Human Renal Proximal Tubular Cells Research Articles

Scopoletin alleviates high glucose-induced toxicity in human renal proximal tubular cells via inhibition of oxidative damage, epithelial-mesenchymal transition, and fibrogenesis.

Recent years of evidence suggest the crucial role of renal tubular cells in developing diabetic kidney disease. Scopoletin (SCOP) is a plant-based coumarin with numerous biological activities. This study aimed to determine the effect of SCOP on renal tubular cells in developing diabetic kidney disease and to elucidate mechanisms. In this study, SCOP was evaluated in vitro using renal proximal tubular (HK-2) cells under hyperglycemic conditions to understand its mechanism of action. In HK-2 cells, SCOP alleviated the high glucose-generated reactive oxygen species (ROS), restored the levels of reduced glutathione, and decreased lipid peroxidation. High glucose-induced alteration in the mitochondrial membrane potential was markedly restored in the SCOP-treated cells. Moreover, SCOP significantly reduced the high glucose-induced apoptotic cell population in the Annexin V-FITC flow cytometry study. Furthermore, high glucose markedly elevated the mRNA expression of fibrotic and extracellular matrix (ECM) components, namely, transforming growth factor (TGF)-β, alfa-smooth muscle actin (α-SMA), collagen I, and collagen III, in HK-2 cells compared to the untreated cells. SCOP treatment reduced these mRNA expressions compared to the high glucose-treated cells. Collagen I and TGF-β protein levels were also significantly reduced in the SCOP-treated cells. Further findings in HK-2 cells revealed that SCOP interfered with the epithelial-mesenchymal transition (EMT) in the high glucose-treated HK-2 cells by normalizing E-cadherin and downregulating the vimentin and α-SMA proteins. In conclusion, SCOP modulates the high glucose-generated renal tubular cell oxidative damage and accumulation of ECM components and may be a promising molecule against diabetic nephropathy.

Identification of endophenotypes supporting outcome prediction in hemodialysis patients based on mechanistic markers of statin treatment

BackgroundStatins are widely used to reduce the risk of cardiovascular disease (CVD). Patients with end-stage renal disease (ESRD) on hemodialysis have significantly increased risk of developing CVD. Statin treatment in these patients however did not show a statistically significant benefit in large trials on a patient cohort level. MethodsWe generated gene expression profiles for statins to investigate the impact on cellular programs in human renal proximal tubular cells and mesangial cells in-vitro. We subsequently selected biomarkers from key statin-affected molecular pathways and assessed these biomarkers in plasma samples from the AURORA cohort, a double-blind, randomized, multi-center study of patients on hemodialysis or hemofiltration that have been treated with rosuvastatin. Patient clusters (phenotypes) were created based on the identified biomarkers using Latent Class Model clustering and the associations with outcome for the generated phenotypes were assessed using Cox proportional hazards regression models. The multivariable models were adjusted for clinical and biological covariates based on previously published data in AURORA. ResultsThe impact of statin treatment on mesangial cells was larger as compared with tubular cells with a large overlap of differentially expressed genes identified for atorvastatin and rosuvastatin indicating a predominant drug class effect. Affected molecular pathways included TGFB-, TNF-, and MAPK-signaling and focal adhesion among others. Four patient clusters were identified based on the baseline plasma concentrations of the eight biomarkers. Phenotype 1 was characterized by low to medium levels of the hepatocyte growth factor (HGF) and high levels of interleukin 6 (IL6) or matrix metalloproteinase 2 (MMP2) and it was significantly associated with outcome showing increased risk of developing major adverse cardiovascular events (MACE) or cardiovascular death. Phenotype 2 had high HGF but low Fas cell surface death receptor (FAS) levels and it was associated with significantly better outcome at 1 year. ConclusionsIn this translational study, we identified patient subgroups based on mechanistic markers of statin therapy that are associated with disease outcome in patients on hemodialysis.

Mitigation of ROS-triggered endoplasmic reticulum stress by upregulating Nrf2 retards diabetic nephropathy

Endoplasmic reticulum stress (ERS) plays a crucial role in the pathogenesis of diabetic nephropathy (DN), and it is often accompanied by an increase in reactive oxygen species (ROS) production. However, the precise relationship between nuclear factor erythroid 2-related factor 2 (Nrf2), a key regulator of ROS balance, and ERS in DN remains elusive. This study aimed to investigate the impact of Nrf2 on ERS and its therapeutic potential in DN. Herein, ERS-related changes, including increased activating transcription factor-6 (ATF6), glucose-regulated protein 78 (GRP78), and transcription factor C/EBP homologous protein (CHOP) expression, were observed in the renal tissues of streptozotocin-induced DN mice and high glucose cultured human renal proximal tubular (HK-2) cells. Nrf2 knockdown increased the sensitivity of HK-2 cells to ERS under high glucose conditions, underscoring the regulatory role of Nrf2 in ERS modulation. Notably, upregulating Nrf2 in ezetimibe-treated diabetic mice restored ERS markers and ameliorated albuminuria, glomerular hypertrophy, mesangial expansion, and tubulointerstitial fibrosis. Furthermore, the inhibition of ERS in HK-2 cells by the ROS scavenger, N-acetylcysteine, highlights the interplay between ROS and ERS. This study, for the first time, elucidates that the upregulation of Nrf2 may alleviate the negative influence of ROS-mediated ERS, presenting a promising therapeutic avenue for delaying the progression of DN. These findings suggest a potential strategy for targeting Nrf2 and ERS in developing novel therapeutic interventions for DN.

Upregulation of UHRF1 Promotes PINK1-mediated Mitophagy to Alleviates Ferroptosis in Diabetic Nephropathy.

Diabetic nephropathy (DN) is a common diabetic complication. Studies show that mitophagy inhibition induced-ferroptosis plays a crucial role in DN progression. UHRF1 is associated with mitophagy and is highly expression in DN patients, however, the effect of UHRF1 on DN is still unclear. Thus, in this study, we aimed to investigate whether UHRF1 involves DN development by the mitophagy/ferroptosis pathway. We overexpressed UHRF1 using an adeno-associated virus 9 (AAV9) system in high-fat diet/streptozotocin-induced diabetic mice. Renal function index, pathological changes, mitophagy factors, and ferroptosis factors were detected in vivo. High-glucose cultured human renal proximal tubular (HK-2) cells were used as in vitro models to investigate the mechanism of UHRF1 in DN. We found that diabetic mice exhibited kidney damage, which was alleviated by UHRF1 overexpression. UHRF1 overexpression promoted PINK1-mediated mitophagy and inhibited the expression of thioredoxin interacting protein (TXNIP), a factor associated with mitochondrial dysfunction. Additionally, UHRF1 overexpression alleviated lipid peroxidation and free iron accumulation, and upregulated the expression of GPX4 and Slc7a11, indicating the inhibition effect of UHRF1 overexpression on ferroptosis. We further investigated the mechanism of UHRF1 in the mitophagy/ferroptosis pathway in DN. We found that UHRF1 overexpression promoted PINK1-mediated mitophagy via inhibiting TXNIP expression, thus suppressing ferroptosis. These findings confirmed that upregulation of UHRF1 expression alleviates DN, indicating that UHRF1 has a reno-protective effect against DN.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury

BackgroundThe mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.MethodsA 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein–Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.ResultsA total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.ConclusionsThe proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

The interplay of mitophagy, autophagy, and apoptosis in cisplatin-induced kidney injury: involvement of ERK signaling pathway

AKI induced by CP chemotherapy remains an obstacle during patient treatments. Extracellular signal-regulated protein kinases 1/2 (ERK), key participants in CP-induced nephrotoxicity, are suggested to be involved in the regulation of mitophagy, autophagy, and apoptosis. Human renal proximal tubular cells (HK-2) and BALB/cN mice were used to determine the role of ERK in CP-induced AKI. We found that active ERK is involved in cell viability reduction during apoptotic events but exerts a protective role in the early stages of treatment. Activation of ERK acts as a maintainer of the mitochondrial population and is implicated in mitophagy initiation but has no significant role in its conduction. In the late stages of CP treatment when ATP is deprived, general autophagy that requires ERK activation is initiated as a response, in addition to apoptosis activation. Furthermore, activation of ERK is responsible for the decrease in reserve respiratory capacity and controls glycolysis regulation during CP treatment. Additionally, we found that ERK activation is also required for the induction of NOXA gene and protein expression as well as FoxO3a nuclear translocation, but not for the regular ERK-induced phosphorylation of FoxO3a on Ser294. In summary, this study gives detailed insight into the involvement of ERK activation and its impact on key cellular processes at different time points during CP-induced kidney injury. Inhibitors of ERK activation, including Mirdametinib, are important in the development of new therapeutic strategies for the treatment of AKI in patients receiving CP chemotherapy.

Dysregulation of the mRNA Expression of Human Renal Drug Transporters by Proinflammatory Cytokines in Primary Human Proximal Tubular Epithelial Cells.

Proinflammatory cytokines, which are elevated during inflammation or infections, can affect drug pharmacokinetics (PK) due to the altered expression or activity of drug transporters and/or metabolizing enzymes. To date, such studies have focused on the effect of cytokines on the activity and/or mRNA expression of hepatic transporters and drug-metabolizing enzymes. However, many antibiotics and antivirals used to treat infections are cleared by renal transporters, including the basal organic cation transporter 2 (OCT2), organic anion transporters 1 and 3 (OAT1 and 3), the apical multidrug and toxin extrusion proteins 1 and 2-K (MATE1/2-K), and multidrug resistance-associated protein 2 and 4 (MRP2/4). Here, we determined the concentration-dependent effect of interleukin-6 (IL-6), IL-1β, tumor necrosis factor (TNF)-α, and interferon-γ (IFN-γ) on the mRNA expression of human renal transporters in freshly isolated primary human renal proximal tubular epithelial cells (PTECs, n = 3-5). PTECs were exposed to either a cocktail of cytokines, each at 0.01, 0.1, 1, or 10 ng/mL or individually at the same concentrations. Exposure to the cytokine cocktail for 48 h was found to significantly downregulate the mRNA expression, in a concentration-dependent manner, of OCT2, the organic anion transporting polypeptides 4C1 (OATP4C1), OAT4, MATE2-K, P-glycoprotein (P-gp), and MRP2 and upregulate the mRNA expression of the organic cation/carnitine transporter 1 (OCTN1) and MRP3. OAT1 and OAT3 also appeared to be significantly downregulated but only at 0.1 and 10 ng/mL, respectively, without a clear concentration-dependent trend. Among the cytokines, IL-1β appeared to be the most potent at down- and upregulating the mRNA expression of the transporters. Taken together, our results demonstrate for the first time that proinflammatory cytokines transcriptionally dysregulate renal drug transporters in PTECs. Such dysregulation could potentially translate into changes in transporter protein abundance or activity and alter renal transporter-mediated drug PK during inflammation or infections.

C/EBPβ mediates expressions of downstream inflammatory factors of the tumor necrosis factor-α signaling pathway in renal tubular epithelial cells with NPHP1 knockdown

To explore the activation of tumor necrosis factor-α (TNF-α) signaling pathway and the expressions of the associated inflammatory factors in NPHP1-defective renal tubular epithelial cells. A human proximal renal tubular cell (HK2) model of lentivirus-mediated NPHP1 knockdown (NPHP1KD) was constructed, and the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors CXCL5, CCL20, IL-1β, IL-6 and MCP-1 were detected using RT-qPCR, Western blotting or enzyme-linked immunosorbent assay. A small interfering RNA (siRNA) was transfected in wild-type and NPHP1KDHK2 cells, and the changes in the expressions of TNF-α, p38, and C/EBPβ and the inflammatory factors were examined. NPHP1KDHK2 cells showed significantly increased mRNA expressions of TNF-α, C/EBPβ, CXCL5, IL-1β, and IL-6 (P < 0.05), protein expressions of phospho-p38 and C/EBPβ (P < 0.05), and IL-6 level in the culture supernatant (P < 0.05), and these changes were significantly blocked by transfection of cells with siRNA-C/EBPβ (P < 0.05). TNF-α signaling pathway is activated and its associated inflammatory factors are upregulated in NPHP1KDHK2 cells, and C/EBPβ may serve as a key transcription factor to mediate these changes.

Altenusin, a fungal metabolite, alleviates TGF-β1-induced EMT in renal proximal tubular cells and renal fibrosis in unilateral ureteral obstruction

Renal fibrosis is a pathological feature of chronic kidney disease (CKD), progressing toward end-stage kidney disease (ESKD). The aim of this study is to investigate the therapeutic potential of altenusin, a farnesoid X receptor (FXR) agonist derived from fungi, on renal fibrosis. The effect of altenusin was determined (i) in vitro using the transforming growth factor β1 (TGF-β1)-induced epithelial to mesenchymal transition (EMT) of human renal proximal tubular cells and (ii) in vivo using mouse unilateral ureteral obstruction (UUO). The findings revealed that incubation of 10 ng/ml TGF-β1 promotes morphological change in RPTEC/TERT1 cells, a human renal proximal tubular cell line, from epithelial to fibroblast-like cells. TGF-β1 markedly increased EMT markers namely α-smooth muscle actin (α-SMA), fibronectin, and matrix metalloproteinase 9 (MMP-9), while decreased the epithelial marker E-cadherin. Co-incubation TGF-β1 with altenusin preserved the epithelial characteristics of the renal epithelial cells by antagonizing TGF-β/Smad signaling pathway, specifically a decreased phosphorylation of Smad2/3 with an increased level of Smad7. Interestingly, the antagonizing effect of altenusin does not require FXR activation. Moreover, altenusin could reverse TGF-β1-induced fibroblast-like cells to epithelial-like cells. Treatment on UUO mice with 30 mg/kg altenusin significantly reduced the expression of α-SMA, fibronectin, and collagen type 1A1 (COL1A1). The reduction in the renal fibrosis markers is correlated with the decreased phosphorylation of Smad2/3 levels but does not improve E-cadherin protein expression. Collectively, altenusin reduces EMT in human renal proximal tubular cells and renal fibrosis by antagonizing the TGF-β/Smad signaling pathway.

Ethanol extract of Abelmoschus manihot suppresses endoplasmic reticulum stress in contrast-induced nephropathy

Objective: To explore the efficacy and potential mechanisms of the ethanol extract of Abelmoschus manihot (L.) Medic in contrast-induced nephropathy (CIN). Methods: CIN rat models and human renal proximal tubular cells (HK-2) with iopromide-induced injury were employed to mimic CIN conditions. The effect of Abelmoschus manihot extract on the rat models and HK-2 cells was evaluated. In rat models, kidney function, histology, oxidative stress and apoptosis were determined. In HK-2 cells, cell viability, apoptosis, mitochondrial membrane potential, and endoplasmic reticulum stress were assessed. Results: Abelmoschus manihot extract significantly improved structural and functional impairments in the kidneys of CIN rats. Additionally, the extract effectively mitigated the decline in cellular viability and reduced iopromide-induced oxidative stress and lipid peroxidation. Mechanistic investigations revealed that Abelmoschus manihot extract prominently attenuated acute endoplasmic reticulum stress-mediated apoptosis by downregulating GRP78 and CHOP protein levels. Conclusions: Abelmoschus manihot extract can be used as a promising therapeutic and preventive agent in the treatment of CIN.

Aurantio-obtusin exerts an anti-inflammatory effect on acute kidney injury by inhibiting NF-κB pathway.

Acute kidney injury (AKI) is one of the major complications of sepsis. Aurantio-obtusin (AO) is an anthraquinone compound with antioxidant and anti-inflammatory activities. This study was developed to concentrate on the role and mechanism of AO in sepsis-induced AKI. Lipopolysaccharide (LPS)-stimulated human renal proximal tubular epithelial cells (HK-2) and BALB/c mice receiving cecal ligation and puncture (CLP) surgery were used to establish in vitro cell model and in vivo mouse model. HK-2 cell viability was measured using MTT assays. Histological alterations of mouse renal tissues were analyzed via hematoxylin and eosin staining. Renal function of mice was assessed by measuring the levels of serum creatinine (SCr) and blood urea nitrogen (BUN). The concentrations of pro-inflammatory cytokines in HK-2 cells and serum samples of mice were detected using corresponding ELISA kits. Protein levels of factors associated with nuclear factor kappa-B (NF-κB) pathway were measured in HK-2 cells and renal tissues by Western blotting. AO exerted no cytotoxic effect on HK-2 cells and AO dose-dependently rescued LPS-induced decrease in HK-2 cell viability. The concentrations of pro-inflammatory cytokines were increased in response to LPS or CLP treatment, and the alterations were reversed by AO treatment. For in vivo experiments, AO markedly ameliorated renal injury and reduced high levels of SCr and BUN in mice underwent CLP operation. In addition, AO administration inhibited the activation of NF-κB signaling pathway in vitro and in vivo. In conclusion, AO alleviates septic AKI by suppressing inflammatory responses through inhibiting the NF-κB pathway.

Ketoconazole-loading strategy to improve antifungal activity and overcome cytotoxicity on human renal proximal tubular epithelial cells

Ketoconazole (KTZ), an antifungal agent used to treat localized or systemic fungal infections by inhibiting ergosterol synthesis, exhibits restricted efficacy within eukaryotic cells owing to its elevated toxicity and limited solubility in water. This study aims to improve the biological activity and overcome cytotoxic effects in the renal system of the hydrophobic KTZ by incorporating it into poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) utilizing biomaterial nano-engineering techniques. KTZ-loaded PLGA NPs (KTZ-NPs) were prepared by single emulsion solvent evaporation method and characterized by using dynamic light scattering (DLS), electrophoretic light scattering (ELS), Fourier transform-infrared (FT-IR) spectroscopy and scanning light microscopy (SEM). Particle size and zeta potential of KTZ-NPs were determined as 182.0 ± 3.27 nm and −27.4 ± 0.56 mV, respectively. Antifungal activity was analyzed with the time-kill and top agar dilution methods on Candida albicans (C. albicans) and Aspergillus flavus (A. flavus). Both KTZ and KTZ-NPs caused a significant decrease in A. flavus cell growth; however, the same effect was only observed in time-killing analysis on C. albicans, indicating a methodological difference in the antifungal analysis. According to the top agar method, the MIC value of KTZ-NPs against A. flavus was 9.1 μg ml−1, while the minimum inhibition concentration (MIC) value of KTZ was 18.2 μg ml−1. The twofold increased antifungal activity indicates that nanoparticular drug delivery systems enhance the water solubility of hydrophobic drugs. In addition, KTZ-NPs were not cytotoxic on human renal proximal tubular epithelial cells (HRPTEpCs) at fungistatic concentration, thus reducing fungal colonization without cytotoxic on renal excretion system cells.

IDENTIFYING SENOLYTIC TARGETS OF DIETARY FLAVONOIDS IN RENAL CELLS BY THERMAL PROTEOME PROFILING

Abstract Senescent cell accumulation is a known driver of aging and age-related pathologies. Clearance of senescent cells is a promising approach to increase longevity and reduce multiple age-related diseases in humans. Flavonoid compounds are present in fruits and vegetables, of which certain flavonoids (e.g., quercetin and fisetin) have demonstrated senolytic, or ‘senescent-cell-killing’, activity in culture and in mice. However, the cellular mechanism of senolysis and the efficacy of these compounds are not known in renal epithelial cells. Here, we combine flavonoid drug screening, targeted metabolomics, and thermal proteome profiling (TPP) with analysis by mass spectrometry (MS) to explore protein targets of senolytic drugs in senescent human renal cortical epithelial cells and human renal proximal tubular epithelial cells. Senescence was induced by exposure to ionizing radiation and the senescence phenotype was validated through a rigorous panel of senescence and viability markers. Proliferating and senescent renal epithelial cells were screened with a panel of 8 flavonoids to identify senolytic drugs and their concentrations for selectively killing senescent cells. Targeted MS assays were developed to assess intracellular drug uptake, a potential mechanism of senescent-specific killing. To identify protein targets of the senolytic flavonoids in senescent cells, we performed a variation of TPP in combination with liquid chromatography (LC)-MS/MS proteomics analysis, carefully controlling for non-senolytic interactions by excluding proteins bound by a non-senolytic flavonoid. The results of this study pave the way for the development of a more specific generation of senolytic compounds and identify novel candidate senolytic pathways engaged by dietary flavonoids.

Mechanism of Huaiqihuang in treatment of diabetic kidney disease based on network pharmacology, molecular docking and in vitro experiment.

This study aimed to investigate the active components, key targets, and potential molecular mechanisms Huaiqihuang (HQH) in the treatment of diabetic kidney disease (DKD) through network pharmacology, molecular docking, and in vitro experiments. The active components and potential targets of HQH were obtained from the TCMSP and HERB databases. The potential targets of DKD were obtained from the GeneCards, OMIM, DrugBank, and TTD databases. Protein interaction relationships were obtained from the STRING database, and a protein interaction network was constructed using Cytoscape software. Gene ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed using the Metascape database. Molecular docking was performed using AutoDock software to verify the binding between key compounds and core target genes. In vitro experiments were conducted using human renal proximal tubular epithelial cells and various methods, such as CCK8, RT-PCR, immunofluorescence, and western blot, to evaluate the effects of HQH on inflammatory factors, key targets, and pathways. A total of 48 active ingredients, 168 potential targets of HQH, and 1073 potential targets of DKD were obtained. A total of 118 potential targets, 438 biological processes, and 187 signal pathways were identified for the treatment of DKD. Gene ontology and Kyoto Encyclopedia of Genes and Genomes analysis indicated that HQH may exert its therapeutic effects on DKD by regulating the expression of inflammatory factors through the nuclear factor kappa B (NF-κB) signaling pathway. The molecular docking results showed that β-sitosterol and baicalein had the highest binding affinity with key targets such as AKT1, IL6, TNF, PTGS2, IL1B, and CASP3, suggesting that they may be the most effective active ingredients of HQH in the treatment of DKD. In vitro experimental results demonstrated that HQH could enhance the viability of human renal proximal tubular epithelial cells inhibited by high glucose, decrease the levels of AKT1, TNF, IL6, PTGS2, IL1B, and CASP3, reduce the expression of NF-κB-P65 (P < .01), inhibit NF-κB-p65 nuclear translocation, and decrease chemokine expression (P < .01). HQH may exert its therapeutic effects on DKD by inhibiting the NF-κB signaling pathway, reducing the level of pro-inflammatory cytokines, and alleviating the high glucose-induced injury of renal tubular epithelial cells.

Interferon-alpha and MxA inhibit BK polyomavirus replication by interaction with polyomavirus large T antigen

IntroductionBK Polyomavirus (BKPyV) infection is a common complication in kidney transplant recipients and can result in poor outcome and graft failure. Currently, there is no known effective antiviral agent. This study investigated the possible antiviral effects of Interferon alpha (IFNα) and its induced protein, MxA, against BKPyV. MethodsIn vitro cell culture experiments were conducted using human primary renal proximal tubular epithelial cells (HRPTECs). We also did animal studies using Balb/c mice with unilateral kidney ischemic reperfusion injury. ResultsOur results demonstrated that IFNα effectively inhibited BKPyV in vitro and murine polyomavirus in animal models. Additionally, IFNα and MxA were found to suppress BKPyV TAg and VP1 production. Silencing MxA attenuated the antiviral efficacy of IFNα.We observed that MxA interacted with BKPyV TAg, causing it to remain in the cytosol and preventing its nuclear translocation. To determine MxA's essential domain for its antiviral activities, different mutant MxA constructs were generated. The MxA mutant K83A retained its interaction with BKPyV TAg, and its antiviral effects were intact. The MxA T103A mutant, on the other hand, abolished GTPase activity and lost its protein-protein interaction with BKPyV TAg, and lost its antiviral effect. ConclusionIFNα and its downstream protein, MxA, have potent antiviral properties against BKPyV. Furthermore, our findings indicate that the interaction between MxA and BKVPyV TAg plays a crucial role in determining the anti-BKPyV effects of MxA.

Size-Dependent Cytotoxicity, Adhesion, and Endocytosis of Micro-/Nano-hydroxyapatite Crystals in HK-2 Cells.

Nano-hydroxyapatite (nano-HAP) is often used as a crystal nest to induce calcium oxalate (CaOx) kidney stone formation, but the mechanism of interaction between HAP crystals of different properties and renal tubular epithelial cells remains unclear. In this study, the adhesion and endocytosis of HAP crystals with sizes of 40 nm, 70 nm, 1 μm, and 2 μm (HAP-40 nm, HAP-70 nm, HAP-1 μm, and HAP-2 μm, respectively) to human renal proximal tubular epithelial cells (HK-2) were comparatively studied. The results showed that HAP crystals of all sizes promoted the expression of osteopontin and hyaluronic acid on the cell surface, destroyed the integrity of the lysosomes, and induced the apoptosis and necrosis of cells. Nano-HAP crystals had a higher specific surface area, a smaller contact angle, a higher surface energy, and a lower Zeta potential than those of micro-HAP. Therefore, the abilities of HK-2 cells to adhere to and endocytose nano-HAP crystals were greater than their abilities to do the same for micro-HAP crystals. The order of the endocytosed crystals was as follows: HAP-40 nm > HAP-70 nm > HAP-1 μm > HAP-2 μm. The endocytosed HAP crystals entered the lysosomes. The more crystal endocytosis and adhesion there is, the more toxic it is to HK-2 cells. The results of this study showed that nanosized HAP crystals greatly promoted the formation of kidney stones than micrometer-sized HAP crystals.

Polystyrene nanoplastics induce apoptosis of human kidney proximal tubular epithelial cells via oxidative stress and MAPK signaling pathways.

Polystyrene nanoplastics (PS-NPs) have recently been found to be present in human blood and kidney. However, the renal toxicity of PS-NPs and the underlying mechanisms have not been fully elucidated. Here, we found that exposure of PS-NPs induced apoptosis of human renal proximal tubular epithelial cells (HK-2) in a size- and dose-dependent manner as revealed by AnnexinV-FITC assay. In addition, PS-NPs promoted ROS production and caused structure changes of mitochondrial and endoplasmic reticulum. Mechanistically, transcriptional sequencing indicated the involvement of MAPK pathway in apoptosis, which was further confirmed by the upregulation of p-p38, p-ERK, CHOP, BAX, cytochrome C, and caspase 3 expression. This study clarified the molecular mechanism underlying PS-NP-induced apoptosis in HK-2 cells and contributed to our risk estimation of PS-NPs in human kidney.

HK-2 cell response to TGF-β highly depends on cell culture medium formulations

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-β) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-β-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-β1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-β1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

NRF2 Deficiency Attenuates Diabetic Kidney Disease in Db/Db Mice via Down-Regulation of Angiotensinogen, SGLT2, CD36, and FABP4 Expression and Lipid Accumulation in Renal Proximal Tubular Cells.

The role(s) of nuclear factor erythroid 2-related factor 2 (NRF2) in diabetic kidney disease (DKD) is/are controversial. We hypothesized that Nrf2 deficiency in type 2 diabetes (T2D) db/db mice (db/dbNrf2 knockout (KO)) attenuates DKD progression through the down-regulation of angiotensinogen (AGT), sodium-glucose cotransporter-2 (SGLT2), scavenger receptor CD36, and fatty -acid-binding protein 4 (FABP4), and lipid accumulation in renal proximal tubular cells (RPTCs). Db/dbNrf2 KO mice were studied at 16 weeks of age. Human RPTCs (HK2) with NRF2 KO via CRISPR-Cas9 genome editing and kidneys from patients with or without T2D were examined. Compared with db/db mice, db/dbNrf2 KO mice had lower systolic blood pressure, fasting blood glucose, kidney hypertrophy, glomerular filtration rate, urinary albumin/creatinine ratio, tubular lipid droplet accumulation, and decreased expression of AGT, SGLT2, CD36, and FABP4 in RPTCs. Male and female mice had similar results. NRF2 KO attenuated the stimulatory effect of the Nrf2 activator, oltipraz, on AGT, SGLT2, and CD36 expression and high-glucose/free fatty acid (FFA)-stimulated lipid accumulation in HK2. Kidneys from T2D patients exhibited markedly higher levels of CD36 and FABP4 in RPTCs than kidneys from non-diabetic patients. These data suggest that NRF2 exacerbates DKD through the stimulation of AGT, SGLT2, CD36, and FABP4 expression and lipid accumulation in RPTCs of T2D.

LRRK2 aggravates kidney injury through promoting MFN2 degradation and abnormal mitochondrial integrity

Mitochondrial dysfunction is one of the key features of acute kidney injury (AKI) and associated fibrosis. Leucine-rich repeat kinase 2 (LRRK2) is highly expressed in kidneys and regulates mitochondrial homeostasis. How it functions in AKI is unclear. Herein we reported that LRRK2 was dramatically downregulated in AKI kidneys. Lrrk2−/− mice exhibited less severity of AKI when compared to wild-type counterparts with less mitochondrial fragmentation and decreased reactive oxygen species (ROS) production in proximal renal tubular cells (PTCs) due to mitofusin 2 (MFN2) accumulation. Overexpression of LRRK2 in human PTC cell lines promoted LRRK2-MKK4/JNK-dependent phosphorylation of MFN2Ser27 and subsequently ubiquitination-mediated MFN2 degradation, which in turn exaggerated mitochondrial damage upon ischemia/reperfusion (I/R) mimicry treatment. Lrrk2 deficiency also alleviated AKI-to-chronic kidney disease (CKD) transition with less fibrosis. In vivo pretreatment of LRRK2 inhibitors attenuated the severity of AKI as well as CKD, potentiating LRRK2 as a novel target to alleviate AKI and fibrosis.