Renal Proximal Tubular Cells Research Articles

Stanniocalcin 1 and 1,25-dihydroxyvitamin D3 cooperatively regulate bone mineralization by osteoblasts

Stanniocalcin 1 (STC1) is a calcium- and phosphate-regulating hormone that is expressed in all tissues, including bone tissues, and is involved in calcium and phosphate homeostasis. Previously, STC1 expression was found to be increased by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administration in renal proximal tubular cells. In this study, we investigated whether STC1 directly regulates osteoblast differentiation or reciprocally controls the effects of 1,25(OH)2D3 on osteoblasts to contribute to bone homeostasis. We found that STC1 inhibited osteoblast differentiation in vitro and bone morphogenetic protein 2 (BMP2)-induced ectopic bone formation in vivo. Moreover, 1,25(OH)2D3 increased STC1 expression through direct binding to the Stc1 promoter of the vitamin D receptor (VDR). STC1 activated the 1,25(OH)2D3–VDR signaling pathway through the upregulation of VDR expression mediated by the inhibition of Akt phosphorylation in osteoblasts. STC1 further increased the effects of 1,25(OH)2D3 on receptor activator of nuclear factor-κB ligand (RANKL) secretion and inhibited osteoblast differentiation by exhibiting a positive correlation with 1,25(OH)2D3. The long-bone phenotype of transgenic mice overexpressing STC1 specifically in osteoblasts was not significantly different from that of wild-type mice. However, compared with that in the wild-type mice, 1,25(OH)2D3 administration significantly decreased bone mass in the STC1 transgenic mice. Collectively, these results suggest that STC1 negatively regulates osteoblast differentiation and bone formation; however, the inhibitory effect of STC1 on osteoblasts is transient and can be reversed under normal conditions. Nevertheless, the synergistic effect of STC1 and 1,25(OH)2D3 through 1,25(OH)2D3 administration may reduce bone mass by inhibiting osteoblast differentiation.

ANKRD1 aggravates renal ischaemia‒reperfusion injury via promoting TRIM25-mediated ubiquitination of ACSL3.

Renal ischaemia‒reperfusion injury (IRI) is the primary cause of acute kidney injury (AKI). To date, effective therapies for delaying renal IRI and postponing patient survival remain absent. Ankyrin repeat domain 1 (ANKRD1) has been implicated in some pathophysiologic processes, but its role in renal IRI has not been explored. The mouse model of IRI-AKI and in vitro model were utilised to investigate the role of ANKRD1. Immunoprecipitation-mass spectrometry was performed to identify potential ANKRD1-interacting proteins. Protein‒protein interactions and protein ubiquitination were examined using immunoprecipitation and proximity ligation assay and immunoblotting, respectively. Cell viability, damage and lipid peroxidation were evaluated using biochemical and cellular techniques. First, we unveiled that ANKRD1 were significantly elevated in renal IRI models. Global knockdown of ANKRD1 in all cell types of mouse kidney by recombinant adeno-associated virus (rAAV9)-mitigated ischaemia/reperfusion-induced renal damage and failure. Silencing ANKRD1 enhanced cell viability and alleviated cell damage in human renal proximal tubule cells exposed to hypoxia reoxygenation or hydrogen peroxide, while ANKRD1 overexpression had the opposite effect. Second, we discovered that ANKRD1's detrimental function during renal IRI involves promoting lipid peroxidation and ferroptosis by directly binding to and decreasing levels of acyl-coenzyme A synthetase long-chain family member 3 (ACSL3), a key protein in lipid metabolism. Furthermore, attenuating ACSL3 in vivo through pharmaceutical approach and in vitro via RNA interference mitigated the anti-ferroptotic effect of ANKRD1 knockdown. Finally, we showed ANKRD1 facilitated post-translational degradation of ACSL3 by modulating E3 ligase tripartite motif containing 25 (TRIM25) to catalyse K63-linked ubiquitination of ACSL3, thereby amplifying lipid peroxidation and ferroptosis, exacerbating renal injury. Our study revealed a previously unknown function of ANKRD1 in renal IRI. By driving ACSL3 ubiquitination and degradation, ANKRD1 aggravates ferroptosis and ultimately exacerbates IRI-AKI, underlining ANKRD1's potential as a therapeutic target for kidney IRI. Ankyrin repeat domain 1 (ANKRD1) is rapidly activated in renal ischaemia‒reperfusion injury (IRI) models in vivo and in vitro. ANKRD1 knockdown mitigates kidney damage and preserves renal function. Ferroptosis contributes to the deteriorating function of ANKRD1 in renal IRI. ANKRD1 promotes acyl-coenzyme A synthetase long-chain family member 3 (ACSL3) degradation via the ubiquitin‒proteasome pathway. The E3 ligase tripartite motif containing 25 (TRIM25) is responsible for ANKRD1-mediated ubiquitination of ACSL3.

Ligustilide alleviates oxidative stress during renal ischemia-reperfusion injury through maintaining Sirt3-dependent mitochondrial homeostasis

BackgroundRenal ischemia-reperfusion (I/R) injury is an inevitable complication during renal transplantation and is closely related to patient prognosis. Mitochondrial damage induced oxidative stress is the core link of renal I/R injury. Ligustilide (LIG), a natural compound extracted from ligusticum chuanxiong hort and angelica sinensis, has exhibited the potential to protect mitochondrial function. However, whether LIG can ameliorate renal I/R injury requires further investigation. Delving deeper into the precise targets and mechanisms of LIG's effect on renal I/R injury is crucial. PurposeThis study aimed to elucidate the specific mechanism of LIG's protective effect on renal I/R injury. MethodsIn this study, an in vivo model of renal ischemia-reperfusion (I/R) injury was developed in mice, along with an in vitro model of hypoxia-reoxygenation (H/R) using human proximal renal tubular epithelial cells (HK-2). To assess the impact of LIG on renal injury, various methods were employed, including serum creatinine (Cr) and blood urea nitrogen (BUN) testing, hematoxylin and eosin (HE) staining, and immunohistochemistry (IHC) for kidney injury molecule-1 (KIM-1). The effects of LIG on oxidative stress were examined using fluorescent probes dihydroethidium (DHE) and dichlorodihydrofluorescein diacetate (DCFH-DA), TdT-mediated dUTP Nick-End Labeling (TUNEL) staining, and flow cytometry. Additionally, the influence of LIG on mitochondrial morphology and function was evaluated through transmission electron microscopy (TEM), Mito Tracker Red CMXRos staining, adenosine triphosphate (ATP) concentration assays, and JC-1 staining. The potential mechanism involving LIG and Sirt3 was explored by manipulating Sirt3 expression through cell transfection. ResultsThe results showed that LIG could provide protective function for mitochondria to alleviate oxidative stress induced by renal I/R. Further mechanistic studies indicated that LIG maintained mitochondrial homeostasis by targeting Sirt3. ConclusionOur findings demonstrated that LIG alleviated oxidative stress during renal I/R injury through maintaining Sirt3-dependent mitochondrial homeostasis. Overall, our data raised the possibility of LIG as a novel therapy for renal I/R injury.

Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats

This study investigated whether chronic undernutrition alters the mitochondrial structure and function in renal proximal tubule cells, thus impairing fluid transport and homeostasis. We previously showed that chronic undernutrition downregulates the renal proximal tubules (Na++K+)ATPase, the main molecular machine responsible for fluid transport and ATP consumption. Male rats received a multifactorial deficient diet, the so-called Regional Basic Diet (RBD), mimicking those used in impoverished regions worldwide, from weaning to a juvenile age (3 months). The diet has a low content (8 %) of poor-quality proteins, low lipids, and no vitamins compared to control (CTR). We investigated citrate synthase activity, mitochondrial respiration (oxygraphy) in phosphorylating and non-phosphorylating conditions with different substrates/inhibitors, potential across the internal membrane (Δψ), and anion superoxide/H2O2 formation. The data were correlated with ultrastructural alterations evaluated using transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM). Citrate synthase activity decreased (∼50 %) in RBD rats, accompanied by a similar reduction in respiration in non-phosphorylating conditions, maximum respiratory capacity, and ATP synthesis. The Δψ generation and its dissipation after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone remained unmodified in the survival mitochondria. H2O2 production increased (∼100 %) after Complex II energization. TEM demonstrated intense matrix vacuolization and disruption of cristae junctions in a subpopulation of RBD mitochondria, which was also demonstrated in the 3D analysis of FIB-SEM tomography. In conclusion, chronic undernutrition impairs mitochondrial functions in renal proximal tubules, with profound alterations in the matrix and internal membrane ultrastructure that culminate with the compromise of ATP supply for transport processes.

Bioenergetics disruption, oxidative stress, and inflammation as underlying mechanisms of tramadol-induced nephrotoxicity.

Tramadol (TR), a commonly prescribed pain reliever for moderate to severe pain, has been associated with kidney damage. This study investigates TR-induced nephrotoxicity mechanisms, focusing on its effects on renal proximal tubular cells (PTCs). The study findings demonstrate that TR disrupts PTC bioenergetic processes, leading to oxidative stress and inflammation. Significant toxicity to PTCs was observed with estimated effective concentration 50 values of 9.8 and 11.5 µM based on 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, respectively. TR also interferes with the function of PTC transporters, including organic cation uptake transporter 1, organic cation transporter 2, P-glycoprotein, and multidrug resistance protein 2. Furthermore, bioenergetics assays showed that TR reduced the activities of mitochondrial complexes I and III, adenosine triphosphate production, mitochondrial membrane potential, and oxygen consumption rate while increasing lactate release. TR also increased the production of reactive oxygen species, lipid peroxidation thiobarbituric acid reactive substances end products, and the expression of the NRf2 gene while decreasing reduced glutathione (GSH-R) stores and catalase and superoxide dismutase antioxidant activities. Additionally, TR increased the production of inflammatory cytokines (TNF-α and IL-6) and their coding genes expression. Interestingly, the study found that antioxidants like GSH-R, inhibitors of IL-6 and TNF-α, and mitochondrial activating Co-Q10 could protect cells against TR-induced cytotoxicity. The study suggests that TR causes nephrotoxicity by disrupting bioenergetic processes, causing oxidative stress and inflammation, but antioxidants and agents targeting mitochondria may have protective and curative potential.

Renogrit selectively protects against cisplatin-induced injury in human renal tubular cells and in Caenorhabditis elegans by harmonizing apoptosis and mitophagy

Cisplatin-induced nephrotoxicity restricts its clinical use against solid tumors. The present study elucidated the pharmacological effects of Renogrit, a plant-derived prescription medicine, using cisplatin-induced human renal proximal tubular (HK-2) cells and Caenorhabditis elegans. Quantification of phytochemicals in Renogrit was performed on HPTLC and UHPLC platforms. Renogrit was assessed in vitro in HK-2 cells post-exposure to clinically relevant concentration of cisplatin. It was observed that renoprotective properties of Renogrit against cisplatin-induced injury stem from its ability to regulate renal injury markers (KIM-1, NAG levels; NGAL mRNA expression), redox imbalance (ROS generation; GST levels), and mitochondrial dysfunction (mitochondrial membrane potential; SKN-1, HSP-60 expression). Renogrit was also found to modulate apoptosis (EGL-1 mRNA expression; protein levels of p-ERK, p-JNK, p-p38, c-PARP1), necroptosis (intracellular calcium accumulation; RIPK1, RIPK3, MLKL mRNA expression), mitophagy (lysosome population; mRNA expression of PINK1, PDR1; protein levels of p-PINK1, LC3B), and inflammation (IL-1β activity; protein levels of LXR-α). More importantly, Renogrit treatment did not hamper normal anti-proliferative effects of cisplatin as observed from cytotoxicity analysis on MCF-7, A549, SiHa, and T24 human cancer cells. Taken together, Renogrit could be a potential clinical candidate to mitigate cisplatin-induced nephrotoxicity without compromising the anti-neoplastic properties of cisplatin.

Caffeine Inhibits Both Basal and Insulin-Activated Urate Transport.

Caffeine, an adenosine receptor antagonist, is a potent central nervous system stimulant that also impairs insulin signaling. Recent studies have suggested that coffee consumption lowers serum urate (SU) and protects against gout by unknown mechanisms. We hypothesized that caffeine lowers SU by affecting activity of urate transporters. We examined the effect of caffeine and adenosine on basal and insulin stimulation of net 14C-urate uptake in the human renal proximal tubule cell line PTC-05 and on individual urate transporters expressed in Xenopus laevis oocytes. We found that caffeine and adenosine efficiently inhibited both basal and insulin stimulation of net 14C-urate uptake mediated by endogenous urate transporters in PTC-05 cells. In oocytes expressing individual urate transporters, caffeine (>0.2 mM) more efficiently inhibited the basal urate transport activity of GLUT9 isoforms, OAT4, OAT1, OAT3, NPT1, ABCG2, and ABCC4 than did adenosine without significantly affecting URAT1 and OAT10. However, unlike adenosine, caffeine at lower concentrations (<0.2 mM) very effectively inhibited insulin activation of urate transport activity of GLUT9, OAT10, OAT1, OAT3, NPT1, ABCG2, and ABCC4 by blocking activation of Akt and extracellular signal-regulated kinase. We postulate that inhibition of urate transport activity of the re-absorptive transporters GLUT9, OAT10, and OAT4 by caffeine is a key mechanism in its urate-lowering effects. Additionally, the ability of caffeine to block insulin-activated urate transport by GLUT9a and OAT10 suggests greater relative inhibition of these transporters in hyperinsulinemia.

USP11 promotes renal tubular cell pyroptosis and fibrosis in UUO mice via inhibiting KLF4 ubiquitin degradation.

The pyroptosis of renal tubular epithelial cells leads to tubular loss and inflammation and then promotes renal fibrosis. The transcription factor Krüppel-like factor 4 (KLF4) can bidirectionally regulate the transcription of target genes. Our previous study revealed that sustained elevation of KLF4 is responsible for the transition of acute kidney injury (AKI) into chronic kidney disease (CKD) and renal fibrosis. In this study, we explored the upstream mechanisms of renal tubular epithelial cell pyroptosis from the perspective of posttranslational regulation and focused on the transcription factor KLF4. Mice were subjected to unilateral ureteral obstruction (UUO) surgery and euthanized on D7 or D14 for renal tissue harvesting. We showed that the pyroptosis of renal tubular epithelial cells mediated by both the Caspase-1/GSDMD and Caspase-3/GSDME pathways was time-dependently increased in UUO mouse kidneys. Furthermore, we found that the expression of the transcription factor KLF4 was also upregulated in a time-dependent manner in UUO mouse kidneys. Tubular epithelial cell-specific Klf4 knockout alleviated UUO-induced pyroptosis and renal fibrosis. In Ang II-treated mouse renal proximal tubular epithelial cells (MTECs), we demonstrated that KLF4 bound to the promoter regions of Caspase-3 and Caspase-1 and directly increased their transcription. In addition, we found that ubiquitin-specific protease 11 (USP11) was increased in UUO mouse kidneys. USP11 deubiquitinated KLF4. Knockout of Usp11 or pretreatment with the USP11 inhibitor mitoxantrone (3 mg/kg, i.p., twice a week for two weeks before UUO surgery) significantly alleviated the increases in KLF4 expression, pyroptosis and renal fibrosis. These results demonstrated that the increased expression of USP11 in renal tubular cells prevents the ubiquitin degradation of KLF4 and that elevated KLF4 promotes inflammation and renal fibrosis by initiating tubular cell pyroptosis.

SET8 inhibition preserves PTEN to attenuate kidney cell apoptosis in cisplatin nephrotoxicity.

The aberrant expression of SET8, a histone methyltransferase that mediates H4 lysine 20 mono-methylation (H4K20me1), is implicated in the pathogenesis of various tumors, however, its role in acute kidney injury (AKI) is unknown. Here we showed that SET8 and H4K20me1 were upregulated in the murine kidney with AKI induced by cisplatin, along with increased renal tubular cell injury and apoptosis and decreased expression of E-cadherin and Phosphatase and Tensin Homolog (PTEN). Suppression of SET8 by UNC0379 improved renal function, attenuated tubule damage, and restored expression of PTEN, but not E-cadherin. UNC0379 was also effective in lessening cisplatin-induced DNA damage response (DDR) as indicated by reduced expression of γ-H2AX, p53, p21, and alleviating cisplatin-impaired autophagy as shown by retained expression of Atg5, Beclin-1, and CHMP2A and enhanced levels of LC3-II in the kidney. Consistently, inhibition of SET8 with either UNC0379 or siRNA mitigated apoptosis and DDR, and restored autophagy, along with PTEN preservation in cultured renal proximal tubular epithelial cell (TKPTs) exposed to cisplatin. Further studies showed that inhibition of PTEN with Bpv or siRNA potentiated cisplatin-induced apoptosis, DDR, and hindered autophagy, and conversely, alleviated by overexpression of PTEN in TKPTs. Finally, blocking PTEN largely abolished the inhibitory effect of UNC0379 on apoptosis. Taken together, these results suggest that SET8 inhibition protects against cisplatin-induced AKI and renal cell apoptosis through a mechanism associated with the preservation of PTEN, which in turn inhibits DDR and restores autophagy.

Involvement of Sirt1-FoxO3a-Bnip3 axis and autophagy mediated mitochondrial turnover in according protection to hyperglycemic NRK-52E cells by Berberine

Aberrant accumulation of dysfunctional mitochondria in renal cells during hyperglycemia signifies perturbed autophagy and mitochondrial turnover. This study aims to focus on the underlying mechanism involved in autophagy and mitophagy inducing efficacy of Berberine (isoquinoline alkaloid) in hyperglycemic NRK-52E cells. Berberine mediated protection to hyperglycemic cells prevented alteration in mitochondrial structure and function. Treatment with SRT-1720 (Sirt1 activator) enhanced autophagy, decreased apoptosis, upregulated expression of downstream moieties (FoxO3a and Bnip3) and ameliorated mitochondria related anomalies while nicotinamide (Sirt1 inhibitor) treatment exhibited reversal of the same. GFP reporter assay ascertained enhanced transcriptional activity of FoxO in Berberine-treated hyperglycemic cells, which was found to be correlated to increased expression of downstream protein Bnip3. Knocking down FoxO3a disrupted autophagy and stimulated apoptosis. N-acetyl-L-cysteine pre-treatment confirmed that generation of ROS intervened high glucose induced toxicity in NRK-52E cells. Berberine co-treatment resulted in differential expressions of key proteins involved in autophagy and mitophagy like LC3B, ATGs, Beclin1, Sirt1, Bnip3, FoxO3a and Parkin. Further, enhanced mitophagy in Berberine-treated cells was confirmed by transmission electron microscopy. Thus, our findings give evidence that the protection accorded by Berberine against hyperglycemia in renal proximal tubular cells (NRK-52E) involves instigation of Sirt1-FoxO3a-Bnip3 axis and autophagy mediated mitophagy induction.

Total Synthesis and Anti-inflammatory Activity of Asperjinone and Asperimide C.

Total syntheses of two γ-butenolide natural products, asperjinone (1) and asperimide C (2) in both racemic and chiral forms have been accomplished utilizing Basavaiah's one-pot Friedel-Crafts/maleic anhydride formation protocol as a key strategy. Our syntheses verified the revised structure of 1 proposed by Williams et al. and the structure and absolute configuration of 2 reported by the Li group. This work also discloses the unprecedented anti-inflammatory activity of 1. Synthetic 1 exhibited significant anti-inflammatory activity in renal proximal tubular epithelial cells (RPTEC) by suppression of gene expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 under LPS-induced renal inflammation condition and was superior to (S)-1, rac-2, 2, and a positive drug control, indomethacin. Moreover, compound 1 inhibited downstream signaling of inflammation by significantly reducing iNOS and COX-2 gene expression and total NO production. The anti-inflammatory activity of asperjinone (1) renders it a potential and promising candidate for developing novel anti-inflammatory agents against inflammation worsening acute kidney injury.

74 Unraveling the Complexities of Obesity in Clear Cell Renal Cell Carcinoma Carcinogenesis

Abstract Background Obesity has been identified as an established risk factor in the development of clear cell renal cell carcinoma (ccRCC). ccRCC arises from the proximal convoluted tubule (PCT) of the kidney which is also the site of obesity-associated chronic kidney disease. Obesity can lead to chronic cellular insults which may lead to DNA injury and whether these mechanisms are critical to cancer initiation in the kidney is unclear. One potential way that obesity can lead to cancer is by altering lipid metabolism, including increasing circulating saturated fatty acids. In particular, palmitic acid is known to be lipotoxic to proximal tubules of the kidney. Understanding how free fatty acids initiate cancer is critical to understanding the role of obesity in cancer initiation and development. We hypothesize that chronic injury and repair due to excess FFA exposure could lead to ccRCC carcinogenesis and aim to define the metabolic phenotype of obesity associated ccRCC. Methods Using publicly available data from The KIRC Cancer Genome Atlas Program (TCGA) and clinicopathologic data, we compared the RNAseq profiles of obese (BMI &amp;gt;= 30) to normal weight (BMI =&amp;lt; 25) sample origins. Of the 337, a total of 333 had BMI available for analysis. We evaluated differences in Hallmark and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. Using DESeq, we adjusted for age and sex. In vitro, HK PCT cells were cultured in DMEM+10%FBS treated with bovine serum albumin (BSA)-palmitic acid complex for 48 hrs. We measured cell viability using Alamar Blue. Results were normalized to controls with BSA only. We extracted protein and performed western blots to identify DNA damage (pATM, H2AX) and endoplasmic reticulum stress (ATF4, eFI2alpha). Results Most of the patients in our clinical data cohort are male (64.4%), while 45.4% of patients are categorized by BMI as obese and 33.2% are categorized as normal. We utilized 333 individuals from the Kyoto Encyclopedia of Genes and Genomes (KEGG) to link genomic information to pathways associated with fatty acid metabolism as well as other cancer-associated hallmark metabolic pathways. We confirmed that excess free fatty acid supplementation leads to decreased cell viability in renal proximal tubule cells and renal embryonal cells and aim to assess differences in gene expression and metabolomic variation with fatty acid supplementation. Conclusions Through the investigation of these questions, we hope to improve understanding of obesity associated ccRCC and ultimately impact oncological outcomes for patients with this cancer. Understanding changes in lipid metabolism around ccRCC initiation will help identify novel pathways for pharmacologic intervention at earlier stages of the disease. DOD CDMRP Funding: yes

Abstract Mo138: Renal NPFFR2-mediated increase in blood pressure is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule

Neuropeptide FF (NPFF), an octapeptide originally found in the brain, participates in the regulation of blood pressure because it is present with its receptors, NPFFR1 and NPFFR2, in the cardiovascular regulatory center in the hypothalamus. However, the role of NPFF and its receptors in the kidney on blood pressure regulation is not known. In both the human and mouse renal proximal tubule, NPFFR2, rather than NPFFR1, is the predominant NPFF receptor determined by RNA in situ hybridization and immunofluorescence microscopy. In mouse renal proximal tubule cells, AC263093 (10 -6 M, 15 min), a specific NPFFR2 agonist and NPFFR1 antagonist, inhibited the forskolin-stimulated cAMP production (Vehicle: 100±6.14%, n=6; AC263093: 72.4±6.53%, n=6, p&lt;0.05), which was reversed by pretreatment with RF-9, an antagonist of both NPFFR1 and NPFFR2 (98.2±9.10%, n=6); RF-9, by itself, had no significant effect (100.8±11.44%, n=6), indicating linkage of NPFFR2 to the inhibitory G protein Gai in renal proximal tubule cells. In human renal proximal tubule cells, NPFF also increased Na + /K + -ATPase protein expression, determined by immunoblotting, in a time- and concentration-dependent manner. Protein expression of Na + /K + -ATPase was decreased in NPFFR2 -deficient human renal proximal tubule cells caused by specific NPFFR2 siRNA (Mock: 100±1.71%, NPFFR2 siRNA: 58.4±4.93%, n=4; P &lt; 0.05). Furthermore, blood pressure, measured by telemetry, was increased and sodium excretion was decreased in conscious C57Bl/6 mice infused with NPFF (9.25 mmol, 0.5 mL/hr/7 days) underneath the renal capsule. The effect was probably via NPFFR2 because the blood pressure of mice was increased by the intraperitoneal injection of AC263093 (20 mg/kg/day, 7 days) and fed a high salt diet (4% NaCl), which is consistent with the decrease in blood pressure (98±4 vs 113±3 mmHg; P &lt; 0.05; n=3-5/group) by the renal subcapsular infusion of Npffr2 siRNA in C57Bl/6 mice also fed the high salt diet. Taken together, NPFFR2 activation increases blood pressure and decreases sodium excretion that is associated with downregulation of cAMP signaling and upregulation of Na + /K + -ATPase protein expression in the renal proximal tubule.

Protective effect of pinostrobin against gentamicin-induced toxicity in human renal proximal tubular cells

Protective effect of pinostrobin against gentamicin-induced toxicity in human renal proximal tubular cells

Targeting alternative splicing of fibronectin in human renal proximal tubule epithelial cells with antisense oligonucleotides to reduce EDA+ fibronectin production and block an autocrine loop that drives renal fibrosis

TGFβ1 is a powerful regulator of fibrosis; secreted in a latent form, it becomes active after release from the latent complex. During tissue fibrosis, the EDA + isoform of cellular fibronectin is overexpressed. In pulmonary fibrosis it has been proposed that the fibronectin splice variant including an EDA domain (FN EDA+) activates latent TGFβ. Our work investigates the potential of blocking the ‘splicing in’ of EDA with antisense oligonucleotides to inhibit TGFβ1-induced EDA + fibronectin and to prevent the cascade of events initiated by TGFβ1 in human renal proximal tubule cells (PTEC).Human primary PTEC were treated with TGFβ1 for 48 h, medium removed and the cells transfected with RNase H-independent antisense oligonucleotides (ASO) designed to block EDA exon inclusion (ASO5). The efficacy of ASO to block EDA exon inclusion was assessed by EDA + fibronectin RNA and protein expression; the expression of TGFβ, αSMA (α smooth muscle actin), MMP2 (matrix metalloproteinse-2), MMP9 (matrix metalloproteinse-9), Collagen I, K Cadherin and connexin 43 was analysed.Targeting antisense oligonucleotides designed to block EDA exon inclusion in fibronectin pre mRNA were effective in reducing the amount of TGFβ1 -induced cellular EDA + fibronectin RNA and secreted EDA + fibronectin protein (assessed by western immunoblotting and immunocytochemistry) in human proximal tubule cells in an in vitro cell culture model. The effect was selective for EDA + exon with no effect on EDB + fibronectin RNA and total fibronectin mRNA.Exogenous TGFβ1 induced endogenous TGFβ, αSMA, MMP2, MMP9 and Col I mRNA. TGFβ1 treatment for 48h reduced the expression of K-Cadherin and increased the expression of connexin-43. These TGFβ1-induced pro-fibrotic changes were attenuated by ASO5 treatment. 48 h after the removal of exogenous TGFβ, further increases in αSMA, MMP2, MMP9 was observed; ASO5 significantly inhibited this subsequent increase. ASO5 treatment also significantly inhibited ability of the cell culture medium harvested at the end of the experiment (96h) to stimulate SMAD3 reporter cells. The role of endogenous TGFβ1 was confirmed by the use of a TGFβ receptor inhibitor.Our results demonstrate a critical role of FN EDA+ in a cycle of TGFβ driven pro-fibrotic responses in human PTEC and blocking its production with ASO technology offers a potential therapy to interrupt this vicious circle and hence limit the progression of renal fibrosis.

Three-dimensional Culture of Human Proximal Tubular Epithelial Cells for an in vitro Evaluation of Drug-induced Kidney Injury

Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC20) was obtained, and the ratio of EC20 values and clinical maximum concentration (Cmax) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels.

Membranous translocation of murine double minute 2 promotes the increased renal tubular immunogenicity in ischemia-reperfusion-induced acute kidney injury.

Kidneys from donors with prolonged warm and cold ischemia are prone to posttransplant T cell-mediated rejection (TCMR) due to ischemia-reperfusion injury (IRI). However, the precise mechanisms still remain obscure. Renal tubular epithelial cells (TECs) are the main target during IRI. Meanwhile, we have previously reported that murine double minute 2 (MDM2) actively participates in TEC homeostasis during IRI. In this study, we established a murine model of renal IRI and a cell model of hypoxia-reoxygenation by culturing immortalized rat renal proximal tubule cells (NRK-52E) in a hypoxic environment for different time points followed by 24 h of reoxygenation and incubating NRK-52E cells in a chemical anoxia-recovery environment. We found that during renal IRI MDM2 expression increased on the membrane of TECs and aggregated mainly on the basolateral side. This process was accompanied by a reduction of a transmembrane protein, programmed death ligand 1 (PD-L1), a coinhibitory second signal for T cells in TECs. Using mutant plasmids of MDM2 to anchor MDM2 on the cell membrane or nuclei, we found that the upregulation of membrane MDM2 could promote the ubiquitination of PD-L1 and lead to its ubiquitination-proteasome degradation. Finally, we set up a coculture system of TECs and CD4+ T cells in vitro; our results revealed that the immunogenicity of TECs was enhanced during IRI. In conclusion, our findings suggest that the increased immunogenicity of TECs during IRI may be related to ubiquitinated degradation of PD-L1 by increased MDM2 on the cell membrane, which consequently results in T-cell activation and TCMR.NEW & NOTEWORTHY Ischemic acute kidney injury (AKI) donors can effectively shorten the waiting time for kidney transplantation but increase immune rejection, especially T cell-mediated rejection (TCMR), the mechanism of which remains to be elucidated. Our study demonstrates that during ischemia-reperfusion injury (IRI), the translocation of tubular murine double minute 2 leads to basolateral programmed death ligand 1 degradation, which ultimately results in the occurrence of TCMR, which may provide a new therapeutic strategy for preventing AKI donor-associated TCMR.

Diosgenin Reduces Acute Kidney Injury and Ameliorates the Progression to Chronic Kidney Disease by Modifying the NOX4/p65 Signaling Pathways.

Acute kidney injury (AKI), if not well controlled, may progress to chronic kidney disease (CKD). Diosgenin is a natural phytosteroid sapogenin from plants. This study aimed to investigate the mechanistic effects of diosgenin on AKI and AKI related development of CKD. The mouse model of ischemia/reperfusion (I/R)-induced AKI was used, and its progressive changes were followed. Human renal proximal tubular epithelial cells were used, and hypoxia stimulation was applied to mimic the in vivo I/R. Diosgenin, given after renal injury, preserved kidney function, as evidenced by a reduction in serum levels of BUN, creatinine, and UACR in both acute and chronic phases of AKI. Diosgenin alleviated I/R-induced tubular injury and prevented macrophage infiltration and renal fibrosis in AKI mice. Furthermore, diosgenin also mitigated the development of CKD from AKI with reduced renal expression of inflammatory, fibrotic, and epithelial-mesenchymal transition markers. In human renal tubular epithelial cells, diosgenin downregulated the hypoxia-induced oxidative stress and cellular damages that were dependent on the NOX4/p65 signaling pathways. Taken together, diosgenin treatment reduced I/R-induced AKI and ameliorated the progression to CKD from AKI probably by modifying the NOX4/p65 signaling pathways.

Nrf2/FSP1/CoQ10 axis-mediated ferroptosis is involved in sodium aescinate-induced nephrotoxicity

Sodium aescinate (SA), an active compound found in horse chestnut seeds, is widely used in clinical practice. Recently, the incidence of SA-induced adverse events, particularly renal impairment, has increased. Our previous work demonstrated that SA causes severe nephrotoxicity via nephrocyte ferroptosis; however, the underlying mechanism remains to be fully elucidated. In the current study, we investigated additional molecular pathways involved in SA-induced nephrotoxicity. Our results showed that SA inhibited cell viability, disrupted cellular membrane integrity, and enhanced reactive oxygen species (ROS), ferrous iron (Fe2+), and malondialdehyde (MDA) levels, as well as lipid peroxidation in rat proximal renal tubular epithelial cell line (NRK-52E) cells. SA also depleted coenzyme Q10 (CoQ10, ubiquinone) and nicotinamide adenine dinucleotide (NADH) and reduced ferroptosis suppressor protein 1 (FSP1) and polyprenyltransferase (coenzyme Q2, COQ2) activity, triggering lipid peroxidation and ROS accumulation in mouse kidneys and NRK-52E cells. The overexpression of COQ2, FSP1, or CoQ10 (ubiquinone) supplementation effectively attenuated SA-induced ferroptosis, whereas iFSP1 or 4-formylbenzoic acid (4-CBA) pretreatment exacerbated SA-induced nephrotoxicity. Additionally, SA decreased nuclear factor-erythroid-2-related factor 2 (Nrf2) levels and inhibited Nrf2 binding to the −1170/-1180 bp ARE site in FSP1 promoter, resulting in FSP1 suppression. Overexpression of Nrf2 or its agonist dimethyl fumarate (DMF) promoted FSP1 expression, thereby improving cellular antioxidant capacity and alleviating SA-induced ferroptosis. These results suggest that SA-triggers renal injury through oxidative stress and ferroptosis, driven by the suppression of the Nrf2/FSP1/CoQ10 axis.

Metformin attenuates PM2.5-induced oxidative stress by inhibiting the AhR/CYP1A1 pathway in proximal renal tubular epithelial cells

The harmful effects of PM2.5 on human health, including an increased risk of chronic kidney disease (CKD), have raised a lot of attention, but the underlying mechanisms are unclear. We used the Shanghai Meteorological and Environmental Animal Exposure System (Shanghai-METAS) to simulate the inhalation of PM2.5 in the real environment and established an animal model by exposing C57BL/6 mice to filtered air (FA) and Particulate Matter (PM2.5) for 8 weeks. PM2.5 impaired the renal function of the mice, and the renal tubules underwent destructive changes. Analysis of NHANES data showed a correlation between reduced kidney function and higher blood levels of PM2.5 components, polychlorinated biphenyls (PCBs) and dioxins, which are Aryl hydrocarbon Receptor (AhR) ligands. PM2.5 exposure induced higher levels of AhR and CYP1A1 and oxidative stress as evidenced by the higher levels of ROS, MDA, and GSSG in kidneys of mice. PM2.5 exposure led to AhR overexpression and nuclear translocation in proximal renal tubular epithelial cells. Inhibition of AhR reduced CYP1A1 expression and PM2.5-increased levels of ROS, MDA and GSSG. Our study suggested metformin can mitigate PM2.5-induced oxidative stress by inhibiting the AhR/CYP1A1 pathway. These findings illuminated the role of AhR/CYP1A1 pathway in PM2.5-induced kidney injury and the protective effect of metformin on PM2.5-induced cellular damage, offering new insights for air pollution-related renal diseases.