Human Proximal Tubular Epithelial Cells Research Articles

The role and mechanism of action of miR-483-3p in mediating the effects of IGF-1 on human renal tubular epithelial cells induced by high glucose

This study aimed to elucidate the influence of miR-483-3p on human renal tubular epithelial cells (HK-2) under high glucose conditions and to understand its mechanism. Human proximal tubular epithelial cells (HK-2) were exposed to 50 mmol/L glucose for 48 h to establish a renal tubular epithelial cell injury model, denoted as the high glucose group (HG group). Cells were also cultured for 48 h in a medium containing 5.5 mmol/L glucose, serving as the low glucose group. Transfection was performed in various groups: HK-2 + low glucose (control group), high glucose (50 mM) (HG group), high glucose + miR-483-3p mimics (HG + mimics group), high glucose +miR-483-3p inhibitor (HG + inhibitor group), and corresponding negative controls. Real-time quantitative polymerase chain reaction (qPCR) assessed the mRNA expression of miR-483-3p, bax, bcl-2, and caspase-3. Western blot determined the corresponding protein levels. Proliferation was assessed using the CCK-8 assay, and cell apoptosis was analyzed using the fluorescence TUNEL method. Western blot and Masson’s staining were conducted to observe alterations in cell fibrosis post miR-483-3p transfection. Furthermore, a dual-luciferase assay investigated the targeting relationship between miR-483-3p and IGF-1. The CCK8 assay demonstrated that the HG + mimics group inhibited HK-2 cell proliferation, while the fluorescent TUNEL method revealed induced cell apoptosis in this group. Conversely, the HG + inhibitor group promoted cell proliferation and suppressed cell apoptosis. The HG + mimics group upregulated mRNA and protein expression of pro-apoptotic markers (bax and caspase-3), while downregulating anti-apoptotic marker (bcl-2) expression. In contrast, the HG + inhibitor group showed opposite effects. Collagen I and FN protein levels were significantly elevated in the HG + mimics group compared to controls (P < 0.05). Conversely, in the HG + inhibitor group, the protein expression of Collagen I and FN was notably reduced compared to the HG group (P < 0.05). The dual luciferase reporter assay confirmed that miR-483-3p could inhibit the luciferase activity of IGF-1’s 3′-UTR region (P < 0.05). miR-483-3p exerts targeted regulation on IGF-1, promoting apoptosis and fibrosis in renal tubular epithelial cells induced by high glucose conditions.

Oridonin ameliorates renal fibrosis in diabetic nephropathy by inhibiting the Wnt/β-catenin signaling pathway

Diabetic nephropathy (DN) is one of the most serious and frequent complications among diabetes patients and presently constitutes vast the cases of end-stage renal disease worldwide. Tubulointerstitial fibrosis is a crucial factor related to the occurrence and progression of DN. Oridonin (Ori) is a diterpenoid derived from rubescens that has diverse pharmacological properties. Our previous study showed that Ori can protect against DN by decreasing the inflammatory response. However, whether Ori can alleviate renal fibrosis in DN remains unknown. Here, we investigated the mechanism through which Ori affects the Wnt/β-catenin signaling pathway in diabetic rats and human proximal tubular epithelial cells (HK-2) exposed to high glucose (HG) levels. Our results revealed that Ori treatment markedly decreased urinary protein excretion levels, improved renal function and alleviated renal fibrosis in diabetic rats. In vitro, HG treatment increased the migration of HK-2 cells while reducing their viability and proliferation rate, and treatment with Ori reversed these changes. Additionally, the knockdown of β-catenin arrested cell migration and reduced the expression levels of Wnt/β-catenin signaling-related molecules (Wnt4, p-GSK3β and β-catenin) and fibrosis-related molecules (α-smooth muscle actin, collagen I and fibronectin), and Ori treatment exerted an effect similar to that observed after the knockdown of β-catenin. Furthermore, the combination of Ori treatment and β-catenin downregulation exerted more pronounced biological effects than treatment alone. These findings may provide the first line of evidence showing that Ori alleviates fibrosis in DN by inhibiting the Wnt/β-catenin signaling pathway and thereby reveal a novel therapeutic avenue for treating tubulointerstitial fibrosis.

LncRNA GABPB1-IT1 is upregulated in ischemia-induced acute kidney injury and downregulates miR-204-5p to promote hypoxia-induced human renal proximal tubular epithelial cell apoptosis.

The present study investigated the role of long non-coding RNA (lncRNA) GABPB1-IT1 in ischemia-induced acute kidney injury (AKI). The expression of GABPB1-IT1 in plasma of patients with ischemia-induced AKI and healthy controls was detected by RT-qPCR. GABPB1-IT1 and miR-204-5p were overexpressed in human renal proximal tubular epithelial cells (HRPTEpCs), followed by RT-qPCR to assess the overexpression effect and the regulatory relationship between GABPB1-IT1 and miR-204-5p. Methylation-specific PCR (MSP) was performed to assess the promoter methylation status of miR-204-5p. Additionally, cell apoptosis assay was carried out to evaluate the correlation between miR-204-5p and GABPB1-IT1 in the context of hypoxia-induced apoptosis of HRPTEpCs. GABPB1-IT1 was upregulated in plasma of patients with ischemia-induced AKI. In HRPTEpCs, hypoxia upregulated the expression of GABPB1-IT1. MiR-204-5p was downregulated in ischemia-induced AKI, and the expression of miR-204-5p was inversely correlated with GABPB1-IT1. In HRPTEpCs, overexpression of GABPB1-IT1 decreased the expression levels of miR-204-5p and increased miR-204-5p gene methylation. In addition, overexpression of GABPB1-IT1 reduced the inhibitory effects of miR-204-5p on the apoptosis of HRPTEpC induced by hypoxia. Furthermore, overexpression of GABPB1-IT1 promoted kidney injury, renal tissue injury scores, and the level of serum creatinine. However, miR-204-5p had the opposite effect. GABPB1-IT1 was upregulated in ischemia-induced AKI and may induce hypoxia-induced apoptosis of HRPTEpC by methylation of miR-204-5p.

All-trans retinoic acid pretreatment of mesenchymal stem cells enhances the therapeutic effect on acute kidney injury.

A promising new therapy option for acute kidney injury (AKI) is mesenchymal stem cells (MSCs). However, there are several limitations to the use of MSCs, such as low rates of survival, limited homing capacity, and unclear differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy of AKI. We established a renal ischemia/reperfusion injury model and treated mice with ATRA-pretreated MSCs via tail vein injection. We found that AKI mice treated with ATRA-MSCs significantly improved renal function compared with DMSO-MSCs treatment. RNA sequencing screened that hyaluronic acid (HA) production from MSCs promoted by ATRA. Further validation by chromatin immunoprecipitation experiments verified that retinoic acid receptor RARα/RXRγ was a potential transcription factor for hyaluronic acid synthase 2. Additionally, an in vitro hypoxia/reoxygenation model was established using human proximal tubular epithelial cells (HK-2). After co-culturing HK-2 cells with ATRA-pretreated MSCs, we observed that HA binds to cluster determinant 44 (CD44) and activates the PI3K/AKT pathway, which enhances the anti-inflammatory, anti-apoptotic, and proliferative repair effects of MSCs in AKI. Inhibition of the HA/CD44 axis effectively reverses the renal repair effect of ATRA-pretreated MSCs. Taken together, our study suggests that ATRA pretreatment promotes HA production by MSCs and activates the PI3K/AKT pathway in renal tubular epithelial cells, thereby enhancing the efficacy of MSCs against AKI.

#1385 Dipeptidyl-peptidase-4 inhibitor teneligliptin attenuates folic acid-induced tubular cell death via inhibiting ferroptosis

Abstract Background and Aims Dipeptidyl peptidase-4 (DPP-4) inhibitors have been shown to attenuate several types of acute kidney injury (AKI) in animal models [1–3]. However, the mechanisms underlying their renoprotective effects remain to be clarified. Previous studies had identified ferroptosis as the primary cell death pathway for folic acid-induced AKI in mice [4]. Referring to that report, we previously reported that teneligliptin, a DPP-4 inhibitor, ameliorates folic acid-induced AKI. In the present study, we investigated whether teneligliptin attenuates folic acid-induced tubular cell death through its anti-ferroptotic properties. Method In in vitro experiments, we used HK-2 cells, human proximal tubular epithelial cells. HK-2 cells were co-cultured with folic acid and z-VAD-FMK (zVAD), an apoptosis inhibitor; Necrostatin-1s (Nec-1s), a necroptosis inhibitor; Ferrostatin-1 (Fer-1), a ferroptosis inhibitor; or teneligliptin. Co-incubation of erastin, a ferroptosis inducer, and teneligliptin was also performed. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was conducted on HK-2 cells to assess cell viability. Results Fer-1, but not z-VAD or Nec-1s, inhibited folic acid-induced cell death (Fig. 1A-C), indicating that ferroptosis contributes to folic acid-induced tubular cell death, rather than apoptosis or necroptosis. Teneligliptin also inhibited folic acid- and erastin-induced tubular cell death (Fig. 1D,E), suggesting that the DPP-4 inhibitor inhibits folic acid-induced tubular cell death via its anti-ferroptotic properties. Conclusion DPP-4 inhibitor prevents folic acid-induced tubular cell death by inhibiting ferroptosis in vitro, which may contribute to the attenuation of AKI in vivo.

#2675 Role of ANXA4 as a modifier in VHL-dependent ccRRC

Abstract Background and Aims The von Hippel-Lindau (VHL) syndrome is a rare genetic disease caused by inactivating mutations of the VHL tumor suppressor gene. VHL syndrome patients frequently develop clear cell renal cell carcinomas (ccRCCs). pVHL acts as the substrate recognition subunit of an E3 ligase involved in the ubiquitinylation of HIFs. Consequently, aberrant HIF-signalling results in a pseudo-hypoxic state characterized by pronounced vascularization in respective tumours. Previous studies have indicated VHL-dependent alterations of the extracellular matrix (ECM). However, to date comprehensive description and understanding of VHL-dependent matrisome signatures is missing. Method Proteomics were used to reveal VHL-dependent protein expression in ccRCC cell lines (A498, 786-O) and CRISPR/Cas9-modified human proximal tubular epithelial cells (hRPTECs) as well as syndromic and sporadic ccRCC patient cohorts. For candidate proteins analysis of proteomic and histological datasets was performed. Further functional analysis was conducted using overexpression, CRISPR/Cas9-mediated knockout (KO) and shRNA-mediated knockdown (KD) approaches (assessment of proliferation, migration, invasion, ECM, cellular membrane-integrity, protein localisation studies and transcriptomics). Results Filtering of proteomic datasets (ccRCC tumors and cell lines) for matrisomal proteins highlighted an enrichment of the ANXA-protein family. Interestingly, functional genetic titration of ANXA4 employing knockdown, overexpression or complete ablation using CRISPR/Cas9 genome editing did not translate into major changes of cellular proliferation or migration in renal cancer or tubular cells. Detailed localization studies revealed that ANXA4 is detectable in distinct cellular compartments, namely the cell membrane and nucleus/nuclear lamina. Various cellular stressors (including cellular densities) initiated shuttling of ANXA4 between these different compartments. Remarkably, similar distribution patterns were detectable in situ in tumours tissue of ccRCC patients. Further transcriptomic analysis of ANXA4 knockout cells demonstrated a major impact on ECM- and cell-adhesion-associated gene sets, reflected by impaired invasive migratory capacity in conditions of reduced ANXA4-expression. Conclusion Loss of VHL distinctly alters the ECM composition in ccRCC. We have identified the ECM-associated ANXA4 as a prominently upregulated protein, although canonical hallmarks of cancer were not directly influenced. Based on RNA-sequencing studies and subcellular localization patterns we assume that ANXA4 (co-)regulates matrisome-related transcriptomic signatures in response to various external stressors. These observations highlight the intricate regulation of ECM-associated signalling pathways in the context of VHL-dependent renal cell carcinoma.

#2339 All-trans retinoic acid enhances the efficacy of mesenchymal stem cells in acute kidney injury

Abstract Background and Aims Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic strategy for acute kidney injury (AKI), but still suffer from the drawbacks of low survival rate, low homing rate, and uncertain differentiation. In search of better therapeutic strategies, we explored all-trans retinoic acid (ATRA) pretreatment of MSCs to observe whether it could improve the therapeutic efficacy on AKI. Method We established a renal ischemia/reperfusion (I/R) injury model induced by clamping of left renal artery for 30 minutes and contralateral nephrectomy. C57BL/6 mice were randomly divided into 4 groups: sham group, IRI group, MSCs group, and ATRA-MSCs group. MSCs or ATRA-pretreated MSCs were injected via tail vein. GFR was measured by transcutaneous monitoring the clearance of FITC-sinistrin. These mice were sacrificed 3 days after surgery, and the serum creatinine (SCr) and blood urea nitrogen (BUN) levels were measured. The renal pathological changes were examined by Periodic Acid-Schiff staining (PAS). The in vitro hypoxia/reoxygenation (H/R) model was established using human proximal tubular epithelial cells (HK-2 cells). Co-culturing HK-2 cells with MSCs or ATRA-pretreated MSCs. Western blot, quantitative real time-PCR (qRT-PCR), and immunohistochemistry were used to detect mRNA or protein expression. We performed RNA sequencing (RNA-seq) analysis in ATRA-pretreated MSCs. The content of hyaluronic acid (HA) in the supernatant of ATRA-pretreated MSCs culture was detected by ELISA. Hyaluronan synthase mRNA expression level in ATRA-pretreated MSCs assessed by qRT-PCR analysis. The HAS2 promoter-binding transcription factors were forecasted by the JASPAR Transcription Factor Binding Site database. Western blot was used to detect whether P13K/AKT pathway was activated by ATRA-pretreated MSCs. Whether this nephroprotective effect could be reversed were explored by silencing HAS2 or CD44 antibodies. Results The AKI mice showed a significant decrease in GFR and even complete loss of renal function. Administration of MSCs significantly increased GFR, and MSCs pretreated with ATRA further increased GFR. Mice injected with ATRA-pretreated MSCs had significantly lower levels of SCr, BUN and ATN scores compared with untreated MSCs. Western blot, quantitative real-time PCR (qRT-PCR), and immunohistochemistry showed that ATRA pretreated MSCs were more effective in reducing AKI inflammation and apoptosis and promoting proliferative repair than untreated MSCs. GO enrichment analysis demonstrated that the hyaluronan biosynthetic process may be a significantly expressed. HAS2 expression and HA content of MSCS were significantly increased after the addition of ATRA. Analysis for potential transcription factor binding sites revealed that the HAS2 promoter region has multiple potential binding sites for the retinoic acid receptor. Based on the predictions from the JASPAR database, the RXR-RAR heterodimer had the highest prediction score. We then performed ChIP analysis to experimentally verify the binding of RXR-RAR heterodimer transcription factor to HAS2 promoters. We further found that ATRA-pretreated MSCs activated the PI3K/AKT signaling pathway in AKI. As expected, we found that ATRA-pretreated MSCs anti-inflammatory, anti-apoptosis, and pro-proliferation effects were substantially reversed by silencing HAS2 or blocking CD44. Conclusion ATRA promotes HA secretion from MSCs and activates the PI3K/AKT pathway, which in turn enhances the therapeutic efficacy of MSCs on AKI.

#1115 TGFβ induced mechanosignaling controls the transcriptome and microenvironmental composition of proximal tubules in chronic kidney disease

Abstract Background and Aims Interstitial fibrosis of the renal parenchyma and progressive atrophy of proximal tubules (PT) are hallmark features of chronic kidney disease (CKD). The latter is characterized by pronounced thickening and multilamellation of the tubular basement membrane, whereas fibrosis is promoted by accumulation of extracellular matrix (ECM) translating into increased matrix rigidity. The role of mechanotransduction via the integrin adhesion complex (IAC) as well as the corresponding signaling programs activated in this context remain largely elusive. Here, we aimed to elucidate the functional role of the ILK-Pinch-Parvin (IPP)-complex as an essential part of the IAC in response to PT damage and CKD. Method Morphometric assessment of a CKD patient biopsy cohort as well as ischaemia-reperfusion injury (IRI) mouse models was performed. Transcriptome studies of human proximal tubular epithelial cells (hRPTECs) under pro-fibrotic and rigid matrix conditions were analyzed. An in vitro model, mimicking CKD, was established and functionally characterized. Reanalysis of single-cell RNA sequencing (scRNA-seq) from a human CKD cohort was employed for cross-correlation and validation. Knockout (KO) models for the IPP-complex were generated by CRISPR/Cas9 genome editing and combined with functional assays (e.g. ECM deposition and sensing). Results In IRI mice, we observed a strong recruitment of the ILK-Pinch-Parvin (IPP)-complex and related-IAC proteins to the tubular basement membrane of proximal tubules (PTs) showing features of tubular atrophy. These findings were translated to CKD patients further assessing markers of active mechanosignaling (YAP) as well as tubular damage (SOX9). Rigidity sensing assays in a hRPTEC model were employed to define the underlying mechanisms. Remarkably, extensive transcriptome analysis revealed that only under pro-fibrotic signaling conditions, hRPTECs sensed rigidity alterations in the underlying substratum, and translated these changes into regulation of matrisome- and adhesome-related gene signatures (including members of the IPP-complex). Further filtering and cross-correlation with available scRNA-seq data from CKD-patients highlighted differential expression of secreted signaling proteins including the matricellular molecule CCN1. Subsequent functional studies demonstrated the underlying pivotal role of the IPP-complex as a central signaling node required for active mechanosignaling in damaged hRPTECs. Conclusion In CKD, IPP and other IAC proteins are recruited to the massively thickened tubular basement membrane of atrophic tubules, indicating a critical role in disease progression. Our results demonstrate that hRPTECs do not express IAC proteins in healthy conditions. However, when inflammation processes (TGFβ treatment) occur, PTs undergo a phenotypic switch, resulting in altered IAC expression signatures. Our data indicate that IAC proteins are crucially involved in sensing of the microenvironmental composition of PTs and thereby reciprocally fine-tune auto-/paracrine signaling properties of epithelial cells.

#1546 Mitochondrial oxidative phosphorylation in human proximal tubular epithelial cells is impaired by iron deficiency

Abstract Background and Aims We recently reported that iron deficiency may precede decline in renal function in patients with diabetes who had normal renal function at baseline but had a subsequent annual rate of eGFR decline of 3.3% or more (J Diabetes Investig. 2023;14:874-882.). Mitochondria contain many mitochondrial respiratory chain (MRC) enzymes that require iron as an active center, and consequently iron deficiency is known to impair mitochondrial function. Since the proximal tubular epithelial cells (PTECs) of the kidney are the cells with the greatest number of mitochondria in the body, we investigated whether iron deficiency impairs the mitochondrial capacity of the PTECs. Method Human cultured PTECs were analyzed in ‘control group’, ‘iron-deficient group ‘(co-cultured with deferiprone (DFP), an iron chelator), and ‘rescue group’ (co-cultured with DFP and FeCl3 which saturates the chelating ability of DFP), each with n ≥ 4 (MRC activities n = 4-5, others n ≥ 5). All analyses were performed after 24 hours of incubation. Values are shown as averages ± SEM. Results A Seahorse Extracellular Flux Analyzer showed that the basal oxygen consumption rates (OCRs) of control, iron-deficiency and rescue groups were 30.5 ± 1.2 pmol/min, 11.5 ± 0.7 pmol/min, and 31.1 ± 1.2 pmol/min, respectively, which revealed that basal OCRs of PTECs significantly decreased in the iron-deficient group. In addition, the analyzer also revealed that the activities of MRC for complexes I, II, and III, which have iron-sulfur clusters as their active centers, were significantly decreased in citrate synthase ratio to 77.6 ± 6.2%, 75.1 ± 6.4%, and 72.8 ± 9.3% of the control group, respectively, while complex IV was not significantly different at 84.9% of the control group. These reductions in MRC activities were not observed in the rescue group. Western blotting results also revealed that complex I, II, and III subunit proteins, NDUF9, SDHA, and UQCR2, were significantly reduced in the iron-deficient group (reduced to 38 ± 5.8%, 87 ± 8.8%, and 55 ± 5.7% of the control group, respectively). On the other hand, lactate concentrations in the culture medium of control group, iron-deficient group and rescue group were 1.73 ± 0.13 mmol, 3.01 ± 0.15 mmol and 1.61 ± 0.12 mmol, which revealed that glycolytic system was increased in the iron-deficient group, presumably to compensate for decreased oxidative phosphorylation. In response to intracellular iron deficiency, the iron regulatory protein 2 (IRP2) was shown in Western blotting to be unchanged in the rescue group (123 ± 4.0% of the control group), but increased in the iron-deficient group (170 ± 20% of the control group). The qRT-PCR analyses showed that mRNA expression of iron-sulfur cluster assembly enzyme (ISCU) was significantly decreased in the iron-deficiency group compared with the control group (41.8%), but not in the rescue group (95.1%), which is presumed that IRP2 suppressed ISCU expression, as has been proved previously. These results should indicate that iron deficiency suppressed iron utilization in the mitochondria of PTECs. The number of viable PTECs in the iron-deficient group after 24 hours was 1.99 ± 0.09 × 10^5 cells/well, which was significantly lower than that of the control (3.15 ± 0.15 × 10^5 cells/well) and rescue (2.68 ± 0.18 × 10^5 cells/well) groups. On the other hand, the number of dead PTECs was 0.042 ± 0.019 × 10^5 cells in the iron-deficient group, which was not significantly different from the control (0.023 ± 0.005 × 10^5 cells) and the rescue (0.020 ± 0.011 × 10^5 cells) groups. These data suggest that cell growth should be suppressed by iron deficiency. Conclusion Mitochondrial oxidative phosphorylation through the MRC was impaired by iron deficiency in PTECs. This impairment of mitochondrial capacity in PTECs may lead to PTECs dysfunction, potentially inducing renal damage such as cell death and interstitial injury. Further analysis of the effects of this mitochondrial dysfunction on ATP production capacity and reactive oxygen species production is needed.

Mammalian target of rapamycin mediates expression and activity of ADAM 17 in diabetic kidney disease

Purpose: Previous data have illustrated a role for the matrix metalloprotease A Disintegrin And Metalloprotease 17 (ADAM17), known to cleave growth factors and cytokines, in renal cell injury in diabetes. The goal of this study was to identify upstream regulators of ADAM17 in the cascade of events contributing to extracellular matrix accumulation in diabetic nephropathy. Rationale: Diabetic kidney disease is a serious complication faced by type 1 and type 2 diabetic patients alike. Albuminuria and extracellular matrix accumulation are prominent features of the disease and this accumulation of extracellular matrix is a contributing factor to renal fibrosis and decline in renal function. The mechanisms involved in the pathogenesis of diabetic kidney disease have not been completely identified. Methodology: Age and weight-matched Sprague Dawley rats were obtained from Harlan Laboratories (Indianapolis, IN). Type 1 diabetes was induced through tail vein injection of streptozotocin. After rats were determined to be diabetic by blood glucose measurement, rapamycin treatments were administered intraperitoneally for two months. Both kidneys were removed and frozen in liquid nitrogen for microscopy and experimental analyses or formalin fixed for morphometric imaging at the experimental endpoint. Kidney cortex homogenates were used for western blot analyses and enzymatic activity assays. Findings: Using the mTOR complex 1 inhibitor rapamycin, it was determined that increased ADAM17 enzymatic activity and ADAM17 protein expression is dependent on mTORC1 in streptozotocin-induced type 1 diabetic rats. Inhibition of mTORC1 with rapamycin abrogated the increase in collagen IV α 2 protein expression observed in diabetic rat cortex. Additionally, this study is the first to provide evidence that mTOR complex 1 activates ADAM17 contributing to extracellular matrix accumulation in diabetic nephropathy. Studies are continuing looking at the molecular mechanisms involved in mTOR activation of ADAM17 resulting in downstream effects in cultured human proximal tubular epithelial cells. Research reported in this poster was supported by the University of the Incarnate Word Offce of Research and Graduate Studies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Modified Zhenwu Tang delays chronic renal failure progression by modulating oxidative stress and hypoxic responses in renal proximal tubular epithelial cells

BackgroundTubulointerstitial fibrosis (TIF) is a critical pathological feature of chronic renal failure (CRF), with oxidative stress (OS) and hypoxic responses in renal proximal tubular epithelial cells playing pivotal roles in disease progression. This study explores the effects of Modified Zhenwu Tang (MZWT) on these processes, aiming to uncover its potential mechanisms in slowing CRF progression. MethodsWe used adenine (Ade) to induce CRF in rats, which were then treated with benazepril hydrochloride (Lotensin) and MZWT for 8 weeks. Assessments included liver and renal function, electrolytes, blood lipids, renal tissue pathology, OS levels, the hypoxia-inducible factor (HIF) pathway, inflammatory markers, and other relevant indicators. In vitro, human renal cortical proximal tubular epithelial cells were subjected to hypoxia and lipopolysaccharide for 72 h, with concurrent treatment using MZWT, FM19G11, and N-acetyl-l-cysteine. Measurements taken included reactive oxygen species (ROS), HIF pathway activity, inflammatory markers, and other relevant indicators. ResultsAde treatment induced significant disruptions in renal function, blood lipids, electrolytes, and tubulointerstitial architecture, alongside heightened OS, HIF pathway activation, and inflammatory responses in rats. In vivo, MZWT effectively ameliorated proteinuria, renal dysfunction, lipid and electrolyte imbalances, and renal tissue damage; it also suppressed OS, HIF pathway activation, epithelial-mesenchymal transition (EMT) in proximal tubular epithelial cells, and reduced the production of inflammatory cytokines and collagen fibers. In vitro findings demonstrated that MZWT decreased apoptosis, reduced ROS production, curbed OS, HIF pathway activation, and EMT in proximal tubular epithelial cells, and diminished the output of inflammatory cytokines and collagen. ConclusionOS and hypoxic responses significantly contribute to TIF development. MZWT mitigates these responses in renal proximal tubular epithelial cells, thereby delaying the progression of CRF.

Kidney-targeted antioxidant salvianolic acid B nanoparticles restoring lysosome homeostasis for acute kidney injury therapy

Acute kidney injury (AKI) is highly correlated with oxidative stress, and the application of antioxidants is one of the promising treatments. Salvianolic acid B (SAB) is a natural polyphenol with powerful antioxidant effects. Due to its inherent characteristic of inferior bioavailability, its clinical application is impeded. In this study, small SAB-incorporated nanoparticles (SFA) were synthesized via the self-assembly of SAB, ferric ions, and bovine serum albumin (BSA). Apart from antioxidant activity, the SFA nanoparticles were found to efficiently restore lysosome dysfunction induced by reactive oxygen species (ROS) in human kidney proximal tubular epithelial HK-2 cells. The small size and BSA incorporation make the SFA nanoparticles to be promising AKI-targeting nanoparticles. They are rapidly accumulated and long-term retained in injured kidneys of glycerol-induced AKI mice, especially in the proximal tubules which are the key lesion location during AKI. In vivo studies indicate that SFA nanoparticles show a superior protective effect compared with the free drug by suppressing oxidative stress and maintaining lysosome homeostasis indicated by enhanced heme oxygenase-1 (HO-1) expression and restored cathepsin B (CTSB) activity, respectively. In summary, this study develops a delivery system for the antioxidant SAB enhancing local delivery and offering an additional lysosome restoring mechanism for AKI therapy.

ACSF2 and lysine lactylation contribute to renal tubule injury in diabetes.

Lactate accumulation is reported to be a biomarker for diabetic nephropathy progression. Lactate drives lysine lactylation, a newly discovered post-translational modification that is involved in the pathogenesis of cancers and metabolic and inflammatory disease. Here, we aimed to determine whether lysine lactylation is involved in the pathogenesis of diabetic nephropathy. Renal biopsy samples from individuals with diabetic nephropathy (n=22) and control samples from individuals without diabetes and kidney disease (n=9) were obtained from the First Affiliated Hospital of Zhengzhou University for immunohistochemical staining. In addition, we carried out global lactylome profiling of kidney tissues from db/m and db/db mice using LC-MS/MS. Furthermore, we assessed the role of lysine lactylation and acyl-CoA synthetase family member 2 (ACSF2) in mitochondrial function in human proximal tubular epithelial cells (HK-2). The expression level of lysine lactylation was significantly increased in the kidneys of individuals with diabetes as well as in kidneys from db/db mice. Integrative lactylome analysis of the kidneys of db/db and db/m mice identified 165 upregulated proteins and 17 downregulated proteins, with an increase in 356 lysine lactylation sites and a decrease in 22 lysine lactylation sites decreased. Subcellular localisation analysis revealed that most lactylated proteins were found in the mitochondria (115 proteins, 269 sites). We further found that lactylation of the K182 site in ACSF2 contributes to mitochondrial dysfunction. Finally, the expression of ACSF2 was notably increased in the kidneys of db/db mice and individuals with diabetic nephropathy. Our study strongly suggests that lysine lactylation and ACSF2 are mediators of mitochondrial dysfunction and may contribute to the progression of diabetic nephropathy. The LC-MS/MS proteomics data have been deposited in the ProteomeXchange Consortium database ( https://proteomecentral.proteomexchange.org ) via the iProX partner repository with the dataset identifier PXD050070.

Carboxymethylated Desmodium styracifolium polysaccharide reduces the risk of calcium oxalate kidney stone formation by inhibiting crystal adhesion and promoting crystal endocytosis.

The inhibition of cell surface crystal adhesion and an appropriate increase in crystal endocytosis contribute to the inhibition of kidney stone formation. In this study, we investigated the effects of different degrees of carboxymethylation on these processes. An injury model was established by treating human renal proximal tubular epithelial (HK-2) cells with 98.3 ± 8.1 nm calcium oxalate dihydrate (nanoCOD) crystals. The HK-2 cells were protected with carboxy (-COOH) Desmodium styracifolium polysaccharides at 1.17% (DSP0), 7.45% (CDSP1), 12.2% (CDSP2), and 17.7% (CDSP3). Changes in biochemical indexes and effects on nanoCOD adhesion and endocytosis were detected. The protection of HK-2 cells from nanoCOD-induced oxidative damage by carboxymethylated Desmodium styracifolium polysaccharides (CDSPs) is closely related to the protection of subcellular organelles, such as mitochondria. CDSPs can reduce crystal adhesion on the cell surface and maintain appropriate crystal endocytosis, thereby reducing the risk of kidney stone formation. CDSP2 with moderate -COOH content showed the strongest protective activity among the CDSPs.

Depression of LncRNA DANCR alleviates tubular injury in diabetic nephropathy by regulating KLF5 through sponge miR-214-5p

ObjectiveDiabetic nephropathy (DN) manifests a critical aspect in the form of renal tubular injury. The current research aimed to determine the function and mechanism of long non-coding ribonucleic acid (LncRNA) differentiation antagonising non-protein coding RNA (DANCR), with a focus on its impact on renal tubular injury.MethodsQuantitative reverse transcription polymerase chain reaction was employed to analyze the RNA levels of DANCR in the serum of patients with DN or human proximal tubular epithelial cells (human kidney 2 [HK2]). The diagnostic significance of DANCR was assessed using a receiver operating characteristic curve. A DN model was established by inducing HK-2 cells with high glucose (HG). Cell proliferation, apoptosis, and the levels of inflammatory factors, reactive oxygen species (ROS), and malondialdehyde (MDA) were detected using the Cell Counting Kit − 8, flow cytometry, and enzyme-linked immunosorbent assay. The interaction between microRNA (miR)-214-5p and DANCR or Krüppel-like factor 5 (KLF5) was investigated using RNA immunoprecipitation and dual-luciferase reporter assays.ResultsElevated levels of DANCR were observed in the serum of patients with DN and HG-inducted HK-2 cells (P < 0.05). DANCR levels effectively identified patients with DN from patients with type 2 diabetes mellitus. Silencing of DANCR protected against HG-induced tubular injury by restoring cell proliferation, inhibiting apoptosis, and reducing the secretion of inflammatory factors and oxidative stress production (P < 0.05). DANCR functions as a sponge for miR-214-5p, and the mitigation of DANCR silencing on HG-induced renal tubular injury was partially attenuated with reduced miR-214-5p (P < 0.05). Additionally, KLF5 was identified as the target of miR-214-5p.ConclusionDANCR was identified as diagnostic potential for DN and the alleviation of renal tubular injury via the miR-214-5p/KLF5 axis, following DANCR silencing, introduces a novel perspective and approach to mitigating DN.

Nlrp2 deletion ameliorates kidney damage in a mouse model of cystinosis.

Cystinosis is a rare autosomal recessive disorder caused by mutations in the CTNS gene that encodes cystinosin, a ubiquitous lysosomal cystine/H+ antiporter. The hallmark of the disease is progressive accumulation of cystine and cystine crystals in virtually all tissues. At the kidney level, human cystinosis is characterized by the development of renal Fanconi syndrome and progressive glomerular and interstitial damage leading to end-stage kidney disease in the second or third decade of life. The exact molecular mechanisms involved in the pathogenesis of renal disease in cystinosis are incompletely elucidated. We have previously shown upregulation of NLRP2 in human cystinotic proximal tubular epithelial cells and its role in promoting inflammatory and profibrotic responses. Herein, we have investigated the role of NLRP2 in vivo using a mouse model of cystinosis in which we have confirmed upregulation of Nlrp2 in the renal parenchyma. Our studies show that double knock out Ctns-/- Nlrp2-/- animals exhibit delayed development of Fanconi syndrome and kidney tissue damage. Specifically, we observed at 4-6 months of age that animals had less glucosuria and calciuria and markedly preserved renal tissue, as assessed by significantly lower levels of inflammatory cell infiltration, tubular atrophy, and interstitial fibrosis. Also, the mRNA expression of some inflammatory mediators (Cxcl1 and Saa1) and the rate of apoptosis were significantly decreased in 4-6-month old kidneys harvested from Ctns-/- Nlrp2-/- mice compared to those obtained from Ctns-/-mice. At 12-14 months of age, renal histological was markedly altered in both genetic models, although double KO animals had lower degree of polyuria and low molecular weight proteinuria and decreased mRNA expression levels of Il6 and Mcp1. Altogether, these data indicate that Nlrp2 is a potential pharmacological target for delaying progression of kidney disease in cystinosis.

Dehydroandrographolide facilitates M2 macrophage polarization by downregulating DUSP3 to inhibit sepsis-associated acute kidney injury.

Sepsis is perceived as lethal tissue damage and significantly increases mortality in combination with acute kidney injury (AKI). M2 macrophages play important roles in the secretion of anti-inflammatory and tissue repair mediators. We aimed to study the role of Dehydroandrographolide (Deh) in sepsis-associated AKI in vitro and in vivo through lipopolysaccharide (LPS)-induced macrophages model and cecal ligation and puncture-induced AKI mice model, and to reveal the mechanism related to M2 macrophage polarization. Enzyme-linked immunosorbent assay kits were used to assess the levels of inflammatory factors. Expression of markers related to M1 macrophages and M2 macrophages were analyzed. Additionally, dual specificity phosphatase 3 (DUSP3) expression was tested. Cell apoptosis was evaluated by flow cytometry analysis and terminal-deoxynucleotidyl transferase-mediated nick end labeling staining. Moreover, renal histological assessment was performed by using hematoxylin and eosin staining. Deh reduced inflammation of THP-1-derived macrophages exposed to LPS. Besides, Deh induced the polarization of M1 macrophages to M2 and downregulated DUSP3 expression in THP-1-derived macrophages under LPS conditions. Further, DUSP3 overexpression reversed the impacts of Deh on the inflammation and M2 macrophages polarization of THP-1-derived macrophages stimulated by LPS. Additionally, human proximal tubular epithelial cells (HK-2) in the condition medium from DUSP3-overexpressed THP-1-derived macrophages treated with LPS and Deh displayed decreased viability and increased apoptosis and inflammation. The in vivo results suggested that Deh improved the renal function, ameliorated pathological injury, induced the polarization of M1 macrophages to M2, suppressed inflammation and apoptosis, and downregulated DUSP3 expression in sepsis-induced mice. Deh facilitated M2 macrophage polarization by downregulating DUSP3 to inhibit septic AKI.

UHPLC-MS-based untargeted metabolomic strategy to reveal the metabolic differences between cisplatin first- and second-generation apoptotic bodies from HK-2 cells

During the process of programmed cell death (apoptosis), the disassembly of apoptotic cells gives rise to apoptotic bodies (ABs) which play an important role in the pathogenesis of different diseases. ABs from renal human proximal tubular cells HK-2 on naïve cells was not always the same: ABs directly generated after exposure of human proximal tubular epithelial HK-2 cells to chemotherapeutic agent cisplatin (1st generation ABs) triggered apoptosis and inhibited cell proliferation, whereas ABs induced by 1st generation ABs (2nd generation ABs) enhanced HK-2 cell proliferation. To shed light on the mechanisms involved in these disparate ABs’ effects, a study of the changes in the metabolome of 1st and 2nd generation ABs produced by HK-2 cells in the cisplatin setting was performed by a reversed-phase ultra-high-performance liquid chromatography-mass spectrometry (RP-UHPLC-MS) untargeted metabolomic approach. Thus, ABs fluid and extracellular fluid from 1st and 2nd generation ABs were analyzed to obtain as much information as possible of the metabolites of these extracellular vesicles. Principal components analysis and partial least square discriminant analysis were conducted to find the statistically significant differences between both groups. A good clustering of each group was observed and variable importance in the projection values were selected to choose the molecular features which were identified according to the Metabolomics Standard Initiative (MSI) guidelines. Finally, the biological relevance of the achieved results was discussed.

Dexmedetomidine attenuates lipopolysaccharide-induced renal cell fibrotic phenotypic changes by inhibiting necroinflammation via activating α2-adrenoceptor: A combined randomised animal and in vitro study

BackgroundAcute kidney injury (AKI) was reported to be one of the initiators of chronic kidney disease (CKD) development. Necroinflammation may contribute to the progression from AKI to CKD. Dexmedetomidine (Dex), a highly selective α2-adrenoreceptor (AR) agonist, has cytoprotective and “anti-” inflammation effects. This study was designed to investigate the anti-fibrotic properties of Dex in sepsis models. MethodsC57BL/6 mice were randomly treated with an i.p. injection of lipopolysaccharides (LPS) (10 mg/kg) alone, LPS with Dex (25 μg/kg), or LPS, Dex and Atipamezole (Atip, an α2-adrenoreceptor antagonist) (500 μg/kg) (n=5/group). Human proximal tubular epithelial cells (HK2) were also cultured and then exposed to LPS (1 μg/ml) alone, LPS and Dex (1 μM), transforming growth factor-beta 1 (TGF-β1) (5 ng/ml) alone, TGF-β1 and Dex, with or without Atip (100 μM) in culture media. Epithelial-mesenchymal transition (EMT), cell necrosis, necroptosis and pyroptosis, and c-Jun N-terminal kinase (JNK) phosphorylation were then determined. ResultsDex treatment significantly alleviated LPS-induced AKI, myofibroblast activation, NLRP3 inflammasome activation, and necroptosis in mice. Atip counteracted its protective effects. Dex attenuated LPS or TGF-β1 induced EMT and also prevented necrosis, necroptosis, and pyroptosis in response to LPS stimulation in the HK2 cells. The anti-EMT effects of Dex were associated with JNK phosphorylation. ConclusionsDex reduced EMT following LPS stimulation whilst simultaneously inhibiting pyroptosis and necroptosis via α2-AR activation in the renal tubular cells. The “anti-fibrotic” and cytoprotective properties and its clinical use of Dex need to be further studied.

Selenium binding protein 1 protects renal tubular epithelial cells from ferroptosis by upregulating glutathione peroxidase 4

Ferroptosis is a form of programmed cell death involved in various types of acute kidney injury (AKI). It is characterized by inactivation of the selenoprotein, glutathione peroxidase 4 (GPX4), and upregulation of acyl-CoA synthetase long-chain family member 4 (ACSL4). Since urinary selenium binding protein 1 (SBP1/SELENBP1) is a potential biomarker for AKI, this study investigated whether SBP1 plays a role in AKI. First, we showed that SBP1 is expressed in proximal tubular cells in normal human kidney, but is significant downregulated in cases of AKI in association with reduced GPX4 expression and increased ACSL4 expression. In mouse renal ischemia-reperfusion injury (I/R), the rapid downregulation of SBP1 protein levels preceded downregulation of GPX4 and the onset of necrosis. In vitro, hypoxia/reoxygenation (H/R) stimulation in human proximal tubular epithelial (HK-2) cells induced ferroptotic cell death in associated with an acute reduction in SBP1 and GPX4 expression, and increased oxidative stress. Knockdown of SBP1 reduced GPX4 expression and increased the susceptibility of HK-2 cells to H/R-induced cell death, whereas overexpression of SBP1 reduced oxidative stress, maintained GPX4 expression, reduced mitochondrial damage, and reduced H/R-induced cell death. Finally, selenium deficiency reduced GPX4 expression and promoted H/R-induced cell death, whereas addition of selenium was protective against H/R-induced oxidative stress. In conclusion, SBP1 plays a functional role in hypoxia-induced tubular cell death. Enhancing SBP1 expression is a potential therapeutic approach for the treatment of AKI.