Proximal Tubule Cells Research Articles

LONP1 alleviates ageing-related renal fibrosis by maintaining mitochondrial homeostasis.

Mitochondrial dysfunction is a pivotal event contributing to the development of ageing-related kidney disorders. Lon protease 1 (LONP1) has been reported to be responsible for ageing-related renal fibrosis; however, the underlying mechanism(s) of LONP1-driven kidney ageing with respect to mitochondrial disturbances remains to be further explored. The level of LONP1 was tested in the kidneys of aged humans and mice. Renal fibrosis and mitochondrial quality control were confirmed in the kidneys of aged mice. Effects of LONP1 silencing or overexpression on renal fibrosis and mitochondrial quality control were explored. In addition, N6-methyladenosine (m6A) modification and methyltransferase like 3 (METTL3) levels, the relationship between LONP1 and METTL3, and the impacts of METTL3 overexpression on mitochondrial functions were confirmed. Furthermore, the expression of insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) and the regulatory effects of IGF2BP2 on LONP1 were confirmed invitro. LONP1 expression was reduced in the kidneys of aged humans and mice, accompanied by renal fibrosis and mitochondrial dysregulation. Overexpression of LONP1 alleviated renal fibrosis and maintained mitochondrial homeostasis, while silencing of LONP1 had the opposite effect. Impaired METTL3-m6A signalling contributed at least in part to ageing-induced LONP1 modification, reducing subsequent degradation in an IGF2BP2-dependent manner. Moreover, METTL3 overexpression alleviated proximal tubule cell injury, preserved mitochondrial stability, inhibited LONP1 degradation, and protected mitochondrial functions. LONP1 mediates mitochondrial function in kidney ageing and that targeting LONP1 may be a potential therapeutic strategy for improving ageing-related renal fibrosis.

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

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

Knockdown of nicotinamide N-methyltransferase ameliorates renal fibrosis caused by ischemia-reperfusion injury and remodels sphingosine metabolism.

CKD currently affects 8.2% to 9.1% of the global population and the CKD mortality rate has increased during recent decades, making it necessary to identify new therapeutic targets. This study investigated the role of nicotinamide N-methyltransferase (NNMT) in renal fibrosis following ischemia-reperfusion injury (IRI), a key factor in chronic kidney disease (CKD) progression. We established a mouse model with a knockdown of NNMT to investigate the impact of this enzyme on renal fibrosis after unilateral IRI. We then utilized histology, immunohistochemistry, and metabolomic analyses to investigate fibrosis markers and sphingolipid metabolism in NNMT-deficient mice. We also utilized an Nnmt lentivirus interference vector or an Nnmt overexpression plasmid to transfect mouse kidney proximal tubule cells, stimulated these cells with TGF-β1, and then measured the pro-fibrotic response and the expression of the methylated and unmethylated forms of Sphk1. The results demonstrated that reducing NNMT expression mitigated fibrosis, inflammation, and lipid deposition, potentially through the modulation of sphingolipid metabolism. Histology, immunohistochemistry, and metabolomic analyses provided evidence of decreased fibrosis and enhanced sphingolipid metabolism in NNMT-deficient mice. NNMT mediated the TGF-β1-induced pro-fibrotic response, knockdown of Nnmt decreased the level of unmethylated Sphk1 and increased the level of methylated Sphk1 in renal tubular epithelial cells. Our findings suggest that NNMT functions in sphingolipid metabolism and has potential as a therapeutic target for CKD. Further research is needed to elucidate the mechanisms linking NNMT to sphingolipid metabolism and renal fibrosis.

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

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

Caffeine Inhibits Both Basal and Insulin-Activated Urate Transport.

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

Sphingosine 1-phosphate protective effect on human proximal tubule cells submitted to an in vitro ischemia model: the role of JAK2/STAT3.

Acute kidney injury is a serious public health problem worldwide, being ischemia and reperfusion (I/R) the main lesion-aggravating factor that contributes to the evolution towards chronic kidney disease. Nonetheless, intervention approaches currently available are just considered palliative options. In order to offer an alternative treatment, it is important to understand key factors involved in the development of the disease including the rescue of the affected cells and/or the release of paracrine factors that are crucial for tissue repair. Bioactive lipids such as sphingosine 1-phosphate (S1P) have significant effects on the modulation of signaling pathways involved in tissue regeneration, such as cell survival, proliferation, differentiation, and migration. The main objective of this work was to explore the protective effect of S1P using human kidney proximal tubule cells submitted to a mimetic I/R lesion, via ATP depletion. We observed that the S1P pre-treatment increases cell survival by 50% and preserves the cell proliferation capacity of injured cells. We showed the presence of different bioactive lipids notably related to tissue repair but, more importantly, we noted that the pre-treatment with S1P attenuated the ischemia-induced effects in response to the injury, resulting in higher endogenous S1P production. All receptors but S1PR3 are present in these cells and the protective and proliferative effect of S1P/S1P receptors axis occur, at least in part, through the activation of the SAFE pathway. To our knowledge, this is the first time that S1PR4 and S1PR5 are referred in these cells and also the first indication of JAK2/STAT3 pathway involvement in S1P-mediated protection in an I/R renal model.

Generation of a conditional cellular senescence model using proximal tubule cells and fibroblasts from human kidneys

Emerging evidence highlights cellular senescence’s pivotal role in chronic kidney disease (CKD). Proximal tubule epithelial cells (PTECs) and fibroblasts are major players in CKD and serve as cellular sources of senescence. The generation of a conditionally immortalized human kidney cell model would allow to better understand the specific mechanisms and factors associated with cellular senescence in a controlled setting, devoid of potential confounding factors such as age and comorbidities. In addition, the availability of human kidney cell lines for preclinical research is sparse and most cell lines do not reflect their in vivo counterparts due to their altered behavior as immortalized cancer-like cells. In this study, PTECs and fibroblasts from human kidneys were isolated and transduced with doxycycline-inducible simian virus 40 large T antigen (SV40LT) vector. By comparing their gene expression with single-cell RNA sequencing data from human kidneys, the newly produced human kidney cell lines demonstrated significant resemblances to their in vivo counterparts. As predicted, PTECs showed functional activity and fibroblasts responded to injury with fibrosis. Withdrawal of the immortalizing factor doxycycline led to p21+ cell-cycle arrest and the key hallmarks of senescence. The obtained senescence gene set largely overlapped between both cell lines and with the previously published SenMayo set of senescence-associated genes. Furthermore, crosstalk experiments showed that senescent PTECs can cause a profibrotic response in fibroblasts by paracrine actions. In 76 human kidney sections, the number of p21+ cells correlated with the degree of fibrosis, age and reduced glomerular filtration, validating the role of senescence in CKD. In conclusion, we provide a novel cellular ex vivo model to study kidney senescence which can serve as a platform for large scale compounds testing.

Regenerative Role of Lrig1+ Cells in Kidney Repair.

In response to severe kidney injury, the kidney epithelium displays remarkable regenerative capabilities driven by adaptable resident epithelial cells. To date, it has been widely considered that the adult kidney lacks multipotent stem cells; thus, the cellular lineages responsible for repairing proximal tubule damage are incompletely understood. Leucine-rich repeats and immunoglobulin-like domains protein 1-expressing cells (Lrig1+ cells) have been identified as a long-lived cell in various tissues that can induce epithelial tissue repair. Therefore, we hypothesized that Lrig1+ cells participate in kidney development and tissue regeneration. We investigated the role of Lrig1+ cells in kidney injury using mouse models. The localization of Lrig1+ cells in the kidney was examined throughout mouse development. The function of Lrig1+ progeny cells in acute kidney injury repair was examined in vivo using a tamoxifen-inducible Lrig1-specific Cre recombinase-based lineage tracing in three different kidney injury mouse models. Additionally, we conducted single-cell RNA-sequencing to characterize the transcriptional signature of Lrig1+ cells and to trace their progeny. Lrig1+ cells were present during kidney development and contributed to formation of the proximal tubule and collecting duct structures in mature mouse kidneys. In three-dimensional culture, single Lrig1+ cells demonstrated long-lasting propagation and differentiated into the proximal tubule and collecting duct lineages. These Lrig1+ proximal tubule cells highly expressed progenitor-like and quiescence-related genes, giving rise to a novel cluster of cells with regenerative potential in adult kidneys. Moreover, these long-lived Lrig1+ cells expanded and repaired damaged proximal tubules in response to three types of acute kidney injury in mice. These findings highlight the critical role of Lrig1+ cells in kidney regeneration.

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

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

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

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

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

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

Endogenous activation of peroxisome proliferator-activated receptor-α in proximal tubule cells in counteracting phosphate toxicity.

Increased dietary phosphate consumption intensifies renal phosphate burden. Several mechanisms for phosphate-induced renal tubulointerstitial fibrosis have been reported. Considering the dual nature of phosphate as both a potential renal toxin and an essential nutrient for the body, kidneys may possess inherent protective mechanisms against phosphate overload, rather than succumbing solely to injury. However, there is limited understanding of such mechanisms. To identify these mechanisms, we conducted single-cell RNA sequencing (scRNA-seq) analysis of the kidneys of control and dietary phosphate-loaded (Phos) mice at a time point when the Phos group had not yet developed tubulointerstitial fibrosis. scRNA-seq analysis identified the highest number of differentially expressed genes in the clusters belonging to proximal tubular epithelial cells (PTECs). Based on these differentially expressed genes, in silico analyses suggested that the Phos group activated peroxisome proliferator-activated receptor-α (PPAR-α) and fatty acid β-oxidation (FAO) in the PTECs. This activation was further substantiated through various experiments, including the use of an FAO activity visualization probe. Compared with wild-type mice, Ppara knockout mice exhibited exacerbated tubulointerstitial fibrosis in response to phosphate overload. Experiments conducted with cultured PTECs demonstrated that activation of the PPAR-α/FAO pathway leads to improved cellular viability under high-phosphate conditions. The Phos group mice showed a decreased serum concentration of free fatty acids, which are endogenous PPAR-α agonists. Instead, experiments using cultured PTECs revealed that phosphate directly activates the PPAR-α/FAO pathway. These findings indicate that noncanonical metabolic reprogramming via endogenous activation of the PPAR-α/FAO pathway in PTECs is essential to counteract phosphate toxicity.NEW & NOTEWORTHY This study revealed the activation of peroxisome proliferator-activated receptor-α and fatty acid β-oxidation in proximal tubular epithelial cells as an endogenous mechanism to protect the kidney from phosphate toxicity. These findings highlight noncanonical metabolic reprogramming as a potential target for suppressing phosphate toxicity in the kidneys.

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

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

Macrophage-derived exosomes promote telomere fragility and senescence in tubular epithelial cells by delivering miR-155

BackgroundChronic kidney disease (CKD) is highly prevalent worldwide, and its global burden is substantial and growing. CKD displays a number of features of accelerated senescence. Tubular cell senescence is a common biological process that contributes to CKD progression. Tubulointerstitial inflammation is a driver of tubular cell senescence and a common characteristic of CKD. However, the mechanism by which the interstitial inflammation drives tubular cell senescence remains unclear. This paper aims to explore the role of exosomal miRNAs derived from macrophages in the development of tubular cell senescence.MethodsAmong the identified inflammation-related miRNAs, miR-155 is considered to be one of the most important miRNAs involved in the inflammatory response. Macrophages, the primary immune cells that mediate inflammatory processes, contain a high abundance of miR-155 in their released exosomes. We assessed the potential role of miR-155 in tubular cell senescence and renal fibrosis. We subjected miR-155−/− mice and wild-type controls, as well as tubular epithelial cells (TECs), to angiotensin II (AngII)-induced kidney injury. We assessed kidney function and injury using standard techniques. TECs were evaluated for cell senescence and telomere dysfunction in vivo and in vitro. Telomeres were measured by the fluorescence in situ hybridization.ResultsCompared with normal controls, miR-155 was up-regulated in proximal renal tubule cells in CKD patients and mouse models of CKD. Moreover, the expression of miR-155 was positively correlated with the extent of renal fibrosis, eGFR decline and p16INK4A expression. The overexpression of miR-155 exacerbated tubular senescence, evidenced by increased detection of p16INK4A/p21expression and senescence-associated β-galactosidase activity. Notably, miR-155 knockout attenuates renal fibrosis and tubule cell senescence in vivo. Interestingly, once released, macrophages-derived exosomal miR-155 was internalized by TECs, leading to telomere shortening and dysfunction through targeting TRF1. A dual-luciferase reporter assay confirmed that TRF1 was the direct target of miR-155. Thus, our study clearly demonstrates that exosomal miR-155 may mediate communication between macrophages and TECs, subsequently inducing telomere dysfunction and senescence in TECs.ConclusionsOur work suggests a new mechanism by which macrophage exosomes are involved in the development of tubule senescence and renal fibrosis, in part by delivering miR-155 to target TRF1 to promote telomere dysfunction. Our study may provide novel strategies for the treatment of AngII-induced kidney injury.

Aberrant proximal tubule DNA methylation underlies phenotypic changes related to kidney dysfunction in patients with diabetes.

Epigenetic mechanisms are considered to contribute to diabetic nephropathy by maintaining memory of poor glycemic control during the early stages of diabetes. However, DNA methylation changes in the human kidney are poorly characterized, because of the lack of cell type-specific analysis. We examined DNA methylation in proximal tubules purified from diabetic nephropathy patients and identified differentially methylated CpG sites, given the critical role of proximal tubules in the kidney injury. Hypermethylation was observed at CpG sites annotated to genes responsible for proximal tubule functions, including gluconeogenesis, nicotinamide adenine dinucleotide synthesis, transporters of glucose, water, phosphate, and drugs, in diabetic kidneys, while genes involved in oxidative stress and the cytoskeleton exhibited demethylation. Methylation levels of CpG sites annotated to ACTN1, BCAR1, MYH9, UBE4B, AFMID, TRAF2, TXNIP, FOXO3, and HNF4A were correlated with the estimated glomerular filtration rate, while methylation of the CpG site in RUNX1 was associated with interstitial fibrosis and tubular atrophy. Hypermethylation of G6PC and HNF4A was accompanied by decreased expression in diabetic kidneys. Proximal tubule-specific hypomethylation of metabolic genes related to HNF4A observed in control kidneys was compromised in diabetic kidneys, suggesting a role for aberrant DNA methylation in the dedifferentiation process. Multiple genes with aberrant DNA methylation in diabetes overlapped genes with altered expressions in maladaptive proximal tubule cells, including transcription factors PPARA and RREB1. In conclusion, DNA methylation derangement in the proximal tubules of patients with diabetes may drive phenotypic changes, characterized by inflammatory and fibrotic features, along with impaired function in metabolism and transport.

Stepwise developmental mimicry generates proximal-biased kidney organoids.

The kidney maintains body fluid homeostasis by reabsorbing essential compounds and excreting waste. Proximal tubule cells, crucial for renal reabsorption of a range of sugars, ions, and amino acids, are highly susceptible to damage, leading to pathologies necessitating dialysis and kidney transplants. While human pluripotent stem cell-derived kidney organoids are used for modeling renal development, disease, and injury, the formation of proximal nephron cells in these 3D structures is incomplete. Here, we describe how to drive the development of proximal tubule precursors in kidney organoids by following a blueprint of in vivo human nephrogenesis. Transient manipulation of the PI3K signaling pathway activates Notch signaling in the early nephron and drives nephrons toward a proximal precursor state. These "proximal-biased" (PB) organoid nephrons proceed to generate proximal nephron precursor cells. Single-cell transcriptional analyses across the organoid nephron differentiation, comparing control and PB types, confirm the requirement of transient Notch signaling for proximal development. Indicative of functional maturity, PB organoids demonstrate dextran and albumin uptake, akin to in vivo proximal tubules. Moreover, PB organoids are highly sensitive to nephrotoxic agents, display an injury response, and drive expression of HAVCR1 / KIM1 , an early proximal-specific marker of kidney injury. Injured PB organoids show evidence of collapsed tubules, DNA damage, and upregulate the injury-response marker SOX9 . The PB organoid model therefore has functional relevance and potential for modeling mechanisms underpinning nephron injury. These advances improve the use of iPSC-derived kidney organoids as tools to understand developmental nephrology, model disease, test novel therapeutics, and for understanding human renal physiology.

The Flavoring Agent Ethyl Vanillin Induces Cellular Stress Responses in HK-2 Cells.

Flavored e-cigarettes are a popular alternative to cigarette smoking; unfortunately, the extrapulmonary effects are not well-characterized. Human proximal tubule cells were cultured for 24 or 48 h with 0-1000 µM ethyl vanillin (ETH VAN) and cytotoxicity evaluated. Mitochondrial health was significantly diminished following 48 h of exposure, accompanied by significantly decreased spare capacity, coupling efficiency, and ATP synthase expression. ETH VAN at 24 h inhibited glycolysis. The endoplasmic reticulum (ER) stress marker C/EBP homologous protein (CHOP) was increased at 100 μM relative to 500-1000 μM. The downstream proapoptotic marker cleaved caspase-3 subsequently showed a decreasing trend in expression after 48 h of exposure. The autophagy biomarkers microtubule-associated proteins 1A/1B light chain 3 (LC3B-I and LC3B-II) were measured by Western blot. LC3B-II levels and the LC3B-II/LC3B-I ratio increased at 24 h, which suggested activation of autophagy. In contrast, by 48 h, the autophagy biomarker LC3B-II decreased, resulting in no change in the LC3B-II/LC3B-I ratio. Mitophagy biomarker PTEN-induced putative kinase 1 (PINK1) expression decreased after 48 h of exposure. The downstream marker Parkin was not significantly changed after 24 or 48 h. These findings indicate that the flavoring ETH VAN can induce energy pathway dysfunction and cellular stress responses in a renal model.

Injury-induced Foxm1 expression in the mouse kidney drives epithelial proliferation by a cyclin F-dependent mechanism.

Acute kidney injury (AKI) strongly upregulates the transcription factor Foxm1 in the proximal tubule in vivo, and Foxm1 drives epithelial proliferation in vitro. Here, we report that deletion of Foxm1 either with a nephron-specific Cre driver or by inducible global deletion reduced proximal tubule proliferation after ischemic injury in vivo. Foxm1 deletion led to increased AKI to chronic kidney disease transition, with enhanced fibrosis and ongoing tubule injury 6 weeks after injury. We report ERK mediated FOXM1 induction downstream of the EGFR in primary proximal tubule cells. We defined FOXM1 genomic binding sites by cleavage under targets and release using nuclease (CUT&RUN) and compared the genes located near FOXM1 binding sites with genes downregulated in primary proximal tubule cells after FOXM1 knockdown. The aligned data sets revealed the cell cycle regulator cyclin F (CCNF) as a putative FOXM1 target. We identified 2 cis regulatory elements that bound FOXM1 and regulated CCNF expression, demonstrating that Ccnf is strongly induced after kidney injury and that Foxm1 deletion abrogates Ccnf expression in vivo and in vitro. Knockdown of CCNF also reduced proximal tubule proliferation in vitro. These studies identify an ERK/FOXM1/CCNF signaling pathway that regulates injury-induced proximal tubule cell proliferation.

PNPLA3 variation and kidney disease.

Accumulating epidemiological evidence shows that the patatin-like phospholipase domain-containing protein-3 (PNPLA3) rs738409 G allele, which is the most robust genetic variant associated with greater susceptibility to metabolic dysfunction-associated steatotic liver disease (MASLD), is significantly associated with impaired kidney function in both adults and children, regardless of the presence of common renal risk factors, MASLD severity, and other potential confounders. Although some prospective studies have reported a significant association between the PNPLA3 rs738409 G allele and the increased risk of developing chronic kidney disease (CKD), the epidemiological evidence about a possible direct effect of the PNPLA3 rs738409 G allele on the risk of developing CKD is still limited. Experimentally, PNPLA3 is expressed in renal podocytes, pericytes, and proximal tubule cells, thus supporting the notion that the mutant PNPLA3 protein may play a role in developing renal steatosis and fibrosis. However, it cannot be ruled out that a part of the adverse effect of the PNPLA3 rs738409 G allele on kidney function may be driven by a direct impact of this genetic variant on the development and progression of MASLD. It is possible to hypothesize that identifying the PNPLA3 genotype might help identify individuals at higher risk of CKD and those at greater risk of advanced MASLD. In this narrative minireview, we summarize the current epidemiological data about the association between the PNPLA3 rs738409 G allele and the risk of CKD and abnormal albuminuria. We also briefly discuss the putative biological mechanisms underpinning this association and its potential and future clinical implications.

Renal handling of albumin in rats with early stage diabetes: A theoretical analysis.

In early diabetic nephropathy (DN), recent studies have shown that albuminuria stems mostly from alterations in tubular function rather than from glomerular damage. Several factors in DN, including hyperfiltration, hypertrophy and reduced abundance of the albumin receptors megalin and cubilin, affect albumin endocytosis in the proximal tubule (PT). To assess their respective contribution, we developed a model of albumin handling in the rat PT that couples the transport of albumin to that of water and solutes. Our simulations suggest that, under basal conditions, ∼75% of albumin is retrieved in the S1segment. The model predicts negligible uptake in S3, as observed experimentally. It also accurately predicts the impact of acute hyperglycaemia on urinary albumin excretion. Simulations reproduce observed increases in albumin excretion in early DN by considering the combined effects of increased glomerular filtration rate (GFR), osmotic diuresis, hypertrophy, and megalin and cubilin downregulation, without stipulating changes in glomerular permselectivity. The results indicate that in isolation, glucose-elicited osmotic diuresis and glucose transporter upregulation raise albumin excretion only slightly. Enlargement of PT diameter not only augments uptake via surface area expansion, but also reduces fluid velocity and thus shear stress-induced stimulation of endocytosis. Overall, our model predicts that downregulation of megalin and cubilin and hyperfiltration both contribute significantly to increasing albumin excretion in rats with early-stage diabetes. The results also suggest that acute sodium-glucose cotransporter 2 inhibition lowers albumin excretion only if GFR decreases sufficiently, and that angiotensin II receptor blockers mitigate urinary albumin loss in early DN in large part by upregulating albumin receptor abundance. KEY POINTS: The urinary excretion of albumin is increased in early diabetic nephropathy (DN). It is difficult to experimentally disentangle the multiple factors that affect the renal handling of albumin in DN. We developed a mathematical model of albumin transport in the rat proximal tubule (PT) to examine the impact of elevated plasma glucose, hyperfiltration, PT hypertrophy and reduced abundance of albumin receptors on albumin uptake and excretion in DN. Our model predicts that glucose-elicited osmotic diuresis per se raises albumin excretion only slightly. Conversely, increases in PT diameter and length favour reduced albumin excretion. Our results suggest that downregulation of the receptors megalin and cubilin in PT cells and hyperfiltration both contribute significantly to increasing albumin excretion in DN. The model helps to better understand the mechanisms underlying urinary loss of albumin in early-stage diabetes, and the impact of specific treatments thereupon.