Renal Tubule Epithelial Cells Research Articles

SFN promotes renal fibrosis via binding with MYH9 in chronic kidney disease

Chronic kidney disease (CKD) is a clinical syndrome characterized by persistent renal function decline. Renal fibrosis is the main pathological process in CKD, but an effective treatment does not exist. Stratifin (SFN) is a highly-conserved, multi-function soluble acidic protein. Therefore, this study explored the effects of SFN on renal fibrosis. First, we found that SFN was highly expressed in patients with CKD, as well as in renal fibrosis animal and cell models. Next, transforming growth factor-beta 1 (TGF-β1) induced injury and fibrosis in human renal tubule epithelial cells, and SFN knockdown reversed these effects. Furthermore, SFN knockdown mitigated unilateral ureteral obstruction (UUO)-induced renal tubular dilatation and renal interstitial fibrosis in mice. Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS), co-immunoprecipitation (Co-IP), and immunofluorescence co-localization assays demonstrated that SFN bound the non-muscle myosin-encoding gene, myosin heavy chain 9 (MYH9), in the cytoplasm of renal tubular epithelial cells. MYH9 knockdown also reduced Col-1 and α-SMA expression, which are fibrosis markers. Finally, silencing SFN decreased MYH9 expression, alleviating renal fibrosis. These results suggest that SFN promotes renal fibrosis in CKD by interacting with MYH9. This study may provide potential strategies for the treatment of CKD.

SFN promotes renal fibrosis via binding with MYH9 in chronic kidney disease

Chronic kidney disease (CKD) is a clinical syndrome characterized by persistent renal function decline. Renal fibrosis is the main pathological process in CKD, but an effective treatment does not exist. Stratifin (SFN) is a highly-conserved, multi-function soluble acidic protein. Therefore, this study explored the effects of SFN on CKD. First, we found that SFN was highly expressed in patients with CKD, as well as in CKD animal and cell models. Next, we induced injury and fibrosis in human renal tubule epithelial cells, and SFN knockdown reversed these effects. Furthermore, SFN knockdown mitigated ureteral obstruction (UUO)-induced renal tubular dilatation and renal interstitial fibrosis in mice. Liquid chromatography-tandem mass spectrometry/mass spectrometry (LC-MS/MS), co-immunoprecipitation (Co-IP), and immunofluorescence co-localization assays demonstrated that SFN bound the non-muscle myosin-encoding gene, myosin heavy chain 9 (MYH9), in the cytoplasm of renal tubular epithelial cells. MYH9 knockdown also reduced Col-1 and α-SMA expression, which are fibrosis markers. Finally, silencing SFN decreased MYH9 expression, alleviating renal fibrosis. These results suggest that SFN promotes kidney fibrosis in CKD by interacting with MYH9. This study may provide potential strategies for the treatment of CKD.

Protective Effect of CXCR7 Against Hypoxia/Reoxygenation Injury in Renal Tubular Epithelial Cells.

Acute kidney injury (AKI) is a multifactorial syndrome with complex pathophysiology and prognosis. Ischaemia‒reperfusion injury (IRI) is a major cause of induced AKI. The aim of this study was to investigate the effect of upregulated CXCR7 expression on renal tubular epithelial cell apoptosis induced by hypoxia/reoxygenation (H/R). HK-2 cells were divided into three groups: control group (pcDNA3.1), hypoxia/reoxygenation + pcDNA3.1 group (H/R+pcDNA3.1) and CXCR7 overexpression + hypoxia/reoxygenation group (H/R+ Flag-CXCR7). Protein levels of renal tubular epithelial cell injury-, apoptosis- and autophagy-related markers were assessed by qRT‒PCR, Western blotting, flow cytometry (FCM), immunofluorescence and transmission electron microscopy (TEM). In addition, HK-2 cells were treated with the autophagy inhibitor 3-MA and divided into 3 groups: control group, 3-MA + pcDNA3.1 group, and 3-MA + Flag-CXCR7 group. Changes in autophagy and apoptosis in renal tubule epithelial cells were assessed by Western blotting and FCM. Compared with those in the control group, the protein and mRNA expression levels of CXCR7 in HK-2 cells were significantly lower under H/R conditions. Under H/R conditions, CXCR7 overexpression in HK-2 cells significantly downregulated the expression of NGAL. Moreover, CXCR7 overexpression significantly decreased H/R-induced cleaved PARP-1 and cleaved Caspase 3 levels, increased the level of the antiapoptotic protein BCL-2 and the autophagy-related molecules ATG5 and LC3B II, and significantly inhibited the expression of P62. Autophagy flow and TEM also showed that CXCR7 significantly promoted autophagy. CXCR7 significantly alleviated the 3-MA-induced inhibition of autophagy and increase in apoptosis. Upregulated CXCR7 expression can inhibit renal tubular epithelial cell apoptosis and damage by regulating autophagy. In conclusion, CXCR7 is a promising target for the prevention and/or treatment of AKI.

#1373 Effect of Astragalus membranaceus normalized acute kidney injury-induced renal conditions corelated with serum carnosine

Abstract Background and Aims Astragalus membranaceus (AM) exhibits various pharmacological effects against several diseases, including chronic kidney disease (CKD) (see http://nccih.nih.gov/health/astragalus). We have previously investigated the correlation between AM and acute kidney injury (AKI), which is an important factor causing CKD pathogenesis or progression. Recently, we demonstrated that old mice showed lethality in response to 20 mg/kg cis-diamminedichloroplatinum (CDDP), which is a general dose used to generate AKI in young mice. AKI also occurred in response to a reduced dose of cisplatin, which was completely reversible in young mice. The administration of AM to mice (20 mg/kg) significantly improved AKI pathology, especially in old mice treated with a reduced dose of cisplatin, thereby demonstrating the therapeutic effect of AM against age-dependent AKI pathogenicity. More recently, we found that serum carnosine levels correlated with renal conditions in AM. Here, we examined the effects associated with serum carnosine levels to prevent AKI and delay CKD progression [1]. Methods Female C57BL/6 mice (52 weeks old) were orally administered either an AM powder mixed with sterilized 0.5% methylcellulose 400 (w/v) (AM group) or only 0.5% methylcellulose 400 (control). Four hours after the most recent dose (20 mg/kg of AM twice a day), 0.5 mg/ml CDDP (reduced dose: 10 mg/kg or 0.9% NaCl for the CG) was injected intraperitoneally into old mice. Subsequently, we measured the blood serum urea nitrogen (BUN) and creatinine (CRE) levels, which are biomarkers of AKI, and assessed histochemical changes in kidney sections stained with anti-CD3 and anti-CD68 antibodies 20 h and 3 d after CDDP injection (n = 6 per group). Thereafter, serum carnosine levels in the samples collected from each sample (n = 6 per group) were determined using a carnosine ELISA kit (Novus, NBP2-75013). Data were analyzed statistically using PRISM software, and statistical significance was set at P = 0.05. Results Higher susceptibility to a half CDDP dose in old mice was indicated by increased BUN levels and the histological damage caused to renal tubule epithelial cells and glomeruli; however, no pathological change was observed in younger mice. Histochemical analysis of old mice showed a significant increase in the number of CD3-positive cells following AKI induction; however, AM pretreatment ameliorated these inflammatory conditions. Our recent findings suggest that a correlation exists between the effects of AM and serum carnosine levels. Fig. 1 shows that different serum carnosine levels were observed in old control mice (white box), probably because of age-dependent pathogenesis. However, serum carnosine was concentrated to lower levels after AM administration (orange box), suggesting an age-dependent AM effect. Moreover, owing to the effect of AM on CDDP-AKI, serum carnosine levels changed transiently but significantly in old mice (blue and yellow boxes) within 20 h; this indicates that age-dependent renal conditions were improved by the AM effect and correlated with serum carnosine levels. Conclusion AM administration normalizes the renal pathology caused by age-dependent susceptibility to CDDP-AKI; this is particularly true for older mice, wherein serum carnosine is downregulated but concentrated. Serum carnosine can be effective in preventing AKI pathology in old mice, regardless of the AM effects. We believe that AM can prevent age-dependent accumulation of AKI pathology, thereby facilitating CKD progression.

#3092 Case report: Renal cystic disease caused to heterozygous NEK8 variant in pediatric patient

Abstract Background and Aims Polycystic kidney diseases (PKDs) are the most prevalent inherited kidney disease [1]. They are caused by pathogenic variants in genes encoding for proteins with roles in the functioning of primary cilium, a sensory organelle at the cell surface of almost all cells regulating key signaling pathways. Therefore, they are also classified as Ciliopathy and can occur with isolated renal involvement with/without systemic manifestations. As 1 of the family of “Never in Mitosis A–related kinases” (NEK), NEK8 is a regulator of Hippo pathway that controls cell growth, regulate the renal tubule epithelial cell cytoskeletal structure, and is involved in ciliary biogenesis/function. Biallelic NEK8 mutations are reported as causing a syndromic ciliopathy with multiorgan developmental defects, referred to as renal-hepatic-pancreatic dysplasia [1]. A recent paper [1] analyzed a total of 21 patients from 12 families with heterozygous NEK8 variants and cystic kidney disease and proposed a dominant negative effect for specific heterozygous variants in the NEK8 kinase domain as a new cause of PKD. Method We describe a case report of PKD associated with NEK8 heterozygous mutation. Results 8 years old female patient accessed to Emergency Department with first evidence of isolated macro-hematuria. Previous medical history was unremarkable. The girl was afebrile, in good clinical condition, with elevated blood pressure values (120/82 mmHg) at the physical examination. The laboratory tests showed normal blood count with hemoglobin 11.7 g/dl, serum creatinine 0.42 mg/dl, no inflammatory markers. Urinalysis revealed proteinuria (100 mg/dL), hemoglobinuria, active urinary sediment with microhematuria (3557 red blood cell/microliter) and leukocyturia (535 white blood cells/microliter). An abdominal ultrasound study observed kidneys of increased size (longitudinal diameter of approximately 14 cm) with presence of multiple cystic formations with a maximum diameter of 2 cm and lost cortico-medullary differentiation; other organs were normal. Ultrasound findings were confirmed with MRI. The macrohematuria resolved quickly, however we recorded the persistence of positive urinary sediment. No other concomitant clinical manifestations were recorded and the cardiological evaluation was normal. Suspecting a cystic renal disease, we proposed a genetic analysis by whole exome sequencing (WES): an heterozygous NEK8 variant (c.133C>T, p. Arg45Trp) was found. Due to persistent proteinuria and hypertension we suggested to initiate RAAS inhibitor treatment. Conclusion The patient's clinical history and onset of disease agree with the data described in the literature for the NEK8 variant c.133C>T, p. Arg45Trp. In literature all patients had an early onset of PKD. Enlarged kidney were present in 79%, followed by hypertension in 67% of cases; no patients had liver involvement, as in our patient. Kidney failure occurred before adulthood in non-mosaic case. The absence of a phenotype with systemic involvement, doesn't exclude the possible subsequent appearance of extra-renal manifestations. Therefore, it is necessary to set up a multidisciplinary follow-up, including cardiological evaluation and radiological study of brain structure. An early nephroprotective therapy and behavioral strategy must be established to prevent disease progression. Thus, it is suggested to include NEK8 in the genetic analysis when PKD is suspected.

MiR-150-5p Alleviates Renal Tubule Epithelial Cell Fibrosis via the Inhibition of Epithelial-Mesenchymal Transition by Targeting ZEB1

Introduction: Although microRNA (miR)-150-5p participates in the progression of renal fibrosis, its mechanism of action remains elusive. Methods: A mouse model of unilateral ureteral obstruction was used. The in vitro renal fibrosis model was established by stimulating human kidney 2 (HK-2) cells with transforming growth factor beta 1 (TGF-β1). The expression profiles of miR-150-5p, zinc finger E-box binding homeobox 1 (ZEB1), and other fibrosis- and epithelial-mesenchymal transition (EMT)-linked proteins were determined using Western blot and quantitative reverse transcription polymerase chain reaction. The relationship between miR-150-5p and ZEB1 in HK-2 cells was confirmed by a dual-luciferase reporter assay. Results: Both in vivo and in vitro renal fibrosis models revealed reduced miR-150-5p expression and elevated ZEB1 level. A significant decrease in E-cadherin levels, as well as increases in alpha smooth muscle actin (α-SMA) and collagen type I (Col-I) levels, was seen in TGF-β1-treated HK-2 cells. The overexpression of miR-150-5p ameliorated TGF-β1-mediated fibrosis and EMT. Notably, miR-150-5p acts by directly targeting ZEB1. A significant reversal of the inhibitory impact of miR-150-5p on TGF-β1-mediated fibrosis and EMT in HK-2 cells was observed upon ZEB1 overexpression. Conclusion: MiR-150-5p suppresses TGF-β1-induced fibrosis and EMT by targeting ZEB1 in HK-2 cells, providing helpful insights into the therapeutic intervention of renal fibrosis.

Astragaloside IV attenuates renal tubule injury in DKD rats via suppression of CD36-mediated NLRP3 inflammasome activation.

In recent years, diabetic kidney disease (DKD) has emerged as a prominent factor contributing to end-stage renal disease. Tubulointerstitial inflammation and lipid accumulation have been identified as key factors in the development of DKD. Earlier research indicated that Astragaloside IV (AS-IV) reduces inflammation and oxidative stress, controls lipid accumulation, and provides protection to the kidneys. Nevertheless, the mechanisms responsible for its protective effects against DKD have not yet been completely elucidated. The primary objective of this research was to examine the protective properties of AS-IV against DKD and investigate the underlying mechanism, which involves CD36, reactive oxygen species (ROS), NLR family pyrin domain containing 3 (NLRP3), and interleukin-1β (IL-1β). The DKD rat model was created by administering streptozotocin along with a high-fat diet. Subsequently, the DKD rats and palmitic acid (PA)-induced HK-2 cells were treated with AS-IV. Atorvastatin was used as the positive control. To assess the therapeutic effects of AS-IV on DKD, various tests including blood sugar levels, the lipid profile, renal function, and histopathological examinations were conducted. The levels of CD36, ROS, NLRP3, Caspase-1, and IL-1β were detected using western blot analysis, PCR, and flow cytometry. Furthermore, adenovirus-mediated CD36 overexpression was applied to explore the underlying mechanisms through in vitro experiments. In vivo experiments demonstrated that AS-IV significantly reduced hyperglycemia, dyslipidemia, urinary albumin excretion, and serum creatinine levels in DKD rats. Additionally, it improved renal structural abnormalities and suppressed the expression of CD36, NLRP3, IL-1β, TNF-α, and MCP-1. In vitro experiments showed that AS-IV decreased CD36 expression, lipid accumulation, and lipid ROS production while inhibiting NLRP3 activation and IL-1β secretion in PA-induced HK-2 cells. AS-IV alleviated renal tubule interstitial inflammation and tubule epithelial cell apoptosis in DKD rats by inhibiting CD36-mediated lipid accumulation and NLRP3 inflammasome activation.

STING antagonist-loaded renal tubule epithelial cell-mimicking nanoparticles ameliorate acute kidney injury by orchestrating innate and adaptive immunity

Inflammatory responses profoundly contribute to the development of renal damage in acute kidney injury (AKI), a frequent clinical syndrome with high morbidity and mortality. However, the effects of existing strategies aiming to suppress the inflammation in AKI remain unsatisfactory due to the inability to simultaneously regulate innate and adaptive immune responses. Here, STING antagonist-loaded renal tubule epithelial cell membrane-coated nanoparticles (SNP@TEC) are developed to ameliorate AKI by orchestrating innate and adaptive immunity. As the reservation of cell membrane αV integrin, biomimetic SNP@TEC are able to target renal tubules via homotypic tropism. The carried antagonist can suppress innate immune responses through relieving STING-triggered inflammatory reaction and repolarizing macrophages to the anti-inflammatory M2 phenotype. Owing to the presence of cell membrane PD-L1, SNP@TEC can also elicit an anti-inflammatory adaptive immune response by blocking the CD80 on dendritic cells to inhibit T-cell activation. In two murine models of AKI induced by cisplatin or ischemia/reperfusion, systemic injection with SNP@TEC significantly alleviates renal damage and restores renal function. In a prophylactic model, SNP@TEC effectively prevent the transition of AKI to chronic kidney disease. SNP@TEC propose a unique approach to orchestrate innate and adaptive immunity, opening a window to remit inflammation for treating renal injury.

Intestinal Absorption of Nanoparticles to Reduce Oxidative Stress and Vasoconstriction for Treating Diabetic Nephropathy.

The etiology of diabetic nephropathy (DN) is complex, and the incidence is increasing year by year. The patient's kidney showed oxidative stress damage, increasing active oxygen species (ROS) content, and vasoconstriction. Due to poor drug solubility and low renal accumulation, the current treatment regimens have not effectively alleviated glomerulopathy and other kidney damage caused by DN. Therefore, it is of great significance to explore new treatment strategies and drug delivery systems. Here, we constructed an oral nanodelivery system (Tel/CAN@CS-DA) that reduced oxidative stress and vasoconstriction. Deoxycholic acid (DA)-modified nanoparticles entered into intestinal epithelial cells (Caco2 cells) via the bile acid biomimetic pathway, then escaped from the lysosomes and eventually spat out the cells, increasing the oral absorption of nanoparticles. Chitosan (CS) nanoparticles could achieve renal targeting through specific binding with a renal giant protein receptor and deliver drugs to renal tubule epithelial cells (HK-2 cells). In vitro studies also proved that telmisartan (Tel) and canagliflozin (CAN) effectively removed cellular reactive oxygen species (ROS) and reduced HK-2 cell apoptosis caused by high glucose. In the in vivo model induced by streptozotocin (STZ), the results showed that the nanosystem not only elevated AMPK protein expression, inhibited angiotensin II (Ang II) protein expression to effectively reduce oxidative stress level, dilated renal blood vessels but also reduced the degree of inflammation and fibrosis. Overall, Tel/CAN@CS-DA multifunctional oral nanosystem can effectively treat DN with low toxicity, which provides a new idea for the treatment of DN.

Lactiplantibacillus plantarum NKK20 Increases Intestinal Butyrate Production and Inhibits Type 2 Diabetic Kidney Injury through PI3K/Akt Pathway.

Nephropathy injury is a prevalent complication observed in individuals with diabetes, serving as a prominent contributor to end-stage renal disease, and the advanced glycation products (AGEs) are important factors that induce kidney injury in patients with diabetes. Addressing this condition remains a challenging aspect in clinical practice. The aim of this study was to explore the effects of Lactiplantibacillus plantarum NKK20 strain (NKK20) which protects against diabetic kidney disease (DKD) based on animal and cell models. The results showed that the NKK20 can significantly reduce renal inflammatory response, serum oxidative stress response, and AGE concentration in diabetic mice. After treatment with NKK20, the kidney damage of diabetic mice was significantly improved, and more importantly, the concentration of butyrate, a specific anti-inflammatory metabolite of intestinal flora in the stool of diabetic mice, was significantly increased. In addition, nontargeted metabolomics analysis showed a significant difference between the metabolites in the mouse serum contents of the NKK20 administration group and those in the nephropathy injury group, in which a total of 24 different metabolites that were significantly affected by NKK20 were observed, and these metabolites were mainly involved in glycerophospholipid metabolism and arachidonic acid metabolism. Also, the administration of butyrate to human kidney- (HK-) 2 cells that were stimulated by AGEs resulted in a significant upregulation of ZO-1, Occludin, and E-cadherin gene expressions and downregulation of α-SMA gene expression. This means that butyrate can maintain the tight junction structure of HK-2 cells and inhibit fibrosis. Butyrate also significantly inhibited the activation of PI3K/Akt pathway. These results indicate that NKK20 can treat kidney injury in diabetic mice by reducing blood glucose and AGE concentration and increasing butyrate production in the intestine. By inhibiting PI3K pathway activation in HK-2 cells, butyrate maintains a tight junction structure of renal tubule epithelial cells and inhibits renal tissue fibrosis. These results suggest that NKK20 is helpful to prevent and treat the occurrence and aggravation of diabetic kidney injury.

Overexpression of long noncoding RNA 4933425B07Rik leads to renal hypoplasia by inactivating Wnt/β-catenin signaling pathway.

Congenital anomalies of the kidney and urinary tract (CAKUT) is a general term for a class of diseases that are mostly caused by intrauterine genetic development limitation. Without timely intervention, certain children with CAKUT may experience progressive decompensation and a rapid decline in renal function, which will ultimately result in end-stage renal disease. At present, a comprehensive understanding of the pathogenic signaling events of CAKUT is lacking. The role of long noncoding RNAs (lncRNAs) in renal development and disease have recently received much interest. In previous research, we discovered that mice overexpressing the lncRNA 4933425B07Rik (Rik) showed a range of CAKUT phenotypes, primarily renal hypoplasia. The current study investigated the molecular basis of renal hypoplasia caused by Rik overexpression. We first used Rapid Amplification of cDNA ends (RACE) to obtain the full-length sequence of Rik in Rik +/+;Hoxb7 mice. Mouse proximal renal tubule epithelial cells (MPTCs) line with Rik overexpression was constructed using lentiviral methods, and mouse metanephric mesenchyme cell line (MK3) with Rik knockout was then constructed by the CRISPR‒Cas9 method. We performed RNA-seq on the Rik-overexpressing cell line to explore possible differentially expressed molecules and pathways. mRNA expression was confirmed by qRT‒PCR. Reduced levels of Wnt10b, Fzd8, and β-catenin were observed when Rik was expressed robustly. On the other hand, these genes were more highly expressed when Rik was knocked out. These results imply that overabundance of Rik might inhibit the Wnt/β-catenin signaling pathway, which may result in renal hypoplasia. In general, such research might help shed light on CAKUT causes and processes and offer guidance for creating new prophylactic and therapeutic strategies.

SIRT2 alleviated renal fibrosis by deacetylating SMAD2 and SMAD3 in renal tubular epithelial cells

Transforming growth factor-β (TGF-β) is the primary factor that drives fibrosis in most, if not all, forms of chronic kidney disease. In kidneys that are obstructed, specific deletion of Sirt2 in renal tubule epithelial cells (TEC) has been shown to aggravate renal fibrosis, while renal tubule specific overexpression of Sirt2 has been shown to ameliorate renal fibrosis. Similarly, specific deletion of Sirt2 in hepatocyte aggravated CCl4-induced hepatic fibrosis. In addition, we have demonstrated that SIRT2 overexpression and knockdown restrain and enhance TGF-β-induced fibrotic gene expression, respectively, in TEC. Mechanistically, SIRT2 reduced the phosphorylation, acetylation, and nuclear localization levels of SMAD2 and SMAD3, leading to inhibition of the TGF-β signaling pathway. Further studies have revealed that that SIRT2 was able to directly interact with and deacetylate SMAD2 at lysine 451, promoting its ubiquitination and degradation. Notably, loss of SMAD specific E3 ubiquitin protein ligase 2 abolishes the ubiquitination and degradation of SMAD2 induced by SIRT2 in SMAD2. Regarding SMAD3, we have found that SIRT2 interact with and deacetylates SMAD3 at lysine 341 and 378 only in the presence of TGF-β, thereby reducing its activation. This study provides initial indication of the anti-fibrotic role of SIRT2 in renal tubules and hepatocytes, suggesting its therapeutic potential for fibrosis.

Cytosine–phosphate–guanine oligodeoxynucleotides alleviate radiation-induced kidney injury in cervical cancer by inhibiting DNA damage and oxidative stress through blockade of PARP1/XRCC1 axis

BackgroundRadiotherapy can cause kidney injury in patients with cervical cancer. This study aims to investigate the possible molecular mechanisms by which CpG-ODNs (Cytosine phosphate guanine-oligodeoxynucleotides) regulate the PARP1 (poly (ADP-ribose) polymerase 1)/XRCC1 (X-ray repair cross-complementing 1) signaling axis and its impact on radiation kidney injury (RKI) in cervical cancer radiotherapy.MethodsThe GSE90627 dataset related to cervical cancer RKI was obtained from the Gene Expression Omnibus (GEO) database. Bioinformatics databases and R software packages were used to analyze the target genes regulated by CpG-ODNs. A mouse model of RKI was established by subjecting C57BL/6JNifdc mice to X-ray irradiation. Serum blood urea nitrogen (BUN) and creatinine levels were measured using an automated biochemical analyzer. Renal tissue morphology was observed through HE staining, while TUNEL staining was performed to detect apoptosis in renal tubular cells. ELISA was conducted to measure levels of oxidative stress-related factors in mouse serum and cell supernatant. An in vitro cell model of RKI was established using X-ray irradiation on HK-2 cells for mechanism validation. RT-qPCR was performed to determine the relative expression of PARP1 mRNA. Cell proliferation activity was assessed using the CCK-8 assay, and Caspase 3 activity was measured in HK-2 cells. Immunofluorescence was used to determine γH2AX expression.ResultsBioinformatics analysis revealed that the downstream targets regulated by CpG-ODNs in cervical cancer RKI were primarily PARP1 and XRCC1. CpG-ODNs may alleviate RKI by inhibiting DNA damage and oxidative stress levels. This resulted in significantly decreased levels of BUN and creatinine in RKI mice, as well as reduced renal tubular and glomerular damage, lower apoptosis rate, decreased DNA damage index (8-OHdG), and increased levels of antioxidant factors associated with oxidative stress (SOD, CAT, GSH, GPx). Among the CpG-ODNs, CpG-ODN2006 had a more pronounced effect. CpG-ODNs mediated the inhibition of PARP1, thereby suppressing DNA damage and oxidative stress response in vitro in HK-2 cells. Additionally, PARP1 promoted the formation of the PARP1 and XRCC1 complex by recruiting XRCC1, which in turn facilitated DNA damage and oxidative stress response in renal tubular cells. Overexpression of either PARP1 or XRCC1 reversed the inhibitory effects of CpG-ODN2006 on DNA damage and oxidative stress in the HK-2 cell model and RKI mouse model.ConclusionCpG-ODNs may mitigate cervical cancer RKI by blocking the activation of the PARP1/XRCC1 signaling axis, inhibiting DNA damage and oxidative stress response in renal tubule epithelial cells.

Modelling BK Polyomavirus dissemination and cytopathology using polarized human renal tubule epithelial cells.

Most humans have a lifelong imperceptible BK Polyomavirus (BKPyV) infection in epithelial cells lining the reno-urinary tract. In kidney transplant recipients, unrestricted high-level replication of donor-derived BKPyV in the allograft underlies polyomavirus-associated nephropathy, a condition with massive epithelial cell loss and inflammation causing premature allograft failure. There is limited understanding on how BKPyV disseminates throughout the reno-urinary tract and sometimes causes kidney damage. Tubule epithelial cells are tightly connected and have unique apical and basolateral membrane domains with highly specialized functions but all in vitro BKPyV studies have been performed in non-polarized cells. We therefore generated a polarized cell model of primary renal proximal tubule epithelial cells (RPTECs) and characterized BKPyV entry and release. After 8 days on permeable inserts, RPTECs demonstrated apico-basal polarity. BKPyV entry was most efficient via the apical membrane, that in vivo faces the tubular lumen, and depended on sialic acids. Progeny release started between 48 and 58 hours post-infection (hpi), and was exclusively detected in the apical compartment. From 72 hpi, cell lysis and detachment gradually increased but cells were mainly shed by extrusion and the barrier function was therefore maintained. The decoy-like cells were BKPyV infected and could transmit BKPyV to uninfected cells. By 120 hpi, the epithelial barrier was disrupted by severe cytopathic effects, and BKPyV entered the basolateral compartment mimicking the interstitial space. Addition of BKPyV-specific neutralizing antibodies to this compartment inhibited new infections. Taken together, we propose that during in vivo low-level BKPyV replication, BKPyV disseminates inside the tubular system, thereby causing minimal damage and delaying immune detection. However, in kidney transplant recipients lacking a well-functioning immune system, replication in the allograft will progress and eventually cause denudation of the basement membrane, leading to an increased number of decoy cells, high-level BKPyV-DNAuria and DNAemia, the latter a marker of allograft damage.

TLR7 activation by miR-21 promotes renal fibrosis by activating the pro-inflammatory signaling pathway in tubule epithelial cells

BackgroundToll-like receptor 7 (TLR7) is an endosomal TLR activated by single-stranded RNA, including endogenous microRNAs. Although TLR7 is known to promote inflammatory responses in pathophysiological conditions, its role in renal fibrosis has not been investigated. Here, we aim to investigate the inflammatory roles of TLR7 in kidney inflammation and fibrosis.MethodsTLR7 knockout mice (Tlr7 −/−) subjected to AD-induced kidney injury were utilized to examine the role of TLR7 in kidney fibrosis. To elucidate the role of TLR7 in renal epithelial cells, NRK52E rat renal tubule epithelial cells were employed.ResultsUnder fibrotic conditions induced by an adenine diet (AD), TLR7 was significantly increased in damaged tubule epithelial cells, where macrophages were highly infiltrated. TLR7 deficiency protected against AD-induced tubular damage, inflammation, and renal fibrosis. Under in vitro conditions, TLR7 activation increased NF-κB activity and induced chemokine expression, whereas TLR7 inhibition effectively blocked NF-κB activation. Furthermore, among the known TLR7 endogenous ligands, miR-21 was significantly upregulated in the tubular epithelial regions. In NRK52E cells, miR-21 treatment induced pro-inflammatory responses, which could be blocked by a TLR7 inhibitor. When the TLR7 inhibitor, M5049, was administered to the AD-induced renal fibrosis model, TLR7 inhibition significantly attenuated AD-induced renal inflammation and fibrosis.ConclusionsOverall, activation of TLR7 by endogenous miR-21 in renal epithelial cells contributes to inflammatory responses in a renal fibrosis model, suggesting a possible therapeutic target for the treatment of renal fibrosis.8V5xz8c9tzre9tDnxLpjTTVideo

Time- and Gender-Dependent Alterations in Mice during the Aging Process.

Compared to young people and adults, there are differences in the ability of elderly people to resist diseases or injuries, with some noticeable features being gender-dependent. However, gender differences in age-related viscera alterations are not clear. To evaluate a potential possibility of gender differences during the natural aging process, we used three age groups to investigate the impact on spleens, kidneys, and adrenal glands. The immunofluorescence results showed that male-specific p21 proteins were concentrated in the renal tubule epithelial cells of the kidney. Histological staining revealed an increase in the frequencies of fat vacuoles located in the renal tubule epithelial cells of the cortex, under the renal capsule in the kidneys of male mice with age. In female mice, we found that the width of the globular zone in the adrenal gland cortex was unchanged with age. On the contrary, the male displayed a reduction in width. Compared to females, the content of epinephrine in adrenal gland tissue according to ELISA analysis was higher in adults, and a greater decline was observed in aged males particularly. These data confirmed the age-dependent differences between female and male mice; therefore, gender should be considered one of the major factors for personalized treatment in clinical diagnosis and treatment.

Compound-42 alleviates acute kidney injury by targeting RIPK3-mediated necroptosis.

Necroptosis plays an essential role in acute kidney injury and is mediated by receptor-interacting protein kinase 1 (RIPK1), receptor-interacting protein kinase 3 (RIPK3), and mixed lineage kinase domain-like pseudokinase (MLKL). A novel RIPK3 inhibitor, compound 42 (Cpd-42) alleviates the systemic inflammatory response. The current study was designed to investigate whether Cpd-42 exhibits protective effects on acute kidney injury and reveal the underlying mechanisms. The effects of Cpd-42 were determined in vivo through cisplatin- and ischaemia/reperfusion (I/R)-induced acute kidney injury and in vitro through cisplatin- and hypoxia/re-oxygenation (H/R)-induced cell damage. Transmission electron microscopy and periodic acid-Schiff staining were used to identify renal pathology. Cellular thermal shift assay and RIPK3-knockout mouse renal tubule epithelial cells were used to explore the relationship between Cpd-42 and RIPK3. Molecular docking and site-directed mutagenesis were used to determine the binding site of RIPK3 with Cpd-42. Cpd-42 reduced human proximal tubule epithelial cell line (HK-2) cell damage, necroptosis and inflammatory responses in vitro. Furthermore, in vivo, cisplatin- and I/R-induced acute kidney injury was alleviated by Cpd-42 treatment. Cpd-42 inhibited necroptosis by interacting with two key hydrogen bonds of RIPK3 at Thr94 and Ser146, which further blocked the phosphorylation of RIPK3 and mitigated acute kidney injury. Acting as a novel RIPK3 inhibitor, Cpd-42 reduced kidney damage, inflammatory response and necroptosis in acute kidney injury by binding to sites Thr94 and Ser146 on RIPK3. Cpd-42 could be a promising treatment for acute kidney injury.

The research of liver failure in Banna miniature pigs caused by amanita exitialis

Objective: To explore the characteristics of Banna miniature pig liver failure induced by amanita exitialis. Methods: From September to October 2020, a reverse high performance liquid chromatography (RP-HPLC) method was used to determine the toxin content of amanita exitialis solution, and 2.0 mg/kg amanita exitialis solution (α-amanitins+β-amanitins) was administered orally to Banna miniature pigs. Toxic symptoms, blood biochemical indexes and histopathological changes of liver, heart and kidney were observed at each time point. Results: All Banna miniature pigs died within 76 h of exposure, and different degrees of digestive tract symptoms such as nausea, vomiting and diarrhea appeared between 6 and 36 h. The biochemical indexes of alanine aminotransferase, aspartate aminotransferase, total bilirubin, lactate dehydrogenase, myoglobin, creatine kinase isoenzyme, blood urea nitrogen and creatinine increased significantly at 52 h after exposure, and the differences were statistically significant compared with 0 h (P<0.05). The bleeding of liver and heart was obvious under macroscopic and microscopic observation, hepatocyte necrosis, renal tubule epithelial cell swelling. Conclusion: Large dose of amanita exitialis can cause acute liver failure of Banna miniature pigs, which is in line with the pathophysiological characteristics of acute liver failure, and lays a foundation for further research on the toxic mechanism and detoxification drugs of amanita exitialis induced liver failure.

#4598 ADMINISTRATION OF ASTRAGALUS MEMBRANACEUS IMPROVES RENAL CONDITIONS AND SERUM CARNOSINE LEVELS IN AN ACUTE KIDNEY INJURY MODEL

Abstract Background and Aims Astragalus membranaceus (AM) exhibits various pharmacological effects against many diseases, including chronic kidney disease (CKD) (see http://nccih.nih.gov/health/astragalus). Acute kidney injury (AKI) is a sudden episode of kidney failure. AKI occurs frequently but is believed to heal completely and is likely an important factor causing CKD pathogenesis or progression. Previously, we demonstrated the lethal dose in old mice is 20 mg/kg cis-diamminedichloroplatinum (CDDP), and the dosage that will cause AKI in young mice is 14 mg/kg of CDDP. Low doses of CDDP caused AKI in old mice. However, the same dosage in young mice would manifest AKI that was subsequently completely reversible. On administration of AM, old mice (50 mg/kg) showed significantly improved AKI pathology, suggesting the therapeutic effect of AM is age-dependent with regard to AKI pathogenicity. Here, we further examined the possible effect of AM in preventing AKI and delaying CKD progression. Method Female C57BL/6 mice were separated into three groups based on age: young, 10 weeks; middle-aged, 35 weeks; and old, 52 weeks. The mice received oral administered of either AM powder mixed with sterilized 0.5% methylcellulose 400 (w/v) (AM group) or 0.5% methylcellulose 400 alone in the control group (CG). Four hours after most recent dose (twice a day at 20 mg/kg AM), 0.5 mg/ml CDDP (reduced dose: 14 mg/kg or 0.9% NaCl for the CG) was injected intraperitoneally. We measured blood serum urea nitrogen (BUN) and creatinine (CRE) levels, which are biomarkers of AKI. Additionally, we assessed histochemical changes in kidney sections stained with anti-CD3 and anti-CD68 antibodies after the mice were sacrificed on day x. Thereafter, serum carnosine levels in the samples collected 4 hours after AM administration (n = 6 per group) were determined using a carnosine ELISA kit (Novus, NBP2-75013). Data were analyzed statistically with PRISM software. Significance was considered P = 0.05. Results The 14 mg/kg CDDP dose in old mice significantly increased BUN and CRE levels and caused histological damage in renal tubule epithelial cells and glomeruli. However, these pathological changes were not observed in young and middle-aged mice receiving the same CDDP dose. Histochemical analysis of the old mice showed a significant increase in the number of CD3- and CD68-positive cells following AKI induction (Fig. 1). However, the increase of these inflammatory markers was clearly ameliorated by the AM pretreatment in the experimental group. Serum carnosine levels were markedly increased in young and middle-aged mice following AM administration (Fig. 2). Moreover, the serum carnosine concentration in old mice was upregulated at levels corresponding to those in young mice with AM administration. However, serum carnosine levels showed no additional increase after AM administration in old mice and remained stable. Our findings suggest that normal renal conditions show a simple upregulation of serum carnosine level after AM administration. Furthermore, AM administration improves renal conditions in old mice with high levels of CD-3 positive cells in particular (Fig. 1, arrow). Figure 2 shows the increased and then subsequent stabilizing of the serum carnosine levels (Fig. 2, arrow). Conclusion AM administration can upregulate serum carnosine in younger mice and shows at least some effectiveness in older mice, where serum carnosine is downregulated but concentrated. Serum carnosine might not be necessary for reducing the pathogenesis of AKI in younger mice, but it becomes important to prevent AKI in old mice. This indicates the effect of AM is probably age-dependent because AKI sequelae manifested spontaneously in all mice but were specifically ameliorated when administered to old mice as opposed to younger mice who recovered completely irrespective of AM administration.

Lead exposure induced developmental nephrotoxicity in Japanese quail (Coturnix japonica) via oxidative stress-based PI3K/AKT pathway inhibition and NF-κB pathway activation

Birds are sensitive to environmental pollution and lead (Pb) contamination could negatively affect nearly all avian organs and systems including kidney of excretive system. Thereby, we used a biological model species-Japanese quail (Coturnix japonica) to examine the nephrotoxic effects of Pb exposure and possible toxic mechanism of Pb on birds. Quail chicks of 7-day-old were exposed to 50 ppm Pb of low dose and high dose of 500 ppm and 1000 ppm Pb in drinking water for five weeks. The results showed that Pb exposure induced kidney weight increase while body weight and length reduction. The increase of uric acid (UA), creatinine (CREA) and cystatin c (Cys C) in the plasma suggested renal dysfunction. Moreover, both microstructural and ultrastructural changes demonstrated obvious kidney damages. In particular, renal tubule epithelial cells and glomeruli swelling indicated renal inflammation. Furthermore, changes in the content and activity of oxidative stress markers suggested that Pb caused excessive oxidative stress in the kidney. Pb exposure also induced abnormal apoptosis in the kidney. In addition, RNA sequencing (RNA-Seq) analysis revealed that Pb disturbed molecular pathways and signaling related with renal function. Especially, Pb exposure resulted in an increase in renal uric acid synthesis by disrupting purine metabolism. Pb caused apoptotic increment by inhibiting the phosphatidylinositol-3-kinase (PI3K)/RAC-alpha serine/threonine-protein kinase (AKT) pathway and induced aggravated inflammation by activating Nuclear Factor kappa B (NF-κB) signaling pathway. The study implied that Pb caused nephrotoxicity through structural damages, uric acid metabolism disorder, oxidation imbalance, apoptosis and inflammatory pathway activation.