Kidneys Of Unilateral Ureteral Obstruction Mice Research Articles

Natural flavonoid pectolinarigenin alleviated kidney fibrosis via inhibiting the activation of TGFβ/SMAD3 and JAK2/STAT3 signaling

Renal fibrosis is a final common manifestation of CKD resulting in progressive loss of kidney function. The activation of SMAD3 and STAT3 played central roles in the pathogenesis of renal fibrosis, which has been recognized as potential targets for antifibrotic therapy. As we known, the potential of natural products as the candidates for drug discovery has been well recognized. Here, we identified that pectolinarigenin (PEC), as a natural flavonoid and a reported STAT3 inhibitor, dose-dependently suppressed TGFβ/SMADs activity in HEK293 cells by luciferase reporter assay. In TGFβ1-stimulated NRK-49F fibroblast, PEC blocked the phosphorylation of SMAD3 and STAT3, and downregulated the major fibrotic gene and protein expression of TGFβ, α-SMA, COL-1, and FN. Notably, oral administration of PEC at a dose of 25 mg/kg/d for 7 days or 14 days effectively ameliorated kidney injury and tubulointerstitial fibrosis after unilateral ureteral obstruction (UUO) surgery in mice. Mechanically, PEC treatment inhibited the phosphorylated activation of SMAD3 and STAT3, which further reduced the protein expression of TGFβ, α-SMA, COL-1, and FN in the obstructed kidneys of UUO mice. In summary, our results suggested that pectolinarigenin alleviated tubulointerstitial fibrosis by inhibiting the activation of SMAD3 and STAT3 signaling.

Inhibition of Fatty Acid-Binding Protein 4 Attenuated Kidney Fibrosis by Mediating Macrophage-to-Myofibroblast Transition.

The macrophage-to-myofibroblast transition (MMT) process is an important pathway that contributing to renal interstitial fibrosis (RIF). Fatty acid–binding protein 4 (FABP4) deteriorated RIF via promoting inflammation in obstructive nephropathy. However, the clinical significance of FABP4 in fibrotic kidney disease remains to be determined and little is known of the FABP4 signaling in MMT. Biopsy specimens of chronic kidney disease patients and kidneys subjected to unilateral ureteral obstruction (UUO) of FABP4-deficient mice or FABP4 inhibitor-treated mice were collected for the investigation of FABP4 mediating MMT of RIF. We conducted kidney RNA-seq transcriptomes and TGF-β1-induced bone marrow–derived macrophage (BMDM) assays to determine the mechanisms of FABP4. We found that FABP4 expression correlated with RIF in biopsy specimens and the injured kidneys of UUO mice where FABP4 was co-expressed with MMT cells. In UUO mice, FABP4 deficiency and a highly selective FABP4 inhibitor BMS309403 treatment both suppressed RIF. FABP4 ablation also attenuated the UUO-induced number of MMT cells and serum amyloid A1 (Saa1) expression. The siRNA-mediated Saa1 knockdown decreased the number of MMT cells in vitro. In conclusion, FABP4 is an important factor contributing to RIF by mediating MMT, and genetic/pharmacological inhibition of FABP4 provides a novel approach for the treatment of kidney fibrosis.

Withaferin A protects against endoplasmic reticulum stress-associated apoptosis, inflammation, and fibrosis in the kidney of a mouse model of unilateral ureteral obstruction

BackgroundWithaferin A is a functional ingredient of a traditional medicinal plant, Withania somnifera, which has been broadly used in India for protecting against chronic diseases. This bioactive steroidal lactone possesses multiple functions such as anti-oxidation, anti-inflammation, and immunomodulation. Chronic kidney disease (CKD) is one of the major health problems worldwide with the high complication, morbidity, and mortality rates. The detailed effects and underlying mechanisms of withaferin A on CKD progression still remain to be clarified. PurposeWe aimed to investigate whether withaferin A treatment ameliorates the development of renal fibrosis and its related mechanisms in a CKD mouse model. MethodsA mouse model of unilateral ureteral obstruction (UUO) was used to mimic the progression of CKD. Male adult C57BL/6J mice were orally administered with 3 mg/kg/day withaferin A for 14 consecutive days after UUO surgery. Candesartan (5 mg/kg/day) was used as a positive control. ResultsBoth Withaferin A and candesartan treatments significantly ameliorated the histopathological changes and collagen deposition in the UUO kidneys. Withaferin A could significantly reverse the increases in the protein levels of pro-fibrotic factors (fibronectin, transforming growth factor-β, and α-smooth muscle actin), inflammatory signaling molecules (phosphorylated nuclear factor-κB-p65, interleukin-1β, and cyclooxygenase-2), and cleaved caspase-3, apoptosis, and infiltration of neutrophils in the UUO kidneys. The protein levels of endoplasmic reticulum (ER) stress-associated molecules (GRP78, GRP94, ATF4, CHOP, phosphorylated eIF2α, and cleaved caspase 12) were increased in the kidneys of UUO mice, which could be significantly reversed by withaferin A treatment. ConclusionWithaferin A protects against the CKD progression that is, at least in part, associated with the moderation of ER stress-related apoptosis, inflammation, and fibrosis in the kidneys of CKD. Withaferin A may serve as a potential therapeutic agent for the development of CKD.

Melatonin ameliorates renal fibroblast-myofibroblast transdifferentiation and renal fibrosis through miR-21-5p regulation.

Fibroblast‐myofibroblast transdifferentiation (FMT) is widely recognized as the major pathological feature of renal fibrosis. Although melatonin has exerted antifibrogenic activity in many diseases, its role in renal FMT remains unclear. In the present study, the aim was to explore the effect of melatonin on renal FMT and the underlying mechanisms. We established the transforming growth factor (TGF)‐β1 stimulated rat renal fibroblast cells (NRK‐49F) model in vitro and unilateral ureteral obstruction (UUO) mice model in vivo. We assessed levels of α‐smooth muscle actin (α‐SMA), col1a1 and fibronectin, STAT3 and AP‐1, as well as miR‐21‐5p and its target genes (Spry1, PTEN, Smurf2 and PDCD4). We found that melatonin reduced the expression of α‐SMA, col1a1 and fibronectin, as well as the formation of α‐SMA filament in TGF‐β1‐treated NRK‐49F cells. Meanwhile, melatonin inhibited STAT3 phosphorylation, down‐regulated miR‐21‐5p expression, and up‐regulated Spry1 and PTEN expression. Moreover, miR‐21‐5p mimics partially antagonized the anti‐fibrotic effect of melatonin. For animal experiments, the results revealed that melatonin remarkably ameliorated UUO‐induced renal fibrosis, attenuated the expression of miR‐21‐5p and pro‐fibrotic proteins and elevated Spry1 and PTEN expression. Nevertheless, agomir of miR‐21‐5p blocked the renoprotective effect of melatonin in UUO mice. These results indicated that melatonin could alleviate TGF‐β1‐induced renal FMT and UUO‐induced renal fibrosis through down‐regulation of miR‐21‐5p. Regulation of miR‐21‐5p/PTEN and/or miR‐21‐5p/Spry1 signal might be involved in the anti‐fibrotic effect of melatonin in the kidneys of UUO mice.

Exogenous miR-29a Attenuates Muscle Atrophy and Kidney Fibrosis in Unilateral Ureteral Obstruction Mice.

Renal fibrosis leads to end-stage renal disease, but antifibrotic drugs are difficult to develop. Chronic kidney disease often results in muscle wasting, and thereby increases morbidity and mortality. In this work, adeno-associated virus (AAV)-mediated overexpressing miR-29a was hypothesized to counteract renal fibrosis and muscle wasting through muscle-kidney crosstalk in unilateral ureteral obstruction (UUO) mice. miR-29a level was downregulated in the kidney and skeletal muscle of UUO mice. The secretion of exosome-encapsulated miR-29a increased in cultured skeletal muscle satellite cells and HEK293 renal cells after stimulation with serum from UUO mice. This result was confirmed by qPCR and microRNA deep sequencing in the serum exosomes of mice with obstructed ureters. A recombinant AAV-miR-29a was generated to overexpress miR-29a and injected into the tibialis anterior muscle of the mice 2 weeks before UUO surgery. AAV-miR-29a abrogated the UUO-induced upregulation of YY1 and myostatin in skeletal muscles. Renal fibrosis was also partially improved in the UUO mice with intramuscular AAV-miR-29a transduction. AAV-miR-29a overexpression reversed the increase in transforming growth factor β, fibronectin, alpha-smooth muscle actin, and collagen 1A1 and 4A1 levels in the kidney of UUO mice. AAV-green fluorescent protein was applied to trace the AAV route in vivo, and fluorescence was significantly visible in the injected/uninjected muscles and in the kidneys. In conclusion, intramuscular AAV-miR-29a injection attenuates muscle wasting and ameliorates renal fibrosis by downregulating several fibrotic-related proteins in UUO mice.

Tamibarotene inhibit the accumulation of fibrocyte and alleviate renal fibrosis by IL-17A

Renal fibrosis is a common pathological process in the progression of chronic kidney disease. Accumulating evidence suggests that interleukin-17A (IL‐17A) and fibrocytes play crucial roles in the pathogenesis of fibrosis. However, the role of IL-17A in the regulation of renal fibrocytes in renal fibrosis has rarely been reported. Here, we report that the plasma IL-17A level is increased in immunoglobulin A nephropathy (IgAN) patients and is correlated with clinical parameters. Using a mouse model of unilateral ureteral obstruction (UUO), we found that both IL-17A expression and fibrocyte infiltration were increased in the kidneys of UUO mice. Besides, IL-17A enhanced fibrosis and fibrocyte-associated chemokine and activator expression in vitro. Furthermore, inhibition of IL-17A using Am80 (Tamibarotene) decreased fibrocytes and fibrocyte-associated chemokine and activator expression and significantly attenuated renal fibrosis in the UUO mice. Our findings suggest that Am80, which inhibits the accumulation of fibrocytes and alleviates renal fibrosis mediated by IL-17A, maybe a novel therapeutic drug for renal fibrosis.

Pioglitazone attenuates kidney fibrosis via miR-21-5p modulation

Pioglitazone has been demonstrated to exert anti-fibrotic and renoprotective effects. But the detailed pharmacological mechanisms have not been clearly revealed. The present study aimed to investigate the possible mechanisms of pioglitazone in these two effects. TGF-β1-stimulated HK-2 cells and unilateral ureteral obstruction (UUO) mice were used as in vitro and in vivo models. The results showed that pioglitazone inhibited Smad-2/3 phosphorylation, upregulated Smad-7 expression and downregulated miR-21-5p expression in TGF-β1-exposed HK-2 cells. In addition, miR-21-5p inhibitors replicated the anti-fibrotic effects of pioglitazone, and miR-21-5p mimics inhibited these effects. In in vivo study, pioglitazone attenuated UUO-induced renal fibrosis and significantly decreased the expressions of pro-fibrotic proteins. Whereas, agomir of miR-21-5p inhibited the renoprotective function of pioglitazone in UUO mice. In conclusion, the present data suggest that modulation of miR-21-5p/Smad-7 signal may be involved in the anti-fibrotic effect of pioglitazone in the kidney of UUO mice.

Petchiether A attenuates obstructive nephropathy by suppressing TGF‐β/Smad3 and NF‐κB signalling

Obstructive nephropathy is the end result of a variety of diseases that block drainage from the kidney(s). Transforming growth factor‐β1 (TGF‐β1)/Smad3‐driven renal fibrosis is the common pathogenesis of obstructive nephropathy. In this study, we identified petchiether A (petA), a novel small‐molecule meroterpenoid from Ganoderma, as a potential inhibitor of TGF‐β1‐induced Smad3 phosphorylation. The obstructive nephropathy was induced by unilateral ureteral obstruction (UUO) in mice. Mice received an intraperitoneal injection of petA/vehicle before and after UUO or sham operation. An in vivo study revealed that petA protected against renal inflammation and fibrosis by reducing the infiltration of macrophages, inhibiting the expression of proinflammatory cytokines (interleukin‐1β and tumour necrosis factor‐α) and reducing extracellular matrix deposition (α‐smooth muscle actin, collagen I and fibronectin) in the obstructed kidney of UUO mice; these changes were associated with suppression of Smad3 and NF‐κB p65 phosphorylation. Petchiether A inhibited Smad3 phosphorylation in vitro and down‐regulated the expression of the fibrotic marker collagen I in TGF‐β1‐treated renal epithelial cells. Further, we found that petA dose‐dependently suppressed Smad3‐responsive promoter activity, indicating that petA inhibits gene expression downstream of the TGF‐β/Smad3 signalling pathway. In conclusion, our findings suggest that petA protects against renal inflammation and fibrosis by selectively inhibiting TGF‐β/Smad3 signalling.

The antifibrotic and anti-inflammatory effects of icariin on the kidney in a unilateral ureteral obstruction mouse model

BackgroundThe pathology change of renal tubulointerstitial fibrosis is a critical feature of chronic kidney disease (CKD), regardless of the primary insults. The infiltration of inflammatory cells and the consecutive secretion of profibrotic factors are frequently and conspicuously observed during the development of renal fibrosis. Icariin, an active polyphenol of the Epimedium genus, has been found to alleviate the symptoms of chronic diseases like diabetes, neurodegeneration, and heart and renal diseases. The effect and mechanism of icariin on the prevention of CKD-associated renal fibrosis still needed clarification. PurposeThe aims of this study were to investigate whether icariin treatment improves the development of CKD-associated renal fibrosis and its possible mechanism. MethodsAn experimental unilateral ureteral obstruction (UUO)-induced chronic renal fibrosis mouse model was used. Mice were orally administered with icariin (20 mg/kg/day) for 3 consecutive days before and 14 consecutive days after UUO surgery. ResultsThe pathological changes, collagen deposition, and protein expressions of profibrotic factors (transforming growth factor-β and connective tissue growth factor) and fibrotic markers (α-smooth muscle actin and fibronectin), which were significantly elevated in the kidneys of UUO mice, could be significantly reversed by icariin treatment. Icariin treatment also significantly inhibited the increased Smad2/3 and decreased E-cadherin protein expressions in the kidneys of UUO mice. Icariin treatment prominently mitigated the protein expression of proinflammatory factors like nuclear factor-κB, cyclooxygenase-2, interleukin 1-β and prooxidative enzyme (NADPH oxidase-4), and it increased the protein expression of antioxidative enzymes (superoxide dismutase and catalase). ConclusionIcariin treatment protects against CKD-associated renal fibrosis via its antifibrotic and anti-inflammatory properties. Icariin may serve as a therapeutic agent in the prevention of CKD-associated renal fibrosis.

Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice.

Our previous study showed that miR-29 attenuates muscle wasting in chronic kidney disease. Other studies found that miR-29 has anti-fibrosis activity. We hypothesized that intramuscular injection of exosome-encapsulated miR-29 would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis. We used an engineered exosome vector, which contains an exosomal membrane protein gene Lamp2b that was fused with the targeting peptide RVG (rabies viral glycoprotein peptide). RVG directs exosomes to organs that express the acetylcholine receptor, such as kidney. The intervention of Exo/miR29 increased muscle cross-sectional area and decreased UUO-induced upregulation of TRIM63/MuRF1 and FBXO32/atrogin-1. Interestingly, renal fibrosis was partially depressed in the UUO mice with intramuscular injection of Exo/miR29. This was confirmed by decreased TGF-β, alpha-smooth muscle actin, fibronectin, and collagen 1A1 in the kidney of UUO mice. When we used fluorescently labeled Exo/miR29 to trace the Exo/miR route invivo and found that fluorescence was visible in un-injected muscle and in kidneys. We found that miR-29 directly inhibits YY1 and TGF-β3, which provided a possible mechanism for inhibition of muscle atrophy and renal fibrosis by Exo/miR29. We conclude that Exo/miR29 ameliorates skeletal muscle atrophy and attenuates kidney fibrosis by downregulating YY1 and TGF-β pathway proteins.

Exogenous miR-29a Attenuates Muscle Atrophy and Kidney Fibrosis in UUO Mice

Our previous study found that miR-29a was decreased in skeletal muscle of mice with renal disease. However, the cause of decreasing miR-29a and the influence other organs is less known. We hypothesized that adeno-associated virus (AAV)-mediated overexpressing miR-29a would counteract unilateral ureteral obstruction (UUO)-induced muscle wasting and renal fibrosis via muscle-kidney crosstalk. The expression of miR-29a was decreased in skeletal muscle and kidney of UUO mice. Serum from UUO mice enhanced secretion of exosome-encapsulated miR-29a from cultured skeletal muscle satellite cells and HEK293 renal cells. This was confirmed by qPCR and microRNA deep sequencing in serum exosomes of mice with obstructed ureters. We generated a recombinant adeno-associated virus (AAV-miR29a) to overexpress miR-29a and injected it into the tibialis anterior (TA) muscle of UUO mice for 14-days. The AAV-miR-29a decreased UUO-induced upregulation of YY1 and myostatin in skeletal muscle. In addition, renal fibrosis was partially depressed in the UUO mice with intramuscular transduction with AAV-miR-29a. We found that TGF, alpha-smooth muscle actin (SMA), fibronectin, collagen 1A1 and 4A1 were increased in the kidney of UUO mice, overexpressing by AAV-miR-29a reversed these changes. When we used AAV-GFP to trace the AAV route in vivo we found that fluorescence was significantly visible in both injected and un-injected muscle and in kidneys. We conclude that intramuscular injection of AAV-miR-29a not only ameliorates skeletal protein metabolism, but also attenuates UUO-induced kidney fibrosis by down-regulating TGF- pathway cascade proteins and extracellular matrix components. Funding Statement: Research reported in this publication was supported by grants from the National Natural Science Foundation of China (Nos. 81700650, 81700618, 31772690), and the National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) of the National Institutes of Health under Award Number R01 AR060268 and American Heart Association Discover and Innovation Grants (17IBDG33780000) to X.H.W. and the Natural Science Foundation of Jiangsu Province (BK20181487), China Young Nephrologist Research Project, and Southeast University High Level Thesis Project to B. W. Declaration of Interests: The authors state no conflicts of interest. Ethics Approval Statement: These experiments were approved by the Emory University IACUC (protocol 141-2008).

Correction: Treatment combining aliskiren with paricalcitol is effective against progressive renal tubulointerstitial fibrosis via dual blockade of intrarenal renin

[This corrects the article DOI: 10.1371/journal.pone.0181757.].

Treatment combining aliskiren with paricalcitol is effective against progressive renal tubulointerstitial fibrosis via dual blockade of intrarenal renin

The aim of this study was to assess any potential additive effects of a treatment combining aliskiren with paricalcitol on reducing renal fibrosis. C57BL/6J mice were treated individually with aliskiren and/or paricalcitol until 7 days after initiation of unilateral ureteral obstruction (UUO).In obstructed kidneys of UUO mice, monotherapy with aliskiren or paricalcitol significantly attenuated interstitial fibrosis, collagen IV accumulation, and α-smooth muscle actin- and terminal deoxynucleotidyl transferase-mediated biotin nick end-labeling-positive cells. The combination treatment showed additive efficacy in inhibition of these parameters. Renal NADPH oxidase (Nox)1 and Nox2 were significantly decreased by aliskiren or paricalcitol alone or in combination, while renal Nox4 expression was significantly reduced by paricalcitol mono- or combination treatment. Increased levels of p-Erk and p-p38 MAPK, and NF-κB in UUO kidneys were also significantly reduced by either aliskiren or paricalcitol treatment alone or in combination. Aliskiren or paricalcitol monotherapy significantly reduced the expression of (pro)renin receptor in UUO kidneys. In addition, aliskiren tended to augment renin expression in UUO kidneys, but paricalcitol reduced its expression level. The combination treatment effectively blocked both (pro)renin receptor and renin expression induced by aliskiren, and resulted in a further reduction of the renal expression of angiotensin II AT1 receptor. Aliskiren failed to increase the expression of vitamin D receptor in UUO kidneys, but the combination treatment restored its expression level. Taken together, a treatment combining aliskiren with paricalcitol better inhibits UUO-induced renal injury. The mechanism of this synergy may involve more profound inhibition of the intrarenal renin-angiotensin system.

PS 04-01] ALISKIREN ENHANCES ANTIFIBROTIC EFFECT OF PARICALCITOL IN OBSTRUCTIVE NEPHROPATHY

Objective: Besides the effect on blood pressure, a previous experiment has reported the renoprotective effect of aliskiren, a direct rennin inhibitor, against unilateral ureteral obstruction (UUO)-induced renal fibrosis and inflammation. Paricalcitol, a vitamin D activator, has also been shown to inhibit renal inflammation and attenuate renal interstitial fibrosis. The aim of this study was to assess any potential additive effects of a therapy combining aliskiren with paricalcitol on reducing renal fibrosis. Design and Method: C57BL6/J mice were treated individually with aliskiren or paricalcitol or their combinations until 7 days after initiation of UUO. Results: In obstructed kidneys of UUO mice, monotherapy with aliskiren or paricalcitol significantly attenuated interstitial fibrosis, collagen deposition and α-smooth muscle actin compared to vehicle-treated UUO control. Levels of renal Nox1 and Nox4 were significantly attenuated with either aliskiren or paricalcitol. The therapy combining aliskiren with paricalcitol showed additive efficacy in inhibition of these parameters. Increased levels of phospho-ERK and phosphor-p38 MAPK in UUO kidneys were also significantly reduced by either aliskiren or paricalcitol treatment alone or in their combinations. Aliskiren seemed to be more potent than paricalcitol in suppressing angiotensin II type 1 receptor expression in obstructed kidneys. Although aliskiren significantly augmented renin expression in UUO kidneys, paricalcitol decreased intrarenal renin expression, and combination therapy with both effectively blocked the renin expression induced by aliskiren. Monotherapy with either aliskiren or paricalcitol significantly reduced the expression of (pro)renin receptor in UUO kidneys and a combination of both resulted in a further reduction of the renal expression of (pro)renin receptor. Conclusions: These results suggest that combination therapy with aliskiren and paricalcitol has a better effect in inhibiting UUO-induced renal fibrosis. This mechanistic basis for this synergy may be attributed to a more profound inhibition of intrarenal RAS activity through interfering with the renin activity by aliskiren and inhibition of renin expression by paricalcitol.

PS 01-09] T-TYPE CALCIUM CHANNEL BLOCKER ATTENUATES UNILATERAL URETERAL OBSTRUCTION-INDUCED RENAL INTERSTITIAL FIBROSIS VIA ACTIVATION OF THE NRF2 ANTIOXIDANT PATHWAY

Objective: Besides the effect on high blood pressure, T-type calcium channel blocker has been reported to exert a renoprotective effect in experimental models with renal fibrosis. However, the exact mechanism of T-type calcium channel blocker on tubulointerstitial fibrosis has not been fully elucidated. In the present study, we investigated whether the renoprotective effect of T-type calcium channel blocker is associated with modulation of the signaling of oxidative stress-induced renal fibrosis. Design and Method: Treatment with a nonhypotensive dose of efonidipine, a T-type calcium channel blocker, or nifedipine, an L-type channel blocker, was initiated one day before unilateral ureteral obstruction (UUO) in C57BL6/J mice, and was continued until 3 and 7 days after UUO. Markers of renal fibrosis and oxidative stress were evaluated. Results: In the obstructed kidneys of UUO mice, treatment with efonidipine significantly attenuated interstitial fibrosis, collagen deposition and inflammation increased by UUO creation compared with treatment with nifedipine. Additionally, efonidipine significantly increased the expression of antioxidant enzymes such as HO-1, NQO1, catalase and SOD1. Increased apoptotic cell death and decreased Bcl-2 expression in the obstructed kidneys were also significantly ameliorated by treatment with efonidipine. Furthermore, the expression of the histone acetyltransferase p300/CBP-associated factor, which is known as a regulator of inflammatory molecules, was significantly inhibited by efonidipine. These beneficial effects of efonipidine were attributed to the increased nuclear expression of nuclear factor-erythroid-2-related factor 2 (Nrf2) on UUO day 3 and the increased expressions of both total and nuclear Nrf2 with elevated Kelch-like ECH-associated protein 1 on UUO day 7, suggesting that efonidipine would promote activation of Nrf2 differently depending time course after UUO. By contrast, nifedipine had little effect on antioxidant enzymes, anti-apoptotic protein and Nrf2 signaling. Conclusions: These results suggest that T-type calcium channel blocker exerts beneficial effects in renal interstitial fibrosis by activating Nrf2 and subsequent antioxidant enzymes.

Effect of amlodipine on mouse renal interstitial fibrosis

Unilateral ureteral obstruction (UUO) is a well-established method to study interstitial fibrosis of the kidney. In this study, we investigated the effects of a calcium channel blocker, amlodipine, on UUO-induced renal interstitial fibrosis in mice. UUO significantly increased the fibrotic area in the obstructed kidney, but this change was inhibited by amlodipine (6.7mg/kg/day in drinking water). mRNA expression of heat shock protein (HSP) 47 and type IV collagen was increased in the kidneys of UUO mice. Amlodipine reduced the expression of both HSP47 and type IV collagen mRNAs. Phosphorylation of c-jun-N-terminal kinase (JNK) was significantly increased by UUO, but the change was inhibited by amlodipine. Collectively, these results suggest that amlodipine may inhibit the expression of HSP47 and type IV collagen by reducing phosphorylation of JNK and ameliorating the renal interstitial fibrosis induced by UUO.

Inhibition of SET Domain-Containing Lysine Methyltransferase 7/9 Ameliorates Renal Fibrosis.

TGF-β1 activity results in methylation of lysine 4 of histone H3 (H3K4) through SET domain-containing lysine methyltransferase 7/9 (SET7/9) induction, which is important for the transcriptional activation of fibrotic genes in vitro. However, in vivo studies utilizing an experimental model of renal fibrosis are required to develop therapeutic interventions that target SET7/9. In this study, we investigated the signaling pathway of TGF-β1-induced SET7/9 expression and whether inhibition of SET7/9 suppresses renal fibrosis in unilateral ureteral obstruction (UUO) mice and kidney cell lines. Among the SET family, SET7/9 was upregulated on days 3 and 7 in UUO mice, and the upregulation was suppressed by TGF-β1 neutralizing antibody. TGF-β1 induced SET7/9 expression via Smad3 in normal rat kidney (NRK)-52E cells. In human kidney biopsy specimens from patients diagnosed with IgA nephropathy and membranous nephropathy, SET7/9 expression was positively correlated with the degree of interstitial fibrosis (r=0.59, P=0.001 in patients with IgA nephropathy; and r=0.58, P<0.05 in patients with membranous nephropathy). In addition, small interfering RNA-mediated knockdown of SET7/9 expression significantly attenuated renal fibrosis in UUO mice. Sinefungin, an inhibitor of SET7/9, also suppressed the expression of mesenchymal markers and extracellular matrix proteins and inhibited H3K4 mono-methylation (H3K4me1) in kidneys of UUO mice. Moreover, sinefungin had an inhibitory effect on TGF-β1-induced α-smooth muscle actin expression and H3K4me1 in both NRK-52E and NRK-49F cells. In conclusion, sinefungin, a SET7/9 inhibitor, ameliorates renal fibrosis by inhibiting H3K4me1 and may be a candidate therapeutic agent.

Epigallocatechin-3-gallate reduces tubular cell apoptosis in mice with ureteral obstruction

BackgroundTubular cell apoptosis plays a crucial role in different kinds of renal diseases. Epigallocatechin-3-gallate (EGCG), a polyphenol extracted from green tea, has been shown to inhibit renal fibrosis in unilateral ureteral obstruction (UUO) mice, but its role in preventing tubular cell apoptosis and the underlying signaling mechanisms still remains unclear. Materials and methodsMice subjected to UUO were intraperitoneally administered EGCG (5 mg/kg) for 14 d. Normal rat kidney proximal tubular epithelial cell line NRK-52E was induced by transforming growth factor β1 (TGF-β1). Periodic acid–schiff and Masson's trichrome staining was used for histologic study. TUNEL, Hoechst staining, and flow cytometry analysis were used to measure the apoptotic status of tubular cells. Western blotting was used to determine the expression of apoptotic-associated proteins and mitogen-activated protein kinase pathway proteins. ResultsEGCG significantly attenuated tubular injury and renal tubulointerstitial fibrosis in the obstructed kidneys of UUO mice. In addition, EGCG prevented UUO and TGF-β1–induced tubular apoptosis in a dose-dependent manner. In parallel, protein expression of B-clell lymphoma-2 (Bcl-2) was upregulated and protein expressions of Bcl-2 accosiated X protein (Bax), cleaved caspase 3, and cleaved poly ADP-ribose polymerase (PARP) were downregulated by EGCG. Furthermore, UUO and TGF-β1–stimulated phosphorylation of mitogen-activated protein kinase was inhibited by EGCG. ConclusionsEGCG effectively reduces tubular cell apoptosis induced by UUO and may have potential as a clinical treatment in patients with chronic kidney disease.

Aliskiren restores renal AQP2 expression during unilateral ureteral obstruction by inhibiting the inflammasome.

Ureteral obstruction is associated with reduced expression of renal aquaporins (AQPs), urinary concentrating defects, and an enhanced inflammatory response, in which the renin-angiotensin system (RAS) may play an important role. We evaluated whether RAS blockade by a direct renin inhibitor, aliskiren, would prevent the decreased renal protein expression of AQPs in a unilateral ureteral obstruction (UUO) model and what potential mechanisms may be involved. UUO was performed for 3 days (3UUO) and 7 days (7UUO) in C57BL/6 mice with or without aliskiren injection. In 3UUO and 7UUO mice, aliskiren abolished the reduction of AQP2 protein expression but not AQP1, AQP3, and AQP4. mRNA levels of renal AQP2 and vasopressin type 2 receptor were decreased in obstructed kidneys of 7UUO mice, which were prevented by aliskiren treatment. Aliskiren treatment was also associated with a reduced inflammatory response in obstructed kidneys of UUO mice. Aliskiren significantly decreased mRNA levels of several proinflammatory factors, such as transforming growth factor-β and tumor necrosis factor-α, seen in obstructed kidneys of UUO mice. Interestingly, mRNA and protein levels of the NOD-like receptor family, pyrin domain-containing 3 (NLRP3) inflammasome components apoptosis-associated speck-like protein containing a caspase recruitment domain, caspase-1, and IL-1β were dramatically increased in obstructed kidneys of 7UUO mice, which were significantly suppressed by aliskiren. In primary cultured inner medullary collecting duct cells, IL-1β significantly decreased AQP2 expression. In conclusions, RAS blockade with the direct renin inhibitor aliskiren increased water channel AQP2 expression in obstructed kidneys of UUO mice, at least partially by preventing NLRP3 inflammasome activation in association with ureteral obstruction.

EW-7197 inhibits hepatic, renal, and pulmonary fibrosis by blocking TGF-β/Smad and ROS signaling.

Fibrosis is an inherent response to chronic damage upon immense apoptosis or necrosis. Transforming growth factor-beta1 (TGF-β1) signaling plays a key role in the fibrotic response to chronic liver injury. To develop anti-fibrotic therapeutics, we synthesized a novel small-molecule inhibitor of the TGF-β type I receptor kinase (ALK5), EW-7197, and evaluated its therapeutic potential in carbon tetrachloride (CCl4) mouse, bile duct ligation (BDL) rat, bleomycin (BLM) mouse, and unilateral ureteral obstruction (UUO) mouse models. Western blot, immunofluorescence, siRNA, and ChIP analysis were carried out to characterize EW-7197 as a TGF-β/Smad signaling inhibitor in LX-2, Hepa1c1c7, NRK52E, and MRC5 cells. In vivo anti-fibrotic activities of EW-7197 were examined by microarray, immunohistochemistry, western blotting, and a survival study in the animal models. EW-7197 decreased the expression of collagen, α-smooth muscle actin (α-SMA), fibronectin, 4-hydroxy-2, 3-nonenal, and integrins in the livers of CCl4 mice and BDL rats, in the lungs of BLM mice, and in the kidneys of UUO mice. Furthermore, EW-7197 extended the lifespan of CCl4 mice, BDL rats, and BLM mice. EW-7197 blocked the TGF-β1-stimulated production of reactive oxygen species (ROS), collagen, and α-SMA in LX-2 cells and hepatic stellate cells (HSCs) isolated from mice. Moreover, EW-7197 attenuated TGF-β- and ROS-induced HSCs activation to myofibroblasts as well as extracellular matrix accumulation. The mechanism of EW-7197 appeared to be blockade of both TGF-β1/Smad2/3 and ROS signaling to exert an anti-fibrotic activity. This study shows that EW-7197 has a strong potential as an anti-fibrosis therapeutic agent via inhibition of TGF-β-/Smad2/3 and ROS signaling.