Proximal Tubule Research Articles

Spns1 is an iron transporter essential for megalin-dependent endocytosis.

Proximal tubule endocytosis is essential to produce protein free urine as well as to regulate system wide metabolic pathways, such as the activation of Vitamin D. We have determined that the proximal tubule expresses an endolysosomal membrane protein, protein spinster homolog1 (Spns1), which engenders a novel iron conductance that is indispensable during embryonic development. Conditional knockout of Spns1 with a novel Cre-LoxP construct specific to megalin-expressing cells led to the arrest of megalin receptor-mediated endocytosis as well as dextran pinocytosis in proximal tubules. The endocytic defect was accompanied by changes in megalin phosphorylation as well as enlargement of lysosomes confirming previous findings in Drosophila and Zebrafish. The endocytic defect was also accompanied by iron overload in proximal tubules. Remarkably, iron levels regulated the Spns1 phenotypes, because feeding an iron deficient diet or mating Spns1 knockout with divalent metal transporter1 (DMT1) knockout rescued the phenotypes. Conversely, iron loading wild type mice reproduced the endocytic defect, These data demonstrate a reversible, negative feedback for apical endocytosis, and raise the possibility that regulation of endocytosis, pinocytosis, megalin activation, and organellar size and function is nutrient-responsive.

Insoluble HIFa protein aggregates by cadmium disrupt hypoxia-prolyl hydroxylase (PHD)-hypoxia inducible factor (HIFa) signaling in renal epithelial (NRK-52E) and interstitial (FAIK3-5) cells.

The kidney is the main organ that senses changes in systemic O2 pressure by hypoxia-PHD-HIFa (HPH) signaling, resulting in adaptive target gene activation, including erythropoietin (EPO). The non-essential transition metal cadmium (Cd) is nephrotoxic and disrupts the renal HPH pathway, which may promote Cd-associated chronic renal disease (CKD). A deeper molecular understanding of Cd interference with renal HPH signaling is missing, and no data with renal cell lines are available. In rat kidney NRK-52E cells, which model the proximal tubule, and murine fibroblastoid atypical interstitial kidney (FAIK3-5) cells, which mimic renal EPO-producing cells, the chemical hypoxia mimetic dimethyloxalylglycine (DMOG; 1 mmol/l) or hypoxia (1% O2) activated HPH signaling. Cd2+ (2.5-20 µmol/l for ≤ 24h) preferentially induced necrosis (trypan blue uptake) of FAIK3-5 cells at high Cd whereas NRK-52E cells specially developed apoptosis (PARP-1 cleavage) at all Cd concentrations. Cd (12.5 µmol/l) abolished HIFa stabilization and prevented upregulation of target genes (quantitative real-time polymerase chain reaction and immunoblotting) induced by DMOG or hypoxia in both cell lines, which was caused by the formation of insoluble HIFa aggregates. Strikingly, hypoxic preconditioning (1% O2 for 18h) reduced apoptosis of FAIK3-5 and NRK-52E cells at low Cd concentrations and decreased insoluble HIFa proteins. Hence, drugs mimicking hypoxic preconditioning could reduce CKD induced by chronic low Cd exposure.

Pik3c3 expression profiling in the mouse kidney and its role in proximal tubule cell physiology.

The class 3 phosphatidylinositol 3-kinase (Pik3c3) plays critical roles in regulating autophagy, endocytosis, and nutrient sensing, but its expression profile in the kidney remains undefined. Recently, we validated a Pik3c3 antibody through immunofluorescence staining of kidney tissues from cell type-specific Pik3c3 knockout mice. Immunohistochemistry unveiled significant disparities in Pik3c3 expression levels across various kidney cell types. Notably, renal interstitial cells exhibit minimal Pik3c3 expression. Further, coimmunofluorescence staining, utilizing nephron segment- or cell type-specific markers, revealed nearly undetectable levels of Pik3c3 expression in glomerular mesangial cells and endothelial cells. Intriguingly, although podocytes exhibit the highest Pik3c3 expression levels among all kidney cell types, the renal proximal tubule cells (RPTCs) express the highest level of Pik3c3 among all renal tubules. RPTCs are known to express the highest level of the epidermal growth factor receptor (EGFR) in adult kidneys; however, the role of Pik3c3 in EGFR signaling within RPTCs remains unexplored. Therefore, we conducted additional cell culture studies. The results demonstrated that Pik3c3 inhibition significantly delayed EGF-stimulated EGFR degradation and the termination of EGFR signaling in RPTCs. Mechanistically, Pik3c3 inhibition surprisingly did not affect the initial endocytosis process but instead impeded the lysosomal degradation of EGFR. In summary, this study defines, for the first time, the expression profile of Pik3c3 in the mouse kidney and also highlights a pivotal role of Pik3c3 in the proximal tubule cells. These findings shed light on the intricate mechanisms underlying Pik3c3-mediated regulation of EGFR signaling, providing valuable insights into the role of Pik3c3 in renal cell physiology. NEW & NOTEWORTHY This is the first report defining the class 3 phosphatidylinositol 3-kinase (Pik3c3) expression profile in the kidney. Pik3c3 is nearly absent in renal interstitial cells, glomerular mesangial cells, and endothelial cells. Remarkably, glomerular podocytes express the highest Pik3c3 level in the kidney. However, the proximal tubule exhibits the highest expression level among all renal tubules. This study also unveils the pivotal role of Pik3c3 in regulating EGFR degradation and signaling termination in RPTCs, furthering our understanding of Pik3c3 in renal cell physiology.

A Focus on the Proximal Tubule Dysfunction in Dent Disease Type 1.

Dent disease type 1 is a rare X-linked recessive inherited renal disorder affecting mainly young males, generally leading to end-stage renal failure and for which there is no cure. It is caused by inactivating mutations in the gene encoding ClC-5, a 2Cl-/H+ exchanger found on endosomes in the renal proximal tubule. This transporter participates in reabsorbing all filtered plasma proteins, which justifies why proteinuria is commonly observed when ClC-5 is defective. In the context of Dent disease type 1, a proximal tubule dedifferentiation was shown to be accompanied by a dysfunctional cell metabolism. However, the exact mechanisms linking such alterations to chronic kidney disease are still unclear. In this review, we gather knowledge from several Dent disease type 1 models to summarize the current hypotheses generated to understand the progression of this disorder. We also highlight some urinary biomarkers for Dent disease type 1 suggested in different studies.

Spatial transcriptomics defines injury specific microenvironments and cellular interactions in kidney regeneration and disease

Kidney injury disrupts the intricate renal architecture and triggers limited regeneration, together with injury-invoked inflammation and fibrosis. Deciphering the molecular pathways and cellular interactions driving these processes is challenging due to the complex tissue structure. Here, we apply single cell spatial transcriptomics to examine ischemia-reperfusion injury in the mouse kidney. Spatial transcriptomics reveals injury-specific and spatially-dependent gene expression patterns in distinct cellular microenvironments within the kidney and predicts Clcf1-Crfl1 in a molecular interplay between persistently injured proximal tubule cells and their neighboring fibroblasts. Immune cell types play a critical role in organ repair. Spatial analysis identifies cellular microenvironments resembling early tertiary lymphoid structures and associated molecular pathways. Collectively, this study supports a focus on molecular interactions in cellular microenvironments to enhance understanding of injury, repair and disease.

Evaluating nephrotoxicity reduction in a novel polymyxin B formulation: insights from a 3D kidney-on-a-chip model.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels (P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase (P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly (P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced (P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics.

Decoding 11-Oxygenated Androgen Synthesis: Insights from Enzyme Gene Expression and LC‒MS/MS Quantification.

Adrenal-origin and peripheral tissue-transformed 11-oxygenated androgens are recognized as significant androgens. However, our current understanding of the synthesis of 11-oxygenated androgens, including the organs and cell types involved, remains limited. We performed comprehensive analyses on an extensive dataset of normal human tissues, which included bulk RNA data from 30 tissues, single-cell RNA sequencing (scRNA) data from 16 tissues and proteomics data from 29 tissues, to characterize the expression profiles of enzyme-encoding genes. To validate the findings, immunohistochemical and liquid chromatography-tandem mass spectrometry (LC‒MS/MS) techniques were employed. Our investigation revealed that the gene expression levels of the enzymes HSD11B2 and AKR1C3 were notably elevated in the kidney and intestines. Intriguingly, within these organs, we observed an increasing trend in enzyme expression with age in women, while a decreasing trend was apparent in men. Sc-RNA analysis revealed that HSD11B2 was predominantly expressed in collecting duct principal cells in the kidney, while AKR1C3 was primarily expressed in the proximal tubules. Intriguingly, nearly all epithelial cells in the intestine expressed these key enzymes. Further analysis using LC‒MS/MS revealed that the kidney exhibited the highest levels of 11-ketoandrostenedione (11KA4) and 11-ketotestosterone (11KT) among the seven tissues examined, and substantial synthesis of 11KA4 and 11KT was also observed in the intestine. Finally, we developed the TransMap website (http://gxmujyzmolab.cn:16245/TransMap/) to provide comprehensive visualization of all currently available transcriptome data. This study offers an overarching perspective on tracing the synthesis of 11-oxygenated androgens in peripheral tissues, thereby providing valuable insights into the potential role of these androgens in humans.

Oridonin Attenuates Diabetes‑induced Renal Fibrosis via the Inhibition of TXNIP/NLRP3 and NF‑κB Pathways by Activating PPARγ in Rats.

Oridonin possesses remarkable anti-inflammatory, immunoregulatory properties. However, the renoprotective effects of oridonin and the underlying molecular mechanisms in diabetic nephropathy (DN). We hypothesized that oridonin could ameliorate diabetes‑induced renal fibrosis. Streptozocin (STZ)-induced diabetic rats were provided with a high-fat diet to establish a type 2 diabetes mellitus (T2DM) animal model, and then treated with Oridonin (10, 20 mg/kg/day) for two weeks. Kidney function and renal fibrosis were assessed. High glucose-induced human renal proximal tubule epithelial cells (HK-2) were also treated with oridonin. The expression of inflammatory factors and fibrotic markers were analyzed. Oridonin treatment preserved kidney function and markedly limited the renal fibrosis size in diabetic rats. The renal fibrotic markers were inhibited in the oridonin 10 mg/kg/day and 20 mg/kg/day groups compared to the T2DM group. The expression of thioredoxin-interacting proteins/ nod-like receptor protein-3 (TXNIP/NLRP3) and nuclear factor (NF)‑κB pathway decreased, while that of peroxisome proliferator-activated receptor-gamma (PPARγ) increased in the oridonin treatment group compared to the non-treated group. In vitro, PPARγ intervention could significantly regulate the effect of oridonin on the high glucose-induced inflammatory changes in HK-2 cells. Oridonin reduces renal fibrosis and preserves kidney function via the inhibition of TXNIP/NLRP3 and NF‑κB pathways by activating PPARγ in rat T2DM model, which indicates potential effect of oridonin in the treatment of DN.

Abstract 08: Genome-wide Maps of Human Kidney Proximal Tubules and Thick Ascending Limbs of Henle reveal an Enrichment for Human Blood Pressure associated Polymorphisms

GWAS has identified &gt;1,000 blood pressure (BP)-associated sentinel SNPs, most of which (&gt;90%) are intronic and intergenic. How they regulate BP-relevant genes remains an open question. We hypothesized many SNPs would reside within cis -regulatory elements specific to nephron segments critical to sodium resorption such as proximal tubule (PT) and thick ascending limb (TAL). To address this, we developed chromatin state (ATAC-seq) and transcriptome (RNA-seq) maps from manually dissected human PT and medullary TAL (mTAL) segments. We identified 216,168 and 179,467 open regions in PT and mTAL, respectively, with the majority (70%) being unique to one tissue. Interestingly, peaks shared between the two segments resided more frequently in promoters, whereas unique peaks were more often found in distal introns or intergenic regions, suggesting they lie within enhancers ( Figure 1A ). Both chromatin state and transcriptomic data demonstrated a correlation between open chromatin peaks within gene promoters and mRNA levels (r=0.11; p&lt;4.6e-16). This correlation was weaker at open regions distal to the promoter, illustrating the difficulty of mapping distal open regions to the gene(s) they regulate. We hypothesized these open regions would be enriched for BP-relevant SNPs. We curated 1,071 bp-associated SNPs and found 87 and 67 in mTAL and PT, open regions respectively, many of which are near BP-relevant genes. This enrichment was significant based on 10 4 randomizations (pValue 10 -4 ) ( Figure 1B ). We conclude that these novel tissue-specific chromatin maps can be used to identify potential mechanisms by which SNPs influence gene expression and BP. Additional studies will be needed to demonstrate both causality and molecular mechanisms.

Abstract P367: Cross-Species Epigenomic Maps of Intergenic Loci for Hypertension and Blood Pressure

Many non-coding SNPs identified in Genome-Wide Association Studies (GWAS) likely affect BP-related gene expression through epigenetic mechanisms. This study analyzes the comparative genomic and epigenomic landscapes in human and rat kidney tissues, including kidney proximal tubule (PT) and medullary thick ascending limb (mTAL), focusing on the major challenge of identifying conserved regulatory elements in intergenic regions. The purpose of our study is to identify BP gene regulatory elements in intergenic regions that are conserved from human to rat for in vivo validation and mechanistic studies. Our ultimate goal is to generate high-resolution, genome-wide epigenomic maps of key BP-relevant tissues, using ATAC-seq, Hi-C, CUT&amp;Tag, DNA methylation profiling and RNA-seq. We are creating visualization hubs for human and rat, integrating our data within the UCSC Genome Browser environment. UCSC contains extensive data for humans but very little for rats, particularly for the updated genome assemblies. To address this gap, we integrated our results with data from the Rat Genome Database (RGD). To date, we harmonized ATAC-seq data from human and rat PT and mTAL and displayed these data in RGD’s JBrowse2 genome browser for unique comparative genome views. Based on our initial analysis of data density and intensity, we are developing a novel pattern recognition algorithm to identify epigenomic marks that are conserved across rat and human genomes. Existing algorithms predominantly focus on the conservation of coding genes, gene families, and genomic sequences. Here, average normalized peak intensities for ATAC-seq data across tissues were subject to canonical correlation between human and rat to identify conserved epigenomic patterns within the BP GWAS loci. Here we present an example of a human BP locus in the intergenic region between NPR3 and TARS1 on chromosome 5 which has a region of conserved synteny with rat chromosome 2. Comparative analysis and visualization of this region show significant correlation between ATAC-seq peaks from rat and human PT and mTAL at this locus, with predicted conserved regulatory elements. These visualizations can uncover novel biological insights and identify potential regulatory targets for intervention. This work underscores the importance of advanced visualization techniques in understanding hypertension pathogenesis, facilitating cross-species comparisons, and enhancing the utility of genomic databases.

Abstract 06: Role of the Atrial Natriuretic Peptide in Mitochondria-mediated Proximal Tubule Epithelial Oxidative Stress in Type 1 Diabetic Kidney Disease

Introduction: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease. Most studies of DKD are glomerulo-centric. The recent progress in the control of renal glucose handling by SGLT2i makes the proximal tubule (PT) a prime candidate. Mitochondrial (mito) dysfunction and mito-induced oxidative stress in DKD is well-studied and is known to accelerate tubular injury. Some studies have suggested the role of the Atrial Natriuretic Peptide (ANP) in DKD. ANP is a hormone that regulates renal salt/water homeostasis and renal hemodynamics. We hypothesize that in DKD, ANP is a beneficial factor that improves proximal tubule mitochondrial function via protection from oxidative stress-induced ferroptosis. Methods: Using male ANP knock-out (SS NPPA-/- ) and wild-type (SS WT ) rats on the Dahl SS background, T1DKD was induced by streptozotocin (STZ; 75mg/kg) at 9-10 weeks of age. Baseline, midpoint, and endpoint blood glucose, urine, and body weights were collected. Western blotting was done on isolated cortical tissue and tissue fibrosis was measured in PicroSirius Red staining. ANOVA through Origin 2019b was used for comparisons. Results: STZ- SS WT and STZ-SS NPPA-/- groups exhibited similar body weight (231.7 ± 14.8 vs. 230.9 ± 8.4 g, respectively), two-kidney weight to body weight ratio (31.8 ± 0.9 vs. 28.4 ± 1.5 mg/g), and endpoint hyperglycemia (with blood glucose levels &gt;650 mg/dL). Both, STZ- SS WT and STZ-SS NPPA-/- groups had high diuresis (59.9 ± 3.1 vs. 52.7 ± 3.2 mL/24hr/100gTBW). Histological analysis revealed elevated cortical fibrosis in the STZ-SS NPPA-/- compared to STZ- SS WT (4.6 ± 0.2 vs. 3.1 ± 0.2 % area, respectively, p&lt;0.05). Moreover, in the kidney cortex, STZ-SS NPPA-/- rats compared to STZ- SS WT rats exhibit a significant increase in OXPHOS complexes IV and V (p&lt;0.05) and a decrease in glutathione peroxidase 4 (GPX4; a core regulator of ferroptosis) abundance (1.5 ± 0.2 vs. 1.9 ± 0.1 a.u., respectively, p&lt;0.05). Conclusion: Data showed that lack of ANP may promote DKD-related tubulointerstitial fibrosis and suggested a relationship with renal cortical mitochondrial function. Future studies will provide mechanistic insight into the role of ANP in mito-related oxidative stress and further assess the role of ANP in PT ferroptosis.

Abstract MP32: Megalin and lysosomal disruption in the kidney epithelial cells of high salt diet fed rats: protective role of angiotensin II type 2 receptor

Earlier we have reported that 2-days high salt diet (HSD) feeding caused proteinuria in obese rats and the AT 2 R agonist treatment prevented proteinuria. Since HSD feeding for 2 days and the AT 2 R activation did not affect blood pressure, GFR, glomerular structure, and inflammation, the mechanism responsible for proteinuria remains unknown. Megalin is a proximal tubule epithelial cells endocytic receptor responsible for filtered protein reabsorption. The absorbed proteins are directed to lysosomes for degradation. Lysosomes is a nutrient sensing subcellular compartment and a critical part of the endocytic machinery and cellular trafficking. We hypothesized that high salt intake caused lysosomal stress, which negatively impacted megalin-protein cellular trafficking and thus caused proteinuria. Therefore, we evaluated the markers of lysosomal stress and megalin expression in the proximal tubules of HSD fed obese Zucker rats. Urinary excretion of N-acetyl-glucosaminidase (NAG), which is a marker of lysosomal injury and oxidative stress, was significantly increased in HSD fed rats compared with normal salt diet (NSD: 16.6 vs HSD: 40.5 RFU/hr/µg Cr). This increase was associated with an increase in another oxidative stress marker H 2 O 2 in the urine (NSD: 0.37 vs HSD: 1.72 ΔRFU/min). Confocal analysis revealed that megalin was localized in the lysosomes in HSD fed rats, as megalin was co-localized with the lysosomal marker LAMP-1 in the proximal tubules. Moreover, in HSD fed rats there was a remarkable increase (2-fold) in the lysosomal size (fig) and number per tubule (NSD: 18 vs HSD: 48). The AT 2 R agonist C21 (0.3 mg/Kg/day, i.p.) treatment of HSD-fed rats restored surface megalin expression, reduced urinary NAG and H 2 O 2 and the lysosomal number and size. Our in vitro studies performed in OK cells (proximal tubule epithelial cells) treated with high NaCl (100 mM) without/with the AT 2 R agonist C21 (1µM) also revealed that compared to NSD, high NaCl reduced megalin surface expression and C21 treatment prevented it. We conclude that high salt intake causes lysosomal stress. Particularly, large lysosomal size potentially is affecting endocytic machinery, including impairing megalin-protein dissociation and megalin recycling, thus causing proteinuria.

Abstract P357: Renal Male and Female Cortical Mitochondria Display Bioenergetic Differences in Response to Varying Substrates

Introduction: In general, renal disease susceptibility is higher in males than in age-matched females until menopause. The damage to renal proximal tubules (PT) is key to the onset of kidney disease development due to their high capacity for reabsorption and intense mitochondrial metabolism. Our previous data identified higher oxygen consumption in the renal cortex of healthy male Sprague Dawley rats and lower H 2 O 2 production compared to females. We hypothesize that sex differences in renal mitochondrial substrate reliance trigger differential bioenergetic responses in PTs, which contributes to kidney disease development later in life. Methods: 11-week-old male and female Sprague Dawley rats were acquired from Charles River Labs. The kidneys were flushed, and the renal cortex was used for ex vivo experiments. Mitochondria were isolated with differential centrifugation, and rovided with different substrate combinations: 10mM pyruvate and 2mM malate (PM), 5mM of glutamate, malate, and succinate (GMS), or 5mM succinate only (S). Spectrofluorimetry was employed to measure membrane potential (TMRM) and H 2 O 2 production (Amplex Red), XF24e Seahorse was used for oxygen consumption rate (OCR). OriginPro was used for statistical analysis (one-way ANOVA with post hoc test) and data displayed as mean ± SEM. Results: Seahorse revealed that male renal cortical mitochondria exhibit higher basal and maximum OCR compared to females when provided with the GMS or PM substrate combinations (39.4 ± 1.18 vs 26.5± 0.88, respectively for basal GMS, and 52.0 ± 2.33 vs 32.6 ± 1.24 maximum respiration GMS, p&lt;0.001). However, similar OCR was observed when mitochondria were provided succinate only (31.2 ± 1.67 vs 28.7 ± 1.30, respectively for basal and 50.5 ± 2.49 vs 51.1 ± 1.76 maximum respiration, p&gt;0.05). Mitochondrial membrane potential was higher in females compared to males when succinate only was provided (p&lt;0.001), whereas similar mitochondrial membrane potential was observed between males and females when GMS or PM were used. Regardless of the substrate combination used, H 2 O 2 production was higher in female renal mitochondria compared to males (p&lt;0.001). Conclusions: We report that male and female renal cortical mitochondria display unique bioenergetic properties in response to varying substrates. These basal bioenergetic differences may contribute to the difference in susceptibility to kidney diseases later in life.

Abstract P427: Novel SNVs of NBCe1, D405E and I803M mutants, reduced cell-surface expression and transport activity.

Introduction: The electrogenic Na + /HCO 3 - cotransporter NBCe1 is mainly expressed in proximal renal tubule, and regulate pH homeostasis and plasma volume. NBCe1 homozygous mutation causes many symptoms, including severe hypotension, proximal renal tubular acidosis (RTA), and extrarenal phenotype (migraine, mental retardation, mineral bone disorder and ocular abnormalities). Hypothesis: Undetermined single nucleotide variants (SNVs) in NBCe1 will be crucial role of sodium and bicarbonate transport. Methods: We identified the missense mutations (D405E and I803M) in NBCe1 variant A (kidney type) from the NCBI database. We conducted a comparative analysis of cellular localization and cell protein expression using confocal microscopy and western blotting. Furthermore, we performed functional analysis in D405E and I803M SNVs using two-electrode voltage clamp method. Results: Immunofluorescence analysis with confocal microscopy revealed that the D405E and I803M variants were present predominantly in the cell-surface in HEK293 cells. Western blotting in HEK293 cells confirmed that the D405E and I803M variants were significantly reduced protein expression level (25% in D405E and 57% in I803M) compared with WT, respectively. Moreover, electrophysiological analysis revealed that D405E and I803M variants were significantly reduced transport activity. These consisting data suggest that cell-expression level and transport activity are comparable. Conclusion: These data illustrate the diverse physiological consequences of distinct SNVs and underscore the importance of functional characterization in membrane transport proteins.

Abstract P391: Role of Renal Dopamine D2 Receptor in Inverse Salt Sensitivity and Salt Sensitivity of Blood Pressure: Insights from Nephron Segment-Specific Deletion Models

The renal dopamine D2 receptor (D2R) is essential in renal and blood pressure (BP) homeostasis. In mice, global Drd2 deletion or renal-selective Drd2 silencing increases the renal expression of proinflammatory factors, renal injury markers, and BP. The D2R is expressed in several nephron segments, including the proximal and distal convoluted tubules. To study the differential effects of D2R in these segments, we generated two mouse models: one with Drd2 deletion only in the S1 and S2 segments of the proximal tubule P SGLT2 ::Cre+ Drd2 fl/fl ( Sglt2 cKO ) and their littermates as controls, P SGLT2 ::Cre- Drd2 fl/fl (Sglt2 WT) and another with deletion of the receptor in the whole nephron P CDHR16 ::Cre+ Drd2 fl/fl ( Cdhr16 cKO ) and their littermates as controls, P CDHR16 ::Cre- Drd2 fl/fl (Cdhr16 WT). In male Sglt2 cKO mice, compared with Sglt2 WT mice, D2R expression in the renal cortex was decreased by 25% (P&lt;0.05, n=5-6/group). In Cdhr16 cKO mice, compared with Cdhr16 WT mice, D2R expression was decreased by 30% (P&lt;0.05; n=3/group). In Sglt2 cKO mice, tubular lumen expansion, an indication of tubular damage, was higher on low salt (0.09%NaCl; LS) than on normal salt (0.4%NaCl; NS) (25.5±.8 vs. 11.4±.0.3; P&lt;0.001) diet. In Cdhr16 cKO mice tubular lumen expansion was also higher on LS than NS (13.9±0.4 vs 8.7±0.2; P&lt;0.001) diet. In Sglt2 cKO mice, BP (carotid artery under anesthesia) was higher on LS (130±2 mm Hg) than on NS (107±2 mm Hg) or HS (4% NaCl) (100±3 mm Hg) (n=10/group) (P&lt;0.05) diet. In Cdhr16 cKO mice, BP (tail cuff, CODA under anesthesia, confirmed by telemetry) was higher on LS (110±8 mm Hg, P&lt;0.02; n=4-5/group) and HS (119±9 mm Hg, P&lt;0.05; n=4/group) diets than on NS diet (84±5 mmHg). On LS diet, UNaV was lower in Sglt2 cKO than Sglt2 WT (1±0.3 vs 7±2 µEq/24h; P&lt; 0.05; n=4-6/group) mice. On LS diet, UNaV was also lower in Cdhr16 cKO than Cdhr16 WT (5±0.9 vs 34±14 µEq/24h; P&lt;0.05; n=3-4/group) mice. These results indicate that a decrease in D2R expression only in the proximal tubule results in inverse salt sensitivity of BP, i.e., BP higher on LS than NS or HS diets, while downregulation of its expression in the whole nephron results in inverse salt sensitivity, as well as salt sensitivity.

Abstract P431: Crosstalk Between Dopamine Receptors and AT1R in Hypertension Induced Cardiac Remodeling

Dopamine receptors are implicated in the regulation of cardiovascular function, yet their specific roles in the modulation of hypertension and cardiac remodeling are not fully understood. The angiotensin II type 1 receptor (AT1R) plays a critical role in the pathophysiology of hypertension by mediating vasoconstriction, sodium retention, and sympathetic nervous system activation. Previous studies from our lab have demonstrated that in Ang II-induced hypertension, there are alterations in D1R and D3R expression in the left ventricle (LV) compared to wild-type (WT) mice. Evidence indicates that AT1R and D1R form heterodimers and inhibition of AT1R has been shown to enhance D1R signaling in proximal tubules, suggesting a significant interaction between these receptor systems. However, the role of intracardiac dopamine receptors on AT1R expression during hypertension remains understudied. Therefore, this study aims to elucidate the crosstalk between intracardiac dopamine receptors and AT1R in the context of hypertension and cardiac remodeling by using in vitro and in vivo pharmacological and molecular methods combined with novel genetic models. Using global D3R deficient (D3KO) and WT mice, we observed that D3KO leads to alterations in AT1R gene expression in LV compared to WT hearts (p&lt;0.001) and in isolated LV cardiac fibroblasts (p&lt;0.0001). D3KO and WT mouse cardiac fibroblasts (mCFb) and human cardiac fibroblasts (hCFb) were stimulated with Ang II (300nM) ± D1R antagonist (SCH, 10µM) or D3R antagonist (SB, 10µM). D1R antagonism significantly increased AT1R (Agtr1 gene) levels in both WT and D3KO mCFb (p&lt;0.01), whereas D3R antagonism reduced AT1R gene expression following Ang II stimulation (p&lt;0.01). Furthermore, these data were confirmed in hCFb. To determine crosstalk between dopamine receptors and AT1R, we used proximity ligation assay (PLA) to examine the close proximity (&lt;40nm) of receptor-receptor expression and interaction. Our results indicate that in mCFb and hCFb, there is an increase in D1R-AT1R interactions following Ang II both with and without D1R antagonism (p&lt;0.05), however, AT1R-D3R interactions were reduced (p&lt;0.05). These data suggest that dopamine receptor activity significantly influences AT1R gene expression, highlighting a potential modulatory role for dopamine receptors in the regulation of AT1R during hypertension.

Altered kidney distribution and loss of ACE2 into the urine in acute kidney injury.

There are diverse pathophysiological mechanisms involved in acute kidney injury (AKI). Among them, overactivity of the renin-angiotensin system (RAS) has been described. Angiotensin-converting enzyme 2 (ACE2) is a tissue RAS enzyme expressed in the apical border of proximal tubules. Given the important role of ACE2 in the metabolism of angiotensin II, this study aimed to characterize kidney and urinary ACE2 in a mouse model of AKI. Ischemia-reperfusion injury (IRI) was induced in C57BL/6 mice by clamping of the left renal artery followed by removal of the right kidney. In kidneys harvested 48 h after IRI, immunostaining revealed a striking maldistribution of ACE2 including spillage into the tubular lumen and the presence of ACE2-positive luminal casts in the medulla. In cortical membranes, ACE2 protein and enzymatic activity were both markedly reduced (37 ± 4 vs. 100 ± 6 ACE2/β-actin, P = 0.0004, and 96 ± 14 vs. 152 ± 6 RFU/μg protein/h, P = 0.006). In urine, full-length membrane-bound ACE2 protein (100 kDa) was markedly increased (1,120 ± 405 vs. 100 ± 46 ACE2/µg creatinine, P = 0.04), and casts stained for ACE2 were recovered in the urine sediment. In conclusion, in AKI caused by IRI, there is a marked loss of ACE2 from the apical tubular border with deposition of ACE2-positive material in the medulla and increased urinary excretion of full-length membrane-bound ACE2 protein. The deficiency of tubular ACE2 in AKI suggests that provision of this enzyme could have therapeutic applications and that its excretion in the urine may also serve as a diagnostic marker of severe proximal tubular injury.NEW & NOTEWORTHY This study provides novel insights into the distribution of kidney ACE2 in a model of AKI by IRI showing a striking detachment of apical ACE2 from proximal tubules and its loss in urine and urine sediment. The observed deficiency of kidney ACE2 protein and enzymatic activity in severe AKI suggests that administration of forms of this enzyme may mitigate AKI and that urinary ACE2 may serve as a potential biomarker for tubular injury.

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.

Irisin preserves mitochondrial integrity and function in tubular epithelial cells after ischemia-reperfusion-induced acute kidney injury.

A myokine secreted by skeletal muscles during exercise called irisin mitigates ischemia-reperfusion (I/R) injury in epithelial cells of various organs by limiting damage to mitochondria. We test whether irisin may preserve the mitochondrial integrity and function in renal tubular epithelial cells and protect against ischemia-reperfusion-induced acute kidney injury (AKI). We correlated serum irisin levels with serum creatinine and BUN levels from both AKI patients and healthy individuals. In mice with irisin administration, various renal injury markers such as serum creatinine, BUN, kidney injury molecule-1 (Kim-1), and neutrophil gelatinase-associated lipocalin (NGAL), and renal histopathology were assessed after I/R. To identify the potential mechanisms of the protective of irisin's protective effect, we perfused proximal tubules under confocal microscopy and analyzed kidney tissues by qPCR, western blot, and immunohistochemistry. Serum irisin correlated inversely with serum creatinine and BUN levels were significantly lower in AKI patients than in healthy subjects. Administering irisin to mice after I/R decreased biomarker levels for AKI including serum creatinine, BUN, Kim-1, NAGL and lessened histological changes. In kidney tissues of mice, irisin upregulated the mitochondrial autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3), the mitochondrial autophagy pathway-related proteins PTEN-induced putative kinase 1 (PINK1) and Parkinson's disease 2 parkin (PARK2) and downregulated the reactive substrate protein sequestosome 1 (P62) and mitochondrial membrane proteins translocase of outer mitochondrial membrane 20 (TOM20) and translocase of inner mitochondrial membrane 23 (TIM23). Irisin protects against renal I/R injury, which may involve the preservation of mitochondrial integrity and function.

P098 CARDIAC REMODELING IN MICE TREATED WITH SODIUM OXALATE AND AORTIC STENOSIS

Background: Cardiovascular Diseases are one of the main global public health problems, with increasing mortality due to population aging and Hypertension (HTN). HTN is a risk factor for other diseases, such as aortic stenosis, renal dysfunction, and cardiac hypertrophy (CH). The hemodynamic overload caused by HTN affects the heart and kidneys, but the exact mechanisms of these alterations are not fully understood. Experimental models help study these mechanisms; however, a well-established model of CH with cardiorenal dysfunction does not yet exist. Objective: to evaluate whether the combination of cardiac hypertrophy, induced by transverse aortic constriction (TAC), and renal injury, induced by soluble sodium oxalate (OXA), can be a new model of CH with cardiorenal dysfunction. Methods: Male C57BL mice were randomized as follows: SHAM, receiving saline or OXA solution, and TAC, receiving saline or OXA solution. Echocardiography, direct blood pressure (BP) recording, metabolic cage, histology were performed to evaluate cardiac and renal function, hemodynamics, autonomic modulation, and baroreflex. Results: The TAC+OXA group showed the highest systolic BP(SBP) (SBP: SHAM: 122±5.24 vs. TAC+OXA: 160±5.24 mmHg, p&lt;0.01), increased double product (DP) (DP: SHAM: 64.65±4.76 mmHg/bpm vs. TAC: 89.53±5.85 mmHg/bpm, p=0.02), significant reduction in autonomic modulation (RMSSD: OXA: 12.5±1.8 vs. TAC+OXA: 2.8±0.2 ms, p&lt;0.01) and reflex tachycardia, eccentric hypertrophy, and worse renal function. The TAC group showed eccentric hypertrophy, increased BP variability (SD-SBP: SHAM: 2.97±0.07 mmHg vs. TAC: 5.63±0.57 mmHg, p≤0.01), diastolic dysfunction, and dilated left ventricle in diastole. The OXA group showed increased autonomic modulation and heart rate variability, renal damage characterized by inflammation, proximal tubule damage, and interstitial fibrosis when associated with TAC. All treated groups showed a significant reduction in reflex tachycardia. Conclusion: Transverse aortic constriction combined with OXA gavage is a model of CH with renal dysfunction, as the combination caused more significant renal damage, systemic hypertension, decreased autonomic modulation and baroreflex sensitivity, and eccentric hypertrophy with subsequent LV diastolic dysfunction.