Human Tubular Epithelial Cells Research Articles

Quercetin prevented diabetic nephropathy by inhibiting renal tubular epithelial cell apoptosis via the PI3K/AKT pathway.

Diabetic nephropathy (DN) is the most common and serious complication of diabetes, posing a significant threat to human health. Currently, safe and effective preventive strategies for DN are lacking. The study aimed to explore the preventive effect and the underlying mechanism of quercetin against DN. In the in vivo experiments, we established a mouse model of type 2 diabetes mellitus (T2DM) induced by a combination of high-fat diet (HFD) and streptozotocin (STZ) to explore the preventive effect of quercetin on DN and its protective role against renal tubular epithelial cell apoptosis. Subsequently, in vitro experiments using human tubular epithelial cells (HK-2 cells) were conducted to further validate the protective effects of quercetin on renal tubular epithelial cell apoptosis. Additionally, we employed RNA sequencing analysis (RNA-seq) and network pharmacology analysis to comprehensively elucidate the molecular mechanisms involved. In vivo, we observed a significant increase in the ratio of urinary microalbumin to creatinine in diabetic mice compared to control mice, accompanied by the activation of renal tubular epithelial cell apoptosis. Remarkably, all of these changes were reversed after quercetin treatment. In vitro, high-glucose-induced apoptosis in HK-2 cells was significantly attenuated by quercetin. Subsequent RNA sequencing analysis and network pharmacology analysis revealed that quercetin was most likely to inhibit high-glucose-induced HK-2 cell apoptosis through the PI3K/AKT signaling pathway. Western Blotting results further demonstrated that quercetin could inhibit the activation of the PI3K/AKT signaling pathway in HK-2 cells induced by high glucose. Our results supported that quercetin could prevent DN by inhibiting tubular epithelial cell apoptosis via the PI3K/AKT pathway. Quercetin might be a promising candidate for the prevention of DN.

Connexin32 gap junction channels deliver miR155-3p to mediate pyroptosis in renal ischemia-reperfusion injury

ObjectivesTo explore whether the gap junction (GJ) composed by connexin32(Cx32) mediated pyroptosis in renal ischemia-reperfusion(I/R) injury via transmitting miR155-3p, with aim to provide new strategies for the prevention and treatment of acute kidney injury (AKI) after renal I/R.Methods8–10 weeks of male C57BL/ 6 wild-type mice and Cx32 knockdown mice were divided into two groups respectively: control group and renal I/R group. MCC950 (50 mg/kg. ip.) was used to inhibit NLRP3 in vivo. Human kidney tubular epithelial cells (HK - 2) and rat kidney tubular epithelial cells (NRK-52E) were divided into high-density group and low-density group, and treated with hypoxia reoxygenation (H/R) to mimic I/R. The siRNA and plasmid of Cx32, mimic and inhibitor of miR155-3p were transfected into HK - 2 cells respectively. Kidney pathological and functional injuries were measured. Western Blot and immunofluorescent staining were used to observe the expression of NLRP3, GSDMD, GSDMD-N, IL - 18, and mature IL-18. The secretion of IL-18 and IL-1β in serum, kidney tissue and cells supernatant were detected by enzyme-linked immuno sorbent assay (ELISA) kit, and the expression of NLPR3 and miR155-3p were detected by RT-qPCR and fluorescence in situ hybridization (FISH).ResultsTubular pyroptosis were found to promote AKI after I/R in vivo and Cx32-GJ regulated pyroptosis by affecting the expression of miR155-3p after renal I/R injury. In vitro, H/R could lead to pyroptosis in HK-2 and NRK-52E cells. When the GJ channels were not formed, and Cx32 was inhibited or knockdown, the expression of miR155-3p was significantly reduced and the pyroptosis was obviously inhibited, leading to the reduction of injury and the increase of survival rate. Moreover, regulating the level of miR155-3p could affect survival rate and pyroptosis in vitro after H/R.ConclusionsThe GJ channels composed of Cx32 regulated tubular pyroptosis in renal I/R injury by transmitting miR155-3p. Inhibition of Cx32 could reduce the level of miR155-3p further to inhibit pyroptosis, leading to alleviation of renal I/R injury which provided a new strategy for preventing the occurrence of AKI.-tWJY53zy_-Wc_anQ2Z7vvVideo

Sacubitril/valsartan ameliorates tubulointerstitial fibrosis by restoring mitochondrial homeostasis in diabetic kidney disease

BackgroundTubulointerstitial fibrosis plays an important role in the progression of diabetic kidney disease (DKD). Sacubitril/valsartan (Sac/Val) exerts a robust beneficial effect in DKD. However, the potential functional effect of Sac/Val on tubulointerstitial fibrosis in DKD is still largely unclear.MethodsStreptozotocin-induced diabetic mice were given Sac/Val or Val by intragastric administration once a day for 12 weeks. The renal function, the pathological changes of tubule injury and tubulointerstitial fibrosis, as well as mitochondrial morphology of renal tubules in mice, were evaluated. Genome-wide gene expression analysis was performed to identify the potential mechanisms. Meanwhile, human tubular epithelial cells (HK-2) were cultured in high glucose condition containing LBQ657/valsartan (LBQ/Val). Further, mitochondrial functions and Sirt1/PGC1α pathway of tubular epithelial cells were assessed by Western blot, Real-time-PCR, JC-1, MitoSOX or MitoTracker. Finally, the Sirt1 specific inhibitor, EX527, was used to explore the potential effects of Sirt1 signaling in vivo and in vitro.ResultsWe found that Sac/Val significantly ameliorated the decline of renal function and tubulointerstitial fibrosis in DKD mice. The enrichment analysis of gene expression indicated metabolism as an important modulator in DKD mice with Sac/Val administration, in which mitochondrial homeostasis plays a pivotal role. Then, the decreased expression of Tfam and Cox IV;, as well as changes of mitochondrial function and morphology, demonstrated the disruption of mitochondrial homeostasis under DKD conditions. Interestingly, Sac/Val administration was found to restore mitochondrial homeostasis in DKD mice and in vitro model of HK-2 cells. Further, we demonstrated that Sirt1/PGC1α, a crucial pathway in mitochondrial homeostasis, was activated by Sac/Val both in vivo and in vitro. Finally, the beneficial effects of Sac/Val on mitochondrial homeostasis and tubulointerstitial fibrosis was partially abolished in the presence of Sirt1 specific inhibitor.ConclusionsTaken together, we demonstrate that Sac/Val ameliorates tubulointerstitial fibrosis by restoring Sirt1/PGC1α pathway-mediated mitochondrial homeostasis in DKD, providing a theoretical basis for delaying the progression of DKD in clinical practice.

Isorhamnetin alleviates cisplatin-induced acute kidney injury via enhancing fatty acid oxidation

Cisplatin is an effective chemotherapy drug widely used in the treatment of various solid tumors. However, the clinical usage of cisplatin is limited by its nephrotoxicity. Isorhamnetin, a natural flavanol compound, displays remarkable pharmacological effects, including anti-inflammatory and anti-oxidation. In this study, we aimed to investigate the potential of isorhamnetin in alleviating acute kidney injury induced by cisplatin. In vitro study showed that isorhamnetin significantly suppressed the cytotoxic effects of cisplatin on human tubular epithelial cells. Furthermore, isorhamnetin exerted significantly inhibitory effects on cisplatin-induced apoptosis and inflammatory response. In acute kidney injury mice induced by a single intraperitoneal injection with 20 mg/kg cisplatin, oral administration of isorhamnetin two days before or 2 h after cisplatin injection effectively ameliorated renal function and renal tubule injury. Transcriptomics RNA-seq analysis of the mice kidney tissues suggested that isorhamnetin treatment may protect against cisplatin-induced nephrotoxicity via PGC-1α mediated fatty acid oxidation. Isorhamnetin achieved significant enhancements in the lipid clearance, ATP level, as well as the expression of PGC-1α and its downstream target genes PPARα and CPT1A, which were otherwise impaired by cisplatin. In addition, the protection effects of isorhamnetin against cisplatin-induced nephrotoxicity were abolished by a PGC-1α inhibitor, SR-18292. In conclusion, our findings indicate that isorhamnetin could protect against cisplatin-induced acute kidney injury by inducing PGC-1α-dependent reprogramming of fatty acid oxidation, which highlights the clinical potential of isorhamnetin as a therapeutic approach for the management of cisplatin-induced nephrotoxicity.

Ascorbate protects human kidney organoids from damage induced by cell-free hemoglobin.

Sepsis-associated acute kidney injury is associated with high morbidity and mortality in critically ill patients. Cell-free hemoglobin (CFH) is released into the circulation of patients with severe sepsis and the levels of CFH are independently associated with mortality. CFH treatment increased cytotoxicity in the human tubular epithelial cell line HK-2. To better model the intact kidney, we cultured human kidney organoids derived from induced pluripotent stem cells. We treated human kidney organoids grown using both three-dimensional and transwell protocols with CFH for 48 h. We found evidence for increased tubular toxicity, oxidative stress, mitochondrial fragmentation, endothelial cell injury and injury-associated transcripts compared to those of the untreated control group. To evaluate the protective effect of clinically available small molecules, we co-treated CFH-injured organoids with ascorbate (vitamin C) or acetaminophen for 48 h. We found significantly decreased toxicity, preservation of endothelial cells and reduced mitochondrial fragmentation in the group receiving ascorbate following CFH treatment. This study provides direct evidence that ascorbate or ascorbic acid protects human kidney cells from CFH-induced damage such as that in sepsis-associated acute kidney injury.

The Protective Role of Tubular Epithelial Cells Produced Fms-like Tyrosine Kinase Receptor 3 Ligand in Acute Kidney Injury

Introduction: Dendritic cells (DC) accumulate in the kidneys of patients with acute kidney injury (AKI), but the Fms-like tyrosine kinase receptor 3 ligand (Flt3L)-dendritic cell axis is poorly defined in kidneys with AKI. Meanwhile it has not been reported whether Flt3L is produced by kidney cells under ischemic-reperfusion injury (IR) induced microenvironment. Methods: (1) Blood specimens and urine were collected from two patient cohorts: a) patients with pre-renal AKI and healthy controls for analysis of serum Flt3L. b) patients with acute myocardial infarction (AMI) developed with or without AKI on admission for analysis of serum and urine Flt3L. (2) Wild type mice and T cell-deficient mice ( Tcra -/- mice) were applied. IR induced AKI mice and hypoxia-reoxygenation stimulated HK-2 cell line (human tubular epithelial cell line) were constructed. The expression of Flt3L in kidney, tubular epithelial cells, and T cells were measured by RT-PCR, ELISA, immunohistochemistry, immunofluorescence staining, RNAscope, and flow cytometry. (3) Wild type mice and type I conventional dendritic cell (cDC1)-deficient mice ( Irf8 -/- mice) were treated with 15 μg rFlt3L or 10mg/kg gilteritinib (Flt3 inhibitor). They were subjected to IR. Serum and kidney tissues were collected. Kidney function, tubular injury, and DC accumulation were quantified. Results: Initially, among patients with pre-renal AKI, we observed an increased level of serum Flt3L within two days after admission. Another patient cohort revealed that this increase was specific for AMI patients with AKI rather than without AKI or healthy controls. Furthermore, among these AMI patients, serum Flt3L levels correlated with Creatinine increase (R 2 = 0.4532, p = 0.0002) and BUN increase (R 2 = 0.3417, p = 0.0017) within 48 hours after admission. Similarly, urine Flt3L levels among these AMI patients also steadily elevated with Creatinine increase (R 2 = 0.6395, p < 0.0001) and BUN increase (R 2 = 0.5115, p = < 0.0001) within 48 hours after admission. Flt3L was also enhanced in wild type mice with IR-AKI but not affected in T cell-deficient mice ( Tcra -/-) with IR-AKI. Among kidney tissue, Flt3L-positive cells scattered within tubules in outer stripe of the outer medulla (OSOM) and mRNA and protein increased within 24 hours after IR-AKD. In vitro, FLT3L mRNA was markedly induced in HK-2 cells during hypoxia and FLT3L protein remained enhanced during reoxygenation. Phenotypically, the numbers of renal cDC1 and CD64 +DC were significantly decreased in gilteritinib-treated IR-AKI mice, which were associated with more severe tubular injury. With reduced kidney cDC1, Irf8 -/- mice also showed worsen kidney injury and aggravated functional failure upon IR-AKI. Therapeutic administration of rFlt3L significantly increased kidney cDC1 and CD64 +DC upon IR-AKI in wild type but not Irf8 -/- mice. This was associated with significantly reduced tubular injury of wild type mice .In addition, pre-treatment of wild type mice with rFlt3L increased kidney cDC1 and protected the mice from severe IR-AKI. Conclusion: Flt3L is upregulated in human and mice and produced by tubular epithelial cells during IR-AKI. It fosters the accumulation of kidney cDC1 and CD64 +DC, thereby limiting the severity of kidney injury in mice. The current study implies the possibility of DC-based immunotherapy for treatment of AKD.

Circ_0068087 knockdown attenuates high-glucose-induced human tubular epithelial cell injury in a microribonucleic acid/progestin and adipoQ receptor 3-dependent manner in diabetic nephropathy.

Previous studies have shown that circular ribonucleic acid mediates the occurrence of diabetic nephropathy. This study aimed to analyze the effects of circ_0068087 on high-glucose (HG)-induced human kidney 2 (HK2) cell dysfunction. Circ_0068087, miR-580-3p, and progestin and adipoQ receptor 3 (PAQR3) expression were detected by quantitative reverse transcription polymerase chain reaction. Cell viability and proliferation were investigated by Cell Counting Kit-8 and EdU assays, respectively. The cell apoptotic rate was assessed by flow cytometry. Inflammatory response was assessed by enzyme-linked immunoassays. Oxidative stress was evaluated by a superoxide dismutase activity assay kit and lipid peroxidation malondialdehyde assay kit. Molecular interaction was identified by dual-luciferase reporter assay. Circ_0068087 and PAQR3 expression were significantly upregulated in diabetic nephropathy patients. HG treatment inhibited HK2 cell proliferation, but induced cell apoptosis, inflammation, oxidative stress and epithelial-mesenchymal transition by regulating circ_0068087. Circ_0068087 acted as a microribonucleic acid-580-3p (miR-580-3p) sponge, and miR-580-3p targeted PAQR3. Furthermore, circ_0068087 depletion repressed PAQR3 expression through miR-580-3p. MiR-580-3p inhibitors or PAQR3 introduction attenuated circ_0068087 silencing mediated-effects in HG-treated HK2 cells. Circ_0068087 promoted HG-induced HK2 cell injuries by the regulation of the miR-580-3p/PAQR3 pathway.

Krüppel-like Factor 15 Suppresses Ferroptosis by Activating an NRF2/GPX4 Signal to Protect against Folic Acid-Induced Acute Kidney Injury.

Acute kidney injury (AKI) is a common and serious disease with high morbidity and mortality, and its pathophysiological mechanisms are not fully understood. Increasing evidence suggests an important role of ferroptosis in AKI. Krüppel-like factor 15 (KLF15) is a transcription factor involved in several metabolic diseases, but its role in AKI and ferroptosis remains unclear. In this study, we explored the potential role of KLF15 using a folic acid-induced AKI model. Our study showed that KLF15 expression was reduced in kidney tissues of AKI mice, and KLF15 knockout exacerbated folic acid-induced ferroptosis and kidney injury. In vitro studies revealed that the ferroptosis inducer erastin significantly suppressed KLF15 expression in human tubular epithelial cells. Notably, the overexpression of KLF15 attenuated ferroptosis, as evidenced by a decrease in the lipid peroxidation marker of malondialdehyde and the upregulation of glutathione peroxidase 4 (GPX4), while KLF15 knockdown with shRNA exerted the opposite effect. Mechanistically, KLF15 stabilized the protein of nuclear factor erythroid 2-related factor 2 (NRF2) and subsequently increased the GPX4 level. Collectively, KLF15 plays an important role in the modulation of ferroptosis in AKI and may be a potential therapeutic target for treating AKI.

Carnosine alleviates cisplatin-induced acute kidney injury by targeting Caspase-1 regulated pyroptosis

Acute kidney injury (AKI) is a syndrome characterized by rapid loss of renal excretory function. Its underlying mechanisms remain unclear. Pyroptosis, a form of programmed cell death, plays an important role in AKI. It is characterized by cell swelling and membrane rupture, triggering the release of cellular contents and activating robust inflammatory responses. Carnosine, a dipeptide with antioxidant and anti-inflammatory properties, has therapeutic effects in AKI. However, the mechanism by which carnosine treats AKI-associated pyroptosis remains unexplored. In this study, we investigated the protective effect of carnosine on renal tubule cells using in vivo and in vitro models of AKI. We found that carnosine therapy significantly alleviated altered serum biochemical markers and histopathological changes in mice with cisplatin-induced AKI. It also reduced the levels of inflammation and pyroptosis. These results were consistent with those seen in human kidney tubular epithelial cells (HK-2) treated with cisplatin. Through molecular docking and cellular thermal shift assay, we identified caspase-1 as a target of carnosine. By knocking down caspase-1 in HK-2 cells using caspase-1 siRNA, we demonstrated that carnosine did not exhibit a protective role in cisplatin-induced HK-2 cells. This study provides the first evidence that carnosine alleviates damage to kidney tubular epithelial cells by targeting caspase-1 and inhibiting pyroptosis. Therefore, carnosine holds promise as a potential therapeutic agent for AKI, with caspase-1 representing an effective therapeutic target in this pathology.

Differences in the Susceptibility of Human Tubular Epithelial Cells for Infection with Orthohantaviruses.

Diseases induced by infection with pathogenic orthohantaviruses are characterized by a pronounced organ-specific manifestation. Pathogenic Eurasian orthohantaviruses cause hemorrhagic fever with renal syndrome (HFRS) with often massive proteinuria. Therefore, the use of a relevant kidney cell culture would be favorable to analyze the underlying cellular mechanisms of orthohantavirus-induced acute kidney injury (AKI). We tested different human tubular epithelial cell lines for their suitability as an in vitro infection model. Permissiveness and replication kinetics of highly pathogenic Hantaan virus (HTNV) and non-/low-pathogenic Tula virus (TULV) were analyzed in tubular epithelial cell lines and compared to human primary tubular epithelial cells. Ana-lysis of the cell line HK-2 revealed the same results for viral replication, morphological and functional effects as observed for HTNV in primary cells. In contrast, the cell lines RPTEC/TERT1 and TH1 demonstrated only poor infection rates after inoculation with HTNV and are unusable as an infection model. While pathogenic HNTV infects primary tubular and HK-2 cells, non-/low-pathogenic TULV infects neither primary tubular cells nor the cell line HK-2. Our results show that permissiveness of renal cells varies between orthohantaviruses with differences in pathogenicity and that HK-2 cells demonstrate a suitable in vitro model to study viral tropism and pathogenesis of orthohantavirus-induced AKI.

Reciprocal regulation between the molecular clock and kidney injury.

Tubulointerstitial fibrosis is the common pathological substrate for many etiologies leading to chronic kidney disease. Although perturbations in the circadian rhythm have been associated with renal disease, the role of the molecular clock in the pathogenesis of fibrosis remains incompletely understood. We investigated the relationship between the molecular clock and renal damage in experimental models of injury and fibrosis (unilateral ureteral obstruction, folic acid, and adenine nephrotoxicity), using genetically modified mice with selective deficiencies of the clock components Bmal1, Clock, and Cry We found that the molecular clock pathway was enriched in damaged tubular epithelial cells with marked metabolic alterations. In human tubular epithelial cells, TGFβ significantly altered the expression of clock components. Although Clock played a role in the macrophage-mediated inflammatory response, the combined absence of Cry1 and Cry2 was critical for the recruitment of neutrophils, correlating with a worsening of fibrosis and with a major shift in the expression of metabolism-related genes. These results support that renal damage disrupts the kidney peripheral molecular clock, which in turn promotes metabolic derangement linked to inflammatory and fibrotic responses.

The therapeutic potential of an allosteric non-competitive CXCR1/2 antagonist for diabetic nephropathy.

Diabetic nephropathy is a major consequence of inflammation developing in type 1 diabetes, with interleukin-8 (IL-8)-CXCR1/2 axis playing a key role in kidney disease progression. In this study, we investigated the therapeutic potential of a CXCR1/2 non-competitive allosteric antagonist (Ladarixin) in preventing high glucose-mediated injury in human podocytes and epithelial cells differentiated from renal stem/progenitor cells (RSC) cultured as nephrospheres. We used human RSCs cultured as nephrospheres through a sphere-forming functional assay to investigate hyperglycemia-mediated effects on IL-8 signalling in human podocytes and tubular epithelial cells. High glucose impairs RSC self-renewal, induces an increase in IL-8 transcript expression and protein secretion and induces DNA damage in RSC-differentiated podocytes, while exerting no effect on RSC-differentiated epithelial cells. Accordingly, the supernatant from epithelial cells or podocytes cultured in high glucose was able to differentially activate leucocyte-mediated secretion of pro-inflammatory cytokines, suggesting that the crosstalk between immune and non-immune cells may be involved in disease progression in vivo. Treatment with Ladarixin during RSC differentiation prevented high glucose-mediated effects on podocytes and modulated either podocyte or epithelial cell-dependent leucocyte secretion of pro-inflammatory cytokines, suggesting CXCR1/2 antagonists as possible pharmacological approaches for the treatment of diabetic nephropathy.

Carnosine alleviates kidney tubular epithelial injury by targeting NRF2 mediated ferroptosis in diabetic nephropathy.

Diabetic nephropathy (DN) can promote the occurrence of end-stage renal disease (ESRD). The injury of renal tubular epithelial cells is a significant reason for the occurrence of ESRD. A recent research demonstrated that ferroptosis was associated with renal tubular injury in DN. Ferroptosis is a kind of cell death brought on by the buildup of iron ions and lipid peroxidation brought on by ROS. Because carnosine (CAR) is a scavenger of iron ions and reactive oxygen species, we investigated whether CAR can improve DN by regulating ferroptosis. The results show that both CAR and Fer-1 significantly reduced kidney damage and inhibited ferroptosis in STZ mice. In addition, ferroptosis caused by HG or erastin (an inducer of ferroptosis) in human kidney tubular epithelial cell (HK2) was also rescued by CAR treatment. It was discovered that the protective effect of CAR against HG-induced ferroptosis was abolished when NRF2 was specifically knocked down in HK2 cells.

Baicalein ameliorates polymyxin B-induced acute renal injury by inhibiting ferroptosis via regulation of SIRT1/p53 acetylation

The polypeptide antibiotic Polymyxin B (PMB) can cause acute kidney injury (AKI), we found that ferroptosis is one of the main mechanisms of renal injury caused by PMB. It was reported that baicalein can inhibit ferroptosis. Therefore, in this study we examined whether baicalein could attenuate PMB-induced renal injury by inhibiting ferroptosis. We confirmed that baicalein could reduce PMB-induced renal injury in vivo and in vitro studies. In the in vitro study, baicalein significantly increased the survival rate of human HK2 tubular epithelial cells. The results of HE staining and electron microscopy in mice also showed that baicalein reduced PMB-induced renal injury, and significantly decreased the levels of BUN and Scr. By detecting ferroptosis-related indicators, we found that pre-incubation of baicalein in HK2 cells down-regulated Fe2+ level, lipid peroxidation, MDA and HO-1 which had been increased by PMB. Furthermore, baicalein up-regulated the levels of SCL7A11, GPX4 and GSH that were decreased by PMB. Moreover, intraperitoneal injection of baicalein in the animal model down-regulated kidney iron level, PTGS2 and 4HNE, and increased the GSH level, which suggested that baicalein could inhibit PMB-induced ferroptosis. Finally, by detecting changes in levels of p53 and p53 K382 acetylation, baicalein was observed to decrease elevated p53 K382 acetylation after PMB treatment, further confirming that baicalein inhibits ferroptosis by reducing p53 K382 acetylation via upregulation of SIRT1 expression. In conclusion, these results suggest that baicalein decreases p53 acetylation level by elevating SIRT1, which can then inhibit PMB-induced ferroptosis and ultimately attenuates AKI.

#4419 THE IMPACT OF TRANSGLUTAMINASE 2 INHIBITION ON HYPERTENSION-INDUCED FIBROSIS IN HYPERTENSIVE NEPHROPATHY

Abstract Background and Aims Hypertensive nephropathy is a chronic disease that requires the novel treatment. Transglutaminase 2 (TG2), linked to various diseases including cardiovascular and kidney diseases, has been suggested to play a role in the development of hypertensive nephropathy through its contribution to fibrous tissue formation, oxidative stress regulation, and inflammation. This study aimed to explore the role of TG2 in the development of hypertensive nephropathy and evaluate its potential as a therapeutic target. Method An in vitro hypertensive model was established using a device that mimics hypertension by applying rotational force. An in vivo hypertensive model was established in rats through subtotal 5/6 nephrectomy to induce fibrosis. The relationship between TG2 expression and the progression of hypertension-induced fibrosis was analyzed, and the impact of TG2 inhibition on fibrosis was evaluated using the TG2 inhibitor, cysteamine. Results In the in vitro study, rotational force was applied to induce hypertension using a hypertensive mimic device, which resulted in fibrosis in primary human glomerular endothelial cells and tubular epithelial cells and increased expression of TG2. The administration of cysteamine at concentrations of 0.5 mM, 1 mM, and 2 mM decreased fibrosis and phosphorylated P65 in a dose-dependent manner. The in vivo hypertensive model demonstrated a significant increase in blood pressure (P<0.001) and kidney fibrosis (P<0.001) over time at 4 and 8 weeks after surgery. A significant increase in TG2 expression was observed as hypertension and fibrosis progressed (P<0.001). Conclusion The progression of hypertension-induced fibrosis was accompanied by an increase in TG2 expression. Inhibition of TG2 showed improvement in fibrosis, suggesting further study of its mechanism may provide new therapeutic options for hypertensive nephropathy.

A myogenic program is enacted by cardiac protein CSRP3 in human and mouse kidney

Introduction: Cardiac injury with renal injury (acute cardiorenal syndrome) can induce renal fibrosis, but mechanisms by which heart injury signals to kidney are poorly understood. The myocardiocyte differentiation factor cardiac LIM protein (CSRP3) is released into plasma after cardiac injury, and taken up by megalin (Lrp2) in renal proximal tubular epithelial cells in vivo. We evaluated the relationship between cardiac injury and renal fibrosis in a mouse model and in human cells by characterizing novel primary culture of human tubular epithelial cells and then testing the hypothesis that CSRP3 alters PTEC transcription of myogenesis, transport, and extracellular matrix genes in vitro and in vivo. Methods: In mice, cardiac arrest (CA) was induced with potassium chloride and resuscitated with epinephrine and chest compressions (CPR) as previously described. SnRNAseq was conducted on mouse kidney cells harvested 24h after CA/CPR and analyzed using Seurat. Research-designated human kidneys were obtained under IRB exemption for non-human subjects research and underwent dissection, digestion and FACS sorting for PTEC selection. hPTEC were passaged up to 4x before transporters were characterized by qPCR, immunoblot, and immunofluorescence. Recombinant human CSRP3 (Origene) was administered to hPTEC followed by RNASeq and qPCR. CSRP3 (5μg) was retroorbitally injected in mice and renal fibrosis was assessed 7 weeks later. Results: In mice, snRNAseq demonstrated that CA/CPR significantly upregulated muscle cell differentiation ontologies in proximal tubule cells, driven by regulation of 110/162 myogenesis genes. Following FACS sorting for CD10 and CD13, hPTEC were 95% pure PTEC and expressed RNA and protein for PT transporters Lrp2, Aqp1, Slc34a1, and Slc5a2. In hPTEC, CSRP3 induced myogenesis differentiation factor 1 (MyoD1)>30 fold, and 20 myogenesis pathway and extracellular matrix genes. 8/20 genes were coordinately regulated between mouse kidney after CA/CPR and hPTEC after CSRP3. CSRP3 injection caused tubulointerstitial fibrosis in mice (V αSMA /V kidney (%): 1.05 (CSRP3) vs 0.68 (control), n=8/group, p<0.01). Conclusion: hPTEC cultured from research-designated donor kidneys express a PT phenotype. The myocardiocyte differentiation factor CSRP3 causes transcriptional change in hPTEC, resulting in myogenic differentiation which is highly similar to that observed in mouse kidney after CA/CPR with coordinate regulation of myogenesis genes. Intravenous administration of CSRP3 to mice causes tubular interstitial fibrosis. Together these data strongly suggest CSRP3 may drive renal fibrosis via the myogenesis pathway. Merit grant I01BX004288-01 This is the full abstract presented at the American Physiology Summit 2023 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.

Hypoxia induces polycystin-1 expression in the renal epithelium.

Mutations in polycystin-1 which is encoded by the PKD1 gene are the main causes for the development of autosomal dominant polycystic kidney disease. However, only little is known about the physiological function of polycystin-1 and even less about the regulation of its expression. Here, we show that expression of PKD1 is induced by hypoxia and compounds that stabilize the hypoxia-inducible transcription factor (HIF) 1α in primary human tubular epithelial cells. Knockdown of HIF subunits confirms HIF-1α-dependent regulation of polycystin-1 expression. Furthermore, HIF ChIP-seq reveals that HIF interacts with a regulatory DNA element within the PKD1 gene in renal tubule-derived cells. HIF-dependent expression of polycystin-1 can also be demonstrated in vivo in kidneys of mice treated with substances that stabilize HIF. Polycystin-1 and HIF-1α have been shown to promote epithelial branching during kidney development. In line with these findings, we show that expression of polycystin-1 within mouse embryonic ureteric bud branches is regulated by HIF. Our finding links expression of one of the main regulators of accurate renal development with the hypoxia signalling pathway and provides additional insight into the pathophysiology of polycystic kidney disease.

Inhibition of N-glycosylation by glucosamine hydrochloride inhibits TGF-β1-induced LOXL2 secretion.

Kidney fibrosis is closely associated with the progression of chronic kidney disease (CKD). Furthermore, copper-containing secretory amine oxidases, such as lysyl oxidase (LOX) and LOX-like 1-4 (LOXL1-4), play pivotal roles in the regulation of extracellular components and facilitate fibrosis. In this study, we investigated the regulation of LOX enzymes in human tubular epithelial HK2 cells to help clarify the role of LOX enzymes in kidney fibrosis. Among 5 LOX enzymes, LOXL2 expression is abundantly expressed in HK2 cells. LOX enzymes inhibitor, β-aminopropionitrile, suppressed transforming growth factor-β1 (TGF-β1)-promoted epithelial-to-mesenchymal transition processes in HK2 cells, indicating that LOX enzymes are involved in TGF-β1-mediated fibrotic processes. Recent studies suggest that LOX enzymes are secreted into the extracellular spaces by exosomes and promote fibrotic processes. Similar to the previous reports, we observed that exosomes secreted from HK2 cells carry LOXL2 into the extracellular spaces. Furthermore, we determined that N-glycosylation on the asparagine residues plays a key role in LOXL2 secretion. Amino acid mutations in three asparagine residues, which can be glycosylated, suppressed the secretion of mutated LOXL2. Moreover, N-acetylglucosaminyltransferase 5, an enzyme used for the biosynthesis of β1,6N-acetylglucosamine-branched N-glycans, participated in LOXL2 secretion, and the N-glycosylation inhibitor, glucosamine hydrochloride (GS), inhibited TGF-β1-mediated LOXL2 secretion and fibrotic processes. Overall, TGF-β1 promotes LOXL2 secretion and may participate in kidney fibrosis. Our results provide novel insight into the antifibrotic properties of GS that contribute to the inhibition of CKD progression.

Metabolomic profiling in kidney cells treated with a sodium glucose-cotransporter 2 inhibitor

We aimed to determine the metabolomic profile of kidney cells under high glucose conditions and following sodium-glucose cotransporter 2 (SGLT2) inhibitor treatment. Targeted metabolomics using the Absolute IDQ-p180 kit was applied to quantify metabolites in kidney cells stimulated with high glucose (25 and 50 mM) and treated with SGLT2 inhibitor, dapagliflozin (2 µM). Primary cultured human tubular epithelial cells and podocytes were used to identify the metabolomic profile in high glucose conditions following dapagliflozin treatment. The levels of asparagine, PC ae C34:1, and PC ae C36:2 were elevated in tubular epithelial cells stimulated with 50 mM glucose and were significantly decreased after 2 µM dapagliflozin treatment. The level of PC aa C32:0 was significantly decreased after 50 mM glucose treatment compared with the control, and its level was significantly increased after dapagliflozin treatment in podocytes. The metabolism of glutathione, asparagine and proline was significantly changed in tubular epithelial cells under high-glucose stimulation. And the pathway analysis showed that aminoacyl-tRNA biosynthesis, arginine and proline metabolism, glutathione metabolism, valine, leucine and isoleucine biosynthesis, phenylalanine, tyrosine, and tryptophan biosynthesis, beta-alanine metabolism, phenylalanine metabolism, arginine biosynthesis, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism were altered in tubular epithelial cells after dapagliflozin treatment following 50 mM glucose compared to those treated with 50 mM glucose.

Rapamycin Does Not Act as a Dietary Restriction Mimetic in the Protection against Ischemia Reperfusion Injury

Introduction: Short-term fasting protects against renal ischemia reperfusion injury (IRI). mTOR signaling is downregulated and may be involved in its protective effect. Rapamycin is considered a possible mimetic as it inhibits the mTOR pathway. This study examines the effect of rapamycin on renal IRI. Material and Methods: Mice were divided into four groups: ad libitum (AL), fasted (F), AL treated with rapamycin (AL+R), and F treated with rapamycin (F+R). Rapamycin was administered intraperitoneally 24 h before bilateral renal IRI was induced. Survival was monitored for 7 days. Renal cell death, regeneration, and mTOR activity were determined 48 h after reperfusion. Oxidative stress resistance of human renal proximal tubular and human primary tubular epithelial cells after rapamycin treatment was determined. Results: All F and F+R mice survived the experiment. Although rapamycin substantially downregulated mTOR activity, survival in the AL+R group was similar to AL (10%). Renal regeneration was significantly reduced in AL+R but not in F+R. After IRI (48 h), pS6K/S6K ratio was lower in F, F+R, and AL+R groups compared to AL fed animals (p = 0.02). In vitro, rapamycin also significantly downregulated mTOR activity (p < 0.001) but did not protect against oxidative stress. Conclusion: Rapamycin pretreatment does not protect against renal IRI. Thus, protection against renal IRI by fasting is not exclusively mediated through inhibition of mTOR activity but may involve preservation of regenerative mechanisms despite mTOR downregulation. Therefore, rapamycin cannot be used as a dietary mimetic to protect against renal IRI.