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Lithium is widely used in psychiatry as the golden standard for more than 60 yr due to its effectiveness. However, its adverse effect has been limiting its long-term use in clinic. About 40% of patients taking lithium develop nephrogenic diabetes insipidus (NDI). Lithium can also induce proliferation of collecting duct cells, leading to microcyst formation in the kidney. Lithium was considered an autophagy inducer that might contribute to the therapeutic benefit of neuropsychiatric disorders. Thus, we hypothesized that autophagy may play a role in lithium-induced kidney nephrotoxicity. To address our hypothesis, we fed mice with a lithium-containing diet with chloroquine (CQ), an autophagy inhibitor, concurrently. Lithium-treated mice presented enhanced autophagy activity in the kidney cortex and medulla. CQ treatment significantly ameliorated lithium-induced polyuria, polydipsia, natriuresis, and kaliuresis accompanied with attenuated downregulation of aquaporin-2 and Na+-K+-2Cl−cotransporter protein. The protective effect of CQ on aquaporin-2 protein abundance was confirmed in cultured cortical collecting duct cells. In addition, we found that lithium-induced proliferation of collecting duct cells was also suppressed by CQ as detected by proliferating cell nuclear antigen staining. Moreover, both phosphorylated mammalian target of rapamycin and β-catenin expression, which have been reported to be increased by lithium and associated with cell proliferation, were reduced by CQ. Taken together, our study demonstrated that CQ protected against lithium-induced NDI and collecting duct cell proliferation possibly through inhibiting autophagy.

Cisplatin is one of the most effective antitumor agents, but its clinical use is highly limited by its severe side effects, especially nephrotoxicity. Recently, the active form of gasdermin D (GSDMD), termed GSDMD-N, was identified to mediate pyroptotic inflammatory cell death in several diseases. However, the role of the GSDMD-N fragment in cisplatin-induced acute kidney injury (AKI) remains unclear. In the present study, we found that pyroptosis was induced by cisplatin in both mouse kidney tissues and renal tubular epithelial cells, accompanied by increased expression of the GSDMD-N fragment. In GSDMD knockout mice with cisplatin-induced AKI, we found that cisplatin-induced loss of renal function, renal tubular injury, and inflammation was significantly attenuated compared with wild-type mice. Furthermore, the GSDMD-N fragment was overexpressed by an established rapid plasmid tail vein injection approach to evaluate the role of this cleaved form of GSDMD in AKI. As expected, mice with GSDMD-N fragment overexpression in the kidney were more susceptible to cisplatin-induced AKI than control mice, as evidenced by further elevated serum levels of blood urea nitrogen and creatinine, aggravated renal pathology, increased expression of neutrophil gelatinase-associated lipocalin and kidney injury molecule-1, and enhanced renal inflammatory cytokine secretion, which indicates a pathogenic role of GSDMD-N in cisplatin-induced AKI by triggering cell pyroptosis. Similar results were also observed in renal tubular epithelial cells overexpressing the GSDMD-N fragment. Thus these findings suggested that the activation of GSDMD contributes to cisplatin-induced AKI, possibly through triggering pyroptosis.

Renal fibrosis is a key pathological phenomenon of chronic kidney disease (CKD) contributing to the progressive loss of renal function. UK383,367 is a procollagen C proteinase inhibitor that has been selected as a candidate for dermal antiscarring agents, whereas its role in renal fibrosis is unclear. In the present study, UK383,367 was applied to a CKD mouse model of unilateral ureteral obstruction (UUO) and cell lines of renal tubular epithelial cells (mouse proximal tubular cells) and renal fibroblast cells (NRK-49F cells) challenged by transforming growth factor-β1. In vivo, bone morphogenetic protein 1, the target of UK383,367, was significantly enhanced in UUO mouse kidneys and renal biopsies from patients with CKD. Strikingly, UK383,367 administration ameliorated tubulointerstitial fibrosis as shown by Masson’s trichrome staining in line with the blocked expression of collagen type I/III, fibronectin, and α-smooth muscle actin in the kidneys from UUO mice. Similarly, the enhanced inflammatory factors in obstructed kidneys were also blunted. In vitro, UK383,367 pretreatment inhibited the induction of collagen type I/III, fibronectin, and α-smooth muscle actin in both mouse proximal tubular cells and NRK-49F cells treated with transforming growth factor-β1. Taken together, these findings indicate that the bone morphogenetic protein 1 inhibitor UK383,367 could serve as a potential drug in antagonizing CKD renal fibrosis by acting on the maturation and deposition of collagen and the subsequent profibrotic response and inflammation.

Diabetic nephropathy (DN) has become the main cause of end-stage renal disease worldwide, but the efficacy of current therapeutic strategies on DN remains unsatisfactory. Recent research has reported the involvement of metabolic rearrangement in the pathological process of DN, and of all the disturbances in metabolism, mitochondria serve as key regulatory hubs. In the present study, high-resolution mass spectrometry-based nontarget metabolomics was used to uncover the metabolic characteristics of the early diabetic kidney with or without the inhibition of mitochondrial activity. At first, we observed a moderate enhancement of mitochondrial complex-1 activity in the diabetic kidney, which was completely normalized by the specific mitochondrial complex-1 inhibitor rotenone (ROT). Meanwhile, metabolomics data indicated an overactivated pentose phosphate pathway, purine and pyrimidine metabolism, hexosamine biosynthetic pathway, and tricarboxylic acid cycle, which were strikingly corrected by ROT. In addition, ROT also strikingly corrected imbalanced redox homeostasis, possibly by increasing the ratio of antioxidant metabolites glutathione and NADPH against their oxidative form. In agreement with the improved metabolic status and oxidative response, ROT attenuated glomerular and tubular injury efficiently. Fibrotic markers (fibronectin, α-smooth muscle actin, collagen type I, and collagen type III), inflammatory factors (TNF-α, IL-1β, and ICAM-1), and oxidative stress were all markedly blocked by ROT. In vitro, ROT dose dependently attenuated high glucose-induced proliferation and extracellular matrix production in mesangial cells. Collectively, these findings revealed that the overactivation of mitochondrial activity in the kidney could contribute to metabolic disorders and the pathogenesis of early DN.

Glomerular diseases are the leading cause of chronic kidney disease, and mesangial cells (MCs) have been demonstrated to be involved in the pathogenesis. Puromycin aminonucleoside (PAN) is a nephrotoxic drug that induces glomerular injury with elusive mechanisms. The present study was undertaken to investigate the role of PAN in MC apoptosis, as well as the underlying mechanism. Here we found that PAN induced MC apoptosis accompanied by declined cell viability and enhanced inflammatory response. The apoptosis was further evidenced by increments of apoptosis regulator BAX (BAX) and caspase-3 expression. In line with the apoptotic response in MCs following PAN treatment, we also found a remarkable induction of estrogen-related receptor-α (ERRα), an orphan nuclear receptor, at both mRNA and protein levels. Interestingly, ERRα silencing by an siRNA approach resulted in an attenuation of the apoptosis and inflammatory response caused by PAN. More importantly, overexpression of ERRα in MCs significantly triggered MC apoptosis in line with increased BAX and caspase-3 expression. In PAN-treated MCs, ERRα overexpression further aggravated PAN-induced apoptosis. In agreement with the in vitro study, we also observed increased ERRα expression in line with enhanced apoptotic response in renal cortex from PAN-treated rats. These data suggest a detrimental effect of ERRα on PAN-induced MC apoptosis and inflammatory response, which could help us to better understand the pathogenic mechanism of MC injury in PAN nephropathy.

Downregulation of aquaporins (AQPs) in obstructive kidney disease has been well demonstrated with elusive mechanisms. Our previous study indicated that mitochondrial dysfunction played a crucial role in this process. However, it is still uncertain how mitochondrial dysfunction affected the AQPs in obstructive kidney disease. This study investigated the role of mitochondria-derived oxidative stress in mediating obstruction-induced downregulation of AQPs. After unilateral ureteral obstruction for 7 days, renal superoxide dismutase 2 (SOD2; mitochondria-specific SOD) was reduced by 85%. Meanwhile, AQP1, AQP2, AQP3, and AQP4 were remarkably downregulated as determined by Western blotting and/or quantitative real-time PCR. Administration of the SOD2 mimic manganese (III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP) significantly attenuated AQP2 downregulation in line with complete blockade of thiobarbituric acid-reactive substances elevation, whereas the reduction of AQP1, AQP3, and AQP4 was not affected. The cyclooxygenase (COX)-2/prostaglandin (PG) E2pathway has been well documented as a contributor of AQP reduction in obstructed kidney; thus, we detected the levels of COX-1/2 and microsomal prostaglandin E synthase 1 (mPGES-1) in kidney and PGE2secretion in urine. Significantly, MnTBAP partially suppressed the elevation of COX-2, mPGES-1, and PGE2. Moreover, a marked decrease of V2receptor was significantly restored after MnTBAP treatment. However, the fibrotic response and renal tubular damage were unaffected by MnTBAP in obstructed kidneys. Collectively, these findings suggested an important role of mitochondrial oxidative stress in mediating AQP2 downregulation in obstructed kidney, possibly via modulating the COX-2/mPGES-1/PGE2/V2receptor pathway.

Aldosterone (Aldo) has been shown as an important contributor of podocyte injury. However, the underlying molecular mechanisms are still elusive. Recently, the pathogenic role of NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in mediating renal tubular damage was identified while its role in podocyte injury still needs evidence. Thus the present study was undertaken to investigate the role of NLRP3 inflammasome in Aldo-induced podocyte damage. In vitro, exposure of podocytes to Aldo enhanced NLRP3, caspase-1, and IL-18 expressions in dose- and time-dependent manners, indicating an activation of NLRP3 inflammasome, which was significantly blocked by the mineralocorticoid receptor antagonist eplerenone or the antioxidant N-acetylcysteine. Silencing NLRP3 by a siRNA approach strikingly attenuated Aldo-induced podocyte apoptosis and nephrin protein downregulation in line with the blockade of caspase-1 and IL-18. In vivo, since day 5 of Aldo infusion, NLRP3 inflammasome activation and podocyte injury evidenced by nephrin reduction occurred concurrently. More importantly, immunofluorescence analysis showed a significant induction of NLRP3 in podocytes of glomeruli following Aldo infusion. In the mice with NLRP3 gene deletion, Aldo-induced downregulation of nephrin and podocin, podocyte foot processes, and albuminuria was remarkably improved, indicating an amelioration of podocyte injury. Finally, we observed a striking induction of NLRP3 in glomeruli and renal tubules in line with an enhanced urinary IL-18 output in nephrotic syndrome patients with minimal change disease or focal segmental glomerular sclerosis. Together, these results demonstrated an important role of NLRP3 inflammasome in mediating the podocyte injury induced by Aldo.

Progressive fibrosis in chronic kidney disease (CKD) is the well-recognized cause leading to the progressive loss of renal function. Emerging evidence indicated a pathogenic role of the NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in mediating kidney injury. However, the role of NLRP3 in the remnant kidney disease model is still undefined. The present study was undertaken to evaluate the function of NLRP3 in modulating renal fibrosis in a CKD model of 5/6 nephrectomy (5/6 Nx) and the potential involvement of mitochondrial dysfunction in the pathogenesis. Employing NLRP3+/+ and NLRP3−/− mice with or without 5/6 Nx, we examined renal fibrotic response and mitochondrial function. Strikingly, tubulointerstitial fibrosis was remarkably attenuated in NLRP3−/− mice as evidenced by the blockade of extracellular matrix deposition. Meanwhile, renal tubular cells in NLRP3−/− mice maintained better mitochondrial morphology and higher mitochondrial DNA copy number, indicating an amelioration of mitochondrial abnormality. Moreover, NLRP3 deletion also blunted the severity of proteinuria and CKD-related hypertension. To further evaluate the direct role of NLRP3 in triggering fibrogenesis, mouse proximal tubular cells (PTCs) were subjected to transforming growth factor β1 (TGF-β1), and the cellular phenotypic changes were detected. As expected, TGF-β1-induced alterations of PTC phenotype were abolished by NLRP3 small interfering RNA, in line with a protection of mitochondrial function. Taken together, NLRP3 deletion protected against renal fibrosis in the 5/6 Nx disease model, possibly via inhibiting mitochondrial dysfunction.

Evidence has demonstrated that aldosterone (Aldo) is involved in the development and progression of chronic kidney diseases. The purpose of the present study was to investigate the role of autophagy in Aldo-induced podocyte damage and the underlying mechanism. Mouse podocytes were treated with Aldo in the presence or absence of 3-methyladenine and N-acetylcysteine. Cell apoptosis was investigated by detecting annexin V conjugates, apoptotic bodies, caspase-3 activity, and alterations of the podocyte protein nephrin. Autophagy was evaluated by measuring the expressions of light chain 3, p62, beclin-1, and autophagy-related gene 5. Aldo (10−7 mol/l) induced podocyte apoptosis, autophagy, and downregulation of nephrin protein in a time-dependent manner. Aldo-induced apoptosis was further promoted by the inhibition of autophagy via 3-methyladenine and autophagy-related gene 5 small interfering RNA pretreatment. Moreover, Aldo time dependently increased ROS generation, and H2O2 (10−4 mol/l) application remarkably elevated podocyte autophagy. After treatment with N-acetylcysteine, the autophagy induced by Aldo or H2O2 was markedly attenuated, suggesting a key role of ROS in mediating autophagy formation in podocytes. Inhibition of ROS could also lessen Aldo-induced podocyte injury. Taken together, our findings suggest that ROS-triggered autophagy played a protective role against Aldo-induced podocyte injury, and targeting autophagy in podocytes may represent a new therapeutic strategy for the treatment of podocytopathy.

Podocyte damage is a common pathological feature in many types of glomerular diseases and is involved in the occurrence and progression of kidney disease. However, the pathogenic mechanisms leading to podocyte injury are still uncertain. The present study was undertaken to investigate the role of microsomal PGE synthase (mPGES)-1 in adriamycin (ADR)-induced podocyte injury as well as the underlying mechanism. In both mouse kidneys and in vitro podocytes, application of ADR remarkably enhanced mPGES-1 expression in line with a stimulation of cyclooxygenase-2. Interestingly, inhibition of mPGES-1 with a small interfering RNA approach significantly attenuated ADR-induced downregualtion of podocin and nephrin. Moreover, ADR-induced podocyte apoptosis was also markedly blocked in parallel with blunted caspase-3 induction. In agreement with the improvement of cell phenotypic alteration and apoptosis, the enhanced inflammatory markers of IL-1β and TNF-α were also significantly suppressed by mPGES-1 silencing. More importantly, in mPGES-1-deficient mice, albuminuria induced by ADR showed a remarkable attenuation in line with decreased urinary output of PGE2and TNF-α, highly suggesting an in vivo role of mPGES-1 in mediating podocyte injury. In summary, findings from the present study offered the first evidence demonstrating a pathogenic role of mPGES-1 in mediating ADR-induced podocyte injury possibly via triggering an inflammatory response.

Proteinuria is involved in the development of tubular lesions and in the progressive loss of renal function in chronic kidney diseases via uncertain mechanisms. Growing evidence suggests a pathogenic role of mitochondrial dysfunction in chronic kidney diseases. Therefore, the present study aimed to define the roles of mitochondria in proteinuria-induced renal tubular injury and their underlying mechanisms. Using the albumin-overload mouse model, we observed severe tubular structure damage and striking tubular cell apoptosis. Furthermore, tubular epithelial cells displayed a loss of E-cadherin expression and gained expression of α-smooth muscle actin and vimentin, indicating a cellular phenotypic alteration. Strikingly, these albumin overload-induced abnormalities were robustly blocked by a mitochondrial SOD2 mimic, Mn(III) tetrakis (4-benzoic acid)porphyrin chloride (MnTBAP). In agreement with these results, we observed a marked change in mitochondrial morphology accompanied by mitochondrial cytochrome c release and a copy number reduction of mitochondrial DNA. These alterations were largely reversed by MnTBAP, suggesting a key role for mitochondria-derived oxidative stress in mediating the albumin effect on mitochondrial dysfunction and subsequent tubular injury. Moreover, the NOD-like receptor family, pyrin domain-containing 3 (NLRP3)/caspase-1/cytokine cascade was activated in the kidney by albumin overload and was entirely abolished by MnTBAP. In albumin-treated mouse proximal tubular cells, albumin directly induced ROS production, mitochondrial dysfunction, NLRP3/caspase-1/cytokine cascade activation, cell apoptosis, and cellular phenotypic transition. Similar to our in vivo results, treatment with either MnTBAP or cyclosporin A, a mitochondrial permeability transition pore inhibitor, remarkably attenuated these abnormalities in cells. Taken together, these novel findings demonstrate a potential role for the mitochondrial dysfunction/NLRP3 inflammasome axis in the pathogenesis of proteinuria-induced renal tubular injury.

Adriamycin (ADR)-induced nephropathy in animals is an experimental analog of human focal segmental glomerulosclerosis, which presents as severe podocyte injury and massive proteinuria and has a poorly understood mechanism. The present study was designed to test the hypothesis that the peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α-mitochondria axis is involved in ADR-induced podocyte injury. Using MPC5 immortalized mouse podocytes, ADR dose dependently induced downregulation of nephrin and podocin, cell apoptosis, and mitochondrial dysfunction based on the increase in mitochondrial ROS production, decrease in mitochondrial DNA copy number, and reduction of mitochondrial membrane potential and ATP content. Moreover, ADR treatment also remarkably reduced the expression of PGC-1α, an important regulator of mitochondrial biogenesis and function, in podocytes. Strikingly, PGC-1α overexpression markedly attenuated mitochondrial dysfunction, the reduction of nephrin and podocin, and the apoptotic response in podocytes after ADR treatment. Moreover, downregulation of PGC-1α and mitochondria disruption in podocytes were also observed in rat kidneys with ADR administration, suggesting that the PGC-1α-mitochondria axis is relevant to in vivo ADR-induced podocyte damage. Taken together, these novel findings suggest that dysfunction of the PGC-1α-mitochondria axis is highly involved in ADR-induced podocyte injury. Targeting PGC-1α may be a novel strategy for the treatment of ADR nephropathy and human focal segmental glomerulosclerosis.

Mesangial cell (MC) proliferation is a key feature in the pathogenesis of a number of renal diseases. Peroxisome proliferator-activated receptor-γ (PPARγ) has attracted considerable attention for its effects on stimulating cell differentiation and on inducing cell cycle arrest. We previously showed that aldosterone (Aldo) stimulates MC proliferation via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway, which was dependent on reactive oxygen species (ROS)-mediated epithelial growth factor receptor (EGFR) transactivation (Huang S, Zhang A, Ding G, and Chen R. Am J Physiol Renal Physiol 296: F1323–F1333, 2009). In this study, we examined whether the PPARγ agonist rosiglitazone inhibited Aldo-induced MC proliferation by modulating ROS-dependent EGFR intracellular signaling. Rosiglitazone at 1–10 μM dose dependently inhibited Aldo-induced MC proliferation of cultured mouse MCs. The inhibitory effect was blocked by the PPARγ antagonist PD-68235, indicating that the rosiglitazone effect acted through PPARγ activation. Rosiglitazone also arrested Aldo-induced cell cycle progression and suppressed expression of cyclins D1 and A. Moreover, rosiglitazone dose dependently blocked Aldo-induced ROS production, EGFR phosphorylation, and PI3K/Akt activation. These results suggest that the PPARγ agonist rosiglitazone may inhibit Aldo-induced MC proliferation directly, by affecting ROS/EGFR/PI3K/Akt signaling pathways and cell cycle-regulatory proteins. PPARγ might be a novel therapeutic target against glomerular diseases.

We previously showed that ANG II induces mesangial cell (MC) proliferation via the JNK-activator protein-1 pathway. The present study attempted to determine the upstream mediators of JNK activation, with emphasis on reactive oxygen species (ROS) and the epidermal growth factor (EGF) receptor (EGFR). In cultured human MCs (HMCs), as early as 3 min, ANG II time dependently increased intracellular ROS production, which was sensitive to 10 μM diphenyleneiodonium sulfate and 500 μM apocynin, two structurally distinct NADPH oxidase inhibitors. In contrast, inhibitors of other oxidant-producing enzymes, including the mitochondrial complex I inhibitor rotenone, the xanthine oxidase inhibitor allopurinol, the cyclooxygenase inhibitor indomethacin, the lipoxygenase inhibitor nordihydroguiaretic acid, the cytochrome P-450 oxygenase inhibitor ketoconazole, and the nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester, were without effect. ANG II-induced ROS generation was inhibited by the angiotensin type 1 receptor antagonist losartan (10 μM) but not the angiotensin type 2 receptor antagonist PD-123319 (10 μM). ANG II induced translocation of p47phoxand p67phoxfrom the cytosol to the membrane. The antioxidants almost abolished the ANG II mitogenic response, as assessed by [3H]thymidine incorporation and cell number, associated with a remarkable blockade of the activation of EGFR (90% inhibition) and JNK (83% inhibition). The EGFR inhibitor AG-1478 was able to mimic the effect of antioxidants, in that it inhibited the mitogenic response and the JNK activation following ANG II treatment. Together, these data suggest that the ROS-EGFR-JNK pathway is involved in transducing the proliferative effect of ANG II in cultured HMCs.

Angiotensin II (ANG II) has been shown to activate c-Jun NH2-terminal kinase (JNK) in cultured mesangial cells, but the functional implication of this phenomenon remains to be determined, largely due to the lack of an effective approach to block JNK. Therefore, the present study was carried out to examine whether JNK is involved in ANG II-induced cell proliferation in cultured human mesangial cells (HMCs) with the use of a newly developed JNK-selective blocker, SP-600125. Within minutes, treatment with 100 nM ANG II activated all three members of MAP kinase family, including extracellular signal-regulated protein kinase (Erk) 1/2, JNK, and p38 in cultured HMCs, as assessed by immunoblotting detection of phosphorylation of MAP kinases. ANG II-dependent activation of JNK was further confirmed by detection of increased phosphorylation and transcription activity of c-Jun after the ANG II treatment. SP-600125 ranging from 5 to 10 μM almost completely abolished the activation of JNK by ANG II without affecting the activities of Erk1/2 and p38. After treatment with 100 ng ANG II, there was a steady increase in [3H]thymidine incorporation that was blocked by SP-60025 in a dose- and time-dependent manner. Similarly, SP-600125 dose dependently reduced the ANG II-induced increase in cell number. The antiproliferative effect of SP-60025 was further determined by cell-cycle analysis with flow cytometry. Twenty-four hours after ANG II treatment, 50% of the quiescent HMCs (G0/G1) progressed into the S phase, and the cell cycle progression was almost completely prevented in the presence of SP-60025. Our data suggest that JNK mediates the proliferative effect of ANG II in cultured HMCs and thus represents a novel therapeutic target for treatment of chronic renal diseases.