Mouse Kidney Research Articles

#1437 Renal expression pattern of the relaxin receptor, RXFP1, and effects of the RXFP1 agonist, AZD5462, on renin in non-human primates and humans

Abstract Background and Aims RXFP1 is a G protein-coupled receptor (GPCR) for the pregnancy hormone, relaxin. While preclinical studies, performed primarily in rodent, describe beneficial effects of this pleiotropic peptide across several organ systems, there has been a disappointing lack of success in translating these observations into clinical benefit. To provide a potential pharmacological explanation accounting for this translational gap, the aim of this study was thus to thoroughly evaluate RXFP1 expression across species and tissues as well as to evaluate the renal-intrinsic effects of the RXFP1 agonist, AZD5462. Method Unbiased bioinformatic analysis of several gene expression databases was used as a first approach to compare RXFP1 tissue-level expression between rodent and human. Experimental validation was subsequently performed using scRNAseq, RNAscope and immunohistochemistry on mouse, rat, non-human primate (NHP) and human kidney. The putative renal target engagement effects of RXFP1 agonism were assessed by determining the acute and chronic effects of the RXFP1-selective small molecule agonist, AZD5462, on plasma renin concentrations in both NHPs and in a Phase 1 randomized, single-blind, placebo-controlled study in healthy humans. Results RXFP1 was identified as one of the most highly expressed GPCRs in human and NHP kidney glomeruli. Specifically, by multiple assays, we identified glomerular endothelial cells as the principal cell type expressing RXFP1. This observation is in stark contrast to mouse and rat kidney, where RXFP1 was not readily detected. AZD5462 administered orally to NHPs and healthy human participants elicited an early and sustained dose-dependent increase in plasma renin concentration, and in humans an additional decline in serum creatinine. Conclusion Human RXFP1 kidney glomerular expression is conserved with NHPs but not rodent, thus highlighting the translational and pharmacological challenges in studying RXFP1/relaxin biology across species. The concordant ability of AZD5462 to induce elevations in renin and reduction in serum creatinine, together with relaxin's reported renal-hemodynamic activity in humans, may underlie a previously unappreciated renal-intrinsic RXFP1 target engagement mechanism driving its hemodynamic and renal effects. The potential therapeutic benefits of RXFP1 agonism are the subject of on-going clinical trials in heart failure patients.

#993 The molecular effects of SGLT2i (Empagliflozin) on α-Klotho and Nephrin/Podocin proteins in type II diabetic mice model

Abstract Background and Aims Type 2 diabetes mellitus (T2DM), especially hyperglycemia, is associated with increased glucose cell toxicity and oxidative stress that can lead to irreversible damage in the kidney such as diabetic nephropathy (DN). DN is one of the most common microvascular complications of diabetes and is the leading cause of end-stage renal disease (ESRD) and replacement therapy. Several works in the setting of early experimental diabetic nephropathy using the recent anti-diabetic drugs such as sodium-glucose transporter type 2 inhibitors in the prevention, development or amelioration of renal injury. The exact mechanisms, by which these drugs ameliorate glomerular injury from hyperglycemia, had not been clarified well. Klotho and Podocin-Nephrin proteins are an important proteins involved in the tubule-glomerular injury. The Klotho is a circulating anti-ageing hormone with anti-oxidative and anti-inflammatory properties with vascular protective effects in animal studies. Podocin & Nephrin are important component of the glomerular foot-process membrane, the slit-diaphragm. Recently, several experimental reports showed decreased α-Klotho, Nephrin and Podocin expression in different models of diabetic nephropathy. Method We used BTBR mouse strain with the ob/ob leptin-deficiency mutation, which develops T2DM with hyperglycemia and DN; C57/BL mice were the control (CON). EMPA was administrated to T2DM mice (T2DM+EMPA) via drinking water for 12 weeks. Blood parameters were measured repeatedly, and at sacrifice, mice kidneys were harvested for histological and biochemical analyses. We evaluated renal glomerular structure of Nephrin, Podocin and α-Klotho proteins expression. Results EMPA treatment reduced T2DM blood glucose vs C57 group (P < 0:01), and increase urine glucose in EMPA+DM group (P < 0:001) EMPA vs. C57BL/6 and DM). Histological analyses in kidney sections, revealed decreased α-Klotho expression in glomeruli of DM mice vs Control mice (15.95 ± 0.64 vs 36 ± 2.42, (P < 0:01), and restored back in DM+ EMPA mice vs control mice (27.64 ± 0.94 vs 15.95 ± 0.64, P < 0:01). Podocin expression in glomeruli: control mice 31.3 ± 3.7 VS DM mice 21.26 ± 1.22 and DM + EMPA mice 28.22 ± 0.50 VS DM mice 21.26 ± 1.22 Conclusion Hyperglycemia (DM type II) reduces Podocin-Nephrin and α-Klotho proteins expression in the glomeruli kidney and upregulated by EMPA treatment. SGLT2i (EMPA) treatment in DM patients can significantly reduce progression of DN and onset of ESRD probably through its effect on α-Klotho/Podocin-Nephrin proteins.

#2349 Lysyl oxidase-like 2 inhibitor ameliorates tubulointerstitial fibrosis and albuminuria in cyclosporine induced nephropathy

Abstract Background and Aims Lysyl oxidase-like 2 (LOXL2), an amine oxidase, contributes to fibrotic scarring in the tissue by facilitating collagen cross-linking. Recent data demonstrated upregulation of lysyl oxidase and LOXL2 in the fibrotic kidneys after administration of cylosporine A (CsA). Thus, inhibition of LOXL2 may render therapeutic effects against CsA-induced nephropathy by ameliorating tubulointerstitial fibrosis. Method Low salt fed CD-1 mice were administered saline or CsA (15 mg/kg/day, intraperitoneally) for 8 weeks. At 4 weeks, CsA-treated animals were divided into 2 groups respectively and treated with: (1) vehicle, (2) LOXL2 inhibitor (10 mg/kg/day, gavage feeding) for another 4 weeks up to 8 weeks. We explored the reduction of tubulointerstitial fibrosis as well as albuminuria with administration of LOXL2 inhibitor in CsA nephropathy mouse model. Results CsA administration significantly increased the levels of serum creatinine (10.0 ± 1.2 vs. 28.4 ± 4.3, p = 0.02), tubular injury scores (0.1 ± 0.2 vs. 3.6 ± 1.3, p = 0.03), tubulointerstitial fibrosis (Sirius red positive area, 14 ± 6% vs. 204 ± 43%, p = 0.01), F4/80 positive inflammatory cell infiltration in mouse kidney (2 ± 1 vs. 21 ± 4, p = 0.001). These changes were associated with upregulated mRNA expression of LOXL2, TGF-β1, and monocyte chemoattractant protein-1 (MCP-1). Administration of LOXL2 inhibitor significantly suppressed the expression of alpha-SMA and collagen 1A, intrarenal F4/80 positive inflammatory cell infiltrations (21 ± 4 vs. 14 ± 2, p = 0.001) and improved tubular injury scores (3.6 ± 1.3 vs. 1.6 ± 1.1, p = 0.02). Moreover, LOXL2 inhibitor effectively reduced albuminuria excretion (46 ± 4 vs. 22 ± 6, p = 0.02). Conclusion Our results suggest that LOXL2 inhibitor treatment can attenuate CsA nephropathy progression and LOXL2 may be a potential therapeutic target in CsA nephropathy.

#102 Increased expression of carbonic anhydrase 4 in megalin deficiency

Abstract Background and Aims Megalin is an endocytic receptor located in the apical membrane of proximal tubule (PT) cells. It prevents low molecular-weight proteinuria by reuptake of a variety of proteins from the filtrate. Megalin is required for normal uptake of Cubilin-bound proteins, but it is unknown if the receptor is important for other apical PT proteins. Carbonic anhydrase 4 (CA4) is an enzyme crucial for PT bicarbonate reuptake. Our aim was to investigate the relationship between the expression patterns of these two proteins. Method A mouse model of kidney-specific mosaic megalin-knockout (M-KO). Liquid chromatography-mass spectrometry (LC-MS) on mouse kidney cortex. Immunohistochemistry (IHC), electron microscopy (EM) and Western blotting (WB) of mouse kidney. Biochemical analysis of mouse blood and urine. Results LC-MS revealed a 4.23-fold increase in CA4 abundance in M-KO mice together with smaller but also statistically significant alterations in other acid-base-associated proteins. IHC shows that the increase in CA4 abundance specifically was in the cells lacking megalin, while cells with megalin retained normal abundance. The same pattern was observed in a biopsy from a patient lacking megalin. WB revealed increased shedding of CA4 in urine from M-KO mice and EM showed mislocalization of CA4. Conclusion We show a hitherto unknown relationship where the expression and subcellular localization of CA4 is altered in PT-cells lacking megalin and show that M-KO mice have altered acid-base handling. Comparable blood parameters at baseline are maintained despite differences in urinary excretion and when challenged with acetazolamide the urinary excretion of the groups becomes alike, however blood parameters become different. While the mechanisms and full extend are unknown, this links megalin to the acid-base regulating systems of the PT.

#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.

#245 Deficiency of GADD45α expression mediated tubulointerstitial injury in diabetic nephropathy

Abstract Background and Aims The tubulointerstitial injury plays crucial roles in the development of diabetic nephropathy (DN). Recently, some studies have reported that disorder of mitochondrial fatty acid β-oxidation in proximal tubules is involved in tubulointerstitial injury of DN. The growth arrest and DNA damage-inducible gene 45 (GADD45) family proteins, especially, GADD45α, plays a crucial role in various cellular processes, such as mitochondrial biogenesis and lipid metabolism. Recent studies have demonstrated that GADD45α facilitates gene activation by inducing DNA demethylation. Therefore, this study aimed to investigate the role of GADD45α in tubulointerstitial injury of DN and its underlying mechanisms. Methods Bioinformatics and machine learning techniques were employed to identify differentially expressed genes between healthy individuals and DN patients. Experiments were conducted by using high glucose-treated HK-2 cells and streptozotocin (STZ)-induced mice. Lentivirus carrying the GADD45α gene was used to induce GADD45α overexpression in HK-2 cells. Adeno-Associated Virus vectors carrying LoxP-flanked GADD45α were intravenously injected into γGT-Cre transgenic mice expressing Cre recombinase in renal proximal tubular cells, specifically leading to GADD45α overexpression in proximal tubules. Results Through bioinformatics and machine learning techniques, GADD45α was identified and found to exhibit a strong association with tubulointerstitial injury in DN. Further analysis revealed that the expression of GADD45α was decreased in the kidneys of DN patients, diabetic mice, and high glucose-treated HK-2 cells. Notably, GADD45α overexpression ameliorated kidney damage and mitochondrial injury, leading to improved fatty acid β-oxidation, restored ATP production, and reduced ROS production. Transcriptomics analyses further demonstrated that GADD45α overexpression enhanced the expression of genes related to mitochondrial biogenesis, dynamics, and electron transport chain, with six transmembrane epithelial antigen of prostate 4 (STEAP4) being a key protein in this process. Knocking down STEAP4 using siRNA in HK-2 cells exacerbated the damage induced by high glucose. In addition, our further results indicated that high glucose increased methylation of the STEAP4 promoter, while GADD45α overexpression decreased methylation and increased STEAP4 expression. Conclusions Deficiency of GADD45α expression mediated tubulointerstitial injury in DN, which was mainly through the decreased STEAP4 expression by demethylating CpG motifs in the STEAP4 promoter region, thereby resulting in reduced fatty acid oxidation, energy synthesis, and increased ROS production, and finally accelerated tubular injuries. These findings suggested that targeting the GADD45α-STEAP4 pathway could be a potential therapeutic strategy for DN.

#2027 Activation of histone deacetylase-1 in fibroblasts and pericytes induces renal interstitial fibrosis after ischemia-reperfusion injury

Abstract Background and Aims Following an acute kidney ischemic event the chromatin remodelling enzyme, histone deacetylase-1 (HDAC1), is activated in many cell types of the kidney including fibroblasts and pericytes. During kidney injury, the fibroblasts/pericytes differentiate into the myofibroblasts and produce extracellular matrix that can lead to a permanent decline in kidney function. Pharmacological inhibition of HDACs can attenuate ischemia-reperfusion-injury (IRI) mediated interstitial fibrosis. The hypothesis of this study was that fibroblast/pericyte HDAC1 activation promotes myofibroblast differentiation and interstitial fibrosis. Method Tamoxifen inducible, fibroblast/pericyte HDAC1 knockout (KO) mice (HDAC1Fl/Fl; Col1a2-CreEr) and littermate controls (HDAC1Fl/Fl) were used. Male and female mice were given tamoxifen by ip at 8-10 weeks of age and IRI or sham surgery completed after a 2-week tamoxifen washout period. A mild, 18 min, bilateral, warm IRI model was used, and samples collected over 4 weeks. Glomerular filtration rate (GFR) was assessed using transdermal FITC-Sinistrin clearance. In vitro experiments with kidney fibroblast cells (NRK49F) overexpressing empty vector or HDAC1 were used for RNA-sequencing to determine the HDAC1-dependent myofibroblast transcriptome. Results We confirmed that HDAC1 was knockdown in myofibroblast cells from KO mice with co-localization of HDAC1 and PDGFRβ or α-smooth muscle actin in the kidneys of IRI mice. Twenty-four hours post-surgery, all IRI mice exhibited 2-3 times increase in serum creatinine and a ∼50% reduction in GFR compared to sham mice (control IRI = 513 ± 129 μl /min/100 g body mass, control sham = 1010 ± 218, n = 4-5, p = 0.02; KO IRI = 529 ± 116, KO sham = 1187 ± 59 μl/min/100 g body mass, n = 4-5, p = 0.004). Subsequent GFR measurements over the next 4 weeks showed a gradual recovery of renal function of IRI mice starting at week 2 and fully restored to sham values by week 4. However, the male control IRI mice had significant focal interstitial fibrosis 2- and 4-weeks post injury (4-week data: sham = 0.3 ± 0.09 % of cortical area, IRI = 1.2 ± 0.2%, n = 6/group, p = 0.0011) but this was attenuated in the KO male IRI mice (sham = 0.4 ± 0.07% n = 4, IRI = 0.5 ± 0.1%, n = 7, p = 0.93). The female mice, regardless of genotype, had very little kidney damage or interstitial fibrosis at 4 weeks. Transcriptomes of NRK49F cells overexpressing HDAC1 had an upregulation of 55 genes related to pro-inflammatory cytokines and tumour necrosis factor signalling and downregulation of 367 genes related to cell junctions, cytoskeleton proteins, and cell adhesion compared to the empty vector control cells. Fibroblast proliferation rates were not statistically different among the groups, suggesting HDAC1 does not affect myofibroblast proliferation but rather pro-inflammatory gene expression. Conclusion In male mice, fibroblast/pericyte activation of HDAC1 leads to the production of pro-inflammatory cytokines that we predict will lead to immune cell infiltration and crosstalk that leads to the excessive production of interstitial fibrosis following IRI.

#2385 Inhibition of histone lysine demethylases 5 attenuates acute kidney injury but not acute kidney disease or chronic kidney disease

Abstract Background and Aims The histone lysine demethylases 5 (KDM5) are members of the family of Jumonji C domain-containing histone demethylases and catalyze the removal of di- and tri-methyl moieties from the fourth lysine of histone 3 (H3K4me2/3). It has been reported that the KDM5 family mediates a range of physiological and pathological events including cell differentiation, motility, senescence and epithelial-mesenchymal transition (EMT) via activating or repressing transcription in demethylase-dependent or independent manners in both homeostasis and disease. This study investigated the potential role of KDM5 in the development and progression of renal fibrosis in acute kidney injury (AKI), chronic kidney disease (CKD) or AKI to CKD transition (acute kidney disease, AKD). Method We evaluated therapeutic effects of KDM5 inhibition in AKI model of unilateral nephrectomy plus contralateral ischemia-reperfusion (IR) injury, AKI to CKD transition model of unilateral IR injury plus removal of contralateral uninjured kidney after 8 days of IR injury, and CKD model of unilateral ureteral obstruction (UUO) in mice. Results KDM5 C70 inhibitor prevented the increase of blood urea nitrogen and positive areas of trichrome, Sirius red, F4/80 and α-smooth muscle actin in injured kidneys 7 days after IR injury. Significant reductions in renal mRNA levels of pro-inflammatory cytokine/chemokine, EMT and pro-fibrotic markers (IL-1β, IL-6, TNF-α, CCL2, CCL3, GM-CSF, MMP9, fibronectin, vimentin, TGF-β1, COL4A1) were also observed with attenuated mRNA levels of KDM5a-c. In addition, renal protein levels of antioxidants such as catalase, SOD1 and SOD2 were increased in injured kidneys of KDM5 inhibitor-treated mice. However, parameters of renal fibrosis and inflammation were similar between mice without and with KDM5 inhibitor in both AKD and CKD models. Conclusion These data demonstrate that inhibition of KDM5 could prevent renal inflammation and fibrosis after acute and severe IR injury. The activation of KDM5 might be an early step in the development of renal fibrosis after AKI insult.

#788 Mitigation of tubular apoptosis and cystogenesis in ADPKD: the impact of TRPM8 through restriction of mitochondrial function

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), characterized by numerous fluid-filled cysts and a progressive decline in renal function, is a prevalent hereditary kidney disorder. Approximately 70% of individuals affected by ADPKD may develop end-stage renal disease, typically manifesting around the median age of 58 years. Despite its prevalence, effective clinical treatments for ADPKD remain elusive. Transient receptor potential melastatin member 8 (TRPM8), serving as a sensor for cold temperatures and activated by the cooling agent menthol, orchestrates cellular Ca2+ signaling by facilitating Ca2+ entry into the cytosol. However, the role of TRPM8 in ADPKD remains undefined. Method The WT 9-12, an epithelial cell line isolated from the proximal tubule of a patient with ADPKD, was utilized for in vitro experiments. Eight-week-old wild-type (WT), Pkd1 knockdown (pkd1kd), and Trpm8, Pkd1 double mutant (T-/-P) mice were used for experiments. Renal Trpm8 was activated by administration of a 0.5% menthol diet for 7 months in mice. The cyst index was measured by H&E stain and calculated using Image J. Apoptosis was evaluated by TUNEL assay (In Situ Cell Death Detection Kit, TMR red). Protein and mRNA expression of mice kidneys was detected by Western blotting and qRT-PCR, respectively. Blood urea nitrogen (BUN) was measured by HITACHI 7600 analyzer. Results The activation of TRPM8 by menthol treatment resulted in an elevation of intracellular Ca2+ levels, subsequently impeding proliferation in WT9-12 cells. Consistently, Trpm8 activation suppressed kidney weight, kidney weight-to-body weight ratio, BUN, fibrosis, and cystogenesis in Pkd1kd mice, while no such effects were observed in WT and T-/-P mice. This suggests a protective role of Trpm8 in ADPKD. Mechanistically, Trpm8 activation mitigated tubular apoptosis in the kidneys of Pkd1kd mice, as evidenced by reduced TUNEL-positive cells and downregulated Bax and cleaved caspase-3, with no such effects observed in WT and T-/-P mice. Intriguingly, the proliferation-associated signaling pathway, ERK, was also suppressed by Trpm8. Proteomic analysis of mice kidneys revealed that Trpm8 regulates mitochondrial dysfunction, EIF2 and Sirtuin signaling pathways. The result was verified in WT9-12 cells showing reduced mitochondrial respiration, particularly impacting oxidative phosphorylation by TRPM8. Conclusion Trpm8 inhibits tubular apoptosis and cyst formation through the restriction of mitochondrial function, subsequently retarding ADPKD development in mice. These findings illuminate a novel and promising strategy for intervening in ADPKD.

#2104 Neutrophil extracellular traps have a pivotal role in tubulointerstitial fibrosis

Abstract Background and Aims Tubulointerstitial fibrosis (TIF) is a typical pathological change in chronic kidney disease (CKD). However, the mechanisms underlying tubulointerstitial fibrosis are still to be fully elucidated. Neutrophil extracellular traps (NETs) participate in the pathophysiological processes of various kidney diseases, but their role in CKD-TIF has not been reported. Here, we investigated the role of NETs in tubulointerstitial fibrosis. Method Immunofluorescence was employed to assess the levels and localization of NETs in the renal tissues of CKD patients. The GSE66494 human CKD microarray dataset was explored to analyze the changes of PAD4 levels. Three CKD mice models, including 14d unilateral ureteral obstruction (UUO), 28d ischemia/reperfusion (I/R) injury, and adenine-induced models were constructed to detect the expression levels of NETs in the kidneys. Then, we established a NETs-deficient (PAD4−/−) CKD mice model and assessed changes in renal function, NETs levels, and renal fibrosis. In vitro, phorbol-12-myristate-13-acetate (the classic inducer of NETs) was used to induce the generation of NETs. Intervention was performed on rat fibroblast NRK49F cells to investigate the mechanism of TIF induced by NETs. Results We first identified NETs levels were significantly elevated in the renal tissues of CKD patients. Additionally, chip data results also indicated elevated PAD4 levels in the renal tissues of CKD patients. NETs levels were elevated in the kidneys of UUO, I/R, and adenine-induced CKD mice, with deposition in the renal interstitium. PAD4-deficient mice did not form neutrophil extracellular traps. Knocking out PAD4 alleviated tubulointerstitial fibrosis in UUO, adenine, and I/R mice after blocking NETs formation. In vitro, NETs intervention in fibroblasts can promote their activation and transformation into myofibroblasts. Conclusion The deletion of NETs by PAD4−/− could significantly improve CKD-TIF. The inhibition of NETs might provide a new molecular target for the prevention and treatment of CKD.

#999 PDK4 triggers ferroptosis in diabetic kidney disease by promoting NRF2 ubiquitination

Abstract Background and Aims Diabetic kidney disease (DKD) is the principal cause of chronic kidney disease and end-stage renal disease. Despite this burden, the detailed understanding of the pathogenic mechanism that promote the initiation and progression of DKD remain to be fully elucidated. This study aims to investigate the role of pyruvate dehydrogenase kinase 4 (PDK4) in diabetic renal tubular cell death from the perspectives of mitochondrial injury, metabolic reprogramming and ferroptosis. Method Cellular transcriptomics was employed to screen the differential genes between normal glucose (as control) and 35 mM glucose combined with 120 uM palmitic acid (as HGL) groups. The vitro experimental groups include Ctl, HGL, HGL/DCA (5 mM PDK4 inhibitor, sodium dichloroacetate), HGL/DMSO, culturing for 48 h. C57BL/6J mice were used to perform the vivo experiments. A total of 40 C57BL/6J mice were randomly divided into 5 groups respectively labeled as control, DKD, DKD/DCA (25 mg/kg, daily), DKD/DMSO (1% DMSO, daily) and DKD/Fer-1 (1 mg/kg, daily) group. Western blot, qRT-PCR, Si-RNA transfection, transmission electron microscopy, ROS probe, FerroOrange/Green probes, liperfluo probe, immunofluorescence and Co-IP assay were used in this study to detect the indicators of metabolic reprogramming, mitochondrial function, oxidative stress and ferroptosis, as well as the NRF2 ubiquitination level. Results Based on the cellular transcriptomics, PDK4 mRNA was significantly elevated in the HK-2 cells treated with HGL. Likewise, mRNA and protein levels were also elevated in the kidneys of mice treated with streptozotocin and high fat diet. In kidneys with PDK4 inhibitor DCA treatment or PDK4 genetic knockdown, glycolipid toxicity-induced metabolic reprogramming and destruction of mitochondrial function were markedly ameliorated. In addition, mitochondrial reactive oxygen species and the oxidative stress markers NADPH oxidase 2 and 4 (NOX2, NOX4) were diminished in DCA-treated or PDK4 knockdown HK-2 cells, while superoxide dismutase 1 and 2 (SOD1, SOD2) and catalase (CAT) expression and glutathione (GSH) levels were restored. DCA intervention or PDK4 knockdown in DKD mice or HK-2 cells alleviated the signs of ferroptosis caused by glycolipid toxicity, demonstrated as up-regulation of SLCA11, GPX4, FSP1 and GSH to maintain the balance of oxidative stress, increase of FTH1 and FPN1 to promote Fe2+ storage and discharge, decrease of TFR1 to reduce Fe2+ intake, as well as reduction of lipid peroxidation products including 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA). PDK4 promoted NRF2 ubiquitination under HGL condition, since inhibition or knockdown of PDK4 in HK-2 cells increased the protein level of NRF2, while the mRNA level was unchanged. Conclusion Collectively, we identified a new ferroptosis inducer gene, PDK4, which is commonly up-regulated in diabetes. PDK4-induced metabolic reprogramming, mitochondrial damage and oxidative stress promote ROS accumulation and ultimately lead to ferroptosis in DKD, this effect can be achieved in part by promoting NRF2 ubiquitination.

Aristolochic Acid Exposure via Dermal Contact or Inhalation of Herbal Powders: Evidence of Occupational Exposure in Herbalists with Urothelial Cancer.

Emerging evidence showing urothelial cancer in herbalists is linked to aristolochic acid (AA) exposure; however, the exposure pathway remains unclear. Here, we show that dermal contact and inhalation of fine powders of AA-containing herbs are significant occupational AA exposure pathways for herbalists. We initiated the study by quantifying the amount of AA in the AA-containing powder deposited on gloves and face masks worn by the operators of an AA-containing herb grinding machine. Then, we measured the kinetics of dermal absorption and dissolution of AA from fine powders of AA-containing herbs into artificial sweat and surrogate lung fluid. Lastly, we quantified the mutagenic AA-DNA adduct levels formed in the kidneys of mice exposed to AA-containing fine powders through dermal contact. Our findings highlight an urgent occupational risk that should demand implementation of safety standards for herbalists exposed to AA-containing fine powders.

CircInpp5b Ameliorates Renal Interstitial Fibrosis by Promoting the Lysosomal Degradation of DDX1.

Renal interstitial fibrosis (RIF) is a classic pathophysiological process of chronic kidney disease (CKD). However, the mechanisms underlying RIF remain unclear. The present study found that a novel circular RNA, cirInpp5b, might be involved in RIF by high-throughput sequencing. Subsequent experiments revealed that circInpp5b was reduced in UUO mouse kidney tissues and TGF-β1-treated proximal tubular cells. The overexpression of circInpp5b inhibited RIF in UUO mice and prevented extracellular matrix (ECM) deposition in TGF-β1-treated proximal tubular cells. Furthermore, overexpression of circInpp5b down-regulated the protein level of DDX1. Mechanistically, circInpp5b bound to the DDX1 protein and promoted its lysosomal degradation. Collectively, the findings of our study demonstrate that circInpp5b ameliorates RIF by binding to the DDX1 protein and promoting its lysosomal degradation.

#2023 The role of neutrophil extracellular traps in cisplatin-induced acute kidney injury

Abstract Background and Aims Acute kidney injury (AKI) is a public health problem that seriously endangers human health. The exact pathogenesis of AKI has not been fully elucidated. Previous studies have shown that the immune system plays a key role in the inflammatory response of AKI in the acute phase, especially innate immunity. Recent discoveries have highlighted that neutrophils, in particular the neutrophil extracellular traps (NETs), have central roles in the occurrence and development of AKI. However, the specific role and mechanism of NETs in AKI of different etiologies are remain unclear. This study aimed to investigate the role of NETs in the development of cisplatin-induced AKI and provide potential therapeutic targets for AKI treatment. Method Wild-type (WT) and NETs-deficient (PAD4-/-) mouse models of AKI were constructed by intraperitoneal injection of cisplatin. Mice were weighed at 24, 48, and 72 hours after modeling, and sacrificed at 72 hours. Serum and kidney specimens were collected for renal function analysis, pathological, immunohistochemical staining, and molecular biology experiments. Mouse bone marrow neutrophils were isolated from healthy mouse. NETs were induced in vitro by adding phorbol 12 myristate 13-acetate (PMA). Primary renal tubular epithelial cells (TECs) of mouse kidney were extracted, and the cells were collected after 24 h of cisplatin and cisplatin combined with NETs intervention, respectively. The inflammatory indicators such as TNF-α, IL-1β, MCP-1, and renal injury indicators such as KIM-1 and Ngal were detected by RT-PCR. Results Compared with the WT-cisplatin group, the PAD4-/- -cisplatin group showed a reduced degree of weight loss after modeling, lower serum creatinine and urea nitrogen levels, and significantly improved renal function. The PAD4-/- -cisplatin group showed a significant decrease in the inflammatory indicators TNF-α, IL-1β, IL-6, and renal injury indicators KIM-1 and Ngal. Cisplatin intervention for 6 hours could induce the generation of NETs. Compared with the cisplatin intervention group, the expression of inflammatory indicators TNF-α, IL-1β, and MCP-1 were significantly higher in the cisplatin combined with NETs intervention TECs group. Conclusion Cisplatin directly induces neutrophils to generate NETs, which can exacerbate cisplatin-induced injury to TECs in vitro. The inhibition of NETs by PAD4-/- improves cisplatin-induced AKI. The inhibition of NETs may be a novel target for the treatment of cisplatin AKI.

#1914 Comprehensive single-cell sequencing and lipidomics identify diverse injury states and cholesterol dynamics of the PTs in AKI-to-CKD transition

Abstract Background and Aims The underlying cellular events and metabolic dysfunction of acute kidney injury (AKI) transformed to chronic kidney disease (CKD) is still largely unclear. Previous studies have suggested lipids and metabolites have a profound impact on acute and chronic inflammation. One of the main sites of renal lipid accumulation is the proximal tubule cells (PTs). Here we demonstrated the injury states and cholesterol dynamics of PTs and its potential function in AKI to CKD in unilateral ischemia-reperfusion injury (UIR) mice model. Method We employed single-cell combinatorial indexing lipidomics to analyze 15 mouse kidneys from a mouse model of failed repaired UIR at five time points. We identified cell types through unsupervised clustering analysis. Differential gene expression, enrichment analysis and main function scoring were applied to define functional heterogeneity and injury states of PTs. Next, pseudotime analysis of differentiation transitions was performed to create the diverse PT lineages. Finally, using SCENIC analysis, we identified the key transcription factors that drive the differentiation of PTs. Results We identified 13 main cell clusters and the dynamic changes in the process of AKI to CKD transition were analysed. A remarkable decrease in PTs after injury at day 1 followed with a recovery at day 3 post AKI were observed. Spatiotemporal profiling of the main cell types showed that the most injured areas in the acute stages of kidney injury are primarily concentrated in the outer stripe, specifically the region where the proximal straight tubules (PSTs) are located. In order to gain a higher resolution of the dynamics of PST subtypes during AKI to CKD, we performed sub-clustering analysis specifically on PSTs, partitioning them into 10 subclusters. Single-cell analysis identified maladaptive PSTs (PST-5) with the highest apoptosis and ferroptosis scores. Notably, pseudotime analysis revealed that PST-5 differentiated from PST-4, which has the highest lipid metabolism score. Indeed, among all the cells in the kidney, the PTs exhibited the most active metabolic reprogramming due to their differentiation and redifferentiation early after AKI. Lipid omics profiling reveals an abnormal elevation of the cholesterol metabolites 27-hydroxycholesterol (27-OH) and 25-hydroxycholesterol (25-OH) following AKI. Interestingly, the PST-4-specific expression of Cyp7b1 served as a key enzyme regulating the concentration of 27-OH. This gene has been found to be significantly absent after AKI. Moreover, the binding of 27HC, a natural ligand, to endogenous estrogen receptor alpha (ERα) leads to the upregulation of heme oxygenase 1 (Hmox1), thereby promoting ferroptosis. Finally, SCENIC analysis revealed that Cyp7b1-mediated accumulation of 27-HC is dependent on the transcription factor Atf3. Conclusion Our results indicate that Cyp7b1 deficiency confers ferroptosis, which are primarily dependent on the upregulation of cellular cholesterol homeostasis and 27-HC secretion, leading to the progression of AKI. These findings suggest that the Cyp7b1/Hmox1/Atf3/27-HC axis may serve as a potential therapeutic target for preventing the development of CKD.

#718 N-Ethylmaleimide Sensitive Factor (NSF) is essential for maintaining podocyte focal adhesion

Abstract Background and Aims Cell-matrix adhesion molecules in podocytes play a critical role in attaching these cells to the glomerular basement membrane, ensuring the proper functioning of the glomerular filtration barrier. Previous studies have shown that patients with proteinuric glomerular diseases often have higher levels of podocyturia. This increase is notably linked to a reduction in focal adhesion proteins, especially in cases of focal segmental glomerulosclerosis. Our study aims to explore the regulation of these focal adhesion molecules in podocytes. Method Doxycycline (Dox) inducible podocyte-specific Tln1 knockout mice (Tln1fl/fl Nphs2-rtTA TetO-Cre (Tln1 KO)) were used as the model for podocyte injury. These mice are genetically modified to lack Talin 1, a key protein in focal adhesion. The absence of Talin 1 in these mice leads to the development of pathological changes akin to focal segmental glomerulosclerosis, characterized by extensive proteinuria, thereby providing a valuable model for understanding this kidney disease (J Clin Invest. 2019 1;129(3):1295-13). As Tln1 KO mice 10 days after Dox induction (Tln1 KO 10 d) showed proteinuria without elevated serum creatinine, these mice were considered as the early phase of podocyte injury. Single-cell RNA sequencing (scRNAseq) was performed using these mice kidney samples, followed by differentially expressed genes (DEG) analyses. Podocyte-specific Nsf KO (Nphs2-Cre Rosa-DTRflox Nsf fl/fl (Nsf cKO)) mice were generated based on the result of the DEG analyses and the effect of NSF on focal adhesion was examined using primary podocyte culture. Results Nsf was identified as one of the most significantly down-regulated DEGs in the podocytes ofTln1 KO 10d mice. Further analysis of public scRNAseq data revealed a decrease in Nsf mRNA expression levels also in lipopolysaccharide-treated mouse podocytes. Notably, proteinuria was observed in Nsf conditional knockout (cKO) mice as early as 2 weeks of age, with all Nsf cKO mice succumbing to end-stage kidney disease by 6 weeks post-birth. Examination of isolated mutant podocytes showed a marked reduction in cell spreading. Additionally, a significant decrease in Integrin β1 expression was observed in primary podocytes of Nsf cKO mice, as evidenced by immunoblot analysis. In vivo studies using transmission electron microscopy revealed endoplasmic reticulum (ER) expansion in the podocytes of Nsf cKO mice. Immunofluorescence staining indicated a substantial increase in the ER and ER-Golgi intermediate compartment (ERGIC) in isolated Nsf cKO podocytes. These findings suggest that the deletion of Nsf in podocytes leads to a disruption in ER-to-Golgi protein transport, subsequently impairing the trafficking of focal adhesion proteins. Conclusion This study provides new insights into the molecular mechanisms underlying podocyte injury and the progression of kidney diseases. NSF plays a pivotal role in maintaining podocyte focal adhesion.

#2372 Investigating the effects of low-level and transient DNA damage on kidney health in a novel mouse model of aging

Abstract Background and Aims Increasing life expectancy worldwide underscores the importance of addressing the effects of aging on organ and cell function. Aging is characterized by a gradual decline in organ function that varies among individuals and is influenced by lifestyle choices and various external factors that induce DNA stress. This mismatch between kidney aging and an individual's lifespan contributes to the increase in end-stage renal disease. Among the kidney's constituent cells, the proximal tubules are frequently damaged by external factors such as ischemia and drugs. Our research has focused on DNA damage and its repair mechanisms (DDR) caused by these injuries, and we have previously shown that extensive DNA damage beyond the capacity of DDR or abnormalities in DDR cause cellular senescence in proximal tubules, resulting in incomplete renal repair after injury (Kishi et al. Sci Transl Med 2019, J Clin Invest 2019). In this study, we extend these studies and test whether acquired and mild DNA stress induces renal senescence using a novel mouse model of aging, the Inducible Changes to the Epigenome (ICE) mouse (Hayano et al. Cell. 2023). Method ICE mice were generated by mating I-PpoI STOP/+ mice with CreERT2/+ mice. I-PpoI, an endonuclease, cleaves DNA only upon administration of tamoxifen and the resulting DNA damage is subsequently repaired by the DDR. To induce transient DNA damage, 4- to 6-month-old mice were fed a tamoxifen-containing diet for 3 weeks and then housed until they reached 14 months of age. A comprehensive renal analysis was then performed, which included renal tissue evaluation, single-nucleus RNA sequencing, q-PCR, and Western blot analysis for phenotypic analysis of mouse kidneys. Results No significant histological changes were observed in the kidney tissues between the ICE mice and the control group, and the levels of interstitial fibrosis were comparable. To ensure unbiased evaluation, we performed single-nucleus RNA-seq. In ICE mice, there was an increased presence of proximal tubules with incomplete repair expressing KIM-1 and VCAM-1. In addition, ICE mice showed increased gene expression of DNA repair-related enzymes (Mgmt) in proximal tubules and evidence of cellular senescence. No differences in mitochondrial number or biosynthetic capacity were observed. Conclusion The consequences of short-term and transient DNA damage were found to have lasting effects on the kidney. In particular, significant changes were observed in the proximal tubules, where DNA instability and accelerated cellular senescence were evident, suggesting an impaired repair phenotype within the proximal tubules. This may be related to increased susceptibility to various external stimuli. For further detailed analysis, studies using proximal tubule-specific ICE mice are currently performed.

#2006 CircMTND5 participated in tubulointerstitial fibrosis progression of lupus nephritis by binding with A1CF

Abstract Background and Aims lupus nephritis (LN) is the most common and severe complication of systemic lupus erythematosus (SLE). CircRNA MTND5 participated in the pathogenesis of LN by sponging miR6812. However, it remains unclear what RNA binding role of circMTND5 play in LN. We aim to investigate whether circMTND5 can interact with RBP in the development and progression in LN, and to clarify the corresponding mechanisms. Method A1CF, a RNA binding protein, E-cadherin, α-SMA, TGF-β, and Fibronectin were analyzed by immunohistochemistry on paraffin-embedded section of LN patients and NC kidney tissues. The above parameters and circMTND5 were also examined in Fcgr2b-/- spontaneous LN mice kidneys by Western blotting and qPCR. In human kidney (HK) -2 cells, the binding of circMTND5 to A1CF protein was examined by RNA immunoprecipitation (RIP) assay. After knockdown in HK-2 cells and overexpression in hTGF-β stimulated HK-2 cells, we examined the all above indices. Results the expression of A1CF was down-regulated in the kidneys of LN patients and was localized in tubular epithelial cells. E-cadherin was down-regulated while α-SMA was up-regulated. The expression of circMTND5 was down-regulated in kidneys of Fcgr2b-/- spontaneous LN mice. In HK-2 cells, the binding of circMTND5 to A1CF was confirmed by RIP. A1CF and E-cadherin were down-regulated, while α-SMA, TGF-β and FN were up-regulated in HK-2 cells with knockdown of circMTND5. The overexpression of circMTND5 reverted the changes of the those above parametered induced by hTGF-β in both mRNA and proteins levels. Conclusion The expression level of circMTND5 in renal tissue of LN mice was downregulated. CircMTND5 might play an important role in tubulointerstitial fibrosis of lupus nephritis by binding with A1CF protein.

The Spermine Oxidase/Spermine Axis Coordinates ATG5-Mediated Autophagy to Orchestrate Renal Senescence and Fibrosis.

Decreased plasma spermine levels are associated with kidney dysfunction. However, the role of spermine in kidney disease remains largely unknown. Herein, it is demonstrated that spermine oxidase (SMOX), a key enzyme governing polyamine metabolism, is predominantly induced in tubular epithelium of human and mouse fibrotic kidneys, alongside a reduction in renal spermine content in mice. Moreover, renal SMOX expression is positively correlated with kidney fibrosis and function decline in patients with chronic kidney disease. Importantly, supplementation with exogenous spermine or genetically deficient SMOX markedly improves autophagy, reduces senescence, and attenuates fibrosis in mouse kidneys. Further, downregulation of ATG5, a critical component of autophagy, in tubular epithelial cells enhances SMOX expression and reduces spermine in TGF-β1-induced fibrogenesis in vitro and kidney fibrosis in vivo. Mechanically, ATG5 readily interacts with SMOX under physiological conditions and in TGF-β1-induced fibrogenic responses to preserve cellular spermine levels. Collectively, the findings suggest SMOX/spermine axis is a potential novel therapy to antagonize renal fibrosis, possibly by coordinating autophagy and suppressing senescence.

Plasma proteome analysis implicates novel proteins as potential therapeutic targets for chronic kidney disease: A proteome-wide association study

Chronic kidney disease (CKD) is prevalent globally with limited therapeutic drugs available. To systemically identify novel proteins involved in the pathogenesis of CKD and possible therapeutic targets, we integrated human plasma proteomes with the genome-wide association studies (GWASs) of CKD, estimated glomerular filtration rate (eGFR) and blood urea nitrogen (BUN) to perform proteome-wide association study (PWAS), Mendelian Randomization and Bayesian colocalization analyses. The single-cell RNA sequencing data of healthy human and mouse kidneys were analyzed to explore the cell-type specificity of identified genes. Functional enrichment analysis was conducted to investigate the involved signaling pathways. The PWAS identified 22 plasma proteins significantly associated with CKD. Of them, the significant associations of three proteins (INHBC, LMAN2, and SNUPN) were replicated in the GWASs of eGFR, and BUN. Mendelian Randomization analyses showed that INHBC and SNUPN were causally associated with CKD, eGFR, and BUN. The Bayesian colocalization analysis identified shared causal variants for INHBC in CKD, eGFR, and BUN (all PP4 > 0.75). The single-cell RNA sequencing revealed that the INHBC gene was sparsely scattered within the kidney cells. This proteomic study revealed that INHBC, LMAN2, and SNUPN may be involved in the pathogenesis of CKD, which represent novel therapeutic targets and warrant further exploration in future research.