Abnormally decreased renal Klotho is linked to endoplasmic reticulum-associated degradation in mice.

Aim: Endoplasmic reticulum-associated degradation (ERAD), which involves degradation of improperly folded proteins retained in the ER, is implicated in various diseases including chronic kidney disease. This study is aimed to determine the role of ERAD in Klotho deficiency of mice and human kidney tubular epithelial cells (HK-2) with renal interstitial fibrosis (RIF). Methods: Following establishment of a mouse RIF model by unilateral ureteral obstruction (UUO), a specific ERAD inhibitor, Eeyarestatin I (EerI), was administered to experimental animals by intraperitoneal injection. Serum and kidney samples were collected for analysis 10 days after operation. Soluble Klotho levels were measured by enzyme-linked immunosorbent assay, while the degree of kidney injury was assessed by renal histopathology. Renal Klotho expression was determined by quantitative real-time PCR, immunohistochemical and western blotting analyses. ERAD and unfolded protein response (UPR) were evaluated by detecting associated components such as Derlin-1, glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and protein disulfide isomerase (PDI). HK-2 cells were exposed to transforming growth factor (TGF)-ß1 with or without EerI, and expressions of related proteins including Klotho, Derlin-1, GRP78, ATF4 and PDI were determined by western blotting analyses. Results: UUO induced severe kidney injuries and RIF. Klotho expression in both serum and kidney tissue was obviously downregulated, while Derlin-1 was notably upregulated, indicating that ERAD was activated to potentially degrade improperly folded Klotho protein in this model. Intriguingly, treatment with EerI led to significantly increased Klotho expression, especially soluble (functional) Klotho. Furthermore, specific inhibition of ERAD increased expression of GRP78, ATF4 and PDI compared with the UUO group. The consistent results in vitro were also obtained in TGF-ß1-treated HK-2 cells exposed to EerI. These observations suggest that UPR was remarkably enhanced in the presence of ERAD inhibition and compensated for excess improperly folded proteins, subsequently contributing to the additional production of mature Klotho protein. Conclusion: ERAD is involved in Klotho deficiency in RIF and its specific inhibition significantly promoted Klotho expression, possibly through enhanced UPR. This may represent a novel regulatory mechanism and new therapeutic target for reversing Klotho deficiency.

15-Lipoxygenase worsens renal fibrosis, inflammation, and metabolism in a murine model of ureteral obstruction.

15-Lipoxygenase (15-LO) is a nonheme iron-containing dioxygenase that has both pro- and anti-inflammatory roles in many tissues and disease states. 15-LO is thought to influence macrophage phenotype, and silencing 15-LO reduces fibrosis after acute inflammatory triggers. The goal of the present study was to determine whether altering 15-LO expression influences inflammation and fibrogenesis in a murine model of unilateral ureteral obstruction (UUO). C57BL/6J mice, 15-LO knockout (Alox15-/-) mice, and 15-LO transgenic overexpressing (15LOTG) mice were subjected UUO, and kidneys were analyzed at 3, 10, and 14 days postinjury. Histology for fibrosis, inflammation, cytokine quantification, flow cytometry, and metabolomics were performed on injured tissues and controls. PD146176, a specific 15-LO inhibitor, was used to complement experiments involving knockout animals. Compared with wild-type animals undergoing UUO, Alox15-/- mouse kidneys had less proinflammatory, profibrotic message along with less fibrosis and macrophage infiltration. PD146176 inhibited 15-LO and resulted in reduced fibrosis and macrophage infiltration similar to Alox15-/- mice. Flow cytometry revealed that Alox15-/- UUO-injured kidneys had a dynamic change in macrophage phenotype, with an early blunting of CD11bHiLy6CHi "M1" macrophages and an increase in anti-inflammatory CD11bHiLy6CInt "M2c" macrophages and reduced expression of the fractalkine receptor chemokine (C-X3-C motif) receptor 1. Many of these findings were reversed when UUO was performed on 15LOTG mice. Metabolomics analysis revealed that wild-type kidneys developed a glycolytic shift postinjury, while Alox15-/- kidneys exhibited increased oxidative phosphorylation. In conclusion, 15-LO manipulation by genetic or pharmacological means induces dynamic changes in the inflammatory microenvironment in the UUO model and appears to be critical in the progression of UUO-induced fibrosis.NEW & NOTEWORTHY 15-Lipoxygenase (15-LO) has both pro- and anti-inflammatory functions in leukocytes, and its role in kidney injury and repair is unexplored. Our study showed that 15-LO worsens inflammation and fibrosis in a rodent model of chronic kidney disease using genetic and pharmacological manipulation. Silencing 15-LO promotes an increase in M2c-like wound-healing macrophages in the kidney and alters kidney metabolism globally, protecting against anaerobic glycolysis after injury.

Deletion of Akt1 Promotes Kidney Fibrosis in a Murine Model of Unilateral Ureteral Obstruction.

We investigated the role of Akt1, one of the three isoforms of Akt, in renal fibrosis using the murine model of unilateral ureteral obstruction (UUO). We subjected wild type and Akt1 -/- mice to UUO. The Akt1 gene was silenced in vitro using short hairpin RNA delivered via a lentiviral vector in human proximal tubular cells (HK2 cells) and kidney fibroblasts (NRK-49F cells). The obstructive kidneys of Akt1 -/- mice showed more severe tubulointerstitial fibrosis than those of wild type mice. The expression of fibronectin and type I collagen was significantly increased in obstructed kidneys of Akt1 -/- mice compared to those of wild type mice. The important finding was that the expression of transforming growth factor ß1 (TGFß1) was significantly increased in the Akt1 -/- mice compared to the wild type mice. The knockdown of Akt1 enhanced the expression of TGFß1 in HK2 cells. Interestingly, the upregulation of TGFß1 due to genetic knockdown of Akt1 was associated with activation of signal transducer and activator of transcript 3 (STAT3) independently of the Smad pathway in NRK-49F and HK2 cells. Immunohistochemical staining also showed that expression of phosphorylated STAT3 was more increased in Akt1 -/- mice than in wild type mice after UUO. Additionally, the deletion of Akt1 led to apoptosis of the renal tubular cells in both in vivo and in vitro studies. Conclusively, these results suggest that the deletion of Akt1 may contribute to renal fibrosis via induction of the TGFß1/STAT3 pathway in a murine model of UUO.

Inhibition of ROCK2 alleviates renal fibrosis and the metabolic disorders in the proximal tubular epithelial cells.

Non-specific inhibition of Rho-associated kinases (ROCKs) alleviated renal fibrosis in the unilateral ureteral obstruction (UUO) model, while genetic deletion of ROCK1 did not affect renal pathology in mice. Thus, whether ROCK2 plays a role in renal tubulointerstitial fibrosis needs to be clarified. In the present study, a selective inhibitor against ROCK2 or genetic approach was used to investigate the role of ROCK2 in renal tubulointerstitial fibrosis. In the fibrotic kidneys of chronic kidney diseases (CKDs) patients, we observed an enhanced expression of ROCK2 with a positive correlation with interstitial fibrosis. In mice, the ROCK2 protein level was time-dependently increased in the UUO model. By treating CKD animals with KD025 at the dosage of 50 mg/kg/day via intraperitoneal injection, the renal fibrosis shown by Masson's trichrome staining was significantly alleviated along with the reduced expression of fibrotic genes. In vitro, inhibiting ROCK2 by KD025 or ROCK2 knockdown/knockout significantly blunted the pro-fibrotic response in transforming growth factor-ß1 (TGF-ß1)-stimulated mouse renal proximal tubular epithelial cells (mPTCs). Moreover, impaired cellular metabolism was reported as a crucial pathogenic factor in CKD. By metabolomics analysis, we found that KD025 restored the metabolic disturbance, including the impaired glutathione metabolism in TGF-ß1-stimulated tubular epithelial cells. Consistently, KD025 increased antioxidative stress enzymes and nuclear erythroid 2-related factor 2 (Nrf2) in fibrotic models. In addition, KD025 decreased the infiltration of macrophages and inflammatory response in fibrotic kidneys and blunted the activation of macrophages in vitro. In conclusion, inhibition of ROCK2 may serve as a potential novel therapy for renal tubulointerstitial fibrosis in CKD.

SerpinB2 Regulates Immune Response in Kidney Injury and Aging.

BACKGROUND: Expression of SerpinB2, a regulator of inflammatory processes, has been described in the context of macrophage activation and cellular senescence. Given that mechanisms for these processes interact and can shape kidney disease, it seems plausible that SerpinB2 might play a role in renal aging, injury, and repair. METHODS: We subjected SerpinB2 knockout mice to ischemia-reperfusion injury or unilateral ureteral obstruction. We performed phagocyte depletion to study SerpinB2's role beyond the effects of macrophages and transplanted bone marrow from knockout mice to wild-type mice and vice versa to dissect cell type-dependent effects. Primary tubular cells and macrophages from SerpinB2 knockout and wild-type mice were used for functional studies and transcriptional profiling. RESULTS: Cultured senescent tubular cells, kidneys of aged mice, and renal stress models exhibited upregulation of SerpinB2 expression. Functionally, lack of SerpinB2 in aged knockout mice had no effect on the magnitude of senescence markers but associated with enhanced kidney damage and fibrosis. In stress models, inflammatory cell infiltration was initially lower in knockout mice but later increased, leading to an accumulation of significantly more macrophages. SerpinB2 knockout tubular cells showed significantly reduced expression of the chemokine CCL2. Macrophages from knockout mice exhibited reduced phagocytosis and enhanced migration. Macrophage depletion and bone marrow transplantation experiments validated the functional relevance of these cell type-specific functions of SerpinB2. CONCLUSIONS: SerpinB2 influences tubule-macrophage crosstalk by supporting tubular CCL2 expression and regulating macrophage phagocytosis and migration. In mice, SerpinB2 expression seems to be needed for coordination and timely resolution of inflammation, successful repair, and kidney homeostasis during aging. Implications of SerpinB2 in human kidney disease deserve further exploration.

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction.

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (αSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfß), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.

Selective inhibition of class IIa histone deacetylases alleviates renal fibrosis.

In this study, we examined the effect of MC1568, a selective class IIa histone deacetylase (HDAC) inhibitor, on the development and progression of renal fibrosis in a murine model of renal fibrosis induced by unilateral ureteral obstruction (UUO). All 4 class IIa HDAC isoforms, in particular HDAC4, were up-regulated in renal epithelial cells of the injured kidney. Administration of MC1568 immediately after UUO injury reduced expression of α-smooth muscle actin (α-SMA), fibronectin, and collagen 1. MC1568 treatment or small interfering RNA-mediated silencing of HDAC4 also suppressed expression of those proteins in cultured renal epithelial cells. Mechanistically, MC1568 abrogated UUO-induced phosphorylation of Smad3, NF-κB, and up-regulation of integrin ɑVß6 in the kidney and inhibited TGF-ß1-induced responses in cultured renal epithelial cells. MC1568 also increased renal expression of klotho, bone morphogenetic protein 7, and Smad7. Moreover, delayed administration of MC1568 at 3 d after ureteral obstruction reversed the expression of α-SMA, fibronectin, and collagen 1 and increased expression of matrix metalloproteinase (MMP)-2 and -9. Collectively, these results suggest that selectively targeting class IIa HDAC isoforms (in particular HDAC4) may inhibit development and progression of renal fibrosis by suppressing activation and expression of multiple profibrotic molecules and increasing expression of antifibrotic proteins and MMPs.-Xiong, C., Guan, Y., Zhou, X., Liu, L., Zhuang, M. A., Zhang, W., Zhang, Y., Masucci, M. V., Bayliss, G., Zhao, T. C., Zhuang, S. Selective inhibition of class IIa histone deacetylases alleviates renal fibrosis.

Neutral endopeptidase inhibitors blunt kidney fibrosis by reducing myofibroblast formation.

Kidney fibrosis is the common pathophysiological mechanism in end-stage renal disease characterized by excessive accumulation of myofibroblast-derived extracellular matrix. Natriuretic peptides have been demonstrated to have cyclic guanosine monophosphate (cGMP)-dependent anti-fibrotic properties likely due to interference with pro-fibrotic tissue growth factor ß (TGF-ß) signaling. However, in vivo, natriuretic peptides are rapidly degraded by neutral endopeptidases (NEP). In a unilateral ureteral obstruction (UUO) mouse model for kidney fibrosis we assessed the anti-fibrotic effects of SOL1, an orally active compound that inhibits NEP and endothelin-converting enzyme (ECE). Mice (n=10 per group) subjected to UUO were treated for 1 week with either solvent, NEP-/ECE-inhibitor SOL1 (two doses), reference NEP-inhibitor candoxatril or the angiotensin II receptor type 1 (AT1)-antagonist losartan. While NEP-inhibitors had no significant effect on blood pressure, they did increase urinary cGMP levels as well as endothelin-1 (ET-1) levels. Immunohistochemical staining revealed a marked decrease in renal collagen (∼55% reduction, P<0.05) and α-smooth muscle actin (α-SMA; ∼40% reduction, P<0.05). Moreover, the number of α-SMA positive cells in the kidneys of SOL1-treated groups inversely correlated with cGMP levels consistent with a NEP-dependent anti-fibrotic effect. To dissect the molecular mechanisms associated with the anti-fibrotic effects of NEP inhibition, we performed a 'deep serial analysis of gene expression (Deep SAGE)' transcriptome and targeted metabolomics analysis of total kidneys of all treatment groups. Pathway analyses linked increased cGMP and ET-1 levels with decreased nuclear receptor signaling (peroxisome proliferator-activated receptor [PPAR] and liver X receptor/retinoid X receptor [LXR/RXR] signaling) and actin cytoskeleton organization. Taken together, although our transcriptome and metabolome data indicate metabolic dysregulation, our data support the therapeutic potential of NEP inhibition in the treatment of kidney fibrosis via cGMP elevation and reduced myofibroblast formation.

Peroxidasin and eosinophil peroxidase, but not myeloperoxidase, contribute to renal fibrosis in the murine unilateral ureteral obstruction model.

Renal fibrosis is the pathological hallmark of chronic kidney disease (CKD) and manifests as glomerulosclerosis and tubulointerstitial fibrosis. Reactive oxygen species contribute significantly to renal inflammation and fibrosis, but most research has focused on superoxide and hydrogen peroxide (H2O2). The animal heme peroxidases myeloperoxidase (MPO), eosinophil peroxidase (EPX), and peroxidasin (PXDN) uniquely metabolize H2O2 into highly reactive and destructive hypohalous acids, such as hypobromous and hypochlorous acid. However, the role of these peroxidases and their downstream hypohalous acids in the pathogenesis of renal fibrosis is unclear. Our study defines the contribution of MPO, EPX, and PXDN to renal inflammation and tubulointerstitial fibrosis in the murine unilateral ureteral obstruction (UUO) model. Using a nonspecific inhibitor of animal heme peroxidases and peroxidase-specific knockout mice, we find that loss of EPX or PXDN, but not MPO, reduces renal fibrosis. Furthermore, we demonstrate that eosinophils, the source of EPX, accumulate in the renal interstitium after UUO. These findings point to EPX and PXDN as potential therapeutic targets for renal fibrosis and CKD and suggest that eosinophils modulate the response to renal injury.

Dicer deficiency in proximal tubules exacerbates renal injury and tubulointerstitial fibrosis and upregulates Smad2/3.

Renal fibrosis is a common pathological feature in chronic kidney disease (CKD), including diabetic kidney disease (DKD) and obstructive nephropathy. Multiple microRNAs have been implicated in the pathogenesis of both DKD and obstructive nephropathy, although the overall role of microRNAs in tubular injury and renal fibrosis in CKD is unclear. Dicer (a key RNase III enzyme for microRNA biogenesis) was specifically ablated from kidney proximal tubules in mice via the Cre-lox system to deplete micoRNAs. Proximal tubular Dicer knockout (PT- Dicer KO) mice and wild-type (WT) littermates were subjected to streptozotocin (STZ) treatment to induce DKD or unilateral ureteral obstruction (UUO) to induce obstructive nephropathy. Renal hypertrophy, renal tubular apoptosis, kidney inflammation, and tubulointerstitial fibrosis were examined. Compared with WT mice, PT- Dicer KO mice showed more severe tubular injury and renal inflammation following STZ treatment. These mice also developed higher levels of tubolointerstitial fibrosis. Meanwhile, PT- Dicer KO mice had a significantly higher Smad2/3 expression in kidneys than WT mice (at 6 mo of age) in both control and STZ-treated mice. Similarly, UUO induced more severe renal injury, inflammation, and interstitial fibrosis in PT- Dicer KO mice than WT. Although we did not detect obvious Smad2/3 expression in sham-operated mice (2-3 mo old), significantly more Smad2/3 was induced in obstructed PT- Dicer KO kidneys. These results supported a protective role of Dicer-dependent microRNA synthesis in renal injury and fibrosis development in CKD, specifically in DKD and obstructive nephropathy. Depletion of Dicer and microRNAs may upregulate Smad2/3-related signaling pathway to enhance the progression of CKD.

Vanin-1 in Renal Pelvic Urine Reflects Kidney Injury in a Rat Model of Hydronephrosis.

Urinary tract obstruction and the subsequent development of hydronephrosis can cause kidney injuries, which results in chronic kidney disease. Although it is important to detect kidney injuries at an early stage, new biomarkers of hydronephrosis have not been identified. In this study, we examined whether vanin-1 could be a potential biomarker for hydronephrosis. Male Sprague-Dawley rats were subjected to unilateral ureteral obstruction (UUO). On day 7 after UUO, when the histopathological renal tubular injuries became obvious, the vanin-1 level in the renal pelvic urine was significantly higher than that in voided urine from sham-operated rats. Furthermore, vanin-1 remained at the same level until day 14. There was no significant difference in the serum vanin-1 level between sham-operated rats and rats with UUO. In the kidney tissue, the mRNA and protein expressions of vanin-1 significantly decreased, whereas there was increased expression of transforming growth factor (TGF)-ß1 and Snail-1, which plays a pivotal role in the pathogenesis of renal fibrosis via epithelial-to-mesenchymal transition (EMT). These results suggest that vanin-1 in the renal pelvic urine is released from the renal tubular cells of UUO rats and reflects renal tubular injuries at an early stage. Urinary vanin-1 may serve as a candidate biomarker of renal tubular injury due to hydronephrosis.

Glomerular hyperpermeability after acute unilateral ureteral obstruction: effects of Tempol, NOS, RhoA, and Rac-1 inhibition.

It is well known that proteinuria following urinary tract obstruction is mainly of a tubular nature. However, it is unknown whether there are also changes in glomerular permeability. In this study, we compared glomerular sieving coefficients (θ) of polydisperse fluorescein isothiocyanate (FITC)-Ficoll 70/400 following a 120- or 180-min unilateral ureteral obstruction (UUO) in anesthetized Sprague-Dawley rats. Samples were collected from the obstructed kidney at 5, 15, and 30 min postrelease and analyzed by means of high-pressure size-exclusion chromatography. After 120-min UUO, mean θ for Ficoll70Å was increased ( P < 0.01) from 2.2 ± 0.5 × 10-5 (baseline) to 10.6 ± 10 × 10-5 15 min postrelease (highest value). After 180-min UUO, mean θ for Ficoll70Å was further increased ( P < 0.001) from 1.4 ± 0.5 × 10-5 (baseline) to 40 ± 10 × 10-5 at 5 min postrelease (highest value). Administration of a reactive oxygen species (ROS) scavenger (Tempol; 1 mg·kg-1·min-1) partly abrogated the permeability effects following 120-min UUO but not after 180 min. Moreover, administration of the RhoA kinase inhibitor Y-27632, the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester, or Rac-1 inhibition did not ameliorate glomerular hyperpermeability following 180-min UUO. We show, for the first time, that acute UUO results in marked elevations in glomerular permeability. In addition, our data suggest a time-dependent pathophysiology of UUO-induced hyperpermeability, where reactive oxygen species generation may play an important role in the early stages.

The proto-oncogene tyrosine protein kinase Src is essential for macrophage-myofibroblast transition during renal scarring.

Src activation has been associated with fibrogenesis after kidney injury. Macrophage-myofibroblast transition is a newly identified process to generate collagen-producing myofibroblasts locally in the kidney undergoing fibrosis in a TGF-ß/Smad3-dependent manner. The potential role of the macrophage-myofibroblast transition in Src-mediated renal fibrosis is unknown. In studying this by RNA sequencing at single-cell resolution, we uncovered a unique Src-centric regulatory gene network as a key underlying mechanism of macrophage-myofibroblast transition. A total of 501 differentially expressed genes associated with macrophage-myofibroblast transition were identified. However, Smad3-knockout largely reduced the transcriptome diversity. More importantly, inhibition of Src largely suppresses ureteral obstruction-induced macrophage-myofibroblast transition in the injured kidney in vivo along with transforming growth factor-ß1-induced elongated fibroblast-like morphology, α-smooth muscle actin expression and collagen production in bone marrow derived macrophages in vitro. Unexpectedly, we further uncovered that Src serves as a direct Smad3 target gene and also specifically up-regulated in macrophages during macrophage-myofibroblast transition. Thus, macrophage-myofibroblast transition contributes to Src-mediated tissue fibrosis. Hence, targeting Src may represent as a precision therapeutic strategy for macrophage-myofibroblast transition-driven fibrotic diseases.

Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis.

Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H2S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H2S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the Cse gene. UUO of wild-type mice reduced the expression of H2S-producing enzymes, CSE, cystathionine ß-synthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H2S and GSH levels. Cse gene deletion lowered H2S and GSH levels in the kidneys. Deleting the Cse gene exacerbated the decrease in H2S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, Cse gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that Cse deficiency impairs renal the H2S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.

Intermedin attenuates renal fibrosis by induction of heme oxygenase-1 in rats with unilateral ureteral obstruction.

BACKGROUND: Intermedin [IMD, adrenomedullin-2 (ADM-2)] attenuates renal fibrosis by inhibition of oxidative stress. However, the precise mechanisms remain unknown. Heme oxygenase-1 (HO-1), an antioxidant agent, is associated with antifibrogenic effects. ADM is known to induce HO-1. Whether IMD has any effect on HO-1 is unclear. Herein, we determined whether the antifibrotic properties of IMD are mediated by induction of HO-1. METHODS: Renal fibrosis was induced by unilateral ureteral obstruction (UUO) performed on male Wistar rats. Rat proximal tubular epithelial cell line (NRK-52E) was exposed to rhTGF-ß1 (10 ng/ml) to establish an in vitro model of epithelial-mesenchymal transition (EMT). IMD was over-expressed in vivo and in vitro using the vector pcDNA3.1-IMD. Zinc protoporphyrin (ZnPP) was used to block HO-1 enzymatic activity. IMD effects on HO-1 expression in the obstructed kidney of UUO rat and in TGF-ß1-stimulated NRK-52E were analyzed by real-time RT-PCR, Western blotting or immunohistochemistry. HO activity in the obstructed kidney, contralateral kidney of UUO rat and NRK-52E was examined by measuring bilirubin production. Renal fibrosis was determined by Masson trichrome staining and collagen I expression. Macrophage infiltration and IL-6 expression were evaluated using immunohistochemical analysis. In vivo and in vitro EMT was assessed by measuring α-smooth muscle actin (α-SMA) and E-cadherin expression using Western blotting or immunofluorescence, respectively. RESULTS: HO-1 expression and HO activity were increased in IMD-treated UUO kidneys or NRK-52E. The obstructed kidneys of UUO rats demonstrated significant interstitial fibrosis on day 7 after operation. In contrast, kidneys that were treated with IMD gene transfer exhibited minimal interstitial fibrosis. The obstructed kidneys of UUO rats also had greater macrophage infiltration and IL-6 expression. IMD restrained infiltration of macrophages and expression of IL-6 in UUO kidneys. The degree of EMT was extensive in obstructed kidneys of UUO rats as indicated by decreased expression of E-cadherin and increased expression of α-SMA. In vitro studies using NRK-52E confirmed these observations. EMT was suppressed by IMD gene delivery. However, all of the above beneficial effects of IMD were eliminated by ZnPP, an inhibitor of HO enzyme activity. CONCLUSION: This study demonstrates that IMD attenuates renal fibrosis by induction of HO-1.

Sphingosine kinase 1 protects renal tubular epithelial cells from renal fibrosis via induction of autophagy.

Autophagy is an important homoeostatic mechanism for the lysosomal degradation of protein aggregates and damaged cytoplasmic components. Recent studies suggest that autophagy which is induced by TGF-ß1 suppresses kidney fibrosis in renal tubular epithelial cells (RTECs) of obstructed kidneys. Sphingosine kinase 1(SK1), converting sphingosine into endogenous sphingosine-1-phosphate (S1P), was shown to modulate autophagy and involved in the processes of fibrotic diseases. Since SK1 activity is also up-regulated by TGF-ß1, we explored its effect on the induction of autophagy and development of renal fibrosis in this study. In vitro, SK1 expression and activity were markedly increased by TGF-ß1 stimulation in a time and concentration dependent manner, and concomitant changes in autophagic response were observed in HK-2 cells. Further, knockdown of SK-1 led to a decrease of autophagy whereas overexpression of SK1 caused a greater induction of autophagy. In addition, overexpression of SK1 resulted in decreased of mature TGF-ß levels through autophagic degradation. In vivo, SK1 enzymatic activity and autophagic response were both up-regulated in a mouse model of kidney fibrosis induced by unilateral ureteral obstruction (UUO); meanwhile, increased of mature TGF-ß1 and deposition of extracellular matrix (ECM) were observed in tubulointerstitial areas compared with sham-operated mice. However, aggravation of renal fibrosis was detected when SK1 inhibitor PF-543 was applied to suppress SK1 enzymatic activity in UUO mice. At the same time, autophagy was also inhibited by PF-543. Thus, our findings suggest that SK1 activation is renoprotective via induction of autophagy in the fibrotic process.

Alpha-lipoic acid ameliorates the epithelial mesenchymal transition induced by unilateral ureteral obstruction in mice.

The epithelial-to-mesenchymal transition (EMT) is one of mechanisms that induce renal interstitial fibrosis. Understanding EMT in renal fibrosis has important therapeutic implications for patients with kidney disease. Alpha-lipoic acid (ALA) is a natural compound with antioxidant properties. Studies for ALA are performed in acute kidney injury with renal tubular apoptosis, renal inflammation, and oxidative stress. We investigated the effects of ALA on EMT-mediated renal interstitial fibrosis in mice with unilateral ureteral obstruction (UUO). UUO mice developed severe tubular atrophy and tubulointerstitial fibrosis, with a robust EMT response and ECM deposition after 7 postoperative days. In contrast, ALA-treated UUO mice showed only moderate injury and minimal fibrosis and also larger reductions in the expression of ECM proteins, inflammatory factors, and EMT markers. ALA was shown to be involved in the suppression of infiltrating macrophages associated with EMT and the progression of interstitial fibrosis. It also lessened the destruction of the tubular basement membrane, by reducing the expression of matrix metalloproteinases. This is the first study to show that ALA modulates EMT in a UUO mouse model. Our results suggest that ALA merits further exploration as a therapeutic agent in the prevention and treatment of chronic kidney disease.

Pirfenidone suppresses MAPK signalling pathway to reverse epithelial-mesenchymal transition and renal fibrosis.

AIM: Recent studies indicate that pirfenidone (PFD) may have anti-fibrotic effects in many tissues, but the potential molecular mechanism remains unknown. The purpose of this study is to investigate the potential effects of PFD on epithelial-to-mesenchymal transition (EMT) and renal fibrosis in a unilateral ureteral obstruction (UUO) rat model and the involved molecular mechanism related to cultured human renal proximal tubular epithelial cells (HK-2). METHODS: Sixty rats were randomly divided into three groups: sham-operated, vehicle-treated UUO, and PFD-treated UUO. Kidney specimens were collected at day 7 or 14 after UUO. PFD treatment was also performed for human HK-2. The tubulointerstitial injury, interstitial collagen deposition, and expression of type I and III collagen, α-SMA, S100A4, fibronection and E-cadherin were assessed. In addition, extracellular signal regulated kinase (ERK1/2), p38 MAPK (p38), and c-Jun N-terminal kinase/stress-activated protein kinase (JNK) were also detected. RESULTS: In vitro, PFD significantly attenuated TGF-ß1-induced EMT and extracellular matrix (ECM) synthesis, as determined by reducing expression of α-SMA, type I and III collagen, S100A4, fibronection, and increased expression of E-cadherin. PFD treatment attenuated TGF-ß1-induced up-regulation of phosphorylation of ERK1/2, p38 and JNK. In vivo, PFD reduced the degree of tubulointerstitial injury and renal fibrosis, which was associated with reduced expression of TGF-ß1, type III collagen, α-SMA, S100A4, fibronection, and increased expression of E-cadherin. CONCLUSION: These results suggest that pirfenidone is able to attenuate EMT and fibrosis in vivo and in vitro through antagonizing the MAPK pathway, providing a potential treatment to alleviate renal tubulointerstitial fibrosis.

Partial deletion of the ROCK2 protein fails to reduce renal fibrosis in a unilateral ureteral obstruction model in mice.

Renal fibrosis is a well­known cause for the progression of chronic kidney disease. Rho/Rho­associated coiled­coil kinase (ROCK) signaling is involved in renal fibrotic processes. Non­selective ROCK1/2 inhibitors have been reported to reduce renal interstitial fibrosis in a rodent unilateral ureteral obstruction (UUO) model. To clarify the role and contribution of ROCK2 in renal fibrosis, the present study used ROCK2 heterozygous knockout (HKO) mice to assess collagen deposition and fibrosis­associated gene expression in the kidney of the UUO model. In the ROCK2 HKO mice, the expression level of ROCK2 in the normal kidney was half of that in the kidney of wild­type (WT) mice. The expression levels of ROCK1 in the ROCK2 HKO mice and WT mice were equivalent. Furthermore, in the ROCK2 HKO and the WT mice, the hydroxyproline content and the gene expression levels of collagen I and transforming growth factor­ß1 in the obstructed kidneys were augmented following UUO. By contrast, the mRNA expression of α­smooth muscle actin decreased in the ROCK2 HKO mice, compared with that in the WT mice. The activity of ROCK in the obstructed kidneys, indicated by the phosphorylation of myosin phosphatase target subunit­1, which is a non­selective substrate of ROCK1 and ROCK2, was equivalent among the ROCK2 HKO and WT mice. In conclusion, no differences in renal interstitial fibrosis or UUO­induced ROCK activity were identified between the ROCK2 HKO and WT mice, indicating that the genetic partial disruption of ROCK2 is insufficient for protecting against renal fibrosis.

Erythropoietin protects the tubular basement membrane by promoting the bone marrow to release extracellular vesicles containing tPA-targeting miR-144.

Renal fibrosis is an inevitable outcome of chronic kidney disease (CKD). Erythropoietin (EPO) has been recently reported to be able to mitigate renal fibrosis. The mechanism underlying the protective effect of EPO, however, remains elusive. In the present study, employing a mouse model of renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction (UUO), we demonstrated that EPO markedly reduced the disruption of the tubular basement membrane (TBM) through attenuating the activation of tissue plasminogen activator (tPA) and matrix metalloproteinase 9 (MMP9), the major matrix proteolytic network in the obstructed kidney. Instead of acting directly on tPA in the kidney, EPO strongly increased the level of circulating microRNA (miR)-144, which was delivered to the injured renal fibroblasts via extracellular vesicles (EVs) to target the tPA 3'-untranslated region and suppress tPA expression. The protective effect of EPO on mouse TBM was inhibited by miR-144 antagomir. Furthermore, in vitro results confirmed that EPO could stimulate bone marrow-derived Sca-1(+)CD44(+)CD11b(-)CD19(-) cells to secrete miR-144-containing EVs, which markedly suppressed tPA expression, as well as metalloproteinase 9 (MMP9) level and activity, in cultured renal fibroblasts. In conclusion, our study provides the first evidence that EPO protects mouse renal TBM through promoting bone marrow cells to generate and secrete miR-144, which, in turn, is efficiently delivered into the mouse kidney via EVs to inhibit the activation of the tPA/MMP9-mediated proteolytic network. This finding thus suggests that EPO, a hormone widely used to treat anemia in CKD, is a potential therapeutic strategy for renal fibrosis.