HK-2 Cells Research Articles

Kidney Injury Evoked by Fine Particulate Matter: Risk Factor, Causation, Mechanism and Intervention Study.

Fine particulate matter (PM2.5) is suggested to pose a severe risk to the kidneys by inducing functional degradation and chronic kidney diseases (CKD). This study aims to explore the nephrotoxicity of PM2.5 exposure and the underlying mechanism. Herein, based on the UK Biobank, it is found that per interquartile range (IQR) increase in PM2.5 is associated with a 6% (95% CI: 1%-11%), 7% (95% CI: 3%-11%), 9% (95% CI: 4%-13%), 11% (95% CI: 9%-13%), and 10% (95% CI: 8%-12%) increase in the risk of nephritis, hydronephrosis, kidney stone, acute renal failure, and CKD, respectively. In experimental study, noticeable kidney injury, which is the initiation of kidney diseases, is observed with PM2.5 exposure in C57BL/6N mice (n = 8), accompanied with oxidative stress, autophagy and pyroptosis. In vitro, HK-2 cells with PM2.5-stimulation exhibit tubulopathy, increased reactive oxygen species (ROS) generation and activated pyroptosis and autophagy. All changes are abolished by ROS scavenger of N-acetyl-L-cysteine (NAC) both in vivo and in vitro. In conclusion, the study provides evidence showing that PM2.5 exposure is associated with 5 kinds of kidney diseases by directly inducing nephrotoxicity, in which ROS may be the potential target by triggering autophagy and pyroptosis.

LINCRNA01094 Promotes Renal Interstitial Fibrosis via the Mir-513b-5p/MELK/Smad3 Axis.

Chronic Kidney Disease (CKD) is a common chronic disease that is a threat to human health. Accumulating evidence showed that long noncoding RNAs (lncRNAs) are associated with various diseases and can function as competing endogenous RNAs (ceRNAs). However, the roles and functions of the lncRNA‒miRNA-mRNA network in CKD are still unclear. In this study, we performed differential expression analysis of lncRNAs, miRNAs, and mRNAs in CKD using the datasets GSE66494 and GSE80247 from the Gene Expression Omnibus. A total of 33 lncRNAs, 20 miRNAs, and 240 mRNAs were differentially expressed between CKD patients and healthy controls. Two ceRNA interaction modules composed of 11 hub nodes, namely, 2 lncRNAs (LINC01086, LINC01094), 2 miRNAs (hsa-miR-197-3p, hsamiR- 513b-5p) and 7 mRNAs (CENPF, TOP2A, ARHGAP11A, CEP55, MELK, DTL, and ANLN) were constructed. In vitro knockdown of LINC01094 expression in renal tubular epithelial HK2 cells significantly attenuated the phenotype of TGFβ1-induced cell fibrosis. The results of RNA immunoprecipitation (RIP) experiments and dual-luciferase reporter experiments based on constructed mutants confirmed that LINC01094 could mediate MELK expression by sponging miR-513b-5p. Our observations indicated that lowering the expression of LINC01094 can significantly attenuate the TGFβ1-induced fibrosis phenotype in HK2 cells and renal inflammation through the miR-513b-5p/MELK/Smad3 signalling axis.

Factors influencing the bioactivity of natural matrices: The case of osmolarity-dependent modulation of cell viability by different dilutions of camel urines

The widespread practice of dromedary urinotherapy as a remedy for various illnesses, including cancer, is well-established in traditional dromedary countries. Researchers attempted to demonstrate anticancer properties of camel urine through in vitro experiments with debated outcomes. Notably, two critical aspects remained unexplored in those assays: (i) the osmolarity of tested urines, which can significantly influence in vitro results; (ii) the potential morphological changes of cells, following exposure to camel urines. In this study, we addressed these gaps by evaluating the osmolarity-dependent modulation of cell viability in human renal cell lines. In this regard, we assessed the impact of hyperosmolar mannitol-based solutions and dromedary urine on the viability and morphology of human non-tumor (HK2) and tumor renal cells (Caki-1). The results indicate that cell viability or morphology in both HK2 and Caki-1 cells are not significantly affected only if mannitol-induced hyperosmolarity is lower than 500 mOsm/L. Notably, when exposed to urine solution, diluted to <500 mOsm/L, statistically significant antiproliferative effects were observed primarily in Caki-1 cells (in presence of two out of ten tested urine samples). Conversely, alterations in cell morphology were observed exclusively in HK2 cells when exposed to the same diluted camel urines. In order to investigate, at molecular level, the observed antiproliferative effects, a preliminary metabolomics analysis of the tested urine samples was performed to identify potential bioactive compounds. The Nuclear Magnetic Resonance (NMR) metabolic profiling revealed the presence of three antioxidant compounds, namely trigonelline, pyruvic acid and N-acetylglucosamine. In conclusion, our results highlight the importance of considering the critical role of osmolarity when evaluating the bioactive properties of camel urine in vitro, which should not be used to treat any illness as it is. Conversely, it can be considered the possibility to use camel urines as a source of bioactive compounds.

The JNK-associated leucine zipper protein exerts a protective effect on renal parenchymal injury by limiting the inflammatory secretome in tubular cells

JNK-associated leucine zipper protein (JLP) is a newly identified renal endogenous anti-fibrotic factor that is selectively enriched in renal tubular epithelial cells (TECs). The loss of JLP by TECs is a landmark event that heralds the progression of kidney fibrosis. JLP deficiency ensues a series of pathogenetic cellular processes that are conducive to fibrotic injury. Inflammatory injury is functionally relevant in fibrotic kidneys, and TECs play an essential role in fueling inflammation through aberrant secretions. It is speculated that the loss of JLP in TECs is associated with the relentless inflammation during the development of kidney fibrosis. This study examined the alteration of a panel of inflammatory signatures in TECs under kidney fibrotic circumstances using a Jlp gene-modified unilateral ureteral obstruction (UUO) mouse model or cultured HK-2 cells. It was found that a deficiency of JLP in TECs led to a significant increase in the secretion of inflammatory cytokines including interleukin-1β (IL-1β), tumor necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), overactivation of the nuclear factor (NF)-κB/c-Jun N-terminal kinase (JNK) pathway, as well as nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome-mediated pyroptosis in response to pro-fibrotic damage. Additionally, the absence of JLP resulted in enhanced macrophage migration and fibroblast activation as paracrine effects elicited by injured TECs. In conclusion, the loss of JLP in TECs catalyses inflammatory injuries in the development of kidney fibrosis.

Clinical significance of miR-625-5p in patients with sepsis-induced acute kidney injury based on bioinformatics analysis.

Sepsis often leads to a cluster of life-threatening symptoms affecting multiple organ systems. Among the organs most vulnerable to damage in this state is the kidney; those afflicted with severe sepsis frequently encounter acute kidney injury (AKI). The study was to investigate the diagnostic role of miR-625-5p in sepsis and sepsis-induced acute kidney injury (SI-AKI) and predict the possible pathways of miR-625-5p involved in SI-AKI by bioinformatics method. RT-qPCR was used to detect the level of miR-625-5p, and the diagnostic value of miR-625-5p was analyzedusing ROC curve. The proliferation and the concentration of inflammatory factors of HK-2 cells induced by LPS were detected by CCK-8 and ELISA. The pathway of miR-625-5p involved in SI-AKI was analyzed by bioinformatics method. The miR-625-5p expression was downregulated in sepsis as well as SI-AKI and has predictive value for sepsis as well as SI-AKI. In addition, miR-625-5p promoted LPS-induced cell proliferation and inhibited the levels of inflammatory cytokines. miR-625-5p may be a diagnostic biomarker for SI-AKI.

4-Octyl itaconate attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis

Objectives: The aim of this study was to investigate the mechanism of itaconate’s potential effect in diabetic kidney disease. Methods: Renal immune responsive gene 1 (IRG1) levels were measured in db/db mice and streptozotocin (STZ) + high-fat diet (HFD)-induced diabetic mice. Irg1 knockout mice were generated. db/db mice were treated with 4-octyl itaconate (4-OI, 50 mg/kg), a derivative of itaconate, for 4 weeks. Renal function and morphological changes were investigated. Ultrastructural alterations were determined by transmission electron microscopy. Results: Renal IRG1 levels were reduced in two diabetic models. STZ+HFD-treated Irg1 knockout mice exhibited aggravated renal tubular injury and worsened renal function. Treatment with 4-OI lowered urinary albumin-to-creatinine ratio and blood urea nitrogen levels, and restored renal histological changes in db/db mice. It improved mitochondrial damage, increased expressions of peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α) and mitochondrial transcription factor A (TFAM) in the renal cortex of db/db mice. These were confirmed in vitro; 4-OI improved high glucose-induced abnormal mitochondrial morphology and TFAM expression in HK-2 cells, effects that were inhibited by PGC-1α silencing. Moreover, 4-OI reduced the number of apoptotic cells in the renal cortex of db/db mice. Further study showed that 4-OI increased renal Nrf2 expression and decreased oxidative stress levels in db/db mice. In HK-2 cells, 4-OI decreased high glucose-induced mitochondrial ROS production, which was reversed by Nrf2 silencing. Nrf2 depletion also inhibited 4-OI-mediated regulation of PGC-1α, TFAM, and mitochondrial apoptotic protein expressions. Conclusions: 4-OI attenuates renal tubular injury in db/db mice by activating Nrf2 and promoting PGC-1α-mediated mitochondrial biogenesis.

Circ_001653 alleviates sepsis associated-acute kidney injury by recruiting BUD13 to regulate KEAP1/NRF2/HO-1 signaling pathway

BackgroundThe kidney is exceptionally vulnerable during sepsis, often resulting in sepsis-associated acute kidney injury (SA-AKI), a condition that not only escalates morbidity but also significantly raises sepsis-related mortality rates. Circular RNA circ_001653 has been previously reported to be upregulated in the serum of SA-AKI patients, while the role and underlying mechanism of circ_001653 in SA-AKI remains unknown. In this study, we aimed to explore the functional role and the molecular mechanism of circ_001653 in the pathogenesis of SA-AKI.MethodsLPS-stimulated HK-2 cells and ligation and perforation of cecum (CLP)-induced rats were used as in vitro and in vivo models of SA-AKI. The target gene expression levels were measured using qRT-PCR and western blot. Renal function (BUN, sCr, uNGAL, and uKIM-1), and renal pathological changes were detected in septic mice. TUNEL and EdU assays were conducted to measure apoptosis and proliferation rates in vitro. DCFH-DA staining was used to detect ROS levels in vitro and in vivo. Oxidative stress markers (SOD, GSH-Px, MDA, and SOD), and inflammation markers (IL-1β, IL-6, and TNF-α) were determined using commercial kits both in vitro and in vivo. Additionally, gain-and-loss-of-function assays and mechanistic experiments were conducted to explore the regulatory role of circ_001653 in SA-AKI pathogenesis.ResultsData showed that circ_001653 expression was high in LPS-stimulated HK-2 cells and CLP-induced rat renal tissue and was mainly localized in the cytoplasm. Notably, circ_001653 silencing alleviated SA-AKI by reducing apoptosis and alleviating oxidative stress and inflammation in HK-2 cells and renal tissue of rats. Mechanistically, it was found that circ_001653 alleviated SA-AKI by recruiting BUD13 to activate the KEAP1/Nrf2/HO-1 signaling pathway.ConclusionsTo summarize, our study is the first to reveal elevated expression of circ_001653 in sepsis-associated AKI, and its downregulation effectively attenuates AKI by reducing apoptosis, inflammation, and oxidative stress. Mechanistically, circ_001653 exerts its effects by recruiting BUD13 to activate the KEAP1/Nrf2/HO-1 signaling pathway. These findings suggest circ_001653 as a potential therapeutic target for the drug development of sepsis-associated AKI.

Molecular mechanism of ALKBH5-mediated m6A demethylation regulating lipopolysaccharide-induced epithelial-mesenchymal transition in sepsis-induced acute kidney injury.

This study explored the mechanism by which the m6A demethylase ALKBH5 mediates epithelial-mesenchymal transition (EMT) in sepsis-associated acute kidney injury (SA-AKI) and AKI-chronic kidney disease (CKD) transition. HK-2 cells were stimulated with lipopolysaccharide (LPS) to establish an in vitro model of SA-AKI. ALKBH5 expression was reduced through the transfection of si-ALKBH5. Cell viability, apoptosis, and migration were detected by CCK-8 assay, TUNEL staining, and Transwell. The levels of TNF-α, IL-1β, and IL-6 were measured by enzyme-linked immunosorbent assay. Quantitative real-time polymerase chain reaction or Western blotting was performed to determine the expressionsof ALKBH5, miR-205-5p, DDX5, E-cadherin, and α-SMA. The m6A level was quantitatively analyzed. The expression of pri-miR-205 bound to DGCR8 and m6A-modified pri-miR-205 after intervention with ALKBH5 expression was detected by RNA immunoprecipitation. A dual-luciferase assay confirmed the binding between miR-205-5p and DDX5. ALKBH5 was highly expressed in LPS-induced HK-2 cells. Inhibition of ALKBH5 increased cell viability, repressed apoptosis, and reduced EMT. Inhibition of ALKBH5 increased the m6A modification level, thereby promoting DGCR8 binding to pri-miR-205 to increase miR-205-5p expression and eventually targeting DDX5 expression. Low expression of miR-205-5p or overexpression of DDX5 partially abolished the inhibitory effect of ALKBH5 silencing on EMT. In conclusion, ALKBH5 represses miR-205-5p expression by removing m6A modification to upregulate DDX5 expression, thereby promoting EMT and AKI-CKD transition after SA-AKI.

Lanthanum chloride exerts therapeutic potential for chronic kidney disease by suppressing nanohydroxyapatite-induced mitophagy and mitochondria-mediated apoptosis

ObjectiveTo investigate the protective effect of lanthanum chloride on kidney injury in chronic kidney disease and its mechanism. Methods1. Patients with CKD stage 2–5 were selected to analyze the effect of lanthanum-containing preparations on CKD. 2. Sixty healthy male Wistar rats were randomly divided into control group, model group, lanthanum chloride groups (0.03 ng/kg, 0.1 ng/kg, 0.3 ng/kg, q.3d., i.v.), and lanthanum carbonate group (0.3 g/kg, q.d., p.o.). The model group was given 2 % adenine suspension (200 mg/kg, q.d., p.o.) for the first two weeks, followed by adenine (200 mg/kg, b.i.d., p.o.) for 2 weeks, and all animals were sacrificed after eight weeks of administration. 3. The serum and kidneys of rats in each group were collected to detect the oxidative stress indicators and the expressions of LC3B-Ⅱ/Ⅰ, p62, Bcl-2, Bax, Caspase-3 and Cleaved Caspase-3. 4. Human renal tubular epithelial cells (HK-2 cells) were divided into control group, model group, lanthanum chloride group, pyrophosphate (PPI) group, chloroquine (CQ) group, rapamycin group, doxorubicin (DOX) group and N-acetyl-L-cysteine (NAC) group. The mitochondrial status, mitophagy and apoptosis levels were detected. Results1.Lanthanum-containing preparations can significantly reduce the biochemical indexes of kidney injury in patients with CKD. 2. In the model group, the glomerular and renal tubular edema, the mitochondria were short and round, and the expression of LC3B-Ⅱ/Ⅰ and Bax increased, while the expression of P62, Bcl-2 and Caspase-3 decreased, and there was a significant improvement in the administration group, especially the 0.1 ng/kg group and lanthanum carbonate group. 3. In the HK-2 cell model group, mitochondrial membrane potential decreased, morphology changed and the results were reversed by lanthanum chloride. ConclusionLanthanum chloride may alter the morphology of nano-hydroxyapatite, thereby inhibiting its induced mitophagy and mitochondria-mediated apoptosis, and ultimately improve CKD renal injury effectively.

Effects and mechanisms of Polygonati Rhizoma polysaccharide on potassium oxonate and hypoxanthine-induced hyperuricemia in mice

Hyperuricemia, a prevalent metabolic disturbance intricately linked to gout and chronic kidney disease (CKD), may be relieved by traditional Chinese medicine Polygonati Rhizoma. It is derived from the rhizomes of Polygonatum sibiricum, Polygonatum kingianum, and Polygonatum cyrtonema, which are rich in polysaccharides and are effective hyperuricemia alleviators. This study investigated the potential of Polygonatum sibiricum polysaccharide (PSP) in managing hyperuricemia. PSP (125, 250, and 500 mg/kg, i.g.) or allopurinol was administered to hyperuricemia mice treated with potassium oxonate and hypoxanthine for two weeks. PSP effectively decreased serum uric acid levels by inhibiting xanthine oxidase and adenosine deaminase activity and expression in the liver and modulating uric acid-related transporters (URAT1, OAT1, and OAT3) in the kidney. PSP lowered serum creatinine and blood urea nitrogen levels, alleviating hyperuricemia-induced renal tubular epithelial-mesenchymal fibrosis. In vitro, PSP promoted mitochondrial biogenesis via the PGC-1α/NRF1/TFAM pathway, suppressed reactive oxygen species production, and prevented cytochrome C and dynamin-related protein 1 dysregulation in HK-2 cells. Furthermore, PSPA (Mw 4.0 kDa) and PSPB (Mw 112.2 kDa) isolated from PSP exhibit different uric acid-lowering mechanisms. In conclusion, our findings highlight the therapeutic potential of PSP and its nephroprotective effects in hyperuricemia, thereby supporting its development as a therapeutic agent for hyperuricemia.

Telomerase reverse transcriptase protects against diabetic kidney disease by promoting AMPK/PGC-1a-regulated mitochondrial energy homeostasis

Disordered glucose and lipid metabolism, coupled with disturbed mitochondrial bioenergetics, are pivotal in the initiation and development of diabetic kidney disease (DKD). While the essential role of telomerase reverse transcriptase (TERT) in regulating mitochondrial function in the cardiovascular system has been recognized, its specific function in maintaining mitochondrial homeostasis in DKD remains unclear. This study aimed to explore how TERT regulates mitochondrial function and the underlying mechanisms. In vitro, human renal proximal tubular HK-2 cells exposed to high glucose/high fat (HG/HF) presented significant downregulation of TERT and AMPK dephosphorylation. This led to decreased ATP production, altered NAD+/NADH ratios, reduced mitochondrial complex activities, increased mitochondrial dysfunction, lipid accumulation, and reactive oxygen species (ROS) production. Knockdown of TERT (si-TERT) further exacerbated mitochondrial dysfunction, decreased mitochondrial membrane potential, and lowered levels of cellular oxidative phosphorylation and glycolysis, as determined via a Seahorse X24 flux analyzer. Conversely, mitochondrial dysfunction was significantly alleviated after pcDNA-TERT plasmid transfection and adeno-associated virus (AAV) 9-TERT gene therapy in vivo. Notably, treatment with an AMPK inhibitor, activator, and si-PGC-1a (peroxisome proliferator-activated receptor γ coactivator-1α), resulted in mitochondrial dysfunction and decreased expression of genes related to energy metabolism and mitochondrial biogenesis. Our findings reveal that TERT protects mitochondrial function and homeostasis by partially activating the AMPK/PGC-1a signaling pathway. These results establish a crucial foundation for understanding TERT's critical role inmitochondrial regulation and its protective effect on DKD.

Trans-cinnamic acid alleviates high-fat diet-induced renal injury via JNK/ERK/P38 MAPK pathway

Obesity-related chronic kidney disease (CKD) poses a significant risk to individuals' health and wellbeing, but the pathological mechanisms and treatment strategies are currently limited. Trans-cinnamic acid (CA) is a key active monomer found in cinnamon bark and is known for its diverse pharmacological activities. However, its effect on obesity-related renal injury remains unknown. In the current study, the in vitro and in vivo experiments were combined to investigate the beneficial effect of CA on renal injury induced by HFD or PA. We found that CA significantly reduced the obesity of zebrafish body and the accumulation of fat in kidney tissues. The histopathological changes and dysfunction induced by HFD were effectively mitigated by CA administration, as evidenced by the detection of Hematoxylin-Eosin straining, NAG activity, creatinine level, and expression of functional-related genes, respectively. Additionally, the in vitro and in vivo findings demonstrated that CA dramatically reduced the oxidative stress, inflammatory, and apoptosis in HFD-induced kidney tissues or PA-treated HEK293T and HK-2 cells. Finally, the results regarding ERK, JNK, and P38 proteins phosphorylation confirmed that CA may alleviate HFD-induced renal injury by inhibiting the phosphorylation of ERK, JNK, and P38 MAPK proteins. This theory was further supported by the results of co-treatment with anisomycin (a JNK activator) or lipopolysaccharide and CA in HEK293T cells. This study proves that CA alleviates the obesity-related CKD probably through inhibition of MAPK signaling pathway.

DMDD, isolated from Averrhoa carambola L., ameliorates diabetic nephropathy by regulating endoplasmic reticulum stress-autophagy crosstalk

BackgroundStudies have shown that Averrhoa carambola L. possesses therapeutic potential for diabetes and related complications. However, the specific beneficial effects and molecular mechanisms of 2-dodecyl-6-meth-oxycyclohexa-2,5-diene-1,4-dione (DMDD) isolated from Averrhoa carambola L. on diabetic nephropathy (DN) require further investigation.Methods80 C57BL/6 J male mice were subjected to a 1-week adaptive feeding, followed by a high-fat diet and intraperitoneal injection of 100 mg/kg streptozotocin (STZ) to construct an in vivo DN model. Additionally, human renal proximal tubular epithelial cells (HK-2) induced by high glucose (HG) were used as an in vitro DN model. The expression levels of epithelial-mesenchymal transition (EMT), endoplasmic reticulum stress (ERS), and autophagy-related proteins in renal tubular cells were detected by Western Blot, flow cytometry, immunofluorescence, and enzyme-linked immunosorbent assay (ELISA) staining. Transcriptome analysis revealed was conducted to elucidate the specific mechanism of by which DMDD mitigates DN by inhibiting ERS and autophagy. HK-2 cells were transfected with IRE1α overexpression lentivirus to reveal the role of IRE1α overexpression in HG-induced HK-2.ResultsThe experimental data showed that DMDD significantly reduced blood glucose levels and improved renal pathological alterations in DN mice. Additionally, DMDD inhibited the calcium (Ca2+) pathway, manifested by decreased autophagosome formation and downregulation of LC3II/I, Beclin-1, and ATG5 expression. Moreover, in HG-induced HK-2 cells, DMDD suppressed the overexpression of GRP78, CHOP, LC3II/I, Beclin1, and ATG5. Notably, IRE1α overexpression significantly increased autophagy incidence; however, DMDD treatment subsequently reduced the expression of LC3II/I, Beclin1, and ATG5.ConclusionDMDD effectively inhibits excessive ERS and autophagy, thereby reducing renal cell apoptosis through the IRE1α pathway and Ca 2+ pathway.

Identification of mitochondria-related genes as diagnostic biomarkers for diabetic nephropathy and their correlation with immune infiltration: New insights from bioinformatics analysis

BackgroundDiabetic nephropathy (DN) is a common and severe microvascular complication of diabetes. Mitochondrial dysfunction and immune inflammation are important factors in the pathogenesis of DN. However, the specific mechanisms and their intricate interactions in DN remain unclear. Besides, there are no effective specific predictive or diagnostic biomarkers for DN so far. Therefore, this study aims to elucidate the role of mitochondrial-related genes and their possibility as predictive or diagnostic biomarkers, as well as their crosstalk with immune infiltration in the progression of DN. MethodsBased on the GEO database and limma R package, the differentially expressed genes (DEGs) of DN were identified. Mitochondrial-related DEGs (MitoDEGs) were then obtained by intersecting these DEGs with mitochondria-related genes from the MitoCarta 3.0 database. Subsequently, the candidate hub genes were further screened by gene co-expression network analysis (WGCNA), and verified mRNA levels of these genes by real-time quantitative PCR (qRT-PCR) in high-glucose-treated human proximal tubular (HK-2) cells. The verified hub genes were utilized to construct a combined diagnostic model for DN, with its diagnostic efficacy assessed across the GSE30122 and GSE96804 datasets. Additionally, the immune infiltration pattern in DN was assessed with the CIBERSORT algorithm, and the Nephroseq v5 database was used to analyze the correlation between hub genes and clinical features of DN. ResultsSeven mitochondria-related candidate hub genes were screened from 56 MitoDEGs. Subsequently, the expression levels of six of them, namely EFHD1, CASP3, AASS, MPC1, NT5DC2, and BCL2A1, exhibited significant inter-group differences in the HK-2 cell model. The diagnostic model based on the six genes demonstrated good diagnostic efficacy in both training and validation sets. Furthermore, correlation analysis indicated that EFHD1 and AASS, downregulated in DN, are positively correlated with eGFR and negatively with serum creatinine. Conversely, CASP3, NT5DC2, and BCL2A1, upregulated in DN, show opposite correlations. In addition, spearman analysis revealed that the six hub genes were significantly associated with the infiltration of immune cells, including M1 and M2 macrophages, mast cells, resting NK cells, gamma delta T cells, and follicular helper T cells. ConclusionThis study elucidated the characteristics of mitochondria-related genes and their correlation with immune cell infiltration in DN, providing new insights for exploring the pathogenesis of DN and facilitating the identification of new potential biomarkers and therapeutic targets.

P4HB regulates the TGFβ/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells

BackgroundDiabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it’s role in HG-induced TECs remains to be elucidated.MethodsThe HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB.ResultsHG induced an increase in the expression of P4HB and TGFβ, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells.ConclusionP4HB regulated the TGFβ/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.

Transcriptional activation of PINK1 by MyoD1 mediates mitochondrial homeostasis to induce renal calcification in pediatric nephrolithiasis

This study aims to uncover the molecular mechanisms underlying pediatric kidney stone formation induced by renal calcium deposition by utilizing high-throughput sequencing data to reveal the regulation of PINK1 by MyoD1. We performed transcriptome sequencing on peripheral blood samples from healthy children and children with kidney stones to obtain differentially expressed genes (DEGs). Genes related to mitochondrial oxidative stress were obtained from the Genecards website and intersected with DEGs to obtain candidate target genes. Additionally, we conducted protein-protein interaction (PPI) analysis using the STRING database to identify core genes involved in pediatric kidney stone disease (KSD) and predicted their transcription factors using the hTFtarget database. We assessed the impact of MyoD1 on the activity of the PINK1 promoter using dual-luciferase reporter assays and investigated the enrichment of MyoD1 on the PINK1 promoter through chromatin immunoprecipitation (ChIP) experiments. To validate our hypothesis, we selected HK-2 cells and established an in vitro kidney stone model induced by calcium oxalate monohydrate (COM). We evaluated the expression levels of various genes, cell viability, volume of adherent crystals in each group, as well as mitochondrial oxidative stress in cells by measuring mitochondrial membrane potential (Δψm), superoxide dismutase (SOD) activity, reactive oxygen species (ROS), and malondialdehyde (MDA) content. Mitochondrial autophagy was assessed using mtDNA fluorescence staining and Western blot analysis of PINK1-related proteins. Apoptosis-related proteins were evaluated using Western blot analysis, and cell apoptosis was measured using flow cytometry. Furthermore, we developed a rat model of KSD and assessed the expression levels of various genes, as well as the pathologic changes in rat renal tissues using H&E and von Kossa staining, transmission electron microscopy (TEM), and the expression of creatinine, blood urea nitrogen, neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) to evaluate the mitochondrial oxidative stress in vivo (through measurement of Δψm, SOD activity, ROS, and MDA content). Mitochondrial autophagy was evaluated by Western blot analysis of PINK1-associated proteins. Apoptosis-related proteins were detected using Western blot analysis, and cellular apoptosis was examined using cell flow cytometry and TUNEL staining. Bioinformatics analysis revealed that the PINK1 gene is upregulated and vital in pediatric kidney stone patients. Our in vitro and in vivo experiments demonstrated that silencing PINK1 could inhibit kidney stone formation by suppressing mitochondrial oxidative stress both in vitro and in vivo. We identified MyoD1 as an upstream transcription factor of PINK1 that contributes to the occurrence of pediatric kidney stones through the activation of PINK1. Our in vivo and in vitro experiments collectively confirmed that silencing MyoD1 could inhibit mitochondrial oxidative stress, mitochondrial autophagy, and cellular apoptosis in a rat model of kidney stones by downregulating PINK1 expression, consequently suppressing the formation of kidney stones. In this study, we discovered that MyoD1 may promote kidney stone formation and development in pediatric patients by transcriptionally activating PINK1 to induce mitochondrial oxidative stress.

PPAR gamma and PGC-1alpha activators protect against diabetic nephropathy by suppressing the inflammation and NF-kappaB activation.

Inflammation plays a critical role in the progression of diabetic nephropathy. Peroxisome proliferator-activated receptor gamma (PPARγ) and its coactivator PPARγ coactivator-1 alpha (PGC-1α) enhance mitochondrial biogenesis and cellular energy metabolism but inhibit inflammation. However, the molecular mechanism through which these two proteins cooperate in the kidney remains unclear. The aim of the present study was to investigate this mechanism. HK-2 human proximal tubular cells were stimulated by inflammatory factors, the expression of PPARγ and PGC-1α were determined via reverse transcription-quantitative polymerase chain reaction (PCR) and western blotting (WB), and DNA binding capacity was measured by an EMSA. Furthermore, db/db mice were used to establish a diabetic nephropathy model and were administered PPARγ and PGC-1α activators. Kidney injury was evaluated microscopically, and the inflammatory response was assessed via WB, immunohistochemistry and immunofluorescence staining. Besides, HK-2 cells were stimulated by high glucose and inflammatory factors with and without ZLN005 treatment, the expression of PPARγ, PGC-1α, p-p65 and p65 were determined via qPCR and WB. Our results revealed that both TNF-α and IL-1β significantly decreased PPARγ and PGC-1 expression in vitro. Cytokines obviously decreased PPARγ DNA binding capacity. Moreover, we detected rapid activation of the NF-κB pathway in the presence of TNF-α or IL-1β. PPARγ and PGC-1α activators effectively protected against diabetic nephropathy and suppressed NF-κB expression both in db/db mice and HK-2 cells. PPARγ and its coactivator PGC-1α actively participate in protecting against renal inflammation by regulating the NF-κB pathway, which highlights their potential as therapeutic targets for renal diseases.

Deficiency of geranylgeranyl biphosphate synthase in kidney tubules causes cystic kidney disease.

Polycystic kidney disease (PKD) is a common hereditary kidney disease. Although PKD occurrence is associated with certain gene mutations, its onset regulatory mechanisms are still not well understood. Here, we first report that the key enzyme geranylgeranyl diphosphate synthase (GGPPS) is specifically expressed in renal tubular epithelial cells of mouse kidneys. We aimed to explore the role of GGPPS in PKD. In this study, we established a Ggppsfl/fl:Cdh16cre mouse model and compared its phenotype with that of wild-type mice. A Ggpps-downregulation HK2 cell model was also used to further determine the role of GGPPS. We found that GGPPS was specifically expressed in renal tubular epithelial cells of mouse kidneys. Its expression also increased with age. Low GGPPS expression was observed in human ADPKD tissues. In the Ggppsfl/fl:Cdh16cre mouse model, Ggpps deletion in renal tubular epithelial cells induced the occurrence and development of renal tubule cystic dilation and caused the death of mice after birth due to abnormal renal function. Enhanced proliferation of cyst-lining epithelial cells was also observed after the knockout of Ggpps. These processes were related to the increased rate of Rheb on membrane/cytoplasm and hyperactivation of mTORC1 signaling. In conclusion, the deficiency of GGPPS in kidney tubules induced the formation of renal cysts. It may play a critical role in PKD pathophysiology. A novel therapeutic strategy could be designed according to this work.

KLF15 ATTENUATES LIPOPOLYSACCHARIDE-INDUCED APOPTOSIS AND INFLAMMATORY RESPONSE IN RENAL TUBULAR EPITHELIAL CELLS VIA PPARΔ.

Background: The kidney is the most commonly affected organ in sepsis patients, and Krüppel-like transcription factor 15 (KLF15) has a kidney-protective effect and is highly enriched in the kidneys. This study aims to explore the role of KLF15 in sepsis-related acute kidney injury. Methods: A septic injury model in HK2 cells was established through the administration of lipopolysaccharide (LPS), followed by the transfection of an overexpression plasmid for KLF15. Cell viability was assessed using Cell Counting Kit-8 assay, and apoptosis was measured via flow cytometry. The levels of inflammatory cytokines were detected using ELISA, and western blot assay was employed to assess the expression of KLF15, PPARδ, as well as inflammatory and apoptosis-related proteins. The interaction between KLF15 and PPARδ was confirmed through the utilization of online databases and immunoprecipitation experiments. The mechanism was further validated using PPARδ agonists and small interfering RNA. Results: LPS-induced HK2 cells showed downregulated expression of KLF15 and PPARδ, along with decreased viability, accompanied by increased levels of apoptosis, TNFα, IL-1β, and IL-6. Additionally, LPS upregulated the expression of Bax, cytoplasmic cytochrome C [Cytc (cyt)], Cox-2, and p-NF-κB-p65 in HK2 cells, while simultaneously downregulating the expression of Bcl2 and mitochondrial cytochrome c [Cytc (mit)]. immunoprecipitation experiment revealed a possible interaction between KLF15 and PPARδ in HK2 cells. Ov-KLF15, Ov-PPARδ, or administration of PPARδ agonists effectively alleviated the aforementioned alterations induced by LPS. However, interference with PPARδ significantly attenuated the protective effect of Ov-KLF15 on HK2 cells. Conclusion: KLF15 attenuates LPS-induced apoptosis and inflammatory responses in HK2 cells via PPARδ.

Quantification of relevant metabolites in apoptotic bodies from HK-2 cells by targeted metabolomics based on liquid chromatography-tandem mass spectrometry

BackgroundApoptotic bodies play an important role in the cellular communication as a consequence of the great variety of biomolecules they harbor. There is evidence that 1st generation apoptotic bodies from HK-2 cells induced by cisplatin or UV light trigger apoptosis in naïve HK-2 cells whereas 2nd generation apoptotic bodies activate cell proliferation showing an opposite effect. Thus, the development of new analytical strategies to quantify the changes in the involved metabolites is imperative to shed light on the biological mechanisms which trigger apoptosis and cell proliferation. ResultsA LC-(Q-Orbitrap)MS method has been developed to quantify the metabolites unequivocally identified in the apoptotic body fluid from HK-2 cells in our previous works based on untargeted metabolomics. Thus, two different columns and gradients were tested and the HILIC column was selected taking into account the retention times and chromatographic separation. Also, different normal collision energies were tested for each metabolite and the parallel reaction monitoring was chosen to carry out the quantitative analysis. Once the method was optimized, it was evaluated in terms of linearity, limits of detection and quantification, matrix effects, accuracy, and precision, for each metabolite. Limits of detection ranged from 0.02 to 1.4 ng mL−1. A total of 9 relevant metabolites proposed as potential biomarkers to reveal metabolic differences among apoptotic bodies from HK-2 cells were quantified and some insights about the biological relevance were discussed. SignificanceThe first targeted metabolomics methodology enabling the quantification of relevant metabolites in apoptotic bodies from HK-2 cells was developed using LC-(Q-Orbitrap)MS. Pyridoxine, kynurenine, and creatine concentrations were determined in apoptotic bodies from HK-2 cells treated with cisplatin and UV light. Phenylacetylglycine, hippuric acid, butyrylcarnitine, acetylcarnitine, carnitine, and phenylalanine were determined in 1st and 2nd generation apoptotic bodies from HK-2 cells treated with cisplatin. Concentrations determined were useful to establish their biological role in the metabolism.