Proximal Tubular Epithelial HK-2 Cells Research Articles

Kidney-targeted antioxidant salvianolic acid B nanoparticles restoring lysosome homeostasis for acute kidney injury therapy

Acute kidney injury (AKI) is highly correlated with oxidative stress, and the application of antioxidants is one of the promising treatments. Salvianolic acid B (SAB) is a natural polyphenol with powerful antioxidant effects. Due to its inherent characteristic of inferior bioavailability, its clinical application is impeded. In this study, small SAB-incorporated nanoparticles (SFA) were synthesized via the self-assembly of SAB, ferric ions, and bovine serum albumin (BSA). Apart from antioxidant activity, the SFA nanoparticles were found to efficiently restore lysosome dysfunction induced by reactive oxygen species (ROS) in human kidney proximal tubular epithelial HK-2 cells. The small size and BSA incorporation make the SFA nanoparticles to be promising AKI-targeting nanoparticles. They are rapidly accumulated and long-term retained in injured kidneys of glycerol-induced AKI mice, especially in the proximal tubules which are the key lesion location during AKI. In vivo studies indicate that SFA nanoparticles show a superior protective effect compared with the free drug by suppressing oxidative stress and maintaining lysosome homeostasis indicated by enhanced heme oxygenase-1 (HO-1) expression and restored cathepsin B (CTSB) activity, respectively. In summary, this study develops a delivery system for the antioxidant SAB enhancing local delivery and offering an additional lysosome restoring mechanism for AKI therapy.

UHPLC-MS-based untargeted metabolomic strategy to reveal the metabolic differences between cisplatin first- and second-generation apoptotic bodies from HK-2 cells

During the process of programmed cell death (apoptosis), the disassembly of apoptotic cells gives rise to apoptotic bodies (ABs) which play an important role in the pathogenesis of different diseases. ABs from renal human proximal tubular cells HK-2 on naïve cells was not always the same: ABs directly generated after exposure of human proximal tubular epithelial HK-2 cells to chemotherapeutic agent cisplatin (1st generation ABs) triggered apoptosis and inhibited cell proliferation, whereas ABs induced by 1st generation ABs (2nd generation ABs) enhanced HK-2 cell proliferation. To shed light on the mechanisms involved in these disparate ABs’ effects, a study of the changes in the metabolome of 1st and 2nd generation ABs produced by HK-2 cells in the cisplatin setting was performed by a reversed-phase ultra-high-performance liquid chromatography-mass spectrometry (RP-UHPLC-MS) untargeted metabolomic approach. Thus, ABs fluid and extracellular fluid from 1st and 2nd generation ABs were analyzed to obtain as much information as possible of the metabolites of these extracellular vesicles. Principal components analysis and partial least square discriminant analysis were conducted to find the statistically significant differences between both groups. A good clustering of each group was observed and variable importance in the projection values were selected to choose the molecular features which were identified according to the Metabolomics Standard Initiative (MSI) guidelines. Finally, the biological relevance of the achieved results was discussed.

N-methyl-d-aspartate receptor 1 activation mediates cadmium-induced epithelial-mesenchymal transition in proximal tubular cells

Cadmium (Cd) is a commonly found environmental pollutant and is known to damage multiple organs with kidneys being the most common one. N-methyl-d-aspartate receptor 1 (NMDAR1) is a ligand-gated ion channel that is highly permeable to calcium ion (Ca2+). Because Cd2+ and Ca2+ have structural and physicochemical similarities, whether and how Cd could interfere NMDAR1 function to cause renal epithelial cells dysfunction remains unknown. In this study, we investigated the role of NMDAR1 in Cd-induced renal damage and found that Cd treatment upregulated NMDAR1 expression and promoted epithelial-mesenchymal transition (EMT) in mouse kidneys in vivo and human proximal tubular epithelial HK-2 cells in vitro, which were accompanied with activation of the inositol-requiring enzyme 1 (IRE-1α) / spliced X box binding protein-1 (XBP-1s) pathway, an indicative of endoplasmic reticulum (ER) stress. Mechanistically, NMDAR1 upregulation by Cd promoted Ca2+ channel opening and Ca2+ influx, resulting in ER stress and subsequently EMT in HK-2 cells. Inhibition of NMDAR1 by pharmacological antagonist MK-801 significantly attenuated Cd-induced Ca2+ influx, ER stress, and EMT. Pretreatment with the IRE-1α/XBP-1s pathway inhibitor STF-083010 also restored the epithelial phenotype of Cd-treated HK-2 cells. Therefore, our findings suggest that NMDAR1 activation mediates Cd-induced EMT in proximal epithelial cells likely through the IRE-1α/XBP-1s pathway, supporting the idea that NMDAR1 could be a potential therapeutic target for Cd-induced renal damage.

Piperlonguminine attenuates renal fibrosis by inhibiting TRPC6

Ethnopharmacological relevanceLiuwei Dihuang (LWDH) is a classic prescription that has been used to the treatment of “Kidney-Yin” deficiency syndrome for more than 1000 years in China. Recent studies have confirmed that LWDH can prevent the progression of renal fibrosis. Numerous studies have demonstrated the critical role that TRPC6 plays in the development of renal fibrosis. Due to the complex composition of LWDH and its remarkable therapeutic effect on renal fibrosis, it is possible to discover new active ingredients targeting TRPC6 for the treatment of renal fibrosis. Aim of studyThis study aimed to identify selective TRPC6 inhibitors from LWDH and evaluate their therapeutical effects on renal fibrosis. Materials and methodsComputer-aided drug design was used to screen the biologically active ingredients of LWDH, and their affinities to human TRPC6 protein were detected by microcalorimetry. TRPC6, TRPC3, and TRPC7 over-expressed HEK293 cells were constructed, and the selective activities of the compounds on TRPC6 were determined by measuring [Ca2+]i in these cells. To establish an in vitro model of renal fibrosis, human renal proximal tubular epithelial HK-2 cells were stimulated with TGF-β1. The therapeutic effects of LWDH compounds on renal fibrosis were then tested by detecting the related proteins. TRPC6 was knocked-down in HK-2 cells to investigate the effects of LWDH active ingredients on TRPC6. Finally, a unilateral ureteral obstruction model of renal fibrosis was established to test the therapeutic effect. ResultsFrom hundreds of LWDH ingredients, 64 active components with oral bioavailability ≥30% and drug-likeness index ≥0.18 were acquired. A total of 10 active components were obtained by molecular docking with TRPC6 protein. Among them, 4 components had an affinity with TRPC6. Piperlonguminine (PLG) had the most potent affinity with TRPC6 and blocking effect on TRPC6-mediated Ca2+ entry. A 100 μM of PLG showed no detectable inhibition on TRPC1, TRPC3, TRPC4, TRPC5, or TRPC7-mediated Ca2+ influx into cells. In vitro results indicated that PLG concentration-dependently inhibited the abnormally high expression of α-smooth muscle actin (α-SMA), collagen I, vimentin, and TRPC6 in TGF-β1-induced HK-2 cells. Consistently, PLG also could not further inhibit TGF-β1-induced expressions of these protein biomarkers in TRPC6 knocked-down HK-2 cells. In vivo, PLG dose-dependently reduced urinary protein, serum creatinine, and blood urea nitrogen levels in renal fibrosis mice and markedly alleviated fibrosis and the expressions of α-SMA, collagen I, vimentin, and TRPC6 in kidney tissues. ConclusionOur results showed that PLG had anti-renal fibrosis effects by selectively inhibiting TRPC6. PLG might be a promising therapeutic agent for the treatment of renal fibrosis.

GBP5 Inhibition Ameliorates the Progression of Lupus Nephritis by Suppressing NLRP3 Inflammasome Activation

ABSTRACT Background The inflammatory response and NLRP3 inflammasome activation are typical characteristics of lupus nephritis (LN). Guanylate-binding protein 5 (GBP5) has effects on the release of proinflammatory cytokines and the activation of NLRP3 inflammasome. However, it is largely unknown whether and how GBP5 contributes to the progression of LN. Methods To detect the role of GBP5 in LN, MRL/lpr mice were administrated with the lentiviral vectors that knockdown GBP5 via tail vein. Proximal tubular epithelial HK-2 cells were treated with LPS and ATP to mimic the inflammatory response of LN in vitro. Results GBP5 expression was increased in the renal cortical tissues of LN mice. The in vivo results showed that GBP5 inhibition prevented the progression of LN, as evidenced by the decreased levels of 24-hour proteinuria, blood urea nitrogen and creatinine, accompanied by the ameliorated renal pathological damages. The increased mRNA and protein levels of proinflammatory factors (IL-6, TNF-α, iNOS and COX-2) in the renal cortex of LN mice were suppressed by GBP5 knockdown. In vitro, we demonstrated that the treatment of LPS combined with ATP induced an increase in GBP5 mRNA and protein expression in HK-2 cells. Mechanically, knockdown of GBP5 inhibited the activation of NLRP3 inflammasome and the secretion of IL-1β and IL-18 both in vivo and in vitro. Conclusion Our findings reveal that GBP5 inhibition prevents the progression of LN, most likely by suppressing NLRP3 inflammasome activation. It provides a novel insight into the therapeutic interventions for LN.

Sirtuin-1 attenuates cadmium-induced renal cell senescence through p53 deacetylation

Cadmium (Cd), the common environmental pollutant, primarily targets at renal proximal tubules and induces nephrotoxicity. Cellular senescence, a phenomenon of cell growth arrest and a characteristics of maladaptive cell self-repair, is associated with renal disease progression. However, whether and how Cd induces renal tubular cells premature senescence is unknown. In our study, we found that Cd induced kidney damage and dysfunctions, which correlated with exacerbated tubular cell senescence, evidenced by increased senescence-associated β-galactosidase activity, the upregulated protein expression of p53 and p21Waf1/Cip1 proteins, and elevated expression and secretion of cytokines in human proximal tubular epithelial HK-2 cells in vitro and in Cd-treated mice in vivo. Moreover, a S-phase arrest and decrease in Edu positive rate were found in Cd-treated HK-2 cells. Mechanistically, Cd suppressed the expression and activity of Sirtuin-1 (SIRT1), an anti-senescence deacetylase, resulting in the accumulation of acetylated p53 and upregulation of p21Waf1/Cip1. Activation of SIRT1 significantly abolished Cd-induced premature senescence and S-phase arrest. Finally, silencing p21Waf1/Cip1 efficiently delayed premature senescence and recovered cell cycle progression. These findings indicate that Cd promotes tubular cells senescence and impairs tubular cells regeneration, resulting in kidney dysfunctions, which could be ameliorated by SIRT1 activation.

Aldehyde dehydrogenase 2 protects against acute kidney injury by regulating autophagy via Beclin-1 pathway

The mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) catalyzes the detoxification of acetaldehyde and endogenous lipid aldehydes. Approximately 40% of East Asians, accounting for 8% of the human population, carry the E504K mutation in ALDH2 that leads to accumulation of toxic reactive aldehydes and increases the risk for cardiovascular disease, cancer, and Alzheimer disease, among others. However, the role of ALDH2 in acute kidney injury (AKI) remains poorly defined and is therefore the subject of the present study using various cellular and organismal sources. In murine models, in which AKI was induced by either the contrast agent iohexol or renal ischemia/reperfusion, KO, activation/overexpression of ALDH2 were associated with increased and decreased renal injury, respectively. In murine renal tubular epithelial cells (RTECs), ALDH2 upregulated Beclin-1 expression, promoted autophagy activation, and eliminated ROS. In vivo and in vitro, both 3-MA and Beclin-1 siRNAs inhibited autophagy and abolished ALDH2-mediated renoprotection. In mice with iohexol-induced AKI, ALDH2 knockdown in RTECs using AAV-shRNA impaired autophagy activation and aggravated renal injury. In human renal proximal tubular epithelial HK-2 cells exposed to iohexol, ALDH2 activation potentiated autophagy and attenuated apoptosis. In mice with AKI induced by renal ischemia/reperfusion, ALDH2 overexpression or pretreatment regulated autophagy mitigating apoptosis of RTECs and renal injury. In summary, our data collectively substantiate a critical role of ALDH2 in AKI via autophagy activation involving the Beclin-1 pathway.

Calciprotein particle-induced cytotoxicity via lysosomal dysfunction and altered cholesterol distribution in renal epithelial HK-2 cells

Dietary phosphate overload induces chronic kidney disease (CKD), and calciprotein particles (CPPs), a form of nanoparticle comprising calcium phosphate and serum proteins, has been proposed to cause renal toxicity. However, the mechanism of CPP cytotoxicity in renal tubular cells is unknown. Here we show that in renal proximal tubular epithelial HK-2 cells, endocytosed CPPs accumulate in late endosomes/lysosomes (LELs) and increase their luminal pH by ~ 1.0 unit. This results in a decrease in lysosomal hydrolase activity and autophagic flux blockage without lysosomal rupture and reactive oxygen species generation. CPP treatment led to vulnerability to H2O2-induced oxidative stress and plasma membrane injury, probably because of autophagic flux blockage and decreased plasma membrane cholesterol, respectively. CPP-induced disruption of lysosomal homeostasis, autophagy flux and plasma membrane integrity might trigger a vicious cycle, leading to progressive nephron loss.

CPUY192018, a potent inhibitor of the Keap1-Nrf2 protein-protein interaction, alleviates renal inflammation in mice by restricting oxidative stress and NF-κB activation

The Keap1-Nrf2-ARE pathway regulates the constitutive and inducible transcription of various genes that encode detoxification enzymes, antioxidant proteins and anti-inflammatory proteins and has pivotal roles in the defence against cellular oxidative stress. In this study, we investigated the therapeutic potential of CPUY192018, a potent small-molecule inhibitor of the Keap1-Nrf2 protein-protein interaction (PPI), in renal inflammation. In human proximal tubular epithelial HK-2 cells, CPUY192018 treatment significantly increased Nrf2 protein level and Nrf2 nuclear translocation, which enhanced Nrf2-ARE transcription capacity and the downstream protein content in a Nrf2 dependent manner. In lipopolysaccharide (LPS)-challenged human HK-2 cells, CPUY192018 exhibited cytoprotective effects by enhancing the Nrf2-ARE regulated antioxidant system and diminished the LPS-induced inflammatory response by hindering the ROS-mediated activation of the NF-κB pathway. In the LPS-induced mouse model of chronic renal inflammation, by activating Nrf2, CPUY192018 treatment balanced renal oxidative stress and suppressed inflammatory responses. Hence, administration of CPUY192018 reduced kidney damage and ameliorated pathological alterations of the glomerulus. Taken together, our study suggested that small-molecule Keap1-Nrf2 PPI inhibitors can activate the Nrf2-based cytoprotective system and protect the kidney from inflammatory injury, raising a potential application of Keap1-Nrf2 PPI inhibitors in the treatment of inflammatory kidney disorders.

Inactivation of TSC1 promotes epithelial-mesenchymal transition of renal tubular epithelial cells in mouse diabetic nephropathy.

Epithelial–mesenchymal transition (EMT) of renal tubular epithelial cells is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic nephropathy (DN). However, the molecular mechanisms of EMT remain largely unknown. Tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling, and the loss of TSC1 or TSC2 function leads to a spectrum of diseases that underlie abnormalities in cell growth, proliferation, differentiation, and migration. In this study, we investigated the effects of TSC1 on high glucose (HG)-induced EMT of human proximal tubular epithelial HK-2 cells in vitro and renal fibrosis in TSC1−/− and db/db mice. We found that the exposure of HK-2 cells to HG (30 mM) time-dependently decreased TSC1 expression, increased the phosphorylation of mTORC1, P70S6K, and 4E-BP-1, and promoted cell migration, resulting in EMT. Transfection of the cells with TSC1 mimic significantly ameliorated HG-induced EMT of HK-2 cells. The tubules-specific TSC1 knockout mice (TSC1−/−) displayed a significant decline in renal function. TSC1−/− mice, similar to db/db mice, showed greatly activated mTORC1 signaling and EMT process in the renal cortex and exacerbated renal fibrosis. Overexpression of TSC1 through LV-TSC1 transfection significantly alleviated the progression of EMT and renal fibrosis in the renal cortex of db/db mice. Taken together, our results suggest that TSC1 plays a key role in mediating HG-induced EMT, and inhibition of TSC1-regulated mTORC1 signaling may be a potential approach to prevent renal fibrosis in DN.

Involvement of Tubular Mas Receptor in Lipid‐Induced Impaired Autophagy and ER Stress in the Kidney

The local renin‐angiotensin system in the kidney plays an important role in development of obesity‐related kidney diseases. The purpose of current study was to investigate the role of Mas receptor in lipid‐induced kidney injuries. In human proximal tubular epithelial HK2 cells, saturated fatty acid palmitic acid (PA, 0.4mM) treatment induced increased LC3B and P62 protein expression and decreased LAMP1 expression, indicating an impaired autophagy flux and accumulation of autophagosome. This was associated with upregulated BIP and CHOP protein expression, two markers of endoplasmic reticulum stress (ER stress), and upregulated expression of cleaved caspase‐3, a marker of apoptosis. PA‐induced autophagosome accumulation, ER stress, and apoptosis was enhanced by Mas receptor agonists Ang (1‐7) or AVE0991, and suppressed by A779, a synthetic specific Mas inhibitor. PA‐induced impaired autophagy flux, increased ER stress and apoptosis were markedly attenuated by Mas receptor gene knockout in HK2 cells. Knockout Mas receptor also improved HK2 cell vitality and proliferation in response to PA treatment. Moreover, PA treatment caused elevated intracellular calcium levels, mitochondrial depolarization which were also markedly prevented by Mas knockout. Mas receptor gene knockout in HK2 cells was associated with markedly improved mitophagy after PA treatment. In mice with high‐fat diet for 12 weeks, Ang (1‐7) treatment caused enhanced autophagosome accumulation, ER stress, and apoptosis in the kidney cortex. In conclusion, Mas receptor mediated lipid‐induced impaired autophagy and ER stress in the kidney, likely contributing to tubular injuries seen in obesity‐related kidney diseases.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Sirtuin-1 ameliorates cadmium-induced endoplasmic reticulum stress and pyroptosis through XBP-1s deacetylation in human renal tubular epithelial cells.

Cadmium (Cd), an occupational and environmental pollutant, induces nephrotoxicity by primarily damaging renal proximal tubular cells. In this study, we hypothesized that pyroptosis, a caspase-1-dependent inflammatory programmed cell death mechanism, mediates Cd-induced nephrotoxicity. Human proximal tubular epithelial HK-2 cells were treated with 0-10µM CdCl2 for 48h. We found that Cd dose-dependently caused cytotoxicity, which correlated with activation of the NLRP3 inflammasome, increases in the expression and secretion of pro-inflammatory cytokines and upregulation of pyroptosis-related genes in HK-2 cells or/and in kidneys of Cd-treated mice. These effects were significantly abrogated by inhibiting caspase-1 activity with inhibitor YVAD or silencing NLRP3 with siRNA in vitro, suggesting that Cd induces caspase-1- and NLRP3-inflammasome-dependent pyroptosis. Moreover, Cd treatment also activated three branches (ATF6, PERK and IRE-1α) of endoplasmic reticulum stress. Selective inhibition of the IRE-1α/XBP-1s branch by a pharmacological inhibitor STF-083010 or by genetic silencing of XBP-1 significantly attenuated Cd-induced NLRP3 inflammasome activation and pyroptosis. Mechanistically, Cd suppressed deacetylase Sirtuin-1 (SIRT-1) protein expression and activity leading to decrease in physical binding with XBP-1s protein, and thus the accumulation of acetylated XBP-1s levels. Activation of SIRT1 using a pharmacological agonist resveratrol or genetic SIRT1 overexpression significantly abolished Cd-induced activation of the IRE-1α/XBP-1s pathway and the NRLP3 inflammasome as well as pyroptosis, which were counteracted by co-overexpression of both SIRT1 and XBP-1s. Collectively, our findings indicate that SIRT1 activity protects against Cd-induced pyroptosis through deacetylating XBP-1s, and thus inhibiting the IRE-1α/XBP-1s pathway in HK-2 cells. These results provide a novel mechanism for Cd-induced nephrotoxicity.

Piperine ameliorated lupus nephritis by targeting AMPK-mediated activation of NLRP3 inflammasome

BackgroundLupus nephritis (LN) is a leading cause of mortality. The activation of NLRP3 inflammasome contributed to LN development and thus became a therapeutic target. Here we assessed the therapeutic potential of piperine, a bioactive compound known to target NLRP3 inflammasome, on LN development both in vivo and in vitro. MethodsLN was induced in BALB/c mice via intraperitoneal injection of pristane. Upon treatment with increasing doses of piperine, we assessed renal lesions, measured serum levels of pro-inflammatory cytokines, and examined expressions of key components of NLRP3 inflammasome in kidney. To explore the molecular mechanisms, we treated the proximal tubular epithelial HK-2 cells with lipopolysaccharide (LPS) and ATP, and examined the effects of piperine on pyroptosis and the activation of NLRP3 inflammasome. Furthermore, we assessed the significance of AMPK signaling in piperine functions in HK-2 cells. ResultsIn pristane-injected mice, piperine significantly ameliorated LN development in a dose-dependent manner, which was associated with the inhibition of NLRP3 inflammasome and the reduction of serum IL-1β, but not of IL-18 level. In HK-2 cells, piperine potently inhibited pyroptosis and the activation of NLRP3 inflammasome in response to LPS + ATP. The effects of piperine were mediated by blocking AMPK activation, and the AMPK agonist metformin bypassed the activities of piperine, and resumed pyroptosis as well as the activation on NLRP3 inflammasome. ConclusionsBy targeting AMPK, piperine significantly suppressed the activation of NLRP3 inflammasome, inhibited the release of pro-inflammatory cytokines, blocked the pyroptosis of tubular epithelial cells, and thus suppressed the development of LN.

Berberine protects HK-2 cells from hypoxia/reoxygenation induced apoptosis via inhibiting SPHK1 expression.

Renal ischemia reperfusion injury (RIRI) refers to the irreversible damage for renal function when blood perfusion is recovered after ischemia for an extended period, which is common in clinical surgeries and has been regarded as a major risk for acute renal failures (ARF) that is accompanied with unimaginably high morbidity and mortality. Hypoxia during ischemia followed by reoxygenation via reperfusion serves as a major event contributing to cell apoptosis, which has been widely accepted as the vital pathogenesis in RIRI. Preventing apoptosis in renal tubular epithelial cell has been considered as effective method for blocking RIRI. In this paper, we established a hypoxia/reoxygenation (H/R) injury model in human proximal tubular epithelial HK-2 cells. Here, we found increased SPHK1 levels in H/R injured HK-2 cells, which could be significantly down regulated after berberine treatment. Berberine has been reported to exert a protective effect on H/R-induced apoptosis of HK-2 cells. So, in our present study, we planned to investigate whether SPHK1 participated in the anti-apoptosis process of berberine in H/R injured HK-2 cells. Our study confirmed the protective effect of berberine against H/R-induced apoptosis in HK-2 cells through promoting cells viability, inhibiting cells apoptosis, and down-regulating p-P38, caspase-3, caspase-9 as well as SPHK1, while up regulating the ratio of Bcl-2/Bax. However, SPHK1 overexpression in HK-2 cells induced severe apoptosis, which can be significantly ameliorated with additional berberine treatment. We concluded that berberine could remarkably prevent H/R-induced apoptosis in HK-2 cells through down-regulating SPHK1 expression levels, and the mechanisms included the suppression of p38 MAPK activation and mitochondrial stress pathways.

The mechanism of oxalate-induced nucleotide binding oligomerization domain-like receptor protein 3 inflammasome activation contributing to the formation of calcium oxalate stone in HK-2 cells

Objective To observe the mechanism of nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome to the formation of calcium oxalate stone after exposure to high concentrate of oxalate. Methods The human kidney proximal tubular epithelial HK-2 cells were cultured and stimulated with different concentrations of soluble oxalate cells. Lactate dehydrogenase (LDH) released assays from cell culture medium and 4’, 6-diamidino-2-phenylindole (DAPI) staining were used to determine the effect of cellular toxicity and injury after exposure to oxalate. All the cells of oxalate group and control group were incubated with calcium oxalate monohydrate (COM) crystals for 24 h, and cell surface adhesion of crystals were observed using an inverted phase-contrast microscope. Western blotting and real-time fluorescent quantitative polymerase chain reaction (FQ-PCR) were applied to detect protein level and mRNA quantity of NLRP3, cysteinyl aspartate-specific protease-1 (Caspase-1), and interleukin-1β (IL-1β) from HK-2 cell lysates after exposure to oxalate. FQ-PCR was applied to analyse mRNA quantity of hyaluronan synthase 1 (HAS1), hyaluronan synthase 2 (HAS2), hyaluronan synthase 3 (HAS3), osteopontin (OPN) and CD44 from HK-2 cell after exposure to oxalate. Intracellular reactive oxygen species (ROS) generation was estimated by the method of using 2’, 7’-dichlorofluorescein diacetate (DCFH-DA) as per constructor’s protocol after exposure to oxalate. Thereafter, N-acetylcysteine (NAC), a scavenger of ROS was pre-treated with HK-2 cells for 2 h, and Western blotting and FQ-PCR were applied to detect protein level and mRNA quantity of NLRP3 gene. HK-2 cells were transfected by NLRP3-small interfering RNA (siRNA). After oxalate stimulation, an inverted phase-contrast microscope was used to detect the number of the binding COM crystals on the surface of the cells, and the levers of HAS1, HAS2, HAS3, OPN and CD44 mRNA through FQ-PCR. Results Exposure of HK-2 cells to different concentrations of oxalate for 24 h, it observed that exposure to the oxalate concentrations (0.8 mmol/L) led to significantly higher in LDH release than the oxalate concentrations (0.6 mmol/L) [(528.37±25.65) vs. (300.55±17.18) mmol/L, P=0.001], and the DAPI staining showed different cell number: 55.67±3.30, 52.33±6.13, 52.33±2.05, 58.00±9.09, 50.00±1.63, 47.33±2.05, 33.67±3.30, 20.33±2.05, and the result showed that exposure to the oxalate concentrations (1.0 mmol/L) led to a meaningful reduction in the number of cells compared with the oxalate concentrations (0.8 mmol/L) (33.67±4.04 vs. 47.33±2.52, P=0.008). Using an inverted phase-contrast microscope observed that a significantly greater number of the binding COM crystals on the surface of the high-oxalate-exposed cells as compared to the controls [(7.34±0.82)% vs. (5.29±0.73)%, P=0.001]. Exposure of HK-2 cells to high concentration oxalate for 24 h significantly increased the expression of protein (1.82±0.04 vs. 1.00±0.02, P=0.000; 1.62±0.23 vs. 1.00±0.06, P=0.021; 1.37±0.16 vs. 1.00±0.18, P=0.029) and mRNA levels (1.15±0.26 vs. 0.81±0.11, P=0.011; 2.16±0.29 vs. 1.04±0.14, P=0.009; 1.80±0.02 vs. 1.00±0.15, P=0.002) of NLRP3, Caspase-1, IL-1β when compared with the control, and the mRNA level (1.35±0.25 vs. 0.81±0.11, P=0.049; 2.46±0.29 vs. 0.84±0.14, P=0.002; 0.90±0.02 vs. 0.60±0.15, P=0.039; 1.68±0.27 vs. 1.00±0.22, P=0.049; 2.03±0.47 vs. 1.00±0.15, P=0.040) of HAS1, HAS2, HAS3, OPN and CD44 significantly up-regulated compared with the control. Exposure to high concentration of oxalate for 24 hours, the intracellular ROS levels of HK-2 were meaningfully increased (736.67±80.21 vs. 208.67±35.73) by the method of using DCFH-DA (P=0.001). Cells were pre-incubated with a ROS scavenger (NAC) for 2 h, followed by treatment with oxalate. According to the western blotting analysis of HK-2 cell lysates, the blockade of ROS by NAC resulted in decreased the protein levels (1.53±0.31 vs. 4.50±0.40)of NLRP3 gene in HK-2 cells (P=0.001), and that in the control and the NAC group were 0.90±0.10 and 1.00±0.26 respectively (P=0.288). The silencing of the NLRP3 gene obviously reduced the number of calcium oxalate crystals adhesion to cells [(1.87±0.21)% vs. (3.57±0.12)%, P=0.003], and the mRNA levels (1.06±0.07 vs. 2.44±0.47, P=0.015; 0.87±0.07 vs. 1.48±0.22, P=0.021; 1.24±0.16 vs. 1.72±0.10, P=0.026; 1.03±0.15 vs. 1.45±0.09, P=0.026; 0.63±0.07 vs. 1.31±0.12, P=0.002) of HAS1, HAS2, HAS3, OPN and CD44 mRNA were also descended. Conclusion Activation of the NLRP3 inflammasome induces by the generation of ROS in the formation of calcium oxalate kidney stone; The NLRP3 inflammasome activation engages by changing the renal tubular epithelial adhesion of crystal and involved in the formation of the stone. Key words: Nucleotide binding oligomerization domain-like receptor protein 3 inflammasome; Oxalate; Reactive oxygen species; Calcium oxalate stone; HK-2

Distribution and Function of PACAP and Its Receptors in the Healthy and Nephrotic Kidney

Background/Aims: Plasma deficiency of pituitary adenylate cyclase-activating polypeptide (PACAP) was recently demonstrated in children with nephrotic syndrome (NS). Previous studies have reported an important protective effect of PACAP on kidney proximal tubules. The aim of this study was to explore the expression of PACAP and its receptors PAC1, VPAC1 and VPAC2 in the healthy and nephrotic kidney and to determine if PACAP has an effect on renal proximal tubular cells exposed to albumin. Methods: Expression of PACAP and its receptors was studied using kidney tissue from healthy and nephrotic children, and in 3 human renal cell lines (glomerular microvascular endothelial cells, podocytes and proximal tubular epithelial HK-2 cells). The functionality of the VPAC1 receptor was tested in HK-2 cells, measuring cyclic adenosine monophosphate levels after PACAP exposure. The influence of PACAP on cell viability and transforming growth factor-β1 (TGF-β1) expression was measured in HK-2 cells exposed to albumin, mimicking proteinuria related damage. Results: VPAC1 expression was detected in the tubular proximal epithelial cells and in the glomerular podocytes of renal tissue from healthy and nephrotic children. Increased staining for PACAP was found in the proximal tubules of renal sections from children with NS compared to healthy renal sections. Expression and functionality of VPAC1 were demonstrated in HK-2 cells. Finally, PACAP did not alter cell viability or TGF-β1 expression of HK-2 cells exposed to albumin. Conclusion: VPAC1 is the predominant receptor in the human kidney. The enhanced presence of PACAP in proximal tubular epithelial cells in nephrotic kidneys points to the reabsorption of filtered PACAP. On short term, PACAP has no in vitro effect on cell viability and TGF-β1 expression of proximal tubular epithelial cells exposed to high concentrations of albumin.

Atrasentan increased the expression of klotho by mediating miR-199b-5p and prevented renal tubular injury in diabetic nephropathy

Atrasentan is a promising therapy for treating diabetic nephropathy (DN). Here we evaluated whether atrasentan down-regulated the miR-199b-5p expression, thereby increasing klotho and preventing renal tubular injury in DN. One-hundred patients with type 2 diabetes mellitus (T2DM) and 40 healthy subjects were included. A DN mice model was established by an injection of streptozotocin (STZ). Human renal proximal tubular epithelial HK-2 cells were exposed to high glucose (20 mmol/L). Treated the mice and HK-2 cells with atrasentan, and we then investigated whether and how miR-199b-5p and Klotho were involved in preventing renal tubular injury in DN. In patients, the serum miR-199b-5p level increased and the klotho concentration decreased in accordance with elevated albuminuria. Atrasentan down-regulated miR-199b-5p and up-regulated klotho of the DN mice and HK-2 cells exposed to high glucose. High glucose promoted the binding of histone H3 to the miR-199b-5p promoter, and atrasentan canceled this effect. MiR-199b-5p targeted the 3′ UTR of klotho. Overexpression of miR-199b-5p canceled the effects of atrasentan on klotho expression and apoptosis of renal tubular cells in both in vivo and in vitro. The increased serum klotho, mediated by miR-199b-5p, is a possible mechanism by which atrasentan prevents renal tubular injury in DN.

The role of IgA in proximal tubular cell activation and tubulointerstitial scarring in IgA nephropathy

BackgroundIgA nephropathy (IgAN) is the commonest form of glomerulonephritis worldwide, and around 30% of patients progress to end-stage renal failure within 20 years. One of the most powerful prognostic factors for progression in IgAN is the development of tubulointerstitial inflammation and fibrosis. The extent of tubulointerstitial damage in IgAN does not however correlate with the degree of mesangial IgA deposition. Changes in glomerular permeability in IgAN result in exposure of the proximal tubular epithelium to IgA. It is therefore possible that a pathogenic interaction between filtered IgA and proximal tubular cells drives tubulointerstitial scarring in IgAN, independent of the effect of albuminuria. We aimed to assess the interaction between IgA and proximal tubular cells to establish whether IgA could independently generate a proinflammatory and profibrotic phenotypic transformation in proximal tubular cells thereby favouring the development of renal fibrosis. MethodsIgA1 was purified from serum from patients with IgAN and healthy individuals by jacalin-agarose affinity chromatography. Human proximal tubular epithelial HK2 cells (PTEC) were exposed for 24 h to IgA1 (100 μg/mL) from patients with IgAN or healthy individuals, or to control conditions IgM (100 μg/mL), IgG (100 μg/mL), albumin (100 μg/mL or 5 mg/mL), or medium alone, and then subjected to protein extraction, with supernatants assayed by sandwich ELISA. FindingsIgA1 from patients with IgAN activated extracellular signal-regulated protein kinase in PTEC to a greater extent than did IgM or IgA1 from healthy individuals. IgA1 from patients with IgAN also stimulated PPAR-response-element-driven luciferase expression, indicating PPAR activation. PTEC synthesis of transforming growth factor β, fibronectin, and interleukin 6 was substantially increased by IgA1 (100 μg/mL) compared with IgM, IgG, and albumin at concentrations up to 50 times higher (5 mg/mL). PTEC production of complement component C3 and activation of the complement cascade with generation of the chemotactic component C5a were induced to a greater extent by IgA1 compared with the other conditions. Finally, IgA1 induced phosphorylation of a megalin cytoplasmic tail plus glutathione-s-transferase fusion protein in HK2 cells suggesting that IgA1 binding to PTEC can trigger the same intracellular signalling pathways as those activated after exposure to albumin. InterpretationWe provide evidence that IgA1 in IgAN triggers a proinflammatory and profibrotic phenotypic transformation of PTECs. Interaction between filtered IgA1 and PTEC might be a key factor in driving tubulointerstitial damage and progression of renal fibrosis in IgAN. Further elucidation of the mechanism of this interaction might reveal novel therapeutic targets relevant in slowing progression of IgA nephropathy. FundingUK Medical Research Council.

Loss of vitamin D receptor in chronic kidney disease: a potential mechanism linking inflammation to epithelial-to-mesenchymal transition

Both peritubular inflammation and tubular epithelial-to-mesenchymal transition (EMT) are critical events during the pathogenesis of renal fibrosis. However, the relationship between these two processes is unclear. Here, we investigated the potential role of the vitamin D receptor (VDR) in coupling peritubular inflammation and EMT. In a mouse model of unilateral ureteral obstruction (UUO), loss of VDR was observed as early as 1 day after surgery. In cultured proximal tubular epithelial HK-2 cells, proinflammatory TNF-α inhibited the expression of VDR in a dose- and time-dependant manner. Treatment with TNF-α sensitized HK-2 cells to EMT stimulated by transforming growth factor (TGF)-β1. Ectopic expression of VDR counteracted the synergistic effect of TNF-α and TGF-β1 on EMT. Furthermore, knockdown of VDR using a small interfering RNA strategy mimicked the effect of TNF-α on facilitating EMT. Either TNF-α treatment or a loss of VDR induced β-catenin activation and its nuclear translocation. The VDR ligand calcitriol reversed the VDR loss and inhibited EMT in the mouse UUO model, and late administration of active vitamin D was effective in restoring VDR expression as well, and reduced collagen accumulation and deposition compared with the vehicle control. β-Catenin expression induced by UUO was also significantly inhibited after the late administration of vitamin D. These results indicate that the early loss of VDR in chronic kidney diseases was likely mediated by proinflammatory TNF-α, which renders tubular cells susceptible to EMT. Our data suggest that loss of VDR couples peritubular inflammation and EMT, two key events in renal fibrogenesis.

Chronic alcohol exposure negatively impacts the physiological and molecular parameters of the renal biotin reabsorption process

Normal body homeostasis of biotin is critically dependent on its renal recovery by kidney proximal tubular epithelial cells, a process that is mediated by the sodium-dependent multivitamin transporter (SMVT; a product of the SLC5A6 gene). Chronic ethanol consumption interferes with the renal reabsorption process of a variety of nutrients, including water-soluble vitamins. To date, however, there is nothing known about the effect of chronic alcohol feeding on physiological and molecular parameters of the renal biotin reabsorption process. We addressed these issues using rats and transgenic mice carrying the human SLC5A6 (P1P2) 5'-regulatory region as an in vivo model systems of alcohol exposure, and cultured human renal proximal tubular epithelial HK-2 cells chronically exposed to alcohol as an in vitro model of alcohol exposure. The [(3)H]biotin uptake results showed that chronic ethanol feeding in rats leads to a significant inhibition in carrier-mediated biotin transport across both renal brush border and basolateral membrane domains. This inhibition was associated with a marked reduction in the level of expression of SMVT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Furthermore, studies with transgenic mice carrying the SLC5A6 5'-regulatory region showed that chronic alcohol feeding leads to a significant decrease in promoter activity. Studies with HK-2 cells chronically exposed to alcohol again showed a marked reduction in carrier-mediated biotin uptake, which was associated with a significant reduction in promoter activity of the human SLC5A6 5'-regulatory region. These findings demonstrate for the first time that chronic ethanol feeding inhibits renal biotin transport and that this effect is, at least in part, being exerted at the transcriptional level.