Human Proximal Tubular Cell Line Research Articles

Mitochondrial calcium uniporter promotes kidney aging in mice through inducing mitochondrial calcium-mediated renal tubular cell senescence.

Renal tubular epithelial cell senescence plays a critical role in promoting and accelerating kidney aging and age-related renal fibrosis. Senescent cells not only lose their self-repair ability, but also can transform into senescence-associated secretory phenotype (SASP) to trigger inflammation and fibrogenesis. Recent studies show that mitochondrial dysfunction is critical for renal tubular cell senescence and kidney aging, and calcium overload and abnormal calcium-dependent kinase activities are involved in mitochondrial dysfunction-associated senescence. In this study we investigated the role of mitochondrial calcium overload and mitochondrial calcium uniporter (MCU) in kidney aging. By comparing the kidney of 2- and 24-month-old mice, we found calcium overload in renal tubular cells of aged kidney, accompanied by significantly elevated expression of MCU. In human proximal renal tubular cell line HK-2, pretreatment with MCU agonist spermine (10 μM) significantly increased mitochondrial calcium accumulation, and induced the production of reactive oxygen species (ROS), leading to renal tubular cell senescence and age-related kidney fibrosis. On the contrary, pretreatment with MCU antagonist RU360 (10 μM) or calcium chelator BAPTA-AM (10 μM) diminished D-gal-induced ROS generation, restored mitochondrial homeostasis, retarded cell senescence, and protected against kidney aging in HK-2 cells. In a D-gal-induced accelerated aging mice model, administration of BAPTA (100 μg/kg. i.p.) every other day for 8 weeks significantly alleviated renal tubuarl cell senescence and fibrosis. We conclude that MCU plays a key role in promoting renal tubular cell senescence and kidney aging. Targeting inhibition on MCU provides a new insight into the therapeutic strategy against kidney aging.

#1453 EZH2 is an epigenetic regulator of renal gluconeogenesis in chronic kidney disease through downregulation of PCK1

Abstract Background and Aims Impaired renal gluconeogenesis has been recently identified as a hallmark of chronic kidney diseases (CKD), and is tightly correlated with the progression of renal tubulointerstitial fibrosis. The methyltransferase enhancer of zeste homolog 2 (EZH2) is an epigenetic regulator playing an important role in renal tubulointerstitial fibrosis. Whether renal gluconeogenesis can be epigenetically regulated is currently unknown. In the current study, we studied EZH2-mediated epigenetic regulation of renal gluconeogenesis in three different mouse models of renal fibrosis. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation, unilateral ischemia reperfusion injury or folic acid (FA) injection. Human proximal tubular cell line (HK2), primary tubular cells or primary tubules was used as an in vitro or ex vivo model. EZH2 was conditional knockout or inhibited by 3-DZNeP in mice. Inhibition of EZH2 by 3-DZNeP or overexpressed by adenovirus was performed in vitro. RNA sequencing and cleavage under targets and tagmentation (CUT&Tag) sequencing analysis were performed and followed by quantitative PCR, CUT&Tag and Western blotting analysis. Glucogenic metabolites were measured and systemic glucose metabolism was assessed by tolerance tests. Results By deletion of Ezh2 gene in mice through a ubiquitous expressed Cre/Esr1 system, we confirmed the pro-fibrotic effect of EZH2 in unilateral ureteral obstruction (UUO) kidneys. Through RNA sequencing and CUT&Tag sequencing analysis, we found that the phosphoenolpyruvate carboxykinase 1 (PCK1), a critical enzyme in gluconeogenesis, is negatively regulated by the methyltransferase EZH2 in fibrotic kidneys, which was further confirmed by quantitative PCR, CUT&Tag and Western blotting analysis in renal cells and UUO or FA kidneys. We further showed that pharmaceutical inhibition of EZH2 by 3-DZNeP enhances PCK1 expression in fibrotic kidneys of three mouse models. Moreover, the concentration of lactate, a glucogenic metabolic intermediate, was increased in mouse fibrotic kidneys, which was reduced by EZH2 inhibition. A further experiment showed that systemic glucose metabolism was impaired in UUO mice and restored by EZH2 inhibition as assessed by insulin or glucose tolerance test. Upregulated EZH2 in UUO kidneys is colocalized with the proximal tubular marker as shown by immunofluorescent staining. The direct anti-gluconeogenesis effect of EZH2 on renal proximal epithelial cells and primary renal tubules was shown by analysis of gluconeogenic gene expression and production of glucose or lactate in human HK2 cells treated with EZH2 inhibitor or exogeneous expression of EZH2 by adenovirus transfection. Importantly, we further confirmed the direct effect of tubular EZH2 on gluconeogenesis in tubular specific deleted EZH2 mice (Ezh2fl/fl;Cdh16-Cre). Moreover, inhibition of PCK1 by 3-mercaptopropionic acid abrogated the pro-gluconeogenic effect and anti-fibrotic effect of EZH2 inhibitor in renal cells and UUO kidneys. Finally, RNA sequencing data from kidney allograft biopsies showed that EZH2 is progressively increased in CKD patients. Conclusion We conclude that EZH2 promotes renal tubulointerstitial fibrosis through epigenetic inhibition of PCK1. Our study suggests that therapeutic restoration of the impaired renal gluconeogenesis is beneficial to CKD.

#1581 Anoctamin 3 inhibits gluconeogenesis and contributes to renal interstitial fibrosis through EZH2

Abstract Background and Aims Renal fibrosis is the common pathological pathway of various chronic kidney diseases progressing to the end stage of renal failure. Anoctamin 3 (ANO3) has been identified as a K-channel regulator and been extensively studied in the field of neurobiology. The role of ANO3 in kidney diseases is currently unknown. Method Mouse models of renal fibrosis were established by unilateral ureteral obstruction (UUO) operation or aristolochic acid I (AAI) injection. Human proximal tubular cell line (HK2) was used as an in vitro model. ANO3 was deleted in vitro by siRNA. Western blotting analysis was performed. Glucogenic metabolites were measured. Results We found that ANO3 is upregulated in mouse kidneys with unilateral ureteral obstruction (UUO) or aristolochic acid nephropathy (AAN), and tightly correlated with the progression of renal fibrosis and negatively correlated with expression of several rate-limiting enzymes of renal gluconeogenesis. Knockdown of ANO3 with siRNA increased the concentration of glucose and reduced the concentration of lactate in the supernatant of TGF-β stimulated renal proximal tubular (HK2) cells. In parallel, transfection of ANO3 siRNA significantly reduced the expression of epithelial-to-mesenchymal transition (EMT) and fibrotic markers in TGF-β stimulated HK2 cells. We further showed that ANO3 expression is positively correlated with the expression of a methyltransferase enhancer of zeste homologue 2 (EZH2) in UUO and AAN mouse kidneys by time. Transfection of ANO3 siRNA significantly reduced the expression of EZH2 in TGF-β stimulated HK2 cells. Overexpression of EZH2 by adenovirus reversed the pro-gluconeogenic and anti-fibrotic effect of ANO3 siRNA in TGF-β stimulated HK2 cells by measuring the concentration of glucose and lactate in supernatant and analyzing the expression of EMT or fibrotic markers. Conclusion In conclusion, ANO3 inhibits renal gluconeogenesis through EZH2 and contributes to renal fibrosis. ANO3 could be promising therapeutic target to inhibit renal fibrosis in chronic kidney disease patients.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury

BackgroundThe mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes.MethodsA 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein–Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms.ResultsA total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway.ConclusionsThe proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Altenusin, a fungal metabolite, alleviates TGF-β1-induced EMT in renal proximal tubular cells and renal fibrosis in unilateral ureteral obstruction

Renal fibrosis is a pathological feature of chronic kidney disease (CKD), progressing toward end-stage kidney disease (ESKD). The aim of this study is to investigate the therapeutic potential of altenusin, a farnesoid X receptor (FXR) agonist derived from fungi, on renal fibrosis. The effect of altenusin was determined (i) in vitro using the transforming growth factor β1 (TGF-β1)-induced epithelial to mesenchymal transition (EMT) of human renal proximal tubular cells and (ii) in vivo using mouse unilateral ureteral obstruction (UUO). The findings revealed that incubation of 10 ng/ml TGF-β1 promotes morphological change in RPTEC/TERT1 cells, a human renal proximal tubular cell line, from epithelial to fibroblast-like cells. TGF-β1 markedly increased EMT markers namely α-smooth muscle actin (α-SMA), fibronectin, and matrix metalloproteinase 9 (MMP-9), while decreased the epithelial marker E-cadherin. Co-incubation TGF-β1 with altenusin preserved the epithelial characteristics of the renal epithelial cells by antagonizing TGF-β/Smad signaling pathway, specifically a decreased phosphorylation of Smad2/3 with an increased level of Smad7. Interestingly, the antagonizing effect of altenusin does not require FXR activation. Moreover, altenusin could reverse TGF-β1-induced fibroblast-like cells to epithelial-like cells. Treatment on UUO mice with 30 mg/kg altenusin significantly reduced the expression of α-SMA, fibronectin, and collagen type 1A1 (COL1A1). The reduction in the renal fibrosis markers is correlated with the decreased phosphorylation of Smad2/3 levels but does not improve E-cadherin protein expression. Collectively, altenusin reduces EMT in human renal proximal tubular cells and renal fibrosis by antagonizing the TGF-β/Smad signaling pathway.

Knockdown of ADAM8 inhibits the proliferation, migration, invasion, and tumorigenesis of renal clear cell carcinoma cells to enhance the immunotherapy efficacy

The current study performed bioinformatics and in vitro and in vivo experiments to explore the effects of ADAM8 on the malignant behaviors and immunotherapeutic efficacy of renal clear cell carcinoma (ccRCC) Cells. The modular genes most associated with immune cells were screened. Then, prognostic risk models were constructed by univariate COX analysis, LASSO regression analysis and multivariate COX analysis, and their diagnostic value was determined. The correlation between tumor mutation load (TMB) scores and the prognosis of ccRCC patients was clarified. Finally, six key genes (ABI3, ADAM8, APOL3, MX2, CCDC69, and STAC3) were analyzed for immunotherapy efficacy. Human and mouse ccRCC cell lines and human proximal tubular epithelial cell lines were used for in vitro cell experiments. The effect of ADAM8 overexpression or knockdown on tumor formation and survival in ccRCC cells was examined by constructing subcutaneous transplanted tumor model. Totally, 636 Black module genes were screened as being most associated with immune cell infiltration. Six genes were subsequently confirmed for the construction of prognostic risk models, of which ABI3, APOL3 and CCDC69 were low-risk factors, while ADAM8, MX2 and STAC3 were high-risk factors. The constructed risk model based on the identified six genes could accurately predict the prognosis of ccRCC patients. Besides, TMB was significantly associated with the prognosis of ccRCC patients. Furthermore, ABI3, ADAM8, APOL3, MX2, CCDC69 and STAC3 might play important roles in treatment concerning CTLA4 inhibitors or PD-1 inhibitors or combined inhibitors. Finally, we confirmed that ADAM8 could promote the proliferation, migration and invasion of ccRCC cells through in vitro experiments, and further found that in in vivo experiments, ADAM8 knockdown could inhibit tumor formation in ccRCC cells, improve the therapeutic effect of anti-PD1, and prolong the survival of mice. Our study highlighted the alleviative role of silencing ADAM8 in ccRCC patients.

HIF-1α activation induces cholesterol homeostasis dysfunction to accelerate progression of diabetic nephropathy in rats

To investigate the effect of hypoxia inducible factor-1α (HIF-1α) activation on cholesterol homeostasis dysfunction in diabetic nephropathy (DN). Rat models of type 1 diabetes established by intraperitoneal STZ injection were treated with intraperitoneal injection of Lificiguat (YC-1, a HIF-1α inhibitor). Human proximal tubular cell line HK-2 was incubated with cobalt chloride (CoCl2, 100 μmol/L) in the presence or absence of 30 mmol/L glucose for 24 h. Renal injury of the rats was assessed by measuring 24-h urinary total protein level and PAS staining of the renal tubules. Cholesterol deposition in rat kidneys and HK-2 cells were observed using a quantitative assay of total cholesterol and Filipin staining, and HIF-1α protein expression was detected using Western blotting, immunohistochemistry or immunofluorescence assay; the expressions of cholesterol metabolism-related proteins HMGCR, LDLr, CXCL16 and profibrogenic factors including TGF-β1 and CTGF were also analyzed. The diabetic rats showed significantly increased 24-h urinary protein level (P<0.001), obvious renal tubular injury, and increased renal cholesterol content (P<0.05) with significantly increased HIF-1α expression in the renal tubular (P<0.01). YC-1 treatment significantly ameliorated tubulointerstitial injury in the diabetic rats as shown by decreased 24-h urinary total protein (P<0.05) and reduced damage area of the tubules, and effectively decreased renal cholesterol levels and renal expression of HIF-1α (P<0.05). In HK-2 cells, CoCl2 stimulation in the presence of high glucose effectively activated HIF-1α expression (P<0.0001), aggravated cholesterol accumulation (P<0.05), and increased the expressions of HMGCR, LDLr, CXCL16, TGF-β1, and CTGF (P<0.05 or 0.01). Consistent with the in vitro study, YC-1 treatment also significantly decreased the expressions of cholesterol metabolism-related proteins and the profibrogenic factors in the renal tubules of the diabetic rats. HIF-1α activation induces cholesterol homeostasis dysregulation possibly by upregulating the de novo synthesis and uptake of cholesterol, thereby aggravating tubulointerstitial injury in DN.

HK-2 cell response to TGF-β highly depends on cell culture medium formulations

The immortalized human renal proximal tubular epithelial cell line HK-2 is most commonly used to study renal cell physiology and human kidney diseases with tubulointerstitial fibrosis such as diabetic nephropathy, obstructive uropathy or allograft fibrosis. Epithelial-to-mesenchymal transition (EMT) is the main pathological process of tubulointerstitial fibrosis in vitro. Transforming growth factor-beta (TGF-β) is a key inducer of EMT. Several pro-fibrotic gene expression differences have been observed in a TGF-β-induced EMT model of HK-2 cells. However, growth conditions and medium formulations might greatly impact these differences. We investigated gene and protein expression of HK-2 cells cultured in six medium formulations. TGF-β1 increased the expression of ACTA2, TGFB1, COL4A1, EGR2, VIM and CTGF genes while reducing PPARG in all medium formulations. Interestingly, TGF-β1 treatment either increased or decreased EGR1, FN, IL6 and C3 gene expression, depending on medium formulations. The cell morphology was slightly affected, but immunoblots revealed TGFB1 and vimentin protein overexpression in all media. However, fibronectin expression as well as the nuclear translocation of EGR1 was medium dependent. In conclusion, our study demonstrates that, using the HK-2 in vitro model of EMT, the meticulous selection of appropriate cell culture medium formulation is essential to achieve reliable scientific results.

Valproic acid treatment attenuates cisplatin-induced kidney injury by suppressing proximal tubular cell damage.

Cisplatin treatment is effective against several types of carcinomas. However, it frequently leads to kidney injury, which warrants effective prevention methods. Sodium valproic acid is a prophylactic drug candidate with a high potential for clinical application against cisplatin-induced kidney injury. Therefore, in this study, we aimed to elucidate the mechanism underlying the prophylactic effect of valproic acid on cisplatin-induced kidney injury in a mouse model and HK2 and PODO cells with cisplatin-induced toxicity. In the mouse model of cisplatin-induced kidney injury, various renal function parameters and tubular damage scores were worsened by cisplatin, but they were significantly improved upon combination with valproic acid. No difference was observed in cisplatin accumulation between the cisplatin-treated and valproic acid-treated groups in whole blood and the kidneys. The mRNA expression levels of proximal tubular damage markers, apoptosis markers, and inflammatory cytokines significantly increased in the cisplatin group 72 h after cisplatin administration but significantly decreased upon combination with valproic acid. In HK2 cells, a human proximal tubular cell line, the cisplatin-induced decrease in cell viability was significantly suppressed by co-treatment with valproic acid. Valproic acid may inhibit cisplatin-induced kidney injury by suppressing apoptosis, inflammatory responses, and glomerular damage throughout the kidneys by suppressing proximal tubular cell damage. However, prospective controlled trials need to evaluate these findings before their practical application.

Limonin mitigates cisplatin-induced acute kidney injury through metabolic reprogramming

BackgroundAcute kidney injury (AKI) is a known complication of cisplatin administration; currently, there are no effective ways to prevent it. Therefore, it largely limited the use of cisplatin in chemotherapy in the clinic. In this study, we reported that Limonin, a triterpenoid compound extracted from citrus, alleviated cisplatin-induced AKI through metabolic reprogramming in the diseased kidneys. MethodsCisplatin was employed to induce AKI in mice. Three groups were set up: Sham, cisplatin + vehicle, and cisplatin + Limonin. Using UHPLC-TOF/MS, we conducted metabolomics to profile the kidneys' endogenous metabolites and metabolic pathways. A network pharmacological method was performed to identify the targets of Limonin on AKI. The human proximal tubular epithelial cell line (HK-2) was applied for in vitro studies. ResultsLimonin preserved serum creatinine and blood urea nitrogen levels after cisplatin-induced AKI. Employing metabolomics, we identified 33 endogenous differentially expressed metabolites and 7 significantly disturbed metabolic pathways in the diseased kidneys within three groups. After AKI, Limonin significantly reduced linoleic acid and its downstream product, arachidonic acid, thus exerting a protective effect on the kidney. The network pharmacological method identified CYP3A4 as a key target of Limonin in treating AKI, while CYP3A4 also serve as a mediator of arachidonic acid metabolism. In vitro, Limonin markedly reduced the level of arachidonic acid and HK-2 cell apoptosis triggered by cisplatin, mainly related to the targeted inhibition of CYP3A4-mediated arachidonic acid metabolism. ConclusionLimonin ameliorates cisplatin-induced AKI by inhibiting CYP3A4 activity to regulate arachidonic acid metabolism, ultimately preserving kidney function.

Impact of hypoxia and reoxygenation on the extra/intracellular metabolome and on transporter expression in a human kidney proximal tubular cell line.

Ischemia-reperfusion injury (IRI) induces several perturbations that alter immediate kidney graft function after transplantation and may affect long-term graft outcomes. Given the IRI-dependent metabolic disturbances previously reported, we hypothesized that proximal transporters handling endo/exogenous substrates may be victims of such lesions. This study aimed to determine the impact of hypoxia/reoxygenation on the human proximal transport system through two semi-targeted omics analyses. Human proximal tubular cells were cultured in hypoxia (6 or 24h), each followed by 2, 24 or 48-h reoxygenation. We investigated the transcriptomic modulation of transporters. Using semi-targeted LC-MS/MS profiling, we characterized the extra/intracellular metabolome. Statistical modelling was used to identify significant metabolic variations. The expression profile of transporters was impacted during hypoxia (y + LAT1 and OCTN2), reoxygenation (MRP2, PEPT1/2, rBAT, and OATP4C1), or in both conditions (P-gp and GLUT1). The P-gp and GLUT1 transcripts increased (FC (fold change) = 2.93 and 4.11, respectively) after 2-h reoxygenation preceded by 24-h hypoxia. We observed a downregulation (FC = 0.42) of y+LAT1 after 24-h hypoxia, and of PEPT2 after 24-h hypoxia followed by 2-h reoxygenation (FC = 0.40). Metabolomics showed that hypoxia altered the energetic pathways. However, intracellular metabolic homeostasis and cellular exchanges were promptly restored after reoxygenation. This study provides insight into the transcriptomic response of the tubular transporters to hypoxia/reoxygenation. No correlation was found between the expression of transporters and the metabolic variations observed. Given the complexity of studying the global tubular transport systems, we propose that further studies focus on targeted transporters.

Urinary Modified C-Reactive Protein is Closely Associated with Tubulointerstitial Lesions in Lupus Nephritis.

Modified C-reactive protein (mCRP) is known to be involved in the upregulation and amplification of the local inflammatory response. This study investigated the circulating and local levels of mCRP and their relevance to clinicopathological features in patients with lupus nephritis. Ninety-five patients with renal biopsy-proven lupus nephritis and 30 normal controls were enrolled in this study. Plasma and urinary mCRP were screened by enzyme-linked immunosorbent assay (ELISA). The renal deposition of mCRP was detected by immunohistochemistry and immunofluorescence staining. A human proximal tubular epithelial cell line (HK2 cells) was incubated with purified IgG from lupus nephritis, and the production of CRP by HK2 cells was further evaluated. Plasma and urinary levels of mCRP increased significantly in patients with lupus nephritis compared with normal controls (P = 0.013, P < 0.001, respectively). The urinary mCRP levels were associated with interstitial inflammatory cell infiltration (r = 0.514, P < 0.001) and interstitial fibrosis (r = 0.270, P = 0.008). The ROC-AUC of the urinary mCRP levels for diagnosing tubulointerstitial lesions was 0.766. The urinary mCRP levels were closely associated with poor outcomes (HR: 1.204, 95% CI: 1.029-1.409, P = 0.020). However, no correlations were found of the plasma mCRP levels with clinicopathological data or the prognosis of lupus nephritis. CRP was mostly deposited in the renal tubules in patients with lupus nephritis, and the expression of CRP was significantly correlated with tubulointerstitial lesion indices. Immunofluorescence staining showed that mCRP could colocalize with IgG in tubules. Lupus nephritis-derived IgG could induce CRP production by HK2 cells. Urinary mCRP levels were significantly increased, and urinary mCRP might be a biomarker for tubulointerstitial lesions in patients with lupus nephritis. Renal CRP could be produced by tubular epithelial cells after stimulation by lupus nephritis-derived IgG, and the local presence of mCRP might play a critical role in the development of tubulointerstitial lesions.

RhMYDGF Alleviates I/R-induced Kidney Injury by Inhibiting Inflammation and Apoptosis via the Akt Pathway.

Renal ischemia/reperfusion (I/R) injury is one of the crucial factors affecting the outcome of renal transplantation. In recent years, myeloid-derived growth factor (MYDGF) has received a lot of attention for its extensive beneficial effects on cardiac repair and protection of cardiomyocytes from cell death. Therefore, we hypothesized that the recombinant human MYDGF (rhMYDGF) protein might play an essential role in safeguarding renal I/R injury. In vivo experiments were conducted using a mouse unilateral I/R model. Mice were pretreated with rhMYDGF by intraperitoneal injection to study the potential mechanism of renal protection. In vitro, we established hypoxia/reoxygenation and H 2 O 2 treatment models to pretreat cells with rhMYDGF. The expression levels of oxidative stress, inflammation, and apoptosis-related factors in tissues and cells were detected. Finally, we explored the role of the protein kinase B (Akt) pathway in the renal protective mechanism of rhMYDGF. In this study, we found that intraperitoneal injection of 1.25 μg rhMYDGF could significantly improve renal function of I/R mice, and reduce oxidative stress, inflammation, and apoptosis. For the human proximal tubular epithelial cell line and human kidney cell line, pretreatment with 0.3 μg/mL rhMYDGF for 24 h significantly downregulated oxidative stress, inflammation, and apoptosis via the phosphorylation of Akt, which could be ameliorated by LY294002. rhMYDGF protects kidney from I/R injury by attenuating oxidative stress, inflammation, and apoptosis through the activation of the Akt pathway.

Transcriptional landscape of mitochondrial electron transport chain inhibition in renal cells

Analysis of the transcriptomic alterations upon chemical challenge, provides in depth mechanistic information on the compound’s toxic mode of action, by revealing specific pathway activation and other transcriptional modulations. Mapping changes in cellular behaviour to chemical insult, facilitates the characterisation of chemical hazard. In this study, we assessed the transcriptional landscape of mitochondrial impairment through the inhibition of the electron transport chain (ETC) in a human renal proximal tubular cell line (RPTEC/TERT1). We identified the unfolded protein response pathway (UPR), particularly the PERK/ATF4 branch as a common cellular response across ETC I, II and III inhibitions. This finding and the specific genes elaborated may aid the identification of mitochondrial liabilities of chemicals in both legacy data and prospective transcriptomic studies.Graphical abstract

DsbA-L alleviates tubular injury in diabetic nephropathy by activating mitophagy through maintenance of MAM integrity.

Mitochondria-associated endoplasmic reticulum membranes (MAMs) regulate ATG14- and Beclin1-mediated mitophagy and play key roles in the development of diabetic nephropathy (DN). DsbA-L is mainly located in MAMs and plays a role in renoprotection, but whether it activates mitophagy by maintaining MAM integrity remains unclear. In the present study, we found that renal tubular damage was further aggravated in diabetic DsbA-L-/- mice compared with diabetic mice and that this damage was accompanied by disrupted MAM integrity and decreased mitophagy. Furthermore, notably decreased expression of ATG14 and Beclin1 in MAMs extracted from the kidneys of diabetic DsbA-L-/- mice was observed. In vitro, overexpression of DsbA-L reversed the disruption of MAM integrity and enhanced mitophagy in HK-2 cells, a human proximal tubular cell line, after exposure to high-glucose (HG) conditions. Additionally, compared with control mice, DsbA-L-/- mice were exhibited down-regulated expression of helicase with zinc finger 2 (HELZ2) in their kidneys according to transcriptome analysis; HELZ2 serves as a cotranscription factor that synergistically functions with PPARα to promote the expression of mitofusin 2 (MFN-2). Treatment of HK-2 cells with MFN-2 siRNA resulted in MAM uncoupling and decreased mitophagy. Moreover, HG notably reduced the expression of HELZ2 and MFN-2 and inhibited mitophagy, and these effects were partially blocked by overexpression of DsbA-L and altered upon cotreatment with HELZ2 siRNA, HELZ2 overexpression or MK886 (PPARα inhibitor) treatment. These data indicate that DsbA-L alleviates diabetic tubular damage by activating mitophagy through maintenance of MAM integrity via the HELZ2/MFN-2 pathway.

LTBP4 Protects Against Renal Fibrosis via Mitochondrial and Vascular Impacts.

As a part of natural disease progression, acute kidney injury (AKI) can develop into chronic kidney disease via renal fibrosis and inflammation. LTBP4 (latent transforming growth factor beta binding protein 4) regulates transforming growth factor beta, which plays a role in renal fibrosis pathogenesis. We previously investigated the role of LTBP4 in chronic kidney disease. Here, we examined the role of LTBP4 in AKI. LTBP4 expression was evaluated in human renal tissues, obtained from healthy individuals and patients with AKI, using immunohistochemistry. LTBP4 was knocked down in both C57BL/6 mice and human renal proximal tubular cell line HK-2. AKI was induced in mice and HK-2 cells using ischemia-reperfusion injury and hypoxia, respectively. Mitochondrial division inhibitor 1, an inhibitor of DRP1 (dynamin-related protein 1), was used to reduce mitochondrial fragmentation. Gene and protein expression were then examined to assess inflammation and fibrosis. The results of bioenergetic studies for mitochondrial function, oxidative stress, and angiogenesis were assessed. LTBP4 expression was upregulated in the renal tissues of patients with AKI. Ltbp4-knockdown mice showed increased renal tissue injury and mitochondrial fragmentation after ischemia-reperfusion injury, as well as increased inflammation, oxidative stress, and fibrosis, and decreased angiogenesis. in vitro studies using HK-2 cells revealed similar results. The energy profiles of Ltbp4-deficient mice and LTBP4-deficient HK-2 cells indicated decreased ATP production. LTBP4-deficient HK-2 cells exhibited decreased mitochondrial respiration and glycolysis. Human aortic endothelial cells and human umbilical vein endothelial cells exhibited decreased angiogenesis when treated with LTBP4-knockdown conditioned media. Mitochondrial division inhibitor 1 treatment ameliorated inflammation, oxidative stress, and fibrosis in mice and decreased inflammation and oxidative stress in HK-2 cells. Our study is the first to demonstrate that LTBP4 deficiency increases AKI severity, consequently leading to chronic kidney disease. Potential therapies focusing on LTBP4-associated angiogenesis and LTBP4-regulated DRP1-dependent mitochondrial division are relevant to renal injury.

Exosomal microRNA-342-5p secreted from adipose-derived mesenchymal stem cells mitigates acute kidney injury in sepsis mice by inhibiting TLR9

BackgroundSepsis-related acute kidney injury (AKI) is an inflammatory disease associated with extremely high mortality and health burden. This study explored the possibility of exosomes secreted by adipose-derived mesenchymal stem cells (AMSCs) serving as a carrier for microRNA (miR)-342-5p to alleviate sepsis-related AKI and investigated the possible mechanism.MethodsSerum was obtained from 30 patients with sepsis-associated AKI and 30 healthy volunteers for the measurement of miR-342-5p, blood urea nitrogen (BUN), and serum creatinine (SCr) levels. For in vitro experiments, AMSCs were transfected with LV-miR-342-5p or LV-miR-67 to acquire miR-342-5p-modified AMSCs and miR-67-modified AMSCs, from which the exosomes (AMSC-Exo-342 and AMSC-Exo-67) were isolated. The human renal proximal tubular epithelial cell line HK-2 was induced by lipopolysaccharide (LPS) to construct a cellular model of sepsis. The expression of Toll-like receptor 9 (TLR9) was also detected in AKI cells and mouse models. The interaction between miR-342-5p and TLR9 was predicted by dual luciferase reporter gene assay.ResultsDetection on clinical serum samples showed that BUN, SCr, and TLR9 were elevated and miR-342-5p level was suppressed in the serum of patients with sepsis-associated AKI. Transfection with LV-miR-342-5p reinforced miR-342-5p expression in AMSCs and AMSC-secreted exosomes. miR-342-5p negatively targeted TLR9. LPS treatment enhanced TLR9 expression, reduced miR-342-5p levels, suppressed autophagy, and increased inflammation in HK-2 cells, while the opposite trends were observed in LPS-induced HK-2 cells exposed to AMSC-Exo-342, Rapa, miR-342-5p mimic, or si-TLR9. Additionally, the effects of AMSC-Exo-342 on autophagy and inflammation in LPS-induced cells could be weakened by 3-MA or pcDNA3.1-TLR9 treatment. Injection of AMSC-Exo-342 enhanced autophagy, mitigated kidney injury, suppressed inflammation, and reduced BUN and SCr levels in sepsis-related AKI mouse models.ConclusionmiR-342-5p transferred by exosomes from miR-342-5p-modified AMSCs ameliorated AKI by inhibiting TLR9 to accelerate autophagy.Graphical

Urinary exosomal miRNA-663a shows variable expression in diabetic kidney disease patients with or without proteinuria

Heterogeneity in the Diabetic Kidney Disease (DKD) diagnosis makes its rational therapeutics challenging. Although albuminuria characterizes DKD, reports also indicate its prevalence among non-proteinuric. Recent understanding of disease progression has thus inclined the focus on proximal tubular cell damage besides the glomeruli. A non-invasive approach exploiting exosomal miRNA derived from human kidney proximal tubular cell line was, hence, targeted. Upon miRNA profiling, three miRNAs, namely, hsa-miR-155-5p, hsa-miR-28-3p, and hsa-miR-425-5p were found to be significantly upregulated, while hsa-miR-663a was downregulated under diabetic conditions. Among these, hsa-miR-663a downregulation was more pronounced in non-proteinuric than proteinuric DKD subjects and was thus selected for the bioinformatics study. Ingenuity Pathway Analysis (IPA) narrowed on to IL-8 signaling and inflammatory response as the most enriched ‘canonical pathway’ and ‘disease pathway’ respectively, during DKD. Further, the putative gene network generated from these enriched pathways revealed experimentally induced diabetes, renal tubular injury, and decreased levels of albumin as part of mapping under ‘disease and function’. Genes target predictions and annotations by IPA reiterated miR-663a’s role in the pathogenesis of DKD following tubular injury. Overall, the observations might offer an indirect reflection of the underlying mechanism between patients who develop proteinuria and non-proteinuria.

ATP/ADP biosensor organoids for drug nephrotoxicity assessment.

Drug nephrotoxicity is a common healthcare problem in hospitalized patients and a major limitation during drug development. Multi-segmented kidney organoids derived from human pluripotent stem cells may complement traditional cell culture and animal experiments for nephrotoxicity assessment. Here we evaluate the capability of kidney organoids to investigate drug toxicity in vitro. Kidney organoids express renal drug transporters, OAT1, OAT3, and OCT2, while a human proximal tubular cell line shows the absence of OAT1 and OAT3. Tenofovir and aristolochic acid (AA) induce proximal tubular injury in organoids which is ameliorated by an OAT inhibitor, probenecid, without damage to podocytes. Similarly, cisplatin causes proximal tubular damage that can be relieved by an OCT inhibitor, cimetidine, collectively suggesting the presence of functional OATs and OCTs in organoid proximal tubules. Puromycin aminonucleoside (PAN) induced segment-specific injury in glomerular podocytes in kidney organoids in the absence of tubular injury. Reporter organoids were generated with an ATP/ADP biosensor, which may be applicable to high-throughput screening in the future. In conclusion, the kidney organoid is a useful tool for toxicity assessment in the multicellular context and may contribute to nephrotoxicity assessment during drug development.

Vitamin D-VDR (vitamin D receptor) alleviates glucose metabolism reprogramming in lipopolysaccharide-induced acute kidney injury.

Background: Our previous study showed that vitamin D (VD)-vitamin D receptor (VDR) plays a nephroprotective role in lipopolysaccharide (LPS)-induced acute kidney injury (AKI). Recently, glucose metabolism reprogramming was reported to be involved in the pathogenesis of AKI. Objective: To investigate the role of VD-VDR in glucose metabolism reprogramming in LPS-induced AKI. Methods: We established a model of LPS-induced AKI in VDR knockout (VDR-KO) mice, renal proximal tubular-specific VDR-overexpressing (VDR-OE) mice and wild-type C57BL/6 mice. In vitro, human proximal tubular epithelial cells (HK-2 cells), VDR knockout and VDR overexpression HK-2 cell lines were used. Results: Paricalcitol (an active vitamin D analog) or VDR-OE reduced lactate concentration, hexokinase activity and PDHA1 phosphorylation (a key step in inhibiting aerobic oxidation) and simultaneously ameliorated renal inflammation, apoptosis and kidney injury in LPS-induced AKI mice, which were more severe in VDR-KO mice. In in vitro experiments, glucose metabolism reprogramming, inflammation and apoptosis induced by LPS were alleviated by treatment with paricalcitol or dichloroacetate (DCA, an inhibitor of p-PDHA1). Moreover, paricalcitol activated the phosphorylation of AMP-activated protein kinase (AMPK), and an AMPK inhibitor partially abolished the protective effect of paricalcitol in LPS-treated HK-2 cells. Conclusion: VD-VDR alleviated LPS-induced metabolic reprogramming in the kidneys of AKI mice, which may be attributed to the inactivation of PDHA1 phosphorylation via the AMPK pathway.