Renal Epithelial Cell Line Research Articles

Renal NOXA1/NOX1 Signaling Regulates Epithelial Sodium Channel and Sodium Retention in Angiotensin II-induced Hypertension.

Aims: NADPH oxidase (NOX)-derived reactive oxygen species (ROS) are implicated in the pathophysiology of hypertension in chronic kidney disease patients. Genetic deletion of NOX activator 1 (Noxa1) subunit of NOX1 decreases ROS under pathophysiological conditions. Here, we investigated the role of NOXA1-dependent NOX1 activity in the pathogenesis of angiotensin II (Ang II)-induced hypertension (AIH) and possible involvement of abnormal renal function. Results: NOXA1 is present in epithelial cells of Henle's thick ascending limb and distal nephron. Telemetry showed lower basal systolic blood pressure (BP) in Noxa1-/-versus wild-type mice. Ang II infusion for 1 and 14 days increased NOXA1/NOX1 expression and ROS in kidney of male but not female wild-type mice. Mean BP increased 30 mmHg in wild-type males, with smaller increases in Noxa1-deficient males and wild-type or Noxa1-/- females. In response to an acute salt load, Na+ excretion was similar in wild-type and Noxa1-/- mice before and 14 days after Ang II infusion. However, Na+ excretion was delayed after 1-2 days of Ang II in male wild-type versus Noxa1-/- mice. Ang II increased epithelial Na+ channel (ENaC) levels and activation in the collecting duct principal epithelial cells of wild-type but not Noxa1-/- mice. Aldosterone induced ROS levels and Noxa1 and Scnn1a expression and ENaC activity in a mouse renal epithelial cell line, responses abolished by Noxa1 small-interfering RNA. Innovation and Conclusion: Ang II activation of renal NOXA1/NOX1-dependent ROS enhances tubular ENaC expression and Na+ reabsorption, leading to increased BP. Attenuation of AIH in females is attributed to weaker NOXA1/NOX1-dependent ROS signaling and efficient natriuresis. Antioxid. Redox Signal. 36, 550-566.

Hirudin, a thrombin inhibitor, attenuates TGF-β-induced fibrosis in renal proximal tubular epithelial cells by inhibition of protease-activated receptor 1 expression via S1P/S1PR2/S1PR3 signaling.

Renal interstitial fibrosis (RIF) is the final common outcome of numerous chronic kidney diseases, contributing to end-stage renal disease. Hirudin, a thrombin inhibitor, has attracted increased attention as a potential treatment approach for renal fibrosis. The present study aimed to investigate the molecular mechanism underlying the effect of hirudin on fibrosis in renal proximal tubular epithelial cells. An in vivo mouse RIF model established using unilateral ureteral obstruction (UUO) and an in vitro of RIF using the renal tubular epithelial cell line HK-2 treated with TGF-β were used. Expressions of sphingosine-1-phosphate (S1P) receptors (S1PR)1-4 and protease-activated receptor 1 (PAR1) were measured by reverse transcription-quantitative PCR and western blotting in mice with UUO and TGF-β induced HK-2 cells. Western blotting was used to detect the expression of N-cadherin, Slug, E-cadherin, Collagen IV, fibronectin, MMP9 and monocyte chemoattractant protein-1. Immunofluorescence staining was conducted to measure α-SMA level expression. The results demonstrated that the expression levels of S1PR1, S1PR2, S1PR3, S1PR4 and PAR1 were upregulated in both TGF-β-induced HK-2 cells and renal tissues from mice with unilateral ureteral ligation. Notably, hirudin inhibited TGF-β-induced PAR1, S1PR2 and S1PR3 upregulation in both HK-2 cells and renal tissues. Additionally, the inhibition of S1PR2 and S1PR3 resulted in PAR1 downregulation. Furthermore, treatment with S1P and PAR1 agonists abolished the effect of hirudin on the expression of EMT, fibrosis-related proteins and monocyte chemoattractant protein 1. In conclusion, hirudin attenuated TGF-β-induced fibrosis in proximal renal tubular epithelial HK-2 cells by inhibiting PAR1 expression via the S1P/S1PR2/S1PR3 signaling pathway. Therefore, hirudin may be considered as a promising therapeutic agent for RIF.

Overexpression of TRIM3 protects against LPS-induced acute kidney injury via repressing IRF3 pathway and NLRP3 inflammasome.

The pathological process of sepsis involves multiple system organs, including kidney. Sepsis-induced acute kidney injury (AKI) has high morbidity and high mortality. Overproduced inflammatory factors contribute to the occurrence and evolvement of AKI. Here, the role and underlying mechanism of tripartite motif containing 3 (TRIM3) and in AKI was explored. Lipopolysaccharide (LPS) was used for constructing AKI model both in vitro and in vivo. RT-PCR and western blot were performed to detect TRIM3, Interferon regulatory factor 3 (IRF3) and NLRP3-ASC-Caspase1 inflammasome. Upon selectively regulating the TRIM3 or IRF3 expression, the proliferation, apoptosis and inflammatory response were detected. The interaction between TRIM3 and IRF3 was verified by Immunoprecipitation (IP). TRIM3 was down-regulated in mediated injury renal tubular epithelial cell line HK-2 treated with LPS. Overexpression of TRIM3 promoted cell viability and reduced apoptosis. In addition, overexpression of TRIM3 inhibited the expression of inflammatory factors (IL-1β, IL-6, TNF-α and IL-18), dampened the phosphorylation of IRF3 and repressed NLRP3 inflammasome activation. Furthermore, TRIM3 overexpression significantly eased the LPS-induced damage on AKI rat model and decreased the serum creatinine and urea nitrogen levels in rat kidney tissues. The results of immunohistochemistry (IHC) and Western blot manifested that TRIM3 was increased dramatically after TRIM3 was overexpressed in the rat kidney tissues, while IRF3 and NLRP3-ASC-Caspase1 inflammasome were significantly repressed following TRIM3 upregulation in the kidney tissues. Mechanistically, TRIM3 interacted with IRF3 and inhibited its phosphorylation. Overexpression of TRIM3 protected against LPS-induced AKI by inhibiting the IRF3 pathway and NLRP3 inflammasome activation.

Experimental Cell Line Models for Nephrotoxicity Screening

The aim of the study was to review literature data on cell models for experimental assessment of drug nephrotoxicity in vitro. Because of nephrotoxicity, 2% of new investigational medicinal products are discarded at the stage of preclinical in vivo studies in laboratory animals, and 19%—after phase 3 clinical trials. Prediction of toxicity in cell models could make drug development more cost-effective and help to reduce/avoid animal testing. At present, there are no official international guidelines for assessment of nephrotoxicity in vitro, but there is a lot of research underway. The main toxicity target in kidneys is renal proximal tubule epithelial cells, therefore the main research is focused on the development of renal proximal tubule epithelial cell lines with stable functional characteristics. Another important aspect in nephrotoxicity modeling is the choice of relevant test methods and end points which would reflect potential toxicity mechanisms. The paper reviews existing human renal proximal tubule epithelial cell lines and current test methods for assessing cytotoxicity. Promising areas for future development of cell models for nephrotoxicity assessment— are optimisation and standardisation of in vitro systems that would help to make preclinical predictions of drug nephrotoxicity in vivo.

MiR-138-5p inhibits proliferation and invasion in kidney renal clear cell carcinoma by targeting SINA3 and regulation of the Notch signaling pathway.

BackgroundThe function of miR‐138‐5p as an oncogenic factor has been reported in certain cancers. This study was performed to analyze the potential involvement of miR‐138‐5p in kidney renal clear cell carcinoma (KIRC).MethodsThe Cancer Genome Atlas (TCGA) database was used to explain the expression of miR‐138‐5p in cancer and paired non‐cancer tissues of KIRC patients. Subsequently, miR‐138‐5p expression in KIRC tissues and cell lines, as well as that in normal tissues and normal renal tubular epithelial cell line, was detected. Artificial overexpressing of miR‐138‐5p was applied to observe its effect on the biological behaviors of KIRC cells. The target mRNA of miR‐138‐5p, SIN3A, was predicted and validated. Altered expression of miR‐138‐5p and SIN3A was introduced to confirm their functions in KIRC proliferation and invasion.ResultsWe showed that miR‐138‐5p was down‐regulated in tumor tissues of KIRC patients comparing to adjacent healthy tissues and linked to dismal prognosis in patients. miR‐138‐5p could hinder KIRC proliferation and invasion, while artificial overexpression of SIN3A led to reversed trends. SIN3A was a target mRNA of miR‐138‐5p. miR‐138‐5p and SIN3A together affect the activation of the Notch signaling pathway.ConclusionThis study evidenced that up‐regulated miR‐138‐5p inhibits proliferation and invasion of KIRC cells involving the transcription of SIN3A and the following regulation of the Notch signaling pathway.

Metabolic and Lipidomic Assessment of Kidney Cells Exposed to Nephrotoxic Vancomycin Dosages.

Vancomycin is a glycopeptide antibiotic used against multi-drug resistant gram-positive bacteria such as Staphylococcus aureus (MRSA). Although invaluable against resistant bacteria, vancomycin harbors adverse drug reactions including cytopenia, ototoxicity, as well as nephrotoxicity. Since nephrotoxicity is a rarely occurring side effect, its mechanism is incompletely understood. Only recently, the actual clinically relevant concentration the in kidneys of patients receiving vancomycin was investigated and were found to exceed plasma concentrations by far. We applied these clinically relevant vancomycin concentrations to murine and canine renal epithelial cell lines and assessed metabolic and lipidomic alterations by untargeted and targeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses. Despite marked differences in the lipidome, both cell lines increased anabolic glucose reactions, resulting in higher sorbitol and lactate levels. To the best of our knowledge, this is the first endometabolic profiling of kidney cells exposed to clinically relevant vancomycin concentrations. The presented study will provide a valuable dataset to nephrotoxicity researchers and might add to unveiling the nephrotoxic mechanism of vancomycin.

Cadmium induces renal inflammation by activating the NLRP3 inflammasome through ROS/MAPK/NF-κB pathway in vitro and in vivo.

Cadmium (Cd) has been reported to induce kidney damage by triggering oxidative stress and inflammation. The NLR family Pyrin Domain Containing 3 (NLRP3) inflammasome has been implicated a role in the pathogenesis of inflammation. However, the connection between Cd and NLRP3 inflammasome in the development of renal inflammation remains unknown. In this study, in vitro experiments based on the telomerase-immortalized human renal proximal-tubule epithelial cell line (RPTEC/TERT1) were carried out. Results revealed that CdCl2 (2-8μM) increased ROS production and activated NLRP3, thereby enhancing secretion of IL-1β and IL-18 (P < 0.05). Knock-down of NLRP3 rescued the RPTEC/TERT1 cells from Cd-induced inflammatory damage. Cd activated the MAPK/NF-κB signaling pathway in RPTEC/TERT1 cells (P < 0.05). In addition, treatment with N-acetylcysteine (NAC) improved inflammation and blocked the upregulation of the MAPK/NF-κB signaling pathway. Pre-treatment with MAPK and NF-κB inhibitors also suppressed NLRP3 inflammasome activation (P < 0.05). Moreover, CdCl2 (25-00mg/L) stimulated the MAPK/NF-κB signaling pathway, activated the NLRP3 inflammasome, and increased inflammatory response (P < 0.05) leading to renal injury in rats. Exposure to cadmium elevated serum levels of NLRP3 and IL-1β in populations (P < 0.05). Further analysis found that serum NLRP3 and IL-1β levels were positively correlated with urine cadmium (UCd) and urine N-acetyl-β-D-glucosaminidase (UNAG). Overall, Cd induced renal inflammation through the ROS/MAPK/NF-κB signaling pathway by activating the NLRP3 inflammasome. Our research thus provides new insights into the molecular mechanism that NLRP3 contributes to Cd-induced kidney damage.

MiR-224-5p Targeting OCLN Promotes the Proliferation, Migration, and Invasion of Clear Cell Renal Cell Carcinoma Cells

A number of studies reported that miR-224-5p is involved in a variety of cancer-related cellular processes, yet its physiological role in clear cell renal cell carcinoma (ccRCC) remains unclear. In order to clarify the function of miR-224-5p in ccRCC, real-time quantitative-PCR was conducted to compare the expression of miR-224-5p in human normal renal tubular epithelial cell lines and ccRCC cell lines first, and a strikingly upregulated expression was observed in ccRCC cell lines. Inhibition of miR-224-5p expression by microRNA inhibitors could inhibit the proliferation, migration, and invasion of ccRCC cells. Besides, it was validated by dual-luciferase assay in which miR-224-5p directly targeted OCLN gene. The expression of OCLN was downregulated in ccRCC cells, and overexpression of miR-224-5p could inhibit the mRNA and protein expression levels of OCLN. Overexpression of OCLN could reduce the proliferation, migration, and invasion of ccRCC cells, while overexpressed miR-224-5p could partially reverse that inhibitory effect. Therefore, the promotive effect of miR-224-5p on the proliferation, invasion, and migration of ccRCC cell lines was at least partly due to the inhibition of OCLN expression. These findings highlighted the important function of miR-224-5p, which was promoting cell proliferation, migration, and invasion by downregulating OCLN, in the pathogenesis of ccRCC, and provided a potential treatment strategy.

β-Endosulfan-mediated induction of pro-fibrotic markers in renal (HK-2) cells in vitro: A new insight in the pathogenesis of chronic kidney disease of unknown etiology.

Chronic kidney disease of unknown etiology (CKDu), manifested clinically as tubulo interstitial fibrosis, has emerged as the second major cause of chronic kidney disease (CKD) in the Indian subcontinent and various agrochemicals have been implicated in its occurance. Among the agrochemicals organochlorine pesticides particularly endosulfan is well known for its toxicity and recent residue analysis have shown its presence in the blood samples of general population. In this present study, we have investigated the consequences of endosulfan exposure at a concentration (0.01 μM) equivalent to their highest reported presence in human blood sample of some CKDu patients, to human renal proximal tubular epithelial (HK-2) cell line with regard to ROS generation and expression of profibrotic and epithelial to mesenchymal (EMT) markers in order to find out endosulfan's ability to induce profibrotic changes in renal cell. We demonstrated a significant increase in intracellular ROS generation and increased expression of TGF-β1 when cells were incubated with β-endosulfan (0.01 μM) indicating occurrence of oxidative stress and fibrotic process. Again, decreased expression of epithelial marker E-cadherin and increase in the expression of mesenchymal marker α-smooth muscle actin (α-SMA) suggest possible onset of EMT process. Pre-treatment with 5 mM concentration of anti-oxidant N-acetyl cysteine partially attenuated the above process. In conclusion, these findings suggest possible involvement of β-endosulfan in the development of CKDu through oxidative stress and profibrotic signaling.

Sodium-glucose cotransporter-2 inhibition reduces cellular senescence in the diabetic kidney by promoting ketone body-induced NRF2 activation.

To evaluate whether sodium-glucose cotransporter-2 (SGLT2) inhibition reduces cellular senescence in the kidney and to investigate the molecular pathways involved in the renoprotective effect. Dapagliflozin (1mg/kg), glimepiride (2.5mg/kg) or vehicle was administered daily via oral gavage for 8 weeks in db/db mice. Expression levels of ageing marker genes (p21, p16, and p53) and oxidative stress were measured in the kidney using real-time RT-PCR, immunohistochemistry, and Western blot analysis. For in vitro analysis, HK-2 cells, a human renal tubular epithelial cell line, were pretreated with H2 O2 to induce cellular senescence, and the levels of ageing markers were measured after treatment with β-hydroxybutyrate (β-HB) or NRF2-specific siRNA. Expression levels of ageing marker genes (p21, p16 and p53) and senescence-associated secretory phenotypes of the kidney were increased in the vehicle-treated db/db (db/db + vehicle) group compared with the db/+ group, and this increase was markedly reversed in the dapagliflozin-treated db/db (db/db + SGLT2 inhibitor) group, but not in the glimepiride-treated db/db (db/db + sulphonylurea [SU]) group. In the kidneys of mice in the db/db + SGLT2 inhibitor group, oxidative stress and DNA damage were also reduced compared with those of mice in the db/db + vehicle and db/db + SU groups. Dapagliflozin increased plasma β-HB, which reduced H2 O2 -induced DNA damage and senescence in HK-2 cells. β-HB-induced NRF2 nuclear translocation mediated anti-senescent effects by inducing antioxidant pathways. Dapagliflozin prevented the progression of diabetic kidney disease by inhibiting cellular senescence and oxidative stress via ketone-induced NRF2 activation.

MiR-150-5p protects against septic acute kidney injury via repressing the MEKK3/JNK pathway

BackgroundSeptic acute kidney injury (AKI) is associated with increased morbidity and mortality in critically ill patients. MicroRNA is reportedly involved in sepsis-induced organ dysfunction, while the role of miR-150 in septic AKI remains ambiguous. MethodsQuantitative real-time PCR (qRT-PCR) was carried out to examine miR-150-5p expression in both septic AKI patients and volunteers without septic AKI. Lipopolysaccharide (LPS) was used to treat renal tubular epithelial cell line HK-2 and C57/BL6 mice to establish in vitro and in vivo sepsis-induced AKI models. Cell apoptosis was determined using TdT-mediated dUTP nick end labeling (TUNEL) staining and flow cytometry. Cell viability was tested using a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Renal pathological changes were examined via Hematoxylin-Eosin (H&E) staining, and renal function was measured via blood urea nitrogen (BUN) and creatinine (Cre) measurements. The MEKK3/JNK profile and oxidative stress markers (including COX2 and iNOS) were examined by immunoblot analysis, and the expression levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) and oxidative stress markers (MDA, SOD, and CAT) were evaluated by ELISA. ResultsMiR-150-5p was down-regulated in the serum of patients with septic AKI (compared to healthy volunteers). Moreover, miR-150-5p levels were lower in LPS-treated HK-2 cell lines and in the septic AKI mouse model. Additionally, Stat-3 activation mediated the decrease of miR-150-5p. Functionally, miR-150-5p agomir attenuated LPS-induced apoptosis in HK-2 cells, in addition to renal inflammatory responses and oxidative stress. In contrast, inhibition of miR-150-5p aggravated LPS-induced apoptosis, inflammatory reactions and oxidative stress. Furthermore, miR-150-5p agomir decreased BUN and Scr levels in the septic AKI mice model repressed TNF-α, IL-6 and IL-1β, and up-regulated SOD and CAT down-regulated MDA in the kidney tissues. Moreover, miR-150-5p was identified as a target gene for Stat3, and the overexpression of Stat3 partially promoted the effect of down-regulating miR-150-5p on LPS-induced HK2 cell injury. Mechanistically, the MEKK3/JNK pathway was identified as a functional target of miR-150-5p, and the knockdown of MEKK3 showed protective effects against LPS mediated HK-2 cell apoptosis. ConclusionStat3-mediated miR-150-5p exerted protective effects in sepsis-induced acute kidney injury by regulating the MEKK3/JNK pathway.

Pharmacologically Inhibiting Glycogen Synthase Kinase-3β Ameliorates Renal Inflammation and Nephrotoxicity in an Animal Model of Cisplatin-Induced Acute Kidney Injury.

The adverse effect of cisplatin administration causes acute kidney injury (AKI) following renal inflammation and nephrotoxicity, characterized by proximal tubular cell apoptosis and necrosis. Pro-apoptotic and pro-inflammatory roles of glycogen synthase kinase (GSK)-3β have been reported. This study investigated the therapeutic blockade of GSK-3β in cisplatin-induced AKI. A renal cisplatin nephrotoxicity model showed activation of GSK-3β in vivo, particularly in proximal tubular epithelial cells. Pharmacologically inhibiting GSK-3β abolished cisplatin nephrotoxicity, including proximal tubular injury, cell cytotoxicity, and biochemical dysfunction. Additionally, GSK-3β inhibitor treatment ameliorated renal inflammation by reducing immune cell infiltration, cell adhesion molecule expression, and pro-inflammatory cytokine/chemokine production. Cisplatin treatment caused GSK-3β activation in vitro in the human renal proximal tubular epithelial cell line HK-2, whereas either pharmacological administration of GSK-3β inhibitors or genetic transduction of GSK-3β short-hairpin RNA impeded cisplatin-induced cytotoxicity. These results indicate that cisplatin activates GSK-3β followed by GSK-3β-mediated renal inflammation and nephrotoxicity, contributing to AKI.

Necrostatins regulate apoptosis, necroptosis, and inflammation in cisplatin-induced nephrotoxicity in LLC-PK1 cells

Acute kidney injury (AKI) is a common clinical problem that is associated with high mortality due to multiple complex mechanisms. Cisplatin is the most important and highly effective chemotherapeutic agent used for the treatment of various solid tumors; however, it is associated with dose-dependent adverse effects, particularly in the kidney where it can cause severe nephrotoxicity. The pathophysiological basis of cisplatin-induced nephrotoxicity has been investigated over the last few decades, and the key pathological occurrences in cisplatin nephrotoxicity include renal tubular cell injury and death. Necrostatin-1 (Nec-1) has been confirmed to act as a specific and potent small-molecule inhibitor of necroptosis. However, the effects of three structurally distinct necrostatins on cisplatin-induced nephrotoxicity remain ambiguous. The aim of this study was to determine if three types of necrostatins (Nec-1, Nec-3-A, and/or Nec-3-B) can exert protective effects in regard to the AKI induced by cisplatin. Our results indicated that necrostatins can prevent cisplatin induced nephrotoxicity via modulating apoptotic pathways through the suppression of cleaved caspase-3 and also by influencing the function of mitogen-activated protein kinase pathway members, including extracellular signal-regulated kinases, c-Jun N-terminal kinases, and p38, in the renal tubular epithelial cell line LLC-PK1. These findings suggest that necrostatins exert beneficial anti-apoptotic effects in the context of AKI induced by cisplatin.

The Emerging Role of Poly (ADP-Ribose) Polymerase Inhibitors as Effective Therapeutic Agents in Renal Cell Carcinoma.

Renal cell carcinoma (RCC) is the sixth most common cancer in the US. However, no significant changes in management have occurred since the tyrosine kinase era until the recent breakthrough with checkpoint inhibitors. Therefore, the need for more therapeutic options is paramount. Our objective was to determine whether PARP inhibition represents a novel therapeutic option for RCC. We used publicly available COSMIC, GDC Data Portal, and cBioPortal databases to explore mutations in DNA repair genes in RCC tissues from the TCGA cohort. We treated a human normal renal epithelial cell line RPTEC/TERT1 and two human renal cancer cell lines ACHN and CAKI-2 with PARPi niraparib, olaparib, rucaparib, veliparib, and talazoparib. Cell survival, cell proliferation, clonogenic ability, and apoptosis were assessed. RCC xenografts in SCID mice were treated with PARPi to evaluate their efficacy in vivo. Data mining revealed that ~27-32% of RCC tissues contain mutations in homologous recombination genes. Niraparib and talazoparib were the most effective at reducing cell survival, proliferation, and clonogenic ability in vitro. Niraparib, talazoparib, and rucaparib were the most effective in reducing RCC xenograft growth in vivo. Agents such as PARPi that exploit mutations in DNA damage repair genes may be effective therapeutic options for RCC.

In Vitro Cell Culture Models of Hyperoxaluric States: Calcium Oxalate and Renal Epithelial Cell Interactions

Urolithiasis is a multifactorial disease with a high incidence and high recurrence rate, characterized by formation of solid deposits in the urinary tract. The most common type of these stones are calcium oxalate stones. Calcium oxalate crystals can, in hyperoxaluric states, interact with renal epithelial cells, causing injury to the renal epithelia. Pathogenesis of urolithiasis is widely investigated, but underlying mechanisms are still not completely clarified. In vitro models offer insight into molecular processes which lead to renal stone formation and are significant for evaluation of prophylactic and therapeutic management of patients with urolithiasis. In this review, we summarize recently published data from in vitro studies investigating interactions of calcium oxalate crystals with renal epithelial cell lines, anti-urolithiatic mechanisms, and the results from studies exploring possible therapeutic and prophylactic options for calcium oxalate urolithiasis in cell cultures.

LncRNA TUG1/miR-29c-3p/SIRT1 axis regulates endoplasmic reticulum stress-mediated renal epithelial cells injury in diabetic nephropathy model in vitro.

Long non-coding RNAs (lncRNAs) are important regulators in diabetic nephropathy. In this study, we investigated the potential role of lncRNA TUG1 in regulating endoplasmic reticulum stress (ERS)-mediated apoptosis in high glucose induced renal tubular epithelial cells. Human renal tubular epithelial cell line HK-2 was challenged with high glucose following transfection with lncRNA TUG1, miR-29c-3p mimics or inhibitor expression plasmid, either alone or in combination, for different experimental purposes. Potential binding effects between TUG1 and miR-29c-3p, as well as between miR-29c-3p and SIRT1 were verified. High glucose induced apoptosis and ERS in HK-2 cells, and significantly decreased TUG1 expression. Overexpressed TUG1 could prevent high glucose-induced apoptosis and alleviated ERS via negatively regulating miR-29c-3p. In contrast, miR-29c-3p increased HK-2 cells apoptosis and ERS upon high glucose-challenge. SIRT1 was a direct target gene of miR-29c-3p in HK-2 cells, which participated in the effects of miR-29c-3p on HK-2 cells. Mechanistically, TUG1 suppressed the expression of miR-29c-3p, thus counteracting its function in downregulating the level of SIRT1. TUG1 regulates miR-29c-3p/SIRT1 and subsequent ERS to relieve high glucose induced renal epithelial cells injury, and suggests a potential role for TUG1 as a promising diagnostic marker of diabetic nephropathy.

LncRNA TUG1 ameliorates diabetic nephropathy via inhibition of PU.1/RTN1 signaling pathway.

Diabetic nephropathy (DN) is a leading cause of end-stage renal failure. The study aimed to investigate whether long noncoding RNA taurine-upregulated gene 1 (TUG1) can ameliorate the endoplasmic reticulum stress (ERS) and apoptosis of renal tubular epithelial cells in DN, and the underlying mechanism. The DN mouse model was established by streptozocin injection, and the human renal tubular epithelial cell line HK-2 was treated with high glucose (HG) to mimic DN in vitro. The molecular mechanism was explored through dual-luciferase activity assay, RNA pull-down assay, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (CHIP) assay. The expression of TUG1 was significantly decreased in the renal tubules of DN model mice. Overexpression of TUG1 reduced the levels of ERS markers and apoptosis markers by inhibiting reticulon-1 (RTN1) expression in HG-induced HK-2 cells. Furthermore, TUG1 down-regulated RTN1 expression by inhibiting the binding of transcription factor PU.1 to the RTN1 promoter, thereby reducing the levels of ERS markers and apoptosis markers. Meanwhile, TUG1-overexpression adenovirus plasmids injection significantly alleviated tubular lesions, and reduced RTN1 expression, ERS markers and apoptosis markers, whereas these results were reversed by injection of PU.1-overexpression adenovirus plasmids. TUG1 restrains the ERS and apoptosis of renal tubular epithelial cells and ameliorates DN through inhibition of transcription factor PU.1.

Mutation of Klotho rs3752472 protect the kidney from the renal epithelial cell injury caused by CaOx crystals through the Wnt/β-catenin signaling pathway.

Calcium oxalate (CaOx) is a major contributor to urolithiasis, one of the most common urological diseases. Our previous study has shown that Klotho rs3752472 polymorphism correlates with an increased risk of CaOx-related urolithiasis in human cohorts. This study aims to identify the effect of Klotho rs3752472 polymorphism on the renal epithelium injury caused by CaOx. A rat urolithiasis model was established and validated. Renal function was assessed, and histological examination was performed. The distribution and expression of Klotho in the rat model were detected by immunohistochemical staining and western blotting analysis. A renal epithelial cell line (HK2) was used and intervened by COM crystals with several concentrations and time points. Expression of Klotho and key mediators in Wnt/β-catenin pathway were assessed by Western blotting analysis. Wide-type and mutated plasmids of Klotho rs3752472 were added in the cell culture, and the activation of Wnt/β-catenin signaling was tested. Finally, Wide-type and mutated plasmids of Klotho rs3752472 were adoptively transferred to the rat model, and the expression of Klotho was verified. In the rat model, Klotho was mainly distributed in the renal tubular area, which significantly declined in the urolithiasis group. In vitro, COM crystals significantly inhibited the expression of Klotho and induced remarkable renal epithelial cell injury. The mutation of Klotho rs3752472 can notably enhance the expression of Klotho, as well as the protection from renal epithelial cell injury and the inhibition of Wnt/β-catenin signaling pathway. After adoptively transferred to the rat urolithiasis model, similar results were observed for the mutation of Klotho rs3752472. Klotho was significantly correlated with the renal epithelial cell injury induced by CaOx crystals. Furthermore, the mutation of Klotho rs3752472 can remarkably enhance the expression of Klotho in renal tissues and cells, and subsequently protect the renal epithelial cell from the formation of CaOx crystals through the inhibition of Wnt/β-catenin signaling pathway.

MO177RENAL PROXIMAL TUBULAR DISORDER AND RENAL FUNCTION LOSS IN HIV-INFECTED PATIENTS TREATED WITH TENOFOVIR DISOPROXIL FUMARATE

Abstract Background and Aims Tenofovir disoproxil fumarate (TDF), as the most common antiviral drug, can cause both proximal tubular transportation dysfunctions and eGFR decline. The relationship between them is not clear due to lack of clinical data from large cohorts, especially in the Chinese population. In this study, we summarized the characteristics of proximal tubular injuries and eGFR decline in the cohort of HIV-infected patients treated with TDF, and explore the the impact of tenofovir on tubular transporters in vitro. Method We enrolled HIV-infected patients treated with TDF, who were regularly followed up in our hospital from Sep 1, 2001 to August 31, 2019. Their baseline and follow-up clinical data were collected. Proximal tubular dysfunction was defined as meeting two or more of the following criteria: hypophosphatemia, hypouricemia, low carbon dioxide binding capacity, positive urine glucose with normal plasma glucose level, and positive urine protein. Rapid deterioration of renal function was defined as the annual decline rate of eGFR exceeding 5ml/min/1.73m2. We also used human renal proximal tubular epithelial cell line (HK2) to further investigate the impact of tenofovir on transporters including SGLT2, NaPi-IIa, and URAT1 through immunofluorescence. Results A total of 375 HIV-infected patients receiving TDF were enrolled, mainly males (90.1%), with a median follow-up duration of 34(17, 58) months. The most common clinical manifestations were proteinuria (20.3%) and hypophosphatemia (12.3%). The prevalence of proximal tubular injury was 6.7%, which was significantly associated with low body weight, but was not associated with age, TDF course, baseline viral load, or baseline CD4+ T lymphocyte count. Their eGFR levels at the end of the follow-up were significantly lower than the baseline levels (104.6±15.2 vs. 110.6±14.2 ml/min/1.73m2, P&amp;lt;0.001). The average annual decline rate of eGFR was 5.0± 22.7 ml/min/1.73m2, and 23.6% of our patients had an annual decline rate of eGFR exceeding 5 ml/min/1.73m2. Rapid deterioration of renal function (≥ 5 ml/min/1.73m2 per year) was significantly associated with female but not related to proximal tubular dysfunction in multivariate logistic regression analysis. In vitro, the survival rate of HK2 cells was more than 95% when treated with tenofovir with a concentration of 1μmol/L for 48h. The expression levels of transporters (SGLT2, URAT1, and NaPi-IIa) were declined under the condition. Conclusion Among the HIV-infected Chinese patients treated with TDF, 6.7% had proximal tubular dysfunction and 23.6% showed accelerated annual decline rate of eGFR (≥ 5 ml/min/1.73m2 per year).

Advanced Sequencing of Kidney Tubule Hypoxia Regulated Epithelial‐to‐Mesenchymal Transition

Hypoxia is a known regulator of epithelial-to-mesenchymal transition in kidney tubule cells. However, culture systems have struggled to observe these transitions at high levels to capture transcriptional signatures due to the detachment of cells from culture vessels. Using the Renal Proximal Tubule Epithelial Cell stabile cell line (RPTEC-TERT1) we developed a shear stress culture system that when exposed to hypoxia (1% oxygen) result in cell dysmorphology instead of detachment, allowing for a large number of cells to be generated for transcriptomic studies. With these cells exposed to hypoxia, we performed both traditional polyA RNAseq using both Illumina and Nanopore technologies and newer direct RNAseq using Nanopore. The polyA RNAseq suggests regulation changes for HIF-1/hypoxia, Nonsense mediated decay, ruffle assembly, and energetic regulated genes. Direct RNAseq, a strategy of running mRNA molecules across Nanopore to detect RNA base modifications transcriptome wide, revealed genes that are acutely modified related to glycolysis, innate immunity, oxidation-reduction, and mitochondrial biology. Cells exposed to hypoxia for 48 hours followed by 48 hours of normoxia, return the modifications back to control levels suggesting there is a major source of hypoxia cell signaling through RNA posttranscriptional modifications. Using the differentially regulated genes of the hypoxia culture model we have also shown that it is possible to filter genetic models systems such as human chronic/acute kidney disease or rat heterogenous stock proteinuria to narrow down novel mechanisms for genetic influence to hypoxia response. In doing this, we nominated and validated the role of SEPTIN8 in hypoxia response cell integrity, where dysregulation of SEPTIN8 can further propagate complications of hypoxia to disease progression.