperfusion (I/R) injury, and adenine-induced models were constructed to detect the expression levels of NETs in the kidneys. Then, we established a NETs-deficient (PAD4−/−) CKD mice model and assessed changes in renal function, NETs levels, and renal fibrosis. In vitro, phorbol-12-myristate-13-acetate (the classic inducer of NETs) was used to induce the generation of NETs. Intervention was performed on rat fibroblast NRK49F cells to investigate the mechanism of TIF induced by NETs. Results We first identified NETs levels were significantly elevated in the renal tissues of CKD patients. Additionally, chip data results also indicated elevated PAD4 levels in the renal tissues of CKD patients. NETs levels were elevated in the kidneys of UUO, I/R, and adenine-induced CKD mice, with deposition in the renal interstitium. PAD4-deficient mice did not form neutrophil extracellular traps. Knocking out PAD4 alleviated tubulointerstitial fibrosis in UUO, adenine, and I/R mice after blocking NETs formation. In vitro, NETs intervention in fibroblasts can promote their activation and transformation into myofibroblasts. Conclusion The deletion of NETs by PAD4−/− could significantly improve CKD-TIF. The inhibition of NETs might provide a new molecular target for the prevention and treatment of CKD.

CRTC2 activates the epithelial–mesenchymal transition of diabetic kidney disease through the CREB-Smad2/3 pathway

BackgroundEpithelial–mesenchymal transition (EMT) plays a key role in tubulointerstitial fibrosis, which is a hallmark of diabetic kidney disease (DKD). Our previous studies showed that CRTC2 can simultaneously regulate glucose metabolism and lipid metabolism. However, it is still unclear whether CRTC2 participates in the EMT process in DKD.MethodsWe used protein‒protein network (PPI) analysis to identify genes that were differentially expressed during DKD and EMT. Then, we constructed a diabetic mouse model by administering STZ plus a high-fat diet, and we used HK-2 cells that were verified to confirm the bioinformatics research results. The effects that were exerted by CRTC2 on epithelial-mesenchymal transition in diabetic kidney disease through the CREB-Smad2/3 signaling pathway were investigated in vivo and in vitro by real-time PCR, WB, IHC and double luciferase reporter gene experiments.ResultsFirst, bioinformatics research showed that CRTC2 may promote EMT in diabetic renal tubules through the CREB-Smad2/3 signaling pathway. Furthermore, the Western blotting and real-time PCR results showed that CRTC2 overexpression reduced the expression of E-cadherin in HK-2 cells. The CRTC2 and α-SMA levels were increased in STZ-treated mouse kidneys, and the E-cadherin level was reduced. The luciferase activity of α-SMA, which is the key protein in EMT, was sharply increased in response to the overexpression of CRTC2 and decreased after the silencing of CREB and Smad2/3. However, the expression of E-cadherin showed the opposite trends. In the real-time PCR experiment, the mRNA expression of α-SMA increased significantly when CRTC2 was overexpressed but partially decreased when CREB and Smad2/3 were silenced. However, E-cadherin expression showed the opposite result.ConclusionThis study demonstrated that CRTC2 activates the EMT process via the CREB-Smad2/3 signaling pathway in diabetic renal tubules.

Upregulation of KLF14 expression attenuates kidney fibrosis by inducing PPARα-mediated fatty acid oxidation

Tubulointerstitial fibrosis (TIF) is essential during the development of end-stage kidney disease (ESKD) and is associated with the impairment of fatty acid oxidation (FAO). Kruppel-like factor 14 (KLF14) is an important gene in lipid metabolism, but its role in TIF remains unknown. TGF-β-stimulated HK-2 cells and mouse unilateral ureteral obstruction (UUO) were used as renal fibrosis models. The role of KLF14 in the process of renal fibrosis was verified by gene knockout mice, genetic or pharmacological interference in animal model and cell model respectively. In the current study, we found that KLF14 expression increased after activation of the TGF-β signaling pathway during TIF. In KLF14−/− mice, more severe fibrosis was observed after unilateral ureteral obstruction (UUO) was induced. In human HK2 cells, knockdown of KLF14 led to more severe fibrosis induced by TGF-β1, while overexpression of KLF14 partially attenuated this process. Specifically, KLF14 deficiency decreased mitochondrial FAO activity, resulting in lipid accumulation. Thus, the energy supply to the cells was insufficient, finally resulting in TIF. We further proved that KLF14 could target peroxisome proliferator activated receptor alpha (PPARα) as a transcriptional activator. This study identified the upregulation of KLF14 expression in response to kidney stress during the process of fibrosis. Upon TIF, the activated TGF-β signaling pathway can enhance KLF14 expression, while the upregulation of KLF14 expression can decrease the degree of TIF by improving FAO activity in tubular epithelial cells and recovering the energy supply mediated by PPARα.

BMP-7 Upregulates Id2 Through the MAPK Signaling Pathway to Improve Diabetic Tubulointerstitial Fibrosis and the Intervention of Oxymatrine.

Diabetic kidney disease is one of the most serious microvascular complications of diabetes. It progresses irreversibly to end-stage renal disease if left untreated. Bone morphogenetic protein (BMP)-7 is a negative regulator of organ fibrosis and may also play an essential role in tubulointerstitial fibrosis. This study aimed to investigate the precise role and potential molecular mechanisms of BMP-7 in the progression of diabetic nephropathy. In this study, BMP-7 was overexpressed in vivo after the replication of the diabetic rat model using streptozotocin. The results showed that BMP-7 inhibited the phosphorylation of related mitogen-activated protein kinase (MAPK) pathways while upregulating the inhibitor of differentiation (Id2) expression and effectively ameliorated pathological renal injury. Further in vitro validation showed that the inhibition of the phosphorylation of MAPKs at a high glucose concentration in renal tubular epithelial cells was followed by the upregulation of Id2 protein expression, suggesting that BMP-7 could improve diabetic nephropathy by upregulating Id2 protein levels through the BMP-7–MAPK signaling pathway. Previous laboratory studies found that oxymatrine improved renal fibrotic lesions. However, the exact mechanism is unclear. The present study showed that oxymatrine treatment in a diabetic rat model upregulated BMP-7 protein expression and inhibited MAPK pathway protein phosphorylation levels. These results suggested that oxymatrine improved the epithelial-to-mesenchymal transition process in the early stage of diabetic kidney disease by regulating the BMP-7–MAPK pathway and ameliorated renal tubulointerstitial fibrosis.

389-P: Pathological Significance of Renal Tubular ROCK2 in Chronic Kidney Disease

The small GTPase Rho and its effector Rho-kinase (ROCK) are implicated in the pathogenesis of diabetic kidney disease. In chronic kidney diseases including diabetic nephropathy, progressive tubulointerstitial fibrosis is the final common pathway to end-stage renal disease and deeply related to renal prognosis. We have previously reported that a chemical ROCK inhibitor suppressed the progression of diabetic nephropathy. ROCK has two isoforms, ROCK1 and ROCK2. ROCK1 is known to be involved in the endocytosis of albumin in tubular epithelial cells via megalin/cubilin-dependent mechanism. On the other hand, the role of renal ROCK2 remains unclear. In the present study, we investigated the role of renal tubular ROCK2 in the pathogenesis of chronic kidney disease using an established model to cause renal fibrosis, unilateral ureteral obstruction (UUO). Masson’s trichrome staining revealed that the collagen deposition was significantly increased in the kidneys of UUO group compared with Sham-operated group. We detected upregulation of ROCK2 at both mRNA and protein levels in UUO group, relative to sham operated group. The kinase activity of ROCK2 was also elevated in UUO group. Based on our research, we suggest that ROCK2 may have an important role in tubulointerstitial fibrosis. ROCK2 may be a potential therapeutic target for the treatment of diabetic nephropathy. Disclosure K. Sekiguchi: None. K. Matoba: Research Support; Self; Eli Lilly Japan K. K. R. Ukichi: None. Y. Nagai: None. Y. Takeda: None. T. Akamine: None. Y. Kanazawa: None. K. Utsunomiya: None. R. Nishimura: Speaker’s Bureau; Self; Abbott Japan Co., Ltd., Boehringer Ingelheim Pharmaceuticals, Inc., Eli Lilly Japan K. K., Kissei Pharmaceutical Co., Ltd., Medtronic, MSD Corporation, Novo Nordisk Inc., Ono Pharmaceutical Co., Ltd., Taisho Pharmaceutical Co., Ltd., Takeda Pharmaceutical Company Limited.

De-Glycyrrhizinated Licorice Extract Attenuates High Glucose-Stimulated Renal Tubular Epithelial-Mesenchymal Transition via Suppressing the Notch2 Signaling Pathway.

Tubulointerstitial fibrosis is a major pathological hallmark of diabetic nephropathy. Increasing evidence has shown that epithelial-to-mesenchymal transition (EMT) of renal proximal tubular cells plays a crucial role in tubulointerstitial fibrosis. Herein, we aimed to elucidate the detailed mechanism of EMT in renal tubular cells under high glucose (HG) conditions, and to investigate the potential of licorice, a medicinal herb, to inhibit HG-induced EMT. Our results showed that renal tubular epithelial cells (normal rat kidney cell clone 52E; NRK-52E) exposed to HG resulted in EMT induction characterized by increased fibronectin and α-SMA (alpha-smooth muscle actin) but decreased E-cadherin. Elevated levels of cleaved Notch2, MAML-1 (mastermind-like transcriptional coactivator 1), nicastrin, Jagged-1 and Delta-like 1 were also concomitantly detected in HG-cultured cells. Importantly, pharmacological inhibition, small interfering RNA (siRNA)-mediated depletion or overexpression of the key components of Notch2 signaling in NRK-52E cells supported that the activated Notch2 pathway is essential for tubular EMT. Moreover, we found that licorice extract (LE) with or without glycyrrhizin, one of bioactive components in licorice, effectively blocked HG-triggered EMT in NRK-52E cells, mainly through suppressing the Notch2 pathway. Our findings therefore suggest that Notch2-mediated renal tubular EMT could be a therapeutic target in diabetic nephropathy, and both LE and de-glycyrrhizinated LE could have therapeutic potential to attenuate renal tubular EMT and fibrosis.

MiR-98-5p Alleviated Epithelial-to-Mesenchymal Transition and Renal Fibrosis via Targeting Hmga2 in Diabetic Nephropathy.

Recently, microRNAs have been recognized as crucial regulators of diabetic nephropathy (DN) development. Epithelial-to-mesenchymal transition (EMT) can play a significant role in tubulointerstitial fibrosis, and it is a hallmark of diabetic nephropathy progression. Nevertheless, the function of miR-98-5p in the modulation of EMT and renal fibrosis during DN remains barely investigated. Hence, identifying the mechanisms of miR-98-5p in regulating EMT and fibrosis is of huge significance. In our present research, decreased miR-98-5p was demonstrated in db/db mice and mice mesangial cells treated with the high dose of glucose. Meanwhile, activated EMT and increased fibrosis was accompanied with the decrease of miR-98-5p in vitro and in vivo. Additionally, to further find out the roles of miR-98-5p in DN development, overexpression of miR-98-5p was applied. Firstly, in vivo investigation exhibited that elevation of miR-98-5p restrained proteinuria, serum creatinine, BUN, the EMT process, and fibrosis. Furthermore, high glucose was able to promote mice mesangial cell proliferation, EMT process, and induced renal fibrosis, which could be prevented by overexpression of miR-98-5p. Moreover, high mobility group A (HMGA2) can exhibit an important role in diverse biological processes. Here, HMGA2 was investigated as a target of miR-98-5p currently. Luciferase reporter assay was conducted and the correlation of miR-98-5p and HMGA2 was validated. Moreover, it was displayed that HMGA2 was remarkably elevated in db/db mice and mice mesangial cells. Furthermore, miR-98-5p strongly depressed HMGA2 protein and mRNA levels in mice mesangial cells. Overall, these revealed miR-98-5p could suppress the EMT process and renal fibrosis through targeting HMGA2 in DN.

Tissue-resident natural killer cells exacerbate tubulointerstitial fibrosis by activating transglutaminase 2 and syndecan-4 in a model of aristolochic acid-induced nephropathy

Despite reports suggesting that tissue-resident natural killer (trNK) cells cause ischemic kidney injury, their contribution to the development of tubulointerstitial fibrosis has not been determined. This study hypothesized that the depletion of trNK cells may ameliorate renal fibrosis by affecting transglutaminase 2/syndecan-4 interactions. Aristolochic acid nephropathy (AAN) was induced in C57BL/6 mice as an experimental model of kidney fibrosis. The mice were treated with anti-asialo GM1 (ASGM1) or anti-NK1.1 antibodies to deplete NK cells. Although both ASGM1 and NK1.1 antibodies suppressed renal NKp46+DX5+ NK cells, renal NKp46+DX5− cells were resistant to suppression by ASGM1 or NK1.1 antibodies during the development of tubulointerstitial fibrosis in the AAN-induced mouse model. Western blot analysis showed that both antibodies increased the expression of fibronectin, transglutaminase 2, and syndecan-4. These findings indicate that trNK cells played an exacerbating role in tubulointerstitial fibrosis by activating transglutaminase 2 and syndecan-4 in the AAN-induced mouse model.

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.

MiR-30c protects diabetic nephropathy by suppressing epithelial-to-mesenchymal transition in db/db mice.

SummaryEpithelial‐to‐mesenchymal transition (EMT) plays a significant role in tubulointerstitial fibrosis, which is a hallmark of diabetic nephropathy. Thus, identifying the mechanisms of EMT activation could be meaningful. In this study, loss of miR‐30c accompanied with increased EMT was observed in renal tubules of db/db mice and cultured HK2 cells exposed to high glucose. To further explore the roles of miR‐30c in EMT and tubulointerstitial fibrosis, recombinant adeno‐associated viral vector was applied to manipulate the expression of miR‐30c. In vivo study showed that overexpression of miR‐30c suppressed EMT, attenuated renal tubulointerstitial fibrosis and reduced proteinuria, serum creatinine, and BUN levels. In addition, Snail1 was identified as a direct target of miR‐30c by Ago2 co‐immunoprecipitation, luciferase reporter, and Western blot assays. Downregulating Snail1 by siRNA reduced high glucose‐induced EMT in HK2 cells, and miR‐30c mimicked the effects. Moreover, miR‐30c inhibited Snail1‐TGF‐β1 axis in tubular epithelial cells undergoing EMT and thereby impeded the release of TGF‐β1; oppositely, knockdown of miR‐30c enhanced the secretion of TGF‐β1 from epitheliums and significantly promoted proliferation of fibroblasts and fibrogenesis of myofibroblasts, aggravated tubulointerstitial fibrosis, and dysfunction of diabetic nephropathy. These results suggest a protective role of miR‐30c against diabetic nephropathy by suppressing EMT via inhibiting Snail1‐TGF‐β1 pathway.

Loss of the Protein Cystathionine β-Synthase During Kidney Injury Promotes Renal Tubulointerstitial Fibrosis

Background/Aims: Renal tubulointerstitial fibrosis (TIF) is the common pathway of progressive chronic kidney disease. Inflammation has been widely accepted as the major driving force of TIF. Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway. CBS is considered to play protective role in liver and pulmonary fibrosis, but its role in TIF remains unknown. The purpose of this study was to investigate the potential role and mechanism of CBS in renal inflammation and TIF. Methods: Renal function, tubulointerstitium damage index score, extracellular matrix (ECM) deposition, and the expressions of collagen I, collagen III, fibronectin, CD3, CD68, IL-1β, TNF-α were measured in sham operation and unilateral ureteral obstruction (UUO) rats. Proteomics and gene array analysis were performed to screen differentially expressed molecules in the development of renal inflammation and TIF in UUO rats. The expression of CBS was detected in patients with obstructive nephropathy and UUO rats. We confirmed the expression of CBS using western blot and real-time PCR in HK-2 cells. Overexpression plasmid and siRNA were transfected specifically to study the possible function of CBS in HK-2 cells. Results: Abundant expression of CBS, localized in renal tubular epithelial cells, was revealed in human and rat renal tissue, which correlated negatively with the progression of fibrotic disease. Expression of CBS was dramatically decreased in the obstructed kidney from UUO rats as compared with the sham group (SHM). In addition, knocking down CBS exacerbated extracellular matrix (ECM) deposition, whereas CBS overexpression attenuated TGF-β1-induced ECM deposition in vitro. Inflammatory and chemotactic factors were also increased in CBS knockdown HK-2 cells stimulated by IL-1β. Conclusions: These findings establish CBS as a novel inhibitor in renal fibrosis and as a new therapeutic target in patients with chronic kidney disease.

Interaction between V-ATPase B2 and (Pro) renin Receptors in Promoting the progression of Renal Tubulointerstitial Fibrosis.

To investigate the levels of (Pro) renin receptor [(P) RR], α-smooth muscle actin (α-SMA), fibronectin (FN), and vacuolar H+-ATPase (V-ATPase) subunits (B2, E, and c) in rat unilateral ureteral obstruction (UUO) models and rat proximal tubular epithelial cells (NRK-52E) treated with prorenin to elucidate the role of V-ATPase in these processes by activating the (P) RR. UUO significantly upregulated (P) RR, V-ATPase subunits, α-SMA and FN expression in tubulointerstitium or tubular epithelial cells. A marked colocalization of (P) RR and the B2 subunit was also observed. Prorenin treatment upregulated α-SMA, FN, (P) RR, and V-ATPase subunits and activity in NRK52E cell in a dose- and time-dependent manner. The V-ATPase inhibitor bafilomycin A1 partially blocked prorenin-induced (P) RR, FN, and α-SMA expression. Co-immunoprecipitate and immunofluorescence results demonstrated that the V-ATPase B2 subunit bound to the (P) RR, which was upregulated after prorenin stimulation. Either siRNA-mediated (P) RR or B2 subunit knockdown partially reduced V-ATPase activity and attenuated prorenin-induced FN and α-SMA expression. From the data we can assume that activation of (P) RR and V-ATPase may play an important role in tubulointerstitial fibrosis with possible involvement of interaction of V-ATPase B2 subunit and (P)RR.

Adenosine Receptors Are Up-Regulated in Unilateral Ureteral Obstructed Rat Kidneys

Despite recent improvements in immunosuppressive regimens, chronic renal allograft rejection remains a major problem. Tubulointerstitial fibrosis is one of the major histological features of chronic renal allograft rejection, but its exact pathogenic mechanisms are not fully understood. Adenosine present in the normal kidney is significantly elevated in response to cellular damage. The cellular effect of adenosine occurs through 4 known adenosine receptor (AR) subtypes; A1AR, A2AAR, A2BAR, and A3AR. All AR subtypes are expressed in the kidney, but the expression of each AR subtype has not been defined under fibrotic conditions. In the present study, we examined AR subtype expression in kidneys that underwent unilateral ureteral obstruction (UUO), a well-characterized model for tubulointerstitial fibrosis. At 5 days after the induction of UUO, we observed α-smooth muscle actin (α-SMA), fibronectin, and collagen I messenger RNA (mRNA) and protein expressions to be significantly up-regulated in the obstructed compared with the sham kidneys, confirming that fibrosis had occurred in the former organs. A1AR mRNA expression in the obstructed kidney cortex was 3.2-fold higher than an the sham kidney cortex. Relative to the sham kidney A2AAR and A2BAR mRNA expressions were also 2.6- and 2.0-fold increased, respectively. A3AR mRNA expression in the obstructed kidney cortex was also up-regulated by 3.3-fold. These data demonstrated that all 4 subtypes of AR were increased in the obstructed kidney, which was accompanied by tubulointerstitial fibrosis. Further studies are needed to determine which subtypes of AR play a protective or pathogenic role in tubulointerstitial fibrosis.

Angiotensin II Induces Epithelial-to-Mesenchymal Transition in Renal Epithelial Cells through Reactive Oxygen Species/Src/Caveolin-Mediated Activation of an Epidermal Growth Factor Receptor–Extracellular Signal-Regulated Kinase Signaling Pathway

Chronic activation of the renin-angiotensin system plays a deleterious role in progressive kidney damage, and the renal proximal tubule is known to play an important role in tubulointerstitial fibrosis; however, the underlying molecular mechanism is unclear. Here we report that in the proximal tubule-like LLCPKcl4 cells expressing angiotensin II (Ang II) type 1 receptor, Ang II induced changes in cell morphology and expression of epithelial-to-mesenchymal transition (EMT) markers, which were inhibited by the miotogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (ERK)-activating kinase (MEK) inhibitor PD98059 or the Src kinase inhibitor PP2. Ang II-stimulated phosphorylation of caveolin-1 (Cav) at Y14 and epidermal growth factor receptor (EGFR) at Y845 and induced association of these phosphoproteins in caveolin-enriched lipid rafts, thereby leading to prolonged EGFR-ERK signaling that was inhibited by Nox4 small interfering RNA (siRNA) and Src siRNA. Two different antioxidants not only inhibited phosphorylation of Src at Y416 but also blocked the EGFR-ERK signaling. Moreover, erlotinib (the EGFR tyrosine kinase inhibitor), EGFR siRNA, and Cav siRNA all inhibited both prolonged EGFR-ERK signaling and phenotypic changes induced by Ang II. Thus, this report provides the first evidence that reactive oxygen species (ROS)/Src-dependent activation of persistent Cav-EGFR-ERK signaling mediates renal tubular cell dedifferentiation and identifies a novel molecular mechanism that may be involved in progressive renal injury caused by chronic exposure to Ang II.

Cordycepin inhibits albumin-induced epithelial-mesenchymal transition of renal tubular epithelial cells by reducing reactive oxygen species production

Albumin induced epithelial-mesenchymal transition (EMT) of renal tubular cells through reactive oxygen species (ROS) pathway plays an important role in tubulointerstitial fibrosis. Cordycepin (3 -deoxyadenosine), a potential antioxidant, was demonstrated to have various pharmacological effects and could inhibit EMT of some cells. However, the role of cordycepin on albumin-induced EMT in renal tubular cells (HK2) is unclear. In this study, we investigated the effect of cordycepin on albumin-induced EMT of HK2 cells and its mechanisms. HK-2 cells were exposed to bovine serum albumin with or without pretreatment with cordycepin. Results showed that albumin significantly induced EMT formation of HK-2 which associated with NADPH oxidase activation and intracellular ROS overproduction through increased Rac1 activity and expression of NOX4, p22phox and p47phox, while these effects were abolished in that pretreated with cordycepin. In conclusion, cordycepin could ameliorate albumin-induced EMT of HK2 cells by decreasing NADPH oxidase activity and inhibiting ROS production.

Cellular and Molecular Basis of Epithelial-Mesenchymal Transition in Renal Fibrosis

Regardless of the underlying etiology, tubulointerstitial fibrosis is a common mechanism in the progression of chronic kidney disease (CKD) to end-stage renal disease. Epithelial-mesenchymal transition (EMT) of renal tubular cells plays an important role in tubulointerstitial fibrosis. Transforming growth factor-?1/Sma and Mad protein (TGF-?1/Smad) is thought to be a main signaling pathway for EMT of renal tubular cells. Progressive renal disease is also characterized histologically by an interstitial infiltrate of mononuclear cells. The chemokines secreted from renal tubular cells can trigger integrin-dependent adhesion of circulating mononuclear cells that leads to infiltration at tubulointerstitial space. The direct interaction of integrin lymphocyte function-associated antigen 1(LFA-1: ?L?2 integrin) on mononuclear cells and its ligand, intracellular adhesion molecule-1(ICAM-1) on renal tubular epithelial cells, contributes to a part of the EMT of renal tubular cells.

Mast cells are required for the development of renal fibrosis in the rodent unilateral ureteral obstruction model

Mast cells are associated with inflammation and fibrosis. Whether they protect against or contribute to renal fibrosis is unclear. Based on our previous findings that mast cells can express and secrete active renin, and that angiotensin (ANG II) is profibrotic, we hypothesized that mast cells play a critical role in tubulointerstitial fibrosis. We tested this hypothesis in the 14-day unilateral ureteral obstruction (UUO) model in rats and mast cell-deficient (MCD) mice (WBB6F1-W/Wv) and their congenic controls (CC). In the 14-day UUO rat kidney, mast cell number is increased and they express active renin. Stabilizing mast cells in vivo with administration of cromolyn sodium attenuated the development of tubulointerstitial fibrosis, which was confirmed by measuring newly synthesized pepsin-soluble collagen and blind scoring of fixed trichrome-stained kidney sections accompanied by spectral analysis. Fibrosis was absent in UUO kidneys from MCD mice unlike that observed in the CC mice. Losartan treatment reduced the fibrosis in the CC UUO kidneys. The effects of mast cell degranulation and renin release were tested in the isolated, perfused kidney preparation. Mast cell degranulation led to renin-dependent protracted flow recovery. This demonstrates that mast cell renin is active in situ and the ensuing ANG II can modulate intrarenal vascular resistance in the UUO kidney. Collectively, the data demonstrate that mast cells are critical to the development of renal fibrosis in the 14-day UUO kidney. Since renin is present in human kidney mast cells, our work identifies potential targets in the treatment of renal fibrosis.

Expression of transcription factor Snai1 and tubulointerstitial fibrosis in progressive nephropathy

Tubulointerstitial fibrosis (TIF) is seen as the final stage of progressive nephropathy, and the degree of TIF is reported to be a major determinant in renal outcomes. In recent years, epithelial-mesenchymal transition (EMT) and the zinc-finger transcription factor snail homolog 1 (Snai1) have each been implicated in the mechanism of TIF. The relationship between EMT and these transcription factors is unclear, however, so in this study we attempted to elucidate the correlation between the expression of Snai1 and clinical markers. We performed immunohistochemical staining on human renal tissue obtained from patients with diabetic nephropathy (DN), IgA nephropathy (IgAN), minimal change disease (MCD) and minor glomerular abnormality (MGA) using anti-Snai1 and anti-vimentin antibodies. We counted Snai1-positive and Snai1/vimentin double positive tubular epithelial cells. Snai1 protein was mainly observed in the nuclei of flattened, damaged tubular epithelial cells, especially in IgAN and DN, and positive cell numbers were significantly higher in IgAN than in MGA, MCD or DN. Snai1/vimentin double staining showed that some vimentin-positive tubular epithelial cells also contained Snai1-positive nuclei, and double positive cell numbers were increased in IgAN and DN. Statistical analysis revealed positive correlations between Snai1/vimentin double positive cell numbers and proteinuria and creatinine in IgAN. Positive correlations were also seen between Snai1/vimentin double positive cell numbers and the severity of proteinuria in DN. The results of this study indicate that Snai1 plays an important role in TIF in patients with progressive nephropathy.