HKC Cells Research Articles

ISL1-overexpressing BMSCs attenuate renal ischemia-reperfusion injury by suppressing apoptosis and oxidative stress through the paracrine action

Ischemia-reperfusion injury (IRI) is a major event in renal transplantation, leading to adverse outcomes. Bone marrow mesenchymal stem cells (BMSCs) are novel promising therapeutics for repairing kidney injuries. The therapeutic efficacy of BMSCs with ISL1 overexpression in renal IRI and its underlying mechanism need to be investigated. The unilateral renal IRI rat model was established to mimic clinical acute kidney injury. Rats were injected with PBS, BMSCs-Scrambled or BMSCs-ISL1 via the tail vein at the timepoint of reperfusion, and then sacrificed after 24 h of reperfusion. The administration of BMSCs-ISL1 significantly improved renal function, inhibited tubular cells apoptosis, inflammation, oxidative stress in rats. In vitro, HKC cells subjected to H2O2 stimulation were pretreated with the conditioned medium (CM) of BMSCs-Scrambled or BMSCs-ISL1. The pretreatment of ISL1-CM attenuated apoptosis and oxidative stress induced by H2O2 in HKC cells. Our proteomic data suggested that haptoglobin (Hp) was one of the secretory proteins in ISL1-CM. Subsequent experiments confirmed that Hp was the important paracrine factor from BMSCs-ISL1 that exerted anti-apoptotic and antioxidant functions. Mechanistically, Hp played a cytoprotective role via the inhibition of ERK signaling pathway, which could be abrogated by Ro 67-7476, the ERK phosphorylation agonist. The results suggested that paracrine action may be the main mechanism for BMSCs-ISL1 to exert protective effects. As an important anti-apoptotic and antioxidant factor in ISL1-CM, Hp may serve as a new therapeutic agent for treating IRI, providing new insights for overcoming the long-term adverse effects of stem cell therapy.

Curcumin inhibits PAT-induced renal ferroptosis via the p62/Keap1/Nrf2 signalling pathway

Patulin (PAT), the most common mycotoxin, is widespread in foods and beverages which poses a serious food safety issue to human health. Our previous research confirmed that exposure to PAT can lead to acute kidney injury (AKI). Curcumin is the most abundant active ingredient in turmeric rhizome with various biological activities. The aim of this study is to investigate whether curcumin can prevent the renal injury caused by PAT, and to explore potential mechanisms. In vivo, supplementation with curcumin attenuated PAT-induced ferroptosis. Mechanically, curcumin inhibited autophagy, led to the accumulation of p62 and its interaction with Keap1, promoted the nuclear translocation of nuclear factor E2 related factor 2 (Nrf2), and increased the expression of antioxidant stress factors in the process of ferroptosis. These results have also been confirmed in HKC cell experiments. Furthermore, knockdown of Nrf2 in HKC cells abrogated the protective effect of curcumin on ferroptosis. In conclusion, we confirmed that curcumin mitigated PAT-induced AKI by inhibiting ferroptosis via activation of the p62/Keap1/Nrf2 pathway. This study provides new potential targets and ideas for the prevention and treatment of PAT.

8-LB: Adipocyte-Derived ANGPTL2 Regulates Abnormal Lipid Metabolism and Inflammation through PirB in Diabetic Kidney Disease

Diabetic kidney disease (DKD) is a disorder of glycolipid metabolism caused by diabetes mellitus, which leads to hemodynamic and inflammatory processes. The aim of this study is to investigate the mechanism of adipocyte-derived angiopoietin-like protein 2 (ANGPTL2) in promoting the progression of DKD. Patients with DKD were included, and plasma ANGPTL2 was detected by ELISA. Type 2 diabetic mouse model induced by HFD and STZ, systemic Angptl2 knockout mouse model and adipose-specific AAV-Angptl2 were constructed. HKC cells stimulated with high glucose and PA in vitro. ELISA was used to measure the levels of IL-6, IL-1β, TNF-α and ANGPTL2 in plasma. RT-qPCR and Western blot were used to detect PirB, lipid metabolism indicators and inflammation, and the proteomics of kidney was performed. HE, oil red O staining and inflammatory factor staining were used to evaluate the degree of renal lipid metabolism and inflammation. The plasma level of ANGPTL2 in DKD patients is significantly higher than that in normal controls. Compared with the normal mice, the DKD mice had increased lipid accumulation and severe inflammation in kidney tissues, with increased PirB, Cybb and pro-inflammatory factors, and decreased CD36 and FATP2 in kidney. Similarly, overexpression of adipose-derived ANGPTL2 in mice caused the same abnormal lipid metabolism and increased inflammation levels, while Angptl2 ko mice significantly improved lipid deposition and inflammation levels and fatty acid transport. Knockdown of ANGPTL2 in HKC cells also inhibited the reduction of CD36 and FATP2, the increase of its receptor PirB, and the increase of Cybb /Nox2-mediated inflammatory cytokines induced by high glucose and high lipid in HKC cells. Adipose-derived ANGPTL2 may play an important role in lipid metabolism and Cybb/ NOX2-regulated inflammatory through its receptor in DKD, suggesting that ANGPTL2 may serve as a potential treatment for preventing the progression of DKD. Disclosure R. Yin: None. L. Yang: None. D. Zhao: None. Funding National Natural Science Foundation of China (82170454)

Klotho inhibits the formation of calcium oxalate stones by regulating the Keap1-Nrf2-ARE signaling pathway.

Oxidative damage is important in calcium oxalate (CaOx) stone development but occurs via multiple pathways. Studies have shown that klotho plays an essential role in ameliorating oxidative damage. This study aims to explore the role of klotho in CaOx stones and whether the underlying mechanism is related to the regulation of Keap1-Nrf2-ARE signaling. The levels of GSH, SOD, CAT, MDA, and ROS were examined by ELISA. The klotho, Bcl-2, caspase-3, Keap1, Nrf2, HO-1, and NQO1 mRNA levels were measured by qRT‒PCR, and their protein levels were detected by Western blotting. Renal tissue apoptosis was examined by TUNEL staining, and crystal cell adherence and apoptosis in HKC cells were assessed based on the Ca2+ concentrations and by flow cytometry. The renal pathological changes and the adhesion of CaOx crystals in the kidneys were examined by hematoxylin-eosin and von Kossa staining, respectively. We constructed a CaOx kidney stone model in vitro. By regulating the klotho gene, klotho overexpression inhibited the CaOx-induced promotion of crystal cell adherence and apoptosis in HKC cells, and these effects were reversed by klotho knockdown. Moreover, our in vivo assay demonstrated that klotho overexpression alleviated glyoxylate administration-induced renal oxidative damage, renal apoptosis, and crystal deposition in the kidneys of mice, and these effects were also associated with activation of the Keap1-Nrf2-ARE pathway. Klotho protein inhibits the oxidative stress response of HKC cells through the Keap1-Nrf2-ARE signaling pathway, reduces the apoptosis of and adhesion of CaOx crystals to HKC cells, and decreases the occurrence of CaOx kidney stones. 20220304.

Polystyrene nanoplastics induce profound metabolic shift in human cells as revealed by integrated proteomic and metabolomic analysis.

Nanoplastics (NPLs) are widespread in our environment. However, their impacts on human health and precise toxicity mechanisms remain poorly understood. Here we studied the internalization, release, and cytotoxicity of polystyrene nanoplastics (PSNPs) using the renal tubular epithelial cell line HKC and human derived liver cell line HL-7702. We also employed an integrated proteomic and metabolomic approach to investigate the potential biological effects of PSNPs on HKC cells. The abundances of 4770 proteins and 100 metabolites were quantified, with 785 proteins and 17 metabolites detected with altered levels in response to PSNPs. Most of the differential proteins and metabolites were enriched in a variety of metabolic pathways, for example, glycolysis, citrate cycle, oxidative phosphorylation, and amino acid metabolism, suggesting the potential effects of NPLs on global cellular metabolism shift in human cells. The altered energy metabolism induced by PSNPs was further confirmed by a Seahorse analysis. Moreover, lysosomal distribution study and western blotting showed that mTORC1 signaling, a central regulator of cellular metabolism, was inhibited upon nanoplastic exposure, likely serving as the link between lysosome dysfunction and metabolic defects. Taken together, our findings systematically mapped the key molecular changes induced by PSNPs in human cells and provide comprehensive biological insights for the risk estimation of NPLs contamination.

Decreased expression of miR-204-3p in peripheral blood and wound margin tissue associated with the onset and poor wound healing of diabetic foot ulcers.

To investigate the relationship between small non-coding RNA-204-3p (miR-204-3p) and the onset and wound healing of diabetic foot ulcers (DFU) and the underlying molecular mechanism, sixty four newly diagnosed patients with T2DM without DFU (T2DM group), 82 T2DM patients with DFU (DFU group), and 60 controls with normal glucose tolerance (NC group) were included. Quantitative real-time PCR (qRT-PCR) method was used to determine miR-204-3p expression levels in peripheral blood and wound margin tissue of subjects, and to analyse the relationship between the expression of miR-204-3p and wound healing. In vitro experiments were also performed to understand the effect of miR-204-3p on high glucose induced injury of HaCaT cells (human keratinocytes). The results showed that miR-204-3p expression level of peripheral blood in the T2DM group was marked lower than that in the NC group [2.38 (1.31-5.04) vs 3.27 (1.51-6.98)] (P < .05). Similarly, the miR-204-3p expression level of peripheral blood in the DFU group was significantly lower than the T2DM group [1.15 (0.78-2.89) vs 2.38 (1.31-5.04)] (P < .01). The expression level of miR-204-3p in peripheral blood and wound margin tissues of DFU patients was positively correlated with the healing rate of foot ulcers after 8 weeks (P < .05). Multifactorial logistic regression analysis showed that decreased expression of miR-204-3p in peripheral blood was an independent risk factor for DFU (OR=2.95, P < .05). The results of in vitro experiments showed that miR-204-3p could improve the proliferation and migration of HKC cells and reduce the proportion of apoptosis of HKC cells by targeted regulation of zinc finger protein Kruppel like factor 6 (KLF6) in high glucose environment. Therefore, the decreased expression of miR-204-3p in peripheral blood and wound tissue of T2DM patients is closely related to the occurrence and poor wound healing of DFU. The down-regulated expression of miR-204-3p can reduce its ability to antagonise the functional damage of keratinocytes induced by high-glucose conditions. These results will provide potential targets for the treatment of DFU.

A geniposide-phospholipid complex ameliorates posthyperuricemia chronic kidney disease induced by inflammatory reactions and oxidative stress

Hyperuricemia is a common metabolic disease and is one of the factors that could induce chronic kidney disease (CKD). Geniposide (GEN) is a typical natural iridoid glucoside compound with a series of biological activities, but the poor bioavailability of GEN limits its clinical application. In this context, the pharmacological activity of the geniposide-phospholipid complex (GEN-PLC) in ameliorating posthyperuricemia CKD was evaluated by in vitro and in vivo experiments in this study. In vitro cell experiments showed that GEN-PLC treatment markedly decreased inflammatory cytokine levels and reactive oxygen species levels compared with those of GEN in uric acid-treated HKC cells. In vivo research results confirmed that a high concentration of uric acid could cause CKD by increasing inflammatory cytokines and reactive oxygen species in hyperuricemic mice. At the same time, GEN-PLC could regulate the PI3K/AKT/NF-κB and Keap1/Nrf2/HO-1 signaling pathways to effectively inhibit the inflammatory response and oxidative stress, thereby ameliorating posthyperuricemia CKD, and the therapeutic effect was better than that of GEN. In addition, the preparation technology of GEN-PLC was optimized, and the physiochemical analysis explained the intermolecular interactions of the two components. Based on the research results, GEN-PLC could enhance the bioavailability of GEN and become a promising candidate for clinical drug development.

Transcriptomics-based analysis of co-exposure of cadmium (Cd) and 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) indicates mitochondrial dysfunction induces NLRP3 inflammasome and inflammatory cell death in renal tubular epithelial cells

Environmental pollution often releases multiple contaminants resulting in as yet largely uncharacterized additive toxicities. Cadmium (Cd) is a widespread pollutant that induces nephrotoxicity in animal models and humans. However, the combined effect of Cd in causing nephrotoxicity with 2,2',4,4'-tetrabromodiphenyl ether (BDE-47), a typical congener of polybrominated diphenyl ethers (PBDEs), has not been evaluated and mechanisms are not completely clear. Here, we applied transcriptome sequencing analysis to investigate the combined toxicity of Cd and BDE-47 in the renal tubular epithelial cell lines HKCs. Cd or BDE-47 exposure decreased cell viability in a dose-dependent manner, and exhibited cell swelling and rounding similar to necrosis, which was exacerbated by co-exposure. Transcriptomic analysis revealed 2191, 1331 and 3787 differentially-expressed genes following treatment with Cd, BDE-47 and co-exposure, respectively. Interestingly, functional annotation and enrichment analyses showed involvement of pathways for oxidative stress, NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome and inflammatory cell death for all three treatments. Examination of indices of mitochondrial function and oxidative stress in HKC cells showed that the levels of reactive oxygen species (ROS), malondialdehyde (MDA) and intracellular calcium ion concentration [Ca2+]i were elevated, while superoxide dismutase (SOD) and mitochondrial membrane potential (MMP) were decreased. The ratio of apoptotic and necrotic cells and intracellular lactate dehydrogenase (LDH) release were increased by Cd or BDE-47 exposure, and was aggravated by co-exposure, and was attenuated by ROS scavenger N-Acetyl-L-cysteine (NAC). NLRP3 inflammasome and pyroptosis pathway-related genes of NLRP3, adaptor molecule apoptosis-associated speck-like protein (ASC), caspase-1, interleukin-18 (IL-18) and IL-1β were elevated, while gasdermin D (GSDMD) was down-regulated, and protein levels of NLRP3, cleaved caspase-1 and cleaved GSDMD were increased, most of which were relieved by NAC. Our data demonstrate that exposure to Cd and BDE-47 induces mitochondrial dysfunction and triggers NLRP3 inflammasome and GSDMD-dependent pyroptosis leading to nephrotoxicity, and co-exposure exacerbates this effect, which could be attenuated by inhibiting ROS. This study provides a further mechanistic understanding of kidney damage, and co-exposure impact is worthy of concern and should be considered to improve the accuracy of environmental health assessment.

Patulin Induces Acute Kidney Injury in Mice through Autophagy-Ferroptosis Pathway.

Patulin (PAT) is a common mycotoxin, widely found in cereals, seafood, nuts, and especially in fruits and their products. Exposure to this mycotoxin has been reported to induce kidney injury. However, the possible mechanism remains unclear. In our study, short-term high-dose intake of PAT caused acute kidney injury (AKI) in mice. We performed high-throughput transcriptional sequencing to identify differentially expressed genes (DEGs) between the treatment and control groups. The ferroptosis signaling pathway had the highest enrichment, suggesting ferroptosis is involved in PAT-induced AKI. Further, the existence of ferroptosis and autophagy was confirmed by observing the changes of mitochondria morphology and the formation of autophagosomes by electron microscopy. And the expression of solute carrier family 7 member 11 (SLC7A11), glutathione peroxidase 4 (GPX4), p62, nuclear receptor coactivator 4 (NCOA4), and ferritin heavy chain 1 (FTH1) were downregulated, whereas acyl-CoA synthase long-chain family member 4 (ACSL4), transferrin (TF), LC3, and ferritin light chain (FTL) expression were upregulated in PAT-exposed mice. These results suggested autophagy-dependent ferroptosis occurred in the animal model. This view has also been confirmed in the human renal tubular epithelial cell (HKC) experiments. Autophagy inhibitor 3-methyladenine (3MA) attenuated PAT-induced ferroptosis and the iron contents in HKC cells. Simultaneous autophagy-dependent ferroptosis can be inhibited by ferroptosis inhibitors ferrostatin-1 (Fer-1) and desferrioxamine (DFO). In general, this study provides a new perspective for exploring the new mechanism of acute kidney injury caused by PAT.

Design, synthesis and bioactive evaluation of geniposide derivatives for antihyperuricemic and nephroprotective effects

Hyperuricemia is a principal factor mediating gout and kidney damage, and xanthine oxidase (XOD) is a key enzyme in the pathogenesis of hyperuricemia. In this context, a series of geniposide derivatives were designed and synthesized, and antihyperuricemic and nephroprotective effects of all derivatives was evaluated in vitro and in vivo. Compound 2e emerged as the most potent XOD inhibitor, with an IC50 value of 6.67 ± 0.46 µM. Simultaneously, cell viability, ROS generation, and SOD levels assay showed that compound 2e could repair the damage of HKC cells by inhibiting the oxidative stress response. The results of the study indicated compound 2e significantly decreased uric acid levels by inhibiting the XOD activity, and repaired kidney damage by inhibiting the expression of TLR4/TLR2/MyD88/NF-κB and NALP3/ASC/caspase-1 signaling pathways. Enzyme inhibition kinetics suggested that compound 2e functioned via reversible mixed competitive inhibition. Moreover, a molecular docking study was performed to gain insight into the binding mode of compound 2e with XOD. These results suggest that geniposide derivatives were potential to be developed into a novel medicine to reveal healthy benefits in natural prevention and reduction risk of hyperuricemia and kidney damage.

HSD17B7 gene in self‐renewal and oncogenicity of keratinocytes from Black versus White populations

Human populations of Black African ancestry have a relatively high risk of aggressive cancer types, including keratinocyte‐derived squamous cell carcinomas (SCCs). We show that primary keratinocytes (HKCs) from Black African (Black) versus White Caucasian (White) individuals have on average higher oncogenic and self‐renewal potential, which are inversely related to mitochondrial electron transfer chain activity and ATP and ROS production. HSD17B7 is the top‐ranked differentially expressed gene in HKCs and Head/Neck SCCs from individuals of Black African versus Caucasian ancestries, with several ancestry‐specific eQTLs linked to its expression. Mirroring the differences between Black and White HKCs, modulation of the gene, coding for an enzyme involved in sex steroid and cholesterol biosynthesis, determines HKC and SCC cell proliferation and oncogenicity as well as mitochondrial OXPHOS activity. Overall, the findings point to a targetable determinant of cancer susceptibility among different human populations, amenable to prevention and management of the disease.

FBXW7 mediates high glucose‑induced SREBP‑1 expression in renal tubular cells of diabetic nephropathy under PI3K/Akt pathway regulation.

Diabetic nephropathy (DN) is a severe complication of diabetes mellitus and lipid metabolism abnormality serves a key role in the pathogenesis of DN. Sterol regulatory element-binding protein 1 (SREBP-1) overexpression mediates aberrant lipid accumulation in renal tubular cells of DN. However, the exact mechanism involved in increased SREBP-1 has not been fully elucidated. The aim of the present study was to explore the mechanism involved in SREBP-1 upregulation. Diabetic mice and high glucose-cultured HKC cells were chosen to detect the expression of FBXW7 and SREBP-1 using immunohistochemistry, western blotting and PCR. The present study demonstrated that F-box and WD repeat domain containing 7 (FBXW7) expression was decreased in renal tubular cells of diabetic mice. Moreover, the co-expression of FBXW7 and SREBP-1 was observed in renal tubular cells, but not in the glomeruli. High glucose-induced the downregulation of FBXW7 expression in in vitro cultured HKC cells, which was accompanied by SREBP-1 upregulation. In addition, overexpression of FBXW7 in HKC cells led to SREBP-1 downregulation. By contrast, knockdown of FBXW7 caused SREBP-1 upregulation in HKC cells. It was found that the PI3K/Akt signaling pathway was activated in high glucose-stimulated HKC cells, and inhibition of PI3K/Akt pathway using LY294002 increased FBXW7 expression and decreased SREBP-1 expression. Taken together, the present results suggested that FBXW7 mediated high glucose-induced SREBP-1 expression in renal tubular cells of DN, under the regulation of the PI3K/Akt signaling pathway.

DNA methylation of Hugl-2 is a prognostic biomarker in kidney renal clear cell carcinoma.

It has been reported that loss of Hugl‐2 contributes to tumour formation and progression in vitro and in vivo. However, whether Hugl‐2 levels decrease during kidney renal clear cell carcinoma (KIRC) and the mechanism involved remain unknown. This study aimed to investigate whether DNA methylation of Hugl‐2 reduces its expression, leading to the progression and poor prognosis of KIRC. Hugl‐2 methylation and mRNA expression and KIRC clinicopathological data were extracted from The Cancer Genome Atlas (TCGA), and relationships among these factors were analyzed using UALCAN, MethHC, Wanderer and LinkedOmics web tools. We found that Hugl‐2 mRNA and protein levels were reduced in KIRC tissues. Moreover, Hugl‐2 mRNA levels were related to tumour grade and overall survival, and Hugl‐2 methylation was increased in KIRC. According to the results of methylation‐specific PCR, KIRC cells had higher Hugl‐2 DNA methylation levels than HKC cells. Moreover, Hugl‐2 DNA methylation correlated negatively with Hugl‐2 mRNA and was also related to the pathology and T stage of KIRC patients. KIRC patients with high Hugl‐2 DNA methylation also had shorter overall survival. Additionally, methylation of cg08827674, a Hugl‐2 probe, was related to pathologic stage, T stage, neoplasm histologic grade, serum calcium level without laterality, M stage, N stage, and ethnicity. Furthermore, treatment with the DNA methylation inhibitor decitabine resulted in upregulation of Hugl‐2 mRNA and protein levels in KIRC cell lines. These results indicate that Hugl‐2 DNA methylation may be both a prognostic marker and a therapeutic target in KIRC.

NF-κB Inhibitor Parthenolide Promotes Renal Tubules Albumin Uptake in Type 2 Diabetic Nephropathy.

Objective Injured tubular reabsorption is highlighted as one of the causes of increased albuminuria in the early stage of diabetic nephropathy; however, the underlying mechanism has not been fully elucidated. In this study, we aimed to explore whether reducing inflammation and remodeling the insulin signaling pathway could improve albumin uptake of renal tubules. Methods 8-week-old male db/db mice (n=8), a type 2 diabetic nephropathy model, administered with nuclear factor kappa-B (NF-κB) inhibitor parthenolide (PTN, 1 mg/kg) intraperitoneally every other day for 8 weeks, were as the treatment group. Meanwhile, the age-matched male db/m mice (n=5) and db/db mice (n=8) were treated with saline as the control group and type 2 diabetic nephropathy group. When the mice were sacrificed, blood and urine were collected to examine homeostasis model assessment of insulin resistance (HOMA-IR) and urine albumin creatinine ratio, and kidney samples were used to analyze histopathologic changes with periodic acid-Schiff (PAS) staining, NF-κB p65, phosphorylation of AKT (p-AKT), amnionless and cubilin expressions with immunohistochemistry as well as western blot, and the albumin uptake of renal tubules by using immunofluorescence. In addition, HKC cells were divided into the insulin group treated with insulin alone, the TNF-α group treated with insulin and tumor necrosis factor (TNF-α), and the TNF-α+PTN group exposed to PTN, insulin and TNF-α. The levels of albumin uptake and expression levels of NF-κB p65, p-IRS-1/IRS-1, p-AKT/AKT, amnionless and cubilin in HKC cells were measured. Results Compared with the db/db group, the db/db+PTN group demonstrated decreased levels of HOMA-IR (36.83±14.09 vs. 31.07±28.05) and urine albumin creatinine ratio (190.3±7.3 vs. 143.0±97.6 mg/mmol); however, the differences were not statistically significant (P>0.05). Periodic acid-Schiff staining showed PTN could alleviate the glomerular hypertrophy and reduce the matrix in mesangial areas of db/db mice. The renal expression of NF-κB p65 was increased and p-AKT (s473) decreased in the db/db group compared with the db/m group (P<0.05). PTN significantly reduced the renal expression of NF-κB p65 and ameliorated the decline of p-AKT (s473) compared with the db/db group (P<0.05). Compared with the db/m group, the expression of amnionless and cubilin decreased and albumin uptake in tubules were reduced in the db/db group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05), and improve albumin uptake in tubules. Insulin promoted albumin uptake and the expression of amnionless and cubilin in HKC cells (P<0.05). TNF-α stimulated the expression of NF-κB p65, increased p-IRS-1 (s307) and reduced p-AKT (s473) in HKC cells (P<0.05). In the TNF-α+PTN group, the expression of NF-κB p65 declined and p-IRS-1 (s307) and p-AKT (s473) were restored, compared with the TNF-α group (P<0.05). The expression of amnionless and cubilin decreased in the TNF-α group (P<0.05), and PTN could significantly increase the expression of cubilin (P<0.05). Conclusions Inflammation caused damage to insulin signaling, which reduced amnionless-cubilin expression and albumin uptake. PTN could reduce inflammation and remodel the impaired insulin signaling pathway, which promoted the expression of cubilin and albumin uptake. Our study can shed light on the role of inflammation in the reduction of albumin uptake of renal tubules in type 2 diabetic nephropathy.

An in vitro attempt at precision toxicology reveals the involvement of DNA methylation alteration in ochratoxin A-induced G0/G1 phase arrest

ABSTRACTPrecision toxicology evaluates the toxicity of certain substances by isolating a small group of cells with a typical phenotype of interest followed by a single cell sequencing-based analysis. In this in vitro attempt, ochratoxin A (OTA), a typical mycotoxin and food contaminant, is found to induce G0/G1 phase cell cycle arrest in human renal proximal tubular HKC cells at a concentration of 20 μM after a 24h-treatment. A small number of G0/G1 phase HKC cells are evaluated in both the presence and absence of OTA. These cells are sorted with a flow cytometer and subjected to mRNA and DNA methylation sequencing using Smart-Seq2 and single-cell reduced-representation bisulfite sequencing (scRRBS) technology, respectively. Integrated analysis of the transcriptome and methylome profiles reveals that OTA causes abnormal expression of the essential genes that regulate G1/S phase transition, act as signal transductors in G1 DNA damage checkpoints, and associate with the anaphase-promoting complex/cyclosome. The alteration of their DNA methylation status is a significant underlying epigenetic mechanism. Furthermore, Notch signaling and Ras/MAPK/CREB pathways are found to be suppressed by OTA. This attempt at precision toxicology paves the way for a deeper understanding of OTA toxicity and provides an innovative strategy to researchers in the toxicology and pharmacology field.

Effect of RNA interference of the expression of HMGA2 on the proliferation and invasion ability of ACHN renal cell carcinoma cells.

This aim of the present study was to observe the effect of high mobility group AT-hook 2 (HMGA2) on the proliferation and invasion ability of ACHN renal cell carcinoma (RCC) cells. Human ACHN cells, an RCC cell line, and HKC normal human renal tubular epithelial cells were cultured. HMGA2 small interfering (si)RNA, Mock-siRNA and their negative control group were designed and synthesized. Subsequently, the ACHN cells were transiently transfected using RNA interference technology. Finally, the mRNA and protein expression levels of HMGA2 were detected using reverse transcription-polymerase chain reaction and western blot analyses. The proliferation ability of the ACHN cells was determined using MTT, and ACHN cell invasion ability was detected using the Transwell method. The results showed that the mRNA and protein expression levels of HMGA2 in the ACHN cells were considerably higher, compared with those in the HKC cells (P<0.01). The RCC cells, in which the expression of HMGA2 was specifically silenced, was successfully constructed. The proliferation rate of cells in the HMGA2-siRNA group was significantly lower, compared with that of cells in the Mock-siRNA group and control group at 24, 48, 72 and 96 h post-transfection (P<0.05). The invasion ability of cells in the HMGA2-siRNA group was significantly lower, compared with that of cells in the Mock-siRNA group and control group (P<0.05) 48 h following transfection. Therefore, the HMGA2 gene may function as an oncogene in the occurrence and development of RCC, and provide specific targets for the targeted therapy of RCC. Further detailed investigations of the HMGA2 gene are important for future gene therapy of RCC.

Crystal adhesion and internalization of sub-micron COM and COD crystals on Human kidney proximal tubular epithelial cells

[Objective] This study aims to compare the internalization of sub-micron calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals in human kidney proximal tubular epi-thelial cells (HKC) so as to explore the mechanism of renal epithelial cell injury caused by urine calcium oxalate crystals. [Methods] Cell viability, malondialdehyde (MDA) content, and propidium iodide (PI) staining were determined to examine cell injury during adhesion. The internalization of COM and COD crystals to HKC cells was observed through scanning electron microscopy. [Results] The injury effect of COM was stronger than that of COD on cells under the same conditions. Both of them injured HKC in a dose-dependent manner. The ability of COM to adhere to the injured HKC cells was higher than that of COD crystals. Compared to the COD group, the crystal adhered on cell surface appeared obvious aggregation in COM treatment group. [Conclusions] Cytotoxicity of sub-micron COM was greater than sub-micron COD. The attached COM crystals aggregated on cell surface. HKC cells swallow COD crystals more easily than COM. These findings provide further insights into kidney stone formation.

Toxicity of sub-micron and micron calcium oxalate monohydrate on renal epithelial cells

[Objective] This study aims to synthesize calcium oxalate monohydrate (COM) crystals with a size of 300 nm and 5 μm, respectively, and to compare their damage and adhesion ability toward human kidney proximal tubule epithelial cells (HKC). [Method] The injury degree of HKC caused by COM crystals was measured by detecting the cell viability, lactate dehydrogenase (LDH) release amounts, reactive oxygen species (ROS), cell death rate, hyaluronic acid (HA) expression and the crystal adhesion amount on cell surface. [Result] HKC cell viability, LDH release quantity, cell death rate and HA expression were closely associated with the size of COM crystals. The damage ability of 300 nm COM crystals on normal cells was higher than that of COM-5 μm crystals. The adhesive amount of small size COM crystals (62.4 μg/cm2) was higher than micron COM crystals (26.7 μg/cm2). The increased adhesion amount of small size COM crystal are related to its larger specific surface area, crystal face changes and larger cell contact probability. [Conclusions] The cytotoxicity of COM crystals toward HKC cells is size dependent. This study is helpful to further clarify the formation mechanism of COM calculi, and provide inspiration for its prevention and treatment.

IGF-1 may predict the severity and outcome of patients with sepsis and be associated with microRNA-1 level changes.

IGF-1 functions as an anti-oxidative stress molecule and some critical patients with sepsis have a lower level of serum IGF-1. However, the association between IGF-1 and the severity or prognosis of sepsis remains unclear. This study aimed to elucidate the relationship between serum IGF-1 levels and the severity and prognosis of sepsis, and the possible mechanism was analyzed. Clinical characteristics of patients with sepsis were recorded and analyzed. Serum IGF-1 levels and micro (mi)RNA-1 levels were tested using radioimmunoassay and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, respectively. The A549 cell line and HKC cell line were cultured in vitro and exposed to H2O2 with or without IGF-1 treatment. Cell death was detected by analyzing cell death markers via ELISA kits, and miRNA-1 levels were detected after H2O2 exposure using RT-qPCR analysis. miRNA-1 in cells was upregulated by transfection and IGF-1 mRNA was detected to determine its relationship with miRNA-1. Once again, cell ELISA kits were used to analyze cell death markers after transfection. Serum IGF-1 levels were reduced in patients with sepsis, whereas miRNA-1 levels were higher (P<0.05 vs. healthy control). Patients in the septic shock subgroup or dead patients had the lowest IGF-1 levels and the highest miRNA-1 levels (P<0.05 vs. sepsis and severe sepsis). IGF-1 levels were inversely proportional to the miRNA-1 level. In vitro, IGF-1 reduced the cell death caused by H2O2. miRNA-1 transfection effectively increased the sensitivity of cells to H2O2 damage by reducing the expression of IGF-1, which was able to prevent cells from injury caused by H2O2. The transfection of negative control miRNA did not influence the level of IGF-1 miRNA and the sensitivity to H2O2 damage. In conclusion, low IGF-1 levels in patients with sepsis may predict increased severity of the condition and poor prognosis. The possible mechanism is that the excessive miRNA-1 levels reduce IGF-1 levels, resulting in insufficient anti-oxidative action by IGF-1 which increases the injury caused by oxidative stress in patients with sepsis.

MiR-330 inhibits IL-22-induced keratinocyte proliferation through targeting CTNNB1

Psoriasis is a common chronic inflammatory skin disease which is characterized by hyperproliferation and aberrant differentiation of keratinocytes; however the exact pathogenesis is largely unknown. Interleukin-22 (IL-22) has demonstrated its vital role in T cell-mediated immune response by interacting with keratinocytes in the pathogenesis of psoriasis. The microRNAs (miRNAs) are a class of small non-coding RNA molecules that play important roles in cellular processes by regulating gene expression at the post-transcriptional level. MiR-330 has been reported to inhibit the proliferation and migration of mouse keratinocytes. In the present study, we indicated that miR-330 expression in lesion tissue of psoriasis patients was specifically down-regulated, and could inhibit IL-22-induced proliferation of HaCaT and HKC cell. Wnt/β-catenin pathway plays an essential role in the pathogenesis of psoriasis. By direct targeting CTNNB1, miR-330 could significantly downregulate IL-22-induced CTNNB1 expression. In addition, we found that the downstream targets of β-catenin, CyclinD1 and Axin2, could be affected by miR-330; miR-330 could suppress CyclinD1 protein expression and rescue Axin2 protein expression. Taken together, we indicated miR-330 inhibits IL-22-induced proliferation of HaCaT and HKC cell by targeting CTNNB1 and subsequently affect the downstream factors, CyclinD1 and Axin2 for the first time, and provide diagnostic markers and a novel target for psoriasis treatment.