Human Proximal Tubular Cell Line Research Articles

Enhancer of zeste homolog 2 modulates oxidative stress-mediated pyroptosis in vitro and in a mouse kidney ischemia-reperfusion injury model.

Enhancer of zeste homolog 2 (EZH2), a well-known methyltransferase, mediates histone H3 lysine 27 trimethylation (H3K27me3) and plays a crucial role in several kidney disease models. However, its role in renal ischemia/reperfusion (I/R) injury still remains unclear. In this study, we found that EZH2 was positively related to renal I/R injury and inhibition of EZH2 with DZNeP alleviated I/R injury and blocked the activation of oxidative stress and pyroptosis in vivo. Similarly, inhibition of EZH2 with either DZNeP or si-RNA also exerted an inhibitory effect on hypoxia/reoxygenation (H/R)-induced oxidative stress and pyroptosis in vitro. Moreover, further study revealed that ablation of reactive oxygen species (ROS) with N-acetyl-cysteine (NAC) suppressed pyroptosis in human renal proximal tubular epithelial cell line cells exposed to H/R stimulation. Furthermore, Nox4, which was positively related to the generation of ROS, was upregulated during H/R process, while it could be reversed by EZH2 inhibition. Consistently, Nox4-mediated ROS generation was attenuated upon inhibition of EZH2 with DZNeP or si-RNA. Additionally, the transcriptional activity of Nox4 was enhanced by the activation of ALK5/Smad2/3 signaling pathway, which was abolished by ALK5 knockdown in vitro. Finally, EZH2 inhibition blocked H/R and I/R-activated ALK5/Smad2/3 pathway and also resulted in an obvious decrease in the transcriptional activity and protein expression levels of Nox4. In conclusion, our results proved that EZH2 inhibition alleviated renal pyroptosis by blocking Nox4-dependent ROS generation through ALK5/Smad2/3 signaling pathway, indicating that EZH2 could be a potential therapeutic target for renal I/R injury.

Metabolomic Changes of Human Proximal Tubular Cell Line in High Glucose Environment

Hyperglycemia causes mitochondrial damage renal tubular cells, which contribute to the progression of diabetic kidney disease. However, the metabolic aberration of renal tubular cells in an hyperglycemic milieu has not been fully elucidated. In this study, human proximal renal tubular cell line (HK-2 cell) are incubated in glucose and mannitol at 5 mM or 25 mM. Cellular metabolome was determined by capillary electrophoresis time of flight mass spectrometer (CE-TOF/MS) and capillary electrophoresis-triple quadrupole mass spectrometry (CE-QqQMS). A total of 116 metabolites were quantified. Principal component analysis (PCA) revealed excellent clustering of metabolomic changes for different treatment conditions, and exposure to glucose at 5 and 25 mM lead to distinct metabolomic profiles as compared to samples treated with serum-free medium or mannitol as osmotic control. Hierarchical clustering analysis showed a number of characteristic changes in metabolic profile following exposure to 5 mM or 25 mM glucose. Notably, lactate-to-pyruvate ratio was significantly increased, while cellular levels of citric acid, α-ketoglutaric acid (i.e. 2-oxoglutaric acid), and fumaric acid were significantly reduced after exposure to glucose at 25 mM but not 5 mM. Moreover, cellular levels of reduced glutathione and total glutathione were significantly decreased, and S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) ratio was significantly increased after exposure to glucose 25 mM but not 5 mM. We conclude that in response to high glucose, HK-2 cells characteristic metabolomic changes, including increase in lactate-to-pyruvate ratio, reduction in Krebs cycle metabolites, reduction in glutathione antioxidant activity, and increase in cellular methylation potential. Our results may shed light on the pathogenesis of diabetic kidney disease, but the expression of glucose metabolism-related protein and enzyme activity in HK-2 cells after hyperglycemia condition need to be confirmed by further studies.

Micro RNA-30b (inhibitor) nanoparticles suppressed the lipopolysaccharide (LPS)-induced acute kidney injury.

The main aim of present study is to evaluate the effect of miR-30b on the function of human proximal tubular epithelial cell line HK-2 cells. For this purpose, miRNA was loaded in an ionically cross-linked polysaccharide nanoparticle. The authors have demonstrated the influence of miR-30b mimic and inhibitor in HK-2 cell killing effect. Lipopolysaccharide (LPS) significantly increased the level of inflammatory cytokines of TNF-α, IL-1β and level was further increased with the treatment of PAg-miR mimic consistent with the cell viability assay. Interestingly, PAg-miR inhibitor significantly downregulated the expression of inflammatory cytokines and thereby reduced the inflammation in the body. Western blot analysis showed that LPS induced severe apoptosis of HK-2 cells and the apoptosis was further promoted by the PAg-miR (mimic). In contrast, PAg-miR (inhibitor) alleviated the apoptosis of HK-2 cells as indicated in the significantly reduced levels of Bax and c-Caspase-3 proteins. Overall, miR-30b promoted LPS-induced HK-2 cell inflammatory injury by inducing the apoptosis and by releasing inflammatory cytokines, as well as by impairing autophagy process.

The mTOR-RUNX1 pathway regulates DC-SIGN expression in renal tubular epithelial cells

Renal tubular epithelial cells (RTECs) play pivotal roles in the innate immune response in kidneys. Dendritic cell specific intracellular adhesion molecule–3 grabbing nonintegrin (DC-SIGN) functions as both the innate immune recognition receptor and the adhesion molecule. In our previous study, we found that DC-SIGN expression was induced in RTECs during renal inflammation. However, the underlying mechanism remains unclear. Here, we used the human renal proximal tubular epithelial cell lines (HK-2) to investigate the mechanism of TNF-α-induced expression of DC-SIGN. Our results showed that TNF-α up-regulated the expressions of DC-SIGN and Runt-related transcription factor 1 (RUNX1) in a time-dependent manner and that it up-regulated DC-SIGN promoter-driven luciferase activity in a dose-dependent manner. The mTOR inhibitor rapamycin and mTOR siRNA blocked the TNF-α-induced up-regulation of DC-SIGN expression. Meanwhile, DC-SIGN expression was also inhibited by RUNX1 siRNA and its inhibitor Ro5-3335. In addition, both mTOR and RUNX1 inhibitors attenuated TNF-α-induced the increase in DC-SIGN promoter-driven luciferase activity. Finally, we found that HK-2 cells exposed to rapamycin or mTOR siRNA reduced the TNF-α-induced up-regulation of RUNX1. In conclusion, these results indicated that the mTOR-RUNX1 pathway participates in the regulation of TNF-α-induced DC-SIGN expression in RTECs.

Empagliflozin Protects HK-2 Cells from High Glucose-Mediated Injuries via a Mitochondrial Mechanism.

Empagliflozin is known to retard the progression of kidney disease in diabetic patients. However, the underlying mechanism is incompletely understood. High glucose induces oxidative stress in renal tubules, eventually leading to mitochondrial damage. Here, we investigated whether empagliflozin exhibits protective functions in renal tubules via a mitochondrial mechanism. We used human proximal tubular cell (PTC) line HK-2 and employed western blotting, terminal deoxynucleotidyl transferase dUTP nick end labelling assay, fluorescence staining, flow cytometry, and enzyme-linked immunosorbent assay to investigate the impact of high glucose and empagliflozin on cellular apoptosis, mitochondrial morphology, and functions including mitochondrial membrane potential (MMP), reactive oxygen species (ROS) production, and adenosine triphosphate (ATP) generation. We found that PTCs were susceptible to high glucose-induced mitochondrial fragmentation, and empagliflozin ameliorated this effect via the regulation of mitochondrial fission (FIS1 and DRP1) and fusion (MFN1 and MFN2) proteins. Empagliflozin reduced the high glucose-induced cellular apoptosis and improved mitochondrial functions by restoring mitochondrial ROS production, MMP, and ATP generation. Our results suggest that empagliflozin may protect renal PTCs from high glucose-mediated injuries through a mitochondrial mechanism. This could be one of the novel mechanisms explaining the benefits demonstrated in EMPA-REG OUTCOME trial.

Optimal Regimen of N-Acetylcysteine on Chromium-Induced Renal Cell Damage.

Chromium (Cr) is a well-known heavy metal that can cause renal damage. The production of reactive oxygen species (ROS) due to chromium-induced toxicity induces cell dysfunction, apoptosis, and death. N-acetylcysteine (NAC) is an antioxidant used as an antidote for chromium-induced toxicity. However, the optimal regimen and protective mechanisms of NAC are not fully understood in human renal cells. Our results showed that exposure to 10 μM K2Cr2O7, a toxic Cr(VI) compound, induced apoptosis and production of intracellular ROS in the human proximal tubular epithelial cell line HK-2. Supplements of 600 or 1000 µg/mL NAC inhibited intracellular ROS in HK-2 cells exposed to Cr(VI) and significantly increased cell viability within 2 h of Cr(VI)-induced cytotoxicity. Moreover, Cr(VI) induced the expression of apoptosis markers, including cleaved-caspase-3, cleaved-poly (ADP-ribose) polymerase, cleaved-caspase 8, and cleaved-caspase 9, and altered the expression ratio of Bax/Bcl-xL. Expression of apoptosis markers within 2 h of Cr(VI)-induced cytotoxicity in cells treated with 600 µg/mL NAC was significantly suppressed. However, delayed treatment with NAC at 4 h and 8 h after exposure to Cr did not suppress the activation of apoptotic pathways. In summary, our study reports the optimum timing and dose of NAC for the protection of human renal proximal tubular cells from Cr(VI)-induced cell death. The NAC treatment strategy described could be applied in clinical practice to suppress renal cell apoptosis, which in turn could rescue renal function.

Silenced miR-21 inhibits renal interstitial fibrosis via targeting ERK1/2 signaling pathway in mice.

To study the influence of micro ribonucleic acid (miR)-21 on the renal interstitial fibrosis (RIF) model mice, and to preliminarily elucidate the mechanism of action of miR-21 in the development of RIF by studying the influences of miR-21 on the expressions of the proteins related to the extracellular signal-regulated kinase (ERK) 1/2 signaling pathway and its downstream proteins. The mouse model of the left unilateral ureteral obstruction (UUO) was established. The experimental mice were divided into the Sham group and UUO group and were normally fed for 3 weeks. Then, they were executed, and their blood was extracted to determine the renal function-related indicators. The left kidney was excised, and the corresponding specimens were reserved for observing the appearance of the kidney and the morphology of the renal tubules and interstitium. The relative expression levels of epithelial (E-)cadherin, α-smooth muscle actin (α-SMA), and ERK1/2 phosphorylated ERK1/2 (p-ERK1/2), the transforming growth factor-β1 (TGF-β1) and the connective tissue growth factor (CTGF) proteins in renal tissues were determined. After the human renal proximal tubular epithelial cell line, the human kidney-2 (HK-2) was treated with high glucose (HG) combined with silenced miR-21 or the ERK1/2 inhibitor PD98059, the relative expression levels of ɑ-SMA and TGF-β1 protein were measured. UUO group had significantly higher content of blood urea nitrogen (BUN), serum creatinine (SCr), and uric acid (UA) than the Sham group, and exhibited the infiltration of renal interstitial monocytes and lymphocytes, renal tubular podocyte damage, phenotypic transformation and atrophy, the activation and proliferation of interstitial fibroblasts, and excessive deposition of extracellular matrix (ECM). Moreover, the expression level of E-cadherin in the renal tissues was decreased, but the relative expression levels of ɑ-SMA, and TGF-β1, CTGF, and p-ERK1/2 proteins were evidently elevated. Lower relative expression levels of ɑ-SMA and TGF-β1 protein were detected in the human renal proximal tubular epithelial cell line HK-2 after the combined treatment with HG and silenced miR-21 or the ERK1/2 inhibitor PD98059. MiR-21 may be related to the occurrence and development of RIF. Silenced miR-21 probably suppresses RIF via the ERK1/2 signaling pathway.

Serum testosterone acts as a prognostic indicator in polycystic ovary syndrome-associated kidney injury.

Polycystic ovary syndrome (PCOS) is closely related with the onset and development of metabolic abnormalities. However, the correlation between PCOS and kidney injury has not been clarified, and the underlying mechanism remains unknown. Herein, we performed a prospective survey in 55 PCOS and 69 healthy participants. Furthermore, the correlation analyses between serum testosterone and renal functional manifestations of patients and healthy subjects, including urinary albumin to creatinine ratio (UACR), urinary κ‐light chains (KapU), urinary λ‐light chains (LamU), urinary α1‐microglobulin (α1‐MU), and urinary β2‐microglobulin (β2‐MU), were analyzed. Compared with that in normal subjects, the levels of serum testosterone and UACR were significantly higher in PCOS patients. Serum testosterone is significantly correlated with the disease severity of PCOS. Although urinary excretions of KapU, LamU, α1‐MU, and β2‐MU did not increase in PCOS patients, they had a significantly positive correlation with the extent of serum testosterone in PCOS patients. IN vitro, primary cultured human ovary granulosa cells (GCs) were isolated from the follicular fluid (FF) extracting from PCOS patients and controls. FF, especially which extracted from PCOS patients with a high expression of serum testosterone, significantly induced cell apoptosis and inflammation in human GCs. To examine the communication between PCOS and kidney injury, a human proximal tubular epithelial cell line (HKC‐8) was cultured and administered FF. Interestingly, FF from PCOS patients with a higher level of serum testosterone induced fibrotic lesions in HKC‐8 cells. These data suggest serum testosterone plays a critical role in PCOS and PCOS‐associated kidney injury. Serum testosterone may serve as a promising indicator for kidney fibrotic injury outcomes in PCOS patients.

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.

SUN-155 Mitochondrial DNA leakage causes inflammation via the cGAS-STING axis in cisplatin-induced acute kidney injury

Cisplatin is an anti-neoplastic drug that induces tubular inflammation. However, its detailed mechanisms are not fully understood. Stimulator of interferon genes (STING) andits upstream or downstream molecules (cGAS-STING axis) play an important role inself/non-self DNA-triggered signal transduction, leading to inflammation. Here, weinvestigated the roles of STING-mediated inflammation in cisplatin-induced tubular injury. The human proximal tubular cell line, HK-2, treated with 20 μM cisplatin or the renal cortex of WT C57BL/6 and STING KO mice injected with 25 mg/kg cisplatin for 48 or 72 h, were analyzed. The changes in cGAS-STING axis activation, mitochondrial DNA (mtDNA)leakage, or BAX expression were evaluated using real-time PCR, western blotting, or immunostaining analysis. Cisplatin and/or STING siRNA-treated HK-2 culture supernatants were analyzed using cytokine arrays and migration assays. ethidium bromide (EtBr) were used for mtDNA depletion, respectively. In HK-2 or kidney cortex of animals treated with cisplatin, cGAS and STING expression was upregulated and translocated from the ER to the Golgi apparatus, indicating STING activation by cisplatin. Subsequently, the cGAS-STING axis(TBK-1 and P65) was activated due to cisplatin-mediated phosphorylation. Cisplatin also induced pro-inflammatory molecule(IL-6, IL-8, ICAM-1, CXCL10, and GM-CSF) production and neutrophil chemotaxis, which were ameliorated by STING knockdown (P < 0.05). Interestingly, cisplatin-mediated mtDNA leakage to the cytosol activated cGAS-STINGaxis-mediated inflammation. In fact, EtBr-mediated mtDNA depletion inhibited the inflammation by cisplatin and mtDNA transfection facilitated cGAS-STING axis and inflammation in HK-2. Cisplatin-inducedBAX expression,which interacted with the mitochondrial permeability transition pore, suggested that mtDNA leakage was caused by the increase in BAX expression. Moreover, STING KO ameliorated kidney dysfunction and tubular injury accompanied with inhibition of inflammation. mtDNA leakage to the cytosol induces tubular inflammationby activating the cGAS-STING axis in cisplatin nephrotoxicity.

Astragaloside IV ameliorates high glucose-induced HK-2 cell apoptosis and oxidative stress by regulating the Nrf2/ARE signaling pathway.

Diabetic nephropathy (DN) has become the major cause of end-stage renal disease increasing the mortality risk of diabetes. Research has demonstrated that the oxidative damage and apoptosis of renal tubular cells is present during DN. Astragaloside IV (AS-IV) has been widely used for the treatment of many diseases, however, the role and mechanism by which AS-IV may ameliorate high glucose-induced apoptosis and oxidative stress of the human proximal tubular cell line HK-2 remains largely unknown. The present study investigated the effect of AS-IV on high glucose-induced apoptosis and oxidative stress in HK-2 cells. Cell viability, apoptosis and protein expression were detected by Trypan blue staining, Cell Counting Kit-8 assay, terminal deoxynucleotidyl transferase 2′-deoxyuridine-5′-triphosphate nick-end labelling, flow cytometry and western blot analyses. In addition, enzymatic activities, including superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), catalase (CAT) and lipid peroxide (LPO), were measured with the corresponding detection kits. DCFH-DA assay and flow cytometry were performed to detect the production of reactive oxygen species (ROS). Western blot analysis and reverse transcription-quantitative polymerase chain reaction were conducted to evaluate protein and mRNA expressions of the nuclear factor erythroid 2 like 2 (Nrf2)/antioxidant response element (ARE) signaling pathway. The results demonstrated that AS-IV significantly enhanced HK-2 cell viability induced by high glucose in a dose-dependent manner. In addition, AS-IV notably inhibited HK-2 cell apoptosis stimulated by high glucose, which may be associated with inhibition of BCL2 associated X protein, Cleaved-caspase-3 and Cleaved-caspase-9, expression and the promotion of Bcl-2. AS-IV significantly increased the activities of antioxidant enzymes SOD, GSH-Px and CAT, and decreased the high-glucose-induced ROS production in HK-2 cells, in a dose-dependent manner. Finally, it was determined that AS-IV regulated the Nrf2/ARE signaling pathway and inhibited the expression of liver-type fatty acid binding protein. In conclusion, these findings may provide evidence that AS-IV has a potential role for the treatment of DN.

β-hydroxybutyrate attenuates renal ischemia-reperfusion injury through its anti-pyroptotic effects

Ketone bodies including β-hydroxybutyrate (β-OHB) have been shown to protect against ischemic tissue injury when present at low concentrations. We evaluated the impact of β-OHB on renal ischemia/reperfusion injury (IRI). Mice were treated with a continuous infusion of β-OHB using an osmotic mini-pump before and after IRI. We also tested the effects of increasing endogenous serum β-OHB levels by fasting. Renal IRI was attenuated by β-OHB treatment compared to saline control, with similar results in the fasting condition. β-OHB treatment reduced the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)-positive cells and increased expression of forkhead transcription factor O3 (FOXO3), an upstream regulator of pyroptosis. Although β-OHB treatment did not impact markers of apoptosis, it decreased the expression of caspase-1 and proinflammatory cytokines, indicating that β-OHB blocked pyroptosis. In a human proximal tubular cell line exposed to hypoxia and reoxygenation, β-OHB reduced cell death in a FOXO3-dependent fashion. Histone acetylation was decreased in kidneys exposed to IRI and in proximal tubular cells exposed to hypoxia and reoxygenation, and this effect was ameliorated by β-OHB through the inactivation of histone deacetylases. Invitro, β-OHB treatment restored histone acetylation at the FOXO3 promoter. Consistent with epigenetic molecular effects, the renoprotective effects of β-OHB were still observed when the continuous infusion was stopped at the time of IRI. Thus, β-OHB attenuates renal IRI through anti-pyroptotic effects, likely mediated by an epigenetic effect on FOXO3 expression.

Disulfide-bond A oxidoreductase-like protein protects against ectopic fat deposition and lipid-related kidney damage in diabetic nephropathy

Ectopic fat deposition (EFD) in the kidney has been shown to play a causal role in diabetic nephropathy; however, the mechanism underlying EFD remains elusive. By transcriptome analysis, we found decreased expression levels of disulfide-bond A oxidoreductase-like protein (DsbA-L) in the kidneys of diabetic mice (induced by high-fat diet plus Streptozotocin) compared with control mice. Increased expression of adipocyte differentiation-related protein and abnormal levels of collagen I, fibronectin, and phosphorylated 5'AMP-activated kinase (p-AMPK), adipose triglyceride lipase (p-ATGL), and HMG-CoA reductase (p-HMGCR) were also observed in diabetic mice. These alterations were accompanied by deposition of lipid droplets in the kidney, and were more pronounced in diabetic DsbA-L knockout mice. Invitro, overexpression of DsbA-L ameliorated high glucose-induced intracellular lipid droplet deposition in a human proximal tubular cell line, and DsbA-L siRNA aggravated lipid droplet deposition and reduced the levels of p-AMPK, p-ATGL, and p-HMGCR. High glucose and palmitic acid treatment enhanced the expression of interleukin-1β and interleukin-18; these enhancements were further increased after treatment with DsbA-L siRNA but alleviated by co-treatment with an AMPK activator. In kidney biopsy tissue from patients with diabetic nephropathy, DsbA-L expression was negatively correlated with EFD and tubular damage. Collectively, these results suggest that DsbA-L has a protective role against EFD and lipid-related kidney damage in diabetic nephropathy. Activation of the AMPK pathway is a potential mechanism underlying DsbA-L action in the kidney.

Characterization of urinary biomarkers and their relevant mechanisms of zoledronate-induced nephrotoxicity using rats and HK-2 cells.

The aim of this study was to identify biomarkers of zoledronate-induced nephrotoxicity and to further characterize the mechanisms underlying this process by analyzing urinary metabolites. Twenty-four rats were randomly divided into four groups containing four (two control groups) or eight rats (two zoledronate groups) per group. The rats were injected intravenously with saline or zoledronate (3 mg/kg) singly (single, 3 weeks) or repeatedly eight times (3 weeks/time, 24 weeks). Serum blood urea nitrogen, serum creatinine, creatinine clearance, and kidney injury observed by hematoxylin and eosin and immunohistochemical staining were changed only in the repeated zoledronate group (3 mg/kg, 3 weeks/time, 24 weeks). Urinary levels of S-adenosylmethionine, S-adenosylhomocysteine, l-cystathionine, l-γ-glutamylcysteine, and glutathione related to glutathione metabolism and fumaric acid and succinic acid related to the tricarboxylic acid cycle in the zoledronate-treated group (3 mg/kg, 3 weeks/time, 24 weeks) were significantly lower than those in the control group, suggesting that zoledronate may cause cellular oxidative stress. Besides, urinary levels of uracil and uridine related to pyrimidine metabolism also decreased after zoledronate treatment (3 mg/kg, 3 weeks/time, 24 weeks), while the levels of hypoxanthine related to purine metabolism, histamine related to histamine metabolism, and several amino acids were significantly increased. Moreover, zoledronate-induced enhanced oxidative stress and histamine overproduction were confirmed by reactive oxygen species (ROS) and histamine measurement in a human proximal tubular cell line. Taken together, zoledronate-induced nephrotoxicity may be attributed to it inducing perturbations in glutathione biosynthesis and the tricarboxylic acid cycle, further causing ROS overproduction, oxidative stress, and cellular inflammation, thereby leading to nephrotoxicity.

Hyperbilirubinemia Induces Pro-Apoptotic Effects and Aggravates Renal Ischemia Reperfusion Injury

Aims: Hyperbilirubinemia is associated with postoperative acute kidney injury in patients undergoing cardiac surgeries. A high concentration of bilirubin could induce oxidative stress and cell apoptosis. The aim of this study was to investigate whether hyperbilirubinemia aggravated the renal tubule cells injury and the pro-apoptotic potential of bilirubin on renal ischemia reperfusion injury (RIRI). Methods: The human proximal tubular epithelial cell line HK-2 cells were challenged with a gradient concentration of bilirubin for 24 h. Cell injury was assessed by flow cytometry and MTT assay. Bilirubin was injected intraperitoneally into male Sprague-Dawley rats once every 12 h (100 mg/kg), 3 times in total. The same solvent volume without bilirubin powder was used as vehicle in non-bilirubin injection groups. The RIRI surgical procedure was a bilateral renal pedicles clamping (45 min) followed by 30 h reperfusion. The rats were divided into 4 groups: negative control (NC), similar surgical procedures without clamping; Bil, bilirubin injection for 36 h, then rats were sacrificed; RIRI, RIRI surgical procedures; Bil + RIRI, RIRI applied 6 h later than the first bilirubin injection, rats were sacrificed after another 30 h. Results: In vitro, bilirubin induced cell apoptosis and significantly decreased the cell viability of HK-2 cells. Bilirubin induced the active caspase 3 and phosphorylation of p38 in HK-2 cells. In vivo, serum creatinine was higher in Bil + RIRI compared with RIRI (p < 0.01). The tubular injury scores of hematoxylin and eosin and tubular necrosis scores of periodic acid-Schiff were higher in Bil + RIRI than these in RIRI (All p < 0.05). The number of Tunel-positive nuclei was higher in Bil + RIRI compared to RIRI (p < 0.001). The active caspase 3 and phosphorylation of p38 were higher and the Bcl2 was lower in Bil + RIRI compared to RIRI. Moreover, the apoptosis level was higher in Bil compared to NC. Conclusions: Our results reveal that the hyperbilirubinemia induces pro-apoptotic effects and aggravates RIRI.

Retracted Article: TMEM88 inhibits fibrosis in renal proximal tubular epithelial cells by suppressing the transforming growth factor-β1/Smad signaling pathway.

Transmembrane protein 88 (TMEM88) belongs to a member of the TMEM family, and was reported to be involved in fibrogenesis. However, the biological role of TMEM88 in renal fibrosis has not been elucidated. Therefore, the objective of this study was to investigate the effect of TMEM88 on cell proliferation and extracellular matrix (ECM) accumulation in a TGF-β1-induced human renal proximal tubular epithelial cell line (HK2). Our results showed that TMEM88 was downregulated in renal fibrotic tissues and TGF-β1-treated HK2 cells. In addition, TMEM88 overexpression inhibited TGF-β1-induced cell proliferation and migration in HK2 cells. Furthermore, TMEM88 overexpression reduced the production of α-SMA, collagen I, and collagen III in TGF-β1-stimulated HK2 cells. Mechanistically, TMEM88 overexpression suppressed the phosphorylation status of Smad2 and Smad3 in TGF-β1-stimulated HK2 cells. In conclusion, data from our experiments demonstrate that TMEM88 plays a pivotal role in the pathological process of renal fibrosis. TMEM88 inhibited fibrosis in renal proximal tubular epithelial cells by suppressing the TGF-β1/Smad signaling pathway.

Sirtuin 3 suppresses the formation of renal calcium oxalate crystals through promoting M2 polarization of macrophages.

This study aims to verify whether the inhibitory effect of Sirtuin 3 (SIRT3) on the formation of renal calcium oxalate crystals was mediated through promoting macrophages (Mϕs) polarization. Identification and quantification of M1 and M2 monocytes were performed using fluorescence-activated cell sorting analysis. SIRT3 protein level and forkhead box O1 (FOXO1) acetylation level were measured using western blot analysis. Cell apoptosis of HK-2 was detected by flow cytometry. Mouse kidney tissues were subjected to Von Kossa staining, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, and immunohistochemical staining for detection of kidney crystals deposition, apoptosis, and expression of crystal-related molecules, respectively. The results showed that human peripheral blood monocytes from patients with kidney stone (KS) exhibited decreased M2 monocytes percentage and SIRT3 expression, whereas increased FOXO1 acetylation compared with the normal controls. In vitro assay revealed that SIRT3 overexpression in bone marrow-derived M0/M1/M2 Mϕs induced M2 polarization and decreased FOXO1 acetylation. Furthermore, FOXO1 knockdown reversed SIRT3-mediated induction of M2 polarization and inhibition of HK-2 (human proximal tubular cell line) apoptosis. Further in vivo experiments demonstrated that SIRT3-overexpressing Mϕs transfusion not only induced M2 polarization, but also alleviated inflammation, apoptosis, and crystals deposition in glyoxylate-induced KS mice. In conclusion, SIRT3 suppresses formation of renal calcium oxalate crystals through promoting M2 polarization via deacetylating FOXO1.

Unraveling reno-protective effects of SGLT2 inhibition in human proximal tubular cells.

Large clinical trials demonstrated that SGLT2 inhibitors (SGLT2i) slow the progression of kidney function decline in type 2 diabetes. Because the underlying molecular mechanisms are largely unknown, we studied the effects of SGLT2i on gene expression in two human proximal tubular (PT) cell lines under normoglycemic conditions, utilizing two SGLT2i, namely empagliflocin and canagliflocin. Genome-wide expression analysis did not reveal substantial differences between these two SGLT2i. Microarray hybridization analysis identified 94 genes that were both upregulated by TGF-β1 and downregulated by either of the two SGLT2i in HK-2 and RPTEC/TERT1 (renal proximal tubular epithelial cells/telomerase reverse transcriptase 1) cells. Extracellular matrix organization showed the highest significance in pathway enrichment analysis. Differential gene expression of three annotated genes of interest within this pathway was verified on mRNA level in both cell lines. Whereas TGF-β1 induced mRNA expression of thrombospondin 1 (THBS1; 4.3-fold), tenascin C (TNC; 8-fold), and platelet-derived growth factor subunit B (PDGF-B; 4.2-fold), SGLT2i downregulated basal mRNA expression of THBS1 (0.2-fold), TNC (0.5 fold), and PDGF-B (0.6-fold). Administration of SGLT2i in the presence of TGF-β1 resulted in a significant inhibition of TGF-β1-induced THBS1 and TNC mRNA expression and TGF-β1-induced THBS1, TNC, and PDGF-BB protein expression. We conclude that SGLT2i block basal and TGF-β1-induced expression of key mediators of renal fibrosis and kidney disease progression in two independent human PT cell lines.

SIRT3 inhibited the formation of calcium oxalate-induced kidney stones through regulating NRF2/HO-1 signaling pathway.

Oxidative stress is important for the calcium oxalate (CaOx)-induced kidney stone formation. Sirtuin 3 (SIRT3) plays an essential role in the amelioration of oxidative damages. This study aims to explore the effect of SIRT3 on the formation of CaOx-induced kidney stones and the underlying mechanism. SIRT3 expression in renal tissues was detected by immunohistochemistry. Apoptosis in renal tissues was examined by TUNEL staining. Crystal-cell adherence and cell apoptosis in HK-2 cells were assessed by analyzing Ca2+ concentration and by the flow cytometry analysis, respectively. Protein expression of SIRT3, nuclear factor erythroid 2-related factor (NRF2), heme oxygenase-1 (HO-1), and Bax in renal tissues or HK-2 cells was examined by Western blot analysis. Renal pathological changes and the adhesion of CaOx crystals in the kidneys were examined by hematoxylin-eosin and von Kossa staining, respectively. Human kidneys with stones showed enhanced renal apoptosis, downregulated SIRT3 expression, and upregulated NRF2/HO-1 expression, compared with the controls. Furthermore, SIRT3 overexpression inhibited the CaOx-induced promotion of crystal-cell adherence and cell apoptosis in human proximal tubular cell line HK-2 cells, which was reversed by the NRF2 knockdown. Moreover, our in vivo assay further confirmed that SIRT3 overexpression alleviated the glyoxylate administration-induced renal damage, renal apoptosis, and crystals deposition in the kidneys from the stone model mice, which was also associated with its activation of the NRF2/HO-1 pathway. Our findings support the notion that overexpression of SIRT3 may inhibit the formation of CaOx-induced kidney stones, at least in part, through regulating the NRF2/HO-1 signaling pathway.

LncRNA MALAT1 modified progression of clear cell kidney carcinoma (KIRC) by regulation of miR-194-5p/ACVR2B signaling.

This investigation was purposed to extrapolate whether and how lncRNA MALAT1, miR-194-5p, and ACVR2B altered development of clear cell kidney carcinoma (KIRC). We totally gathered 318 pairs of KIRC tissues and adjacent normal tissues, and also purchased human KIRC cell lines and normal human proximal tubular epithelial cell line. Besides, si-MALAT1, pcDNA-MALAT1, miR-194-5p mimic, miR-194-5p inhibitor, and negative control (NC) were, respectively, transfected into KIRC cells. The viability, proliferation, and apoptosis of the cells were determined with CCK-8 assay, colony formation assay, and flow cytometry. Dual-luciferase reporter gene assay was implemented to validate the targeted relationships between MALAT1 and miR-194-5p, as well as between miR-194-5p and ACVR2B. The results showed that highly expressed MALAT1, ACVR2B, and lowly expressed miR-194-5p were associated with larger tumor size (≥4 cm), advanced TNM stage and poor prognosis of KIRC patients, when, respectively, compared with lowly expressed MALAT1, ACVR2B, and highly expressed miR-194-5p (P < 0.05). Transfection of pcDNA-MALAT1, miR-194-5p inhibitor, and pcDNA-ACVR2B conferred the KIRC cells with promoted viability and proliferation, as well as reduced apoptosis (P < 0.05). Treatment of rats with pcDNA-MALAT1, miR-194-5p inhibitor, or pcDNA-ACVR2B also contributed to larger tumor size growing in them (P < 0.05). Moreover, MALAT1 could directly target miR-194-5p to suppress its expression, and ACVR2B was the targeted molecule of miR-194-5p (P < 0.05). Finally, ACVR2B could reverse the effects exerted by miR-194-5p on viability, proliferation, and apoptosis of KIRC cells (P < 0.05). In conclusion, LncRNA MALAT1/miR-194-5p/ACVR2B signaling was regarded as a candidate pathway for modulating KIRC progression.