Hypochlorite modified albumins promote cell death in the tubule interstitium in rats via mitochondrial damage in obstructive nephropathy and the protective effects of antioxidant peptides

A major feature of the injury sustained by the kidney during obstructive nephropathy is a profound induction of apoptosis in the tubular epithelium. In this study, we explored the central roles of mitochondria and the mechanism of the protective effect of the mitochondrial targeted peptides in tubular cell apoptosis and interstitial fibrosis during obstructive nephropathy. Unilateral ureter obstruction (UUO) was performed on rats, and the animals were randomly assigned to intravenous treatment with normal saline, rat serum albumin (RSA), or HOCl-rat serum albumin (HOCl-RSA) in the presence or absence of SS-31. A sham-operation control group was set up by left ureteral dissociation but not ligation. Compared with the control group, UUO animals displayed fibrotic abnormalities, accompanied by increased expression of collagen-I, fibronectin, α-SMA protein and mRNA in the renal interstitium. They also displayed oxidative stress, as evidenced by increased levels of HOCl-alb, TBARS, and mitochondrial reactive oxygen species (ROS) and a decrease in MnSOD activity in the renal homogenate. Damage to mitochondrial structure and functions was observed, as evidenced by a decrease in the mitochondrial membrane potential (MMP), ATP production, mtDNA copy number alterations and release of cytochrome C (cyto C) from the mitochondria to the cytoplasm. These changes were accompanied by activation of caspase-3, caspase-7, caspase-9, and PARP-1 and increased apoptotic cells in the proximal tubules. HOCl-RSA challenge further exacerbated the above biological effects in UUO animals, but these effects were prevented by administration of SS-31. These data suggested that accumulation of HOCl-alb may promote tubular cell apoptosis and interstitial fibrosis, probably related to mitochondrial oxidative stress and damage, and that SS-31 might contribute to apoptotic pathway suppression via scavenging of ROS in the mitochondria.     

Diabetes-induced up-regulation of uncoupling protein-2 results in increased mitochondrial uncoupling in kidney proximal tubular cells

We have previously reported increased O(2) consumption unrelated to active transport by tubular cells and up-regulated mitochondrial uncoupling protein (UCP)-2 expressions in diabetic kidneys. It is presently unknown if the increased UCP-2 levels in the diabetic kidney results in mitochondrial uncoupling and increased O(2) consumption, which we therefore investigated in this study. The presence of UCP-2 in proximal tubular cells was confirmed by immunohistochemistry and found to be increased (western blot) in homogenized tissue and isolated mitochondria from kidney cortex of diabetic rats. Isolated proximal tubular cells had increased total and ouabain-insensitive O(2) consumption compared to controls. Isolated mitochondria from diabetic animals displayed increased glutamate-stimulated O(2) consumption (in the absence of ADP and during inhibition of the ATP-synthase by oligomycin) compared to controls. Guanosine diphosphate, an UCP inhibitor, and bovine serum albumin which removes fatty acids that are essential for UCP-2 uncoupling activity, independently prevented the increased glutamate-stimulated O(2) consumption in mitochondria from diabetic animals. In conclusion, diabetic rats have increased mitochondrial UCP-2 expression in renal proximal tubular cells, which results in mitochondrial uncoupling and increased O(2) consumption. This mechanism may be protective against diabetes-induced oxidative stress, but will increase O(2) usage. The subsequently reduced O(2) availability may contribute to diabetes-induced progressive kidney damage.     

An in vivo and in vitro model on the protective effect of cilnidipine on contrast-induced nephropathy via regulation of apoptosis and CaMKⅡ/mPTP pathway

Contrast-induced nephropathy (CIN) is an acute kidney injury (AKI) observed after the administration of contrast media. Calcium channel blockers (CCBs) have been reported to exert a renal protective effect. This study aims to investigate the role of cilnidipine, a novel CCBs, on CIN by regulating the calcium/calmodulin-dependent protein kinase Ⅱ(CaMKⅡ)/mitochondrial permeability transition pore (mPTP) pathway. Here, iohexol, a representative contrast media, was used to establish CIN model. KN-93 (CaMKⅡ inhibitor) and atractyloside (mPTP opener) were administered in rats, and CaMKⅡ overexpression was used in Human proximal tubular epithelial cells. Markers of renal injury (serum creatinine, blood urea nitrogen, and urinary NAGL), hematoxylin-eosin stain, oxidative stress (ROS, superoxide dismutase [SOD], and malondialdehyde [MDA] levels), cell death (MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling [TUNEL]), mitochondrial function (mPTP, mitochondrial membrane potential [MMP], and ATP) were assessed. Western blots were used to measure the expression levels of Bax/Bcl-2, caspase-3, CaMKⅡ/mPTP signaling pathways. Results showed that cilnidipine markedly improved kidney function, and alleviated tubular cell apoptosis, oxidative stress and mitochondrial damage induced by iohexol in vitro and in vivo. The underlying mechanism may be that cilnidipine relieved CaMKⅡ activation and mPTP opening induced by iohexol. All of these protective effects of cilnidipine were attenuated by CaMKⅡ overexpression and atractyloside (mPTP opener) pretreatment. Moreover, KN-93 (CaMKⅡ inhibitor) treatment showed a similar renal protective effect with cilnidipine, while the protective effect of cilnidipine on kidney in CIN rats was not further suppressed by KN-93 cotreatment. These in vitro and in vivo results point toward the fact that cilnidipine might be a novel therapeutic drug against contrast-induced nephrotoxicity in a CaMKⅡ-dependent manner.     

Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction

Renal fibrosis is the common pathological feature in a variety of chronic kidney diseases. Aging is highly associated with the progression of renal fibrosis. Among several determinants, mitochondrial dysfunction plays an important role in aging. However, the underlying mechanisms of mitochondrial dysfunction in age‐related renal fibrosis are not elucidated. Herein, we found that Wnt/β‐catenin signaling and renin–angiotensin system (RAS) activity were upregulated in aging kidneys. Concomitantly, mitochondrial mass and functions were impaired with aging. Ectopic expression of Klotho, an antagonist of endogenous Wnt/β‐catenin activity, abolished renal fibrosis in d ‐galactose (d ‐gal)‐induced accelerated aging mouse model and significantly protected renal mitochondrial functions by preserving mass and diminishing the production of reactive oxygen species. In an established aging mouse model, dickkopf 1, a more specific Wnt inhibitor, and the mitochondria‐targeted antioxidant mitoquinone restored mitochondrial mass and attenuated tubular senescence and renal fibrosis. In a human proximal tubular cell line (HKC‐8), ectopic expression of Wnt1 decreased biogenesis and induced dysfunction of mitochondria, and triggered cellular senescence. Moreover, d ‐gal triggered the transduction of Wnt/β‐catenin signaling, which further activated angiotensin type 1 receptor (AT1), and then decreased the mitochondrial mass and increased cellular senescence in HKC‐8 cells and primary cultured renal tubular cells. These effects were inhibited by AT1 blocker of losartan. These results suggest inhibition of Wnt/β‐catenin signaling and the RAS could slow the onset of age‐related mitochondrial dysfunction and renal fibrosis. Taken together, our results indicate that Wnt/β‐catenin/RAS signaling mediates age‐related renal fibrosis and is associated with mitochondrial dysfunction.     

Oleate and eicosapentaenoic acid attenuate palmitate-induced inflammation and apoptosis in renal proximal tubular cell

Free fatty acid (FFA)-bound albumin, which is filtrated through the glomeruli and reabsorbed into proximal tubular cells, is one of the crucial mediators of tubular damage in proteinuric kidney disease. In this study, we examined the role of each kind of FFA on renal tubular damage in vitro and tried to identify its molecular mechanism. In cultured proximal tubular cells, a saturated fatty acid, palmiate, increased the expression of monocyte chemoattractant protein-1 (MCP-1), but this effect was abrogated by co-incubation of monounsaturated fatty acid, oleate, or ω-3 polyunsaturated fatty acid, eicosapentaenoic acid (EPA). Palmitate led to intracellular accumulation of diacylglycerol (DAG) and subsequent activation of protein kinase C protein family. Among the several PKC inhibitors, rottlerin, a PKCθ inhibitor, prevented palmitate-induced MCP-1 expression via inactivation of NFB pathway. Overexpression of dominant-negative PKCθ also inhibited palmitate-induced activation of MCP-1 promoter. Furthermore, palmitate enhanced PKCθ-dependent mitochondrial apoptosis, which was also prevented by co-incubation with oleate or EPA through restoration of pro-survival Akt pathway. Moreover, oleate and EPA inhibited palmitate-induced PKCθ activation through the conversion of intracellular DAG to triglyceride with the restoration of diacylglycerol acyltransferase 2 expression. These results suggest that oleate and EPA have protective effects against the palmitate-induced renal tubular cell damage by inhibiting PKCθ activation.     

The mechanism of inhibition of the sarco/endoplasmic reticulum Ca2+ ATPase by paxilline

Although cis-diamminedichloroplatinum (II) (cisplatin) is a potent anticancer drug, clinical use of this agent is highly limited predominantly because of its strong side effects on the kidney and gastrointestinal tracts. We found that cisplatin impaired respiratory function and DNA of mitochondria in renal proximal tubules and small intestinal mucosal cells, thereby inducing apoptosis of epithelial cells. Cisplatin-induced mitochondrial dysfunction and DNA (mtDNA) injury, lipid peroxidation, and apoptosis of epithelial cells in the kidney and small intestine were strongly inhibited by L-carnitine. However, carnitine had no appreciable effect on the tumoricidal action of cisplatin against cancer cells inoculated in the peritoneal cavity. These results indicate that L-carnitine may have therapeutic potential for inhibiting the side effects of cisplatin and other anticancer agents in the kidney and small intestine.     

CaMKII as a key regulator of contrast-induced nephropathy through mPTP opening in HK-2 cells

Contrast-induced nephropathy (CIN), refers to acute kidney injury observed after administration of contrast media during angiographic or other medical procedures such as urography, and accounting for 12% of all causes of acute renal failure, but no specific prevention or treatment strategy exists for its obscure pathophysiology. The aim of our study was to explore the influence of calcium/calmodulin-dependent protein kinase II (CaMKII) in CIN by using HK-2 cells. Knockdown of CypD was achieved by lentivirus, and CaMKII overexpression by transfection with the plasmid. In this study, we have demonstrated that CypD-mediated mPTP opening triggered mitochondrial dysfunction and tubule cells apoptosis in CIN. We also found that iohexol treatment was associated with mitochondrial ROS overloading, ATP depletion and LDH release. Inhibition of CypD with the pharmacologic inhibitor or knockdown of CypD abrogated mPTP opening, oxidative stress, mitochondria damage, and cell apoptosis induced by iohexol. In addition, we found that inhibition of the CaMKII activity alleviated iohexol-induced CypD expression, whereas also decreased mPTP opening, oxidative stress, mitochondria damage, and cell apoptosis, similarly to the inhibition of CypD did. Moreover, CaMKII overexpression enhanced iohexol-induced mPTP opening, mitochondrial damage and renal tubular epithelial cells apoptosis. These findings first identified the novel role of CaMKII in iohexol-induced tubular cells apoptosis and delineated the CaMKII-CypD/mPTP pathway during contrast-induced tubular cell damage. Hence, these results could provide a new strategy for CIN protection.     

Pirfenidone inhibits fibrocyte accumulation in the lungs in bleomycin-induced murine pulmonary fibrosis

Background

Bone marrow-derived fibrocytes reportedly play important roles in the pathogenesis of idiopathic pulmonary fibrosis. Pirfenidone is an anti-fibrotic agent; however, its effects on fibrocytes have not been investigated. The aim of this study was to investigate whether pirfenidone inhibits fibrocyte pool size in the lungs of bleomycin-treated mice.

Methods

Bleomycin (100 mg/kg) was infused with osmotic pumps into C57BL/6 mice, and pirfenidone (300 mg/kg/day) was orally administered daily for 2 wk. The lungs were removed, and single-cell suspensions were subjected to fluorescence-activated cell sorter (FACS) analysis to detect fibrocytes, which were defined as CD45 and collagen-I double-positive cells. Immunohistochemistry was performed on the lung specimens to quantify fibrocytes. Chemokines in the lung digests were measured with enzyme-linked immunosorbent assay. The effect of pirfenidone on alveolar macrophages was evaluated with bronchoalveolar lavage (BAL). In a therapeutic setting, pirfenidone administration was initiated 10 days after bleomycin treatment. For chemotaxis assay, lung fibrocytes were isolated with immunomagnetic selection (CD45-positive mesenchymal cells) after culture and allowed to migrate toward chemokines in the presence or absence of pirfenidone. Moreover, the effect of pirfenidone on the expression of chemokine receptors on fibrocytes was evaluated.

Results

Pirfenidone significantly ameliorated bleomycin-induced pulmonary fibrosis as assessed with quantitative histology and collagen measurement. Fibrocyte pool size in bleomycin-treated mice lungs was attenuated from 26.5% to 13.7% by pirfenidone on FACS analysis. This outcome was also observed in a therapeutic setting. Immunohistochemistry revealed that fibrocytes were significantly decreased by pirfenidone administration compared with those in bleomycin-treated mice (P = 0.0097). Increased chemokine (CC motif) ligand-2 (CCL2) and CCL12 production in bleomycin-treated mouse lungs was significantly attenuated by pirfenidone (P = 0.0003 and P < 0.0001, respectively). Pirfenidone also attenuated macrophage counts stimulated by bleomycin in BAL fluid. Fibrocyte migration toward CCL2 and chemokine (CC motif) receptor-2 expression on fibrocytes was significantly inhibited by pirfenidone in vitro.

Conclusions

Pirfenidone attenuated the fibrocyte pool size in bleomycin-treated mouse lungs via attenuation of CCL2 and CCL12 production in vivo, and fibrocyte migration was inhibited by pirfenidone in vitro. Fibrocyte inhibition is considered a mechanism of anti-fibrotic action of pirfenidone.     

Ghrelin Protects against Renal Damages Induced by Angiotensin-II via an Antioxidative Stress Mechanism in Mice

We explored the renal protective effects by a gut peptide, Ghrelin. Daily peritoneal injection with Ghrelin ameliorated renal damages in continuously angiotensin II (AngII)-infused C57BL/6 mice as assessed by urinary excretion of protein and renal tubular markers. AngII-induced increase in reactive oxygen species (ROS) levels and senescent changes were attenuated by Ghrelin. Ghrelin also inhibited AngII-induced upregulations of transforming growth factor-β (TGF-β) and plasminogen activator inhibitor-1 (PAI-1), ameliorating renal fibrotic changes. These effects were accompanied by concomitant increase in mitochondria uncoupling protein, UCP2 as well as in a key regulator of mitochondria biosynthesis, PGC1α. In renal proximal cell line, HK-2 cells, Ghrelin reduced mitochondria membrane potential and mitochondria-derived ROS. The transfection of UCP2 siRNA abolished the decrease in mitochondria-derived ROS by Ghrelin. Ghrelin ameliorated AngII-induced renal tubular cell senescent changes and AngII-induced TGF-β and PAI-1 expressions. Finally, Ghrelin receptor, growth hormone secretagogue receptor (GHSR)-null mice exhibited an increase in tubular damages, renal ROS levels, renal senescent changes and fibrosis complicated with renal dysfunction. GHSR-null mice harbored elongated mitochondria in the proximal tubules. In conclusion, Ghrelin suppressed AngII-induced renal damages through its UCP2 dependent anti-oxidative stress effect and mitochondria maintenance. Ghrelin/GHSR pathway played an important role in the maintenance of ROS levels in the kidney.     

Expression of metallothionein in renal tubules of rats exposed to acute and endurance exercise

The induction of exercise-induced apoptosis in not actively involved in exercise organs, such as kidney could be a result of oxidative stress. Metallothionein (MT) exerts a protective effect in the cell against oxidative stress and apoptosis. We have previously demonstrated an increased incidence of apoptosis in distal tubular cells and collecting ducts in rat kidney after acute exercise. The present study was designed to test the hypothesis that MT may play a protective role in rat renal tubules against exercise-induced apoptosis after the acute exercise and regular training. Male Wistar rats were divided into control, acute exercised and 8-wk regularly trained groups. The kidneys were removed after a rest period of 6 h and 96 h. The ultrastructure of renal tubular cells was examined by electron microscopy. Apoptosis was detected in paraffin sections by the TUNEL technique. Expression of MT was examined by immunohistochemistry. The level of lipid peroxidation (thiobarbituric acid reactive substances - TBARS) was assayed in renal tissue homogenates. After acute exercise, the occurrence of apoptosis was restricted to distal tubules and collecting ducts of rat kidney, whereas the proximal tubules remained unaffected. The 8-wk training did not result in increased apoptosis in tubular cell. MT expression was confined exclusively to proximal tubules in all groups. However, it was significantly increased in acutely exercised animals, as compared to control and trained rats. After the 8-wk training, MT expression remained unaltered as compared to the control group. TBARS levels were significantly increased after acute exercise, while after regular training they remained unchanged. A significant correlation between TBARS level and MT expression was demonstrated. The findings could suggest a protective role of MT against oxidative stress and apoptosis in proximal tubular cells.     

NLRP3 Inflammasome Mediates Albumin-induced Renal Tubular Injury through Impaired Mitochondrial Function

Proteinuria serves as a direct causative factor of renal tubular cell injury and is highly associated with the progression of chronic kidney disease via uncertain mechanisms. Recently, evidence demonstrated that both NLRP3 inflammasome and mitochondria are involved in the chronic kidney disease progression. The present study was undertaken to examine the role of NLRP3 inflammasome/mitochondria axis in albumin-induced renal tubular injury. In patients with proteinuria, NLRP3 was significantly up-regulated in tubular epithelial cells and was positively correlated with the severity of proteinuria. In agreement with these results, albumin remarkably activated NLRP3 inflammasome in both in vitro renal tubular cells and in vivo kidneys in parallel with significant epithelial cell phenotypic alteration and cell apoptosis. Genetic disruption of NLRP3 inflammasome remarkably attenuated albumin-induced cell apoptosis and phenotypic changes under both in vitro and in vivo conditions. In addition, albumin treatment resulted in a significant mitochondrial abnormality as evidenced by the impaired function and morphology, which was markedly reversed by invalidation of NLRP3/caspase-1 signaling pathway. Interestingly, protection of mitochondria function by Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) or cyclosporin A (CsA) robustly attenuated albumin-induced injury in mouse proximal tubular cells. Collectively, these findings demonstrated a pathogenic role of NLRP3 inflammasome/caspase-1/mitochondria axis in mediating albumin-induced renal tubular injury. The discovery of this novel axis provides some potential targets for the treatment of proteinuria-associated renal injury.     

Cannabinoid receptor 2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing

Kidney is one of the most important organs in maintaining the normal life activities. With the high abundance of mitochondria, renal tubular cell plays the vital role in functioning in the reabsorption and secretion of kidney. Reports have shown that mitochondrial dysfunction is of great importance to renal tubular cell senescence and subsequent kidney ageing. However, the underlying mechanisms are not elucidated. Cannabinoid receptor 2 is one of the two receptors responsible for the activation of endocannabinoid system. CB2 is primarily upregulated in renal tubular cells in chronic kidney diseases and mediates fibrogenesis. However, the role of CB2 in tubular mitochondrial dysfunction and kidney ageing has not been clarified. In this study, we found that CB2 was upregulated in kidneys in 24-month-old mice and d -galactose (d -gal)-induced accelerated ageing mice, accompanied by the decrease in mitochondrial mass. Furthermore, gene deletion of CB2 in d -gal-treated mice could greatly inhibit the activation of β-catenin signalling and restore the mitochondrial integrity and Adenosine triphosphate (ATP) production. In CB2 knockout mice, renal tubular cell senescence and kidney fibrosis were also significantly inhibited. CB2 overexpression or activation by the agonist AM1241 could sufficiently induce the decrease in PGC-1α and a variety of mitochondria-related proteins and trigger cellular senescence in cultured human renal proximal tubular cells. CB2-activated mitochondrial dysfunction and cellular senescence could be blocked by ICG-001, a blocker for β-catenin signalling. These results show CB2 plays a central role in renal tubular mitochondrial dysfunction and kidney ageing. The intrinsic mechanism may be related to its activation in β-catenin signalling.     

Role of mitofusin 2 in the renal stress response

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function. However, despite a small (20%) decrease in nephron number, newborn cKO pups had organ or tubular function that did not differ from littermate Cre-negative pups. MFN2 deficiency in proximal tubule epithelial cells in primary culture induced mitochondrial fragmentation but did not significantly alter ATP turnover, maximal mitochondrial oxidative reserve capacity, or the low level of oxygen consumption during cyanide exposure. MFN2 deficiency also did not increase apoptosis of tubule epithelial cells under non-stress conditions. In contrast, metabolic stress caused by ATP depletion exacerbated mitochondrial outer membrane injury and increased apoptosis by 80% in MFN2 deficient vs. control cells. Despite similar stress-induced Bax 6A7 epitope exposure in MFN2 deficient and control cells, MFN2 deficiency significantly increased mitochondrial Bax accumulation and was associated with greater release of both apoptosis inducing factor and cytochrome c. In conclusion, MFN2 deficiency in the kidney causes mitochondrial fragmentation but does not affect kidney or tubular function during development or under non-stress conditions. However, MFN2 deficiency exacerbates renal epithelial cell injury by promoting Bax-mediated mitochondrial outer membrane injury and apoptosis.     

Farnesoid X Receptor Protects against Kidney Injury in Uninephrectomized Obese Mice

Activation of the farnesoid X receptor (FXR) has indicated a therapeutic potential for this nuclear bile acid receptor in the prevention of diabetic nephropathy and obesity-induced renal damage. Here, we investigated the protective role of FXR against kidney damage induced by obesity in mice that had undergone uninephrectomy, a model resembling the clinical situation of kidney donation by obese individuals. Mice fed a high-fat diet developed the core features of metabolic syndrome, with subsequent renal lipid accumulation and renal injury, including glomerulosclerosis, interstitial fibrosis, and albuminuria. The effects were accentuated by uninephrectomy. In human renal biopsies, staining of 4-hydroxynonenal (4-HNE), glucose-regulated protein 78 (Grp78), and C/EBP-homologous protein, markers of endoplasmic reticulum stress, was more prominent in the proximal tubules of 15 obese patients compared with 16 non-obese patients. In mice treated with the FXR agonist obeticholic acid, renal injury, renal lipid accumulation, apoptosis, and changes in lipid peroxidation were attenuated. Moreover, disturbed mitochondrial function was ameliorated and the mitochondrial respiratory chain recovered following obeticholic acid treatment. Culturing renal proximal tubular cells with free fatty acid and FXR agonists showed that FXR activation protected cells from free fatty acid-induced oxidative stress and endoplasmic reticulum stress, as denoted by a reduction in the level of reactive oxygen species staining and Grp78 immunostaining, respectively. Several genes involved in glutathione metabolism were induced by FXR activation in the remnant kidney, which was consistent with a decreased glutathione disulfide/glutathione ratio. In summary, FXR activation maintains endogenous glutathione homeostasis and protects the kidney in uninephrectomized mice from obesity-induced injury.     

Irisin is induced in renal ischemia-reperfusion to protect against tubular cell injury via suppressing p53

Renal ischemia-reperfusion is a major cause of acute kidney injury, a disease currently without effective treatments. Irisin was initially identified as an important factor produced by muscles to mediate the health benefits of exercise, and recent work has further suggested its protective effect against lung and liver injury. However, the role of Irisin in kidney diseases, including renal ischemia-reperfusion injury (IRI), remains unknown. In the present study, we found that the Irisin precursor, fibronectin type III domain-containing protein 5 (Fndc5), was induced in renal tubules in a mouse model of renal IRI and in cultured mouse renal proximal tubular cells subjected ATP depletion injury. Functionally, silencing Fndc5 in cultured proximal tubular cells increased the sensitivity to ATP depletion-induced apoptosis, whereas both Fndc5 overexpression and supplementation of recombinant Irisin alleviated ATP depletion-induced apoptosis. In vivo, administration of recombinant Irisin dramatically attenuated kidney dysfunction, tissue damage, tubular cell apoptosis, and inflammation during renal IRI in mice. Mechanistically, Irisin suppressed the activation of p53 in renal IRI, a critical factor in tubular cell death. Together, these results indicate that Irisin is induced in renal IRI as a protective mechanism for renal tubular cells, suggesting the therapeutic potential of recombinant Irisin in renal IRI and related kidney diseases.     

Pirfenidone inhibits the expression of HSP47 in TGF-beta1-stimulated human lung fibroblasts

Pirfenidone (5-methyl-1-phenyl-2-(1H)-pyridone) is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and patients with idiopathic pulmonary fibrosis (IPF). Heat shock protein (HSP) 47, a collagen-specific molecular chaperone, is involved in the processing and/or secretion of procollagen and plays an important role in the pathogenesis of IPF. The present study evaluated the in vitro effects of pirfenidone on expression of HSP47 and collagen type I in cultured normal human lung fibroblasts (NHLF). Expression levels of HSP47 and collagen type I in NHLF stimulated by transforming growth factor (TGF)-beta1 were evaluated genetically, immunologically and immunocytochemically. Treatment with TGF-beta1 stimulated both mRNA and protein expressions of both HSP47 and collagen type I in NHLF, and pirfenidone significantly inhibited this TGF-beta1-enhanced expression in a dose-dependent manner. We concluded that the anti-fibrotic effect of pirfenidone may be mediated not only through direct inhibition of collagen type I expression but also at least partly through inhibition of HSP47 expression in lung fibroblasts, with a resultant reduction of collagen synthesis in lung fibrosis.     

Mitochondrial and Metabolic Dysfunction in Renal Convoluted Tubules of Obese Mice: Protective Role of Melatonin

Obesity is a common and complex health problem, which impacts crucial organs; it is also considered an independent risk factor for chronic kidney disease. Few studies have analyzed the consequence of obesity in the renal proximal convoluted tubules, which are the major tubules involved in reabsorptive processes. For optimal performance of the kidney, energy is primarily provided by mitochondria. Melatonin, an indoleamine and antioxidant, has been identified in mitochondria, and there is considerable evidence regarding its essential role in the prevention of oxidative mitochondrial damage. In this study we evaluated the mechanism(s) of mitochondrial alterations in an animal model of obesity (ob/ob mice) and describe the beneficial effects of melatonin treatment on mitochondrial morphology and dynamics as influenced by mitofusin-2 and the intrinsic apoptotic cascade. Melatonin dissolved in 1% ethanol was added to the drinking water from postnatal week 5–13; the calculated dose of melatonin intake was 100 mg/kg body weight/day. Compared to control mice, obesity-related morphological alterations were apparent in the proximal tubules which contained round mitochondria with irregular, short cristae and cells with elevated apoptotic index. Melatonin supplementation in obese mice changed mitochondria shape and cristae organization of proximal tubules, enhanced mitofusin-2 expression, which in turn modulated the progression of the mitochondria-driven intrinsic apoptotic pathway. These changes possibly aid in reducing renal failure. The melatonin-mediated changes indicate its potential protective use against renal morphological damage and dysfunction associated with obesity and metabolic disease.     

Reciprocal regulation between ER stress and autophagy in renal tubular fibrosis and apoptosis

Both endoplasmic reticulum (ER) stress and autophagy have been implicated in chronic kidney injury and renal fibrosis. However, the relationship and regulatory mechanisms between ER stress and autophagy under this condition remain largely unknown. In this study, we first established a mouse model of ER stress-induced chronic kidney injury by 2 weekly injections of a low dose of tunicamycin (TM), a classical ER stress inducer. This model showed the induction of ER stress, autophagy, fibrosis and apoptosis in kidney tissues. In vitro, TM also induced ER stress, autophagy, fibrosis and apoptosis in HK-2 human kidney proximal tubular cells and BUMPT-306 mouse kidney proximal tubular cells. In these cells, autophagy inhibitor suppressed TM-induced fibrotic changes and apoptosis, suggesting an involvement of autophagy in ER stress-associated chronic kidney injury. PERK inhibitor ameliorated autophagy, fibrotic protein expression and apoptosis in TM-treated cells, indicating a role of the PERK/eIF2α pathway in autophagy activation during ER stress. Similar results were shown in TGF-β1-treated HK-2 cells. Interestingly, in both TM- or TGF-β1-treated kidney proximal tubular cells, inhibition of autophagy exaggerated ER stress, suggesting that autophagy induced by ER stress provides a negative feedback mechanism to reduce the stress. Together, these results unveil a reciprocal regulation between ER stress and autophagy in chronic kidney injury and fibrosis.Subject terms: Acute kidney injury, Chronic kidney disease     

Persistent activation of autophagy in kidney tubular cells promotes renal interstitial fibrosis during unilateral ureteral obstruction

Renal fibrosis is the final, common pathway of end-stage renal disease. Whether and how autophagy contributes to renal fibrosis remains unclear. Here we first detected persistent autophagy in kidney proximal tubules in the renal fibrosis model of unilateral ureteral obstruction (UUO) in mice. UUO-associated fibrosis was suppressed by pharmacological inhibitors of autophagy and also by kidney proximal tubule-specific knockout of autophagy-related 7 (PT-Atg7 KO). Consistently, proliferation and activation of fibroblasts, as indicated by the expression of ACTA2/α-smooth muscle actin and VIM (vimentin), was inhibited in PT-Atg7 KO mice, so was the accumulation of extracellular matrix components including FN1 (fibronectin 1) and collagen fibrils. Tubular atrophy, apoptosis, nephron loss, and interstitial macrophage infiltration were all inhibited in these mice. Moreover, these mice showed a specific suppression of the expression of a profibrotic factor FGF2 (fibroblast growth factor 2). In vitro, TGFB1 (transforming growth factor β 1) induced autophagy, apoptosis, and FN1 accumulation in primary proximal tubular cells. Inhibition of autophagy suppressed FN1 accumulation and apoptosis, while enhancement of autophagy increased TGFB1-induced-cell death. These results suggest that persistent activation of autophagy in kidney proximal tubules promotes renal interstitial fibrosis during UUO. The profibrotic function of autophagy is related to the regulation on tubular cell death, interstitial inflammation, and the production of profibrotic factors.     

Proximal tubular RAGE mediated the renal fibrosis in UUO model mice via upregulation of autophagy

Previous studies reported that RAGE participated in the process of kidney fibrosis, but the function and regulation pathway of RAGE in proximal tubular cells in this process remains unclear. Here, we found that expression of RAGE was increased by TGF-β1 treatment and unilateral ureteral obstruction (UUO). Knock down of RAGE ameliorated renal fibrosis by TGF-β1 treatment, the expression of vimentin, Collagen I&III, and fibronectin are decreased. Mechanistically, RAGE mediated TGF-β1-induced phosphorylation of Stat3 and directly upregulated the Atg7 to increase the level of autophagy, and ultimately resulting in renal fibrosis. Furthermore, PT-RAGE-KO mice reduced kidney fibrosis in UUO model via inhibiting Stat3/Atg7 axis by knocking down RAGE. Furthermore, the above findings were confirmed in kidney of patients with obstructive nephropathy. Collectively, RAGE in proximal tubular cells promotes the autophagy to increase renal fibrosis via upregulation of Stat3/Atg7 axis.Subject terms: Autophagy, Urogenital diseases