Gene Expression In Kidney Research Articles

Dietary intake of xylose impacts the transcriptome and proteome of tissues involved in xylose metabolism in swine

Xylose is a primary component of arabinoxylan in swine diets. As arabinoxylan is a significant component of fiber, and fiber is generally rising in practical pig diets globally, the study of arabinoxylan and xylose is of increasing interest. However, the mechanisms by which free xylose may be absorbed and the pathways impacted by xylose have yet to be elucidated in pigs. The objective of this study was to determine the impact of xylose supplementation on gene expression and protein abundance in jejunum, kidney, liver, and muscle tissues which have previously been identified as possible sites of xylose absorption or metabolism. This study aimed to expand the preliminary understanding of dietary xylose metabolism and utilization in pigs. One study, replicated twice with 24 crossbred gilts, was used to assess two dietary treatments: a xylose-free (0%) control and 8% D-xylose. The impact of xylose on growth was monitored by measuring initial and final body weight, serum IGF-1, and liver glycogen concentrations. The rate and efficiency of weight gain were reduced on the xylose diet but not to a level that would occur if xylose was not used at all; the detection of xylose systemically further supports this conclusion. This study confirmed that pigs can utilize dietary xylose. To determine the impact of xylose on tissue metabolism, samples were collected from all four tissues for gene expression analysis by RNA-sequencing, and kidney and liver samples were subjected to proteomic analysis using 2D-DIGE and mass spectrometry. The majority of differentially expressed (DE) genes were identified in the kidney samples (n = 157), with a few identified in the jejunum (n = 16), liver (n = 1), and muscle (n = 20) samples. The DE genes in the kidney were mainly identified as being involved in lipid biosynthesis and fatty acid metabolism. Proteomic results corroborated these findings. Although the inclusion of xylose in a diet at practical levels is shown to impact energy metabolic processes, it has been confirmed that this five-carbon sugar can support levels of growth only slightly below those of glucose, a six-carbon sugar that is more commonly utilized as an energy source in pig diets.

#5554 ROLE OF THE TUBULAR EPITHELIAL CELL NLRP3 INFLAMMASOME DURING HYPERGLYCEMIA AND GLUCOSURIA IN DIABETES

Abstract Background and Aims Diabetes is a prominent risk factor for the progression of CKD because hyperglycemia and glucose filtration increase the hemodynamic and metabolic workload to the nephrons, which imposes adaptive mechanisms. Tubules adapt to diabetes by enlargement and epithelial cell hypertrophy but an exceeding metabolic workload promotes oxidative stress, cytokine signaling and tubular epithelial (TEC) cell demise leading to tubular atrophy and interstitial fibrosis. Several studies propose a role of the NLRP3 inflammasome in tubulointerstitial inflammation in diabetic and non-diabetic kidney disease. The NLRP3 inflammasome is a sensor of cellular damage responsible for regulate the IL-1β and IL-18 production, playing important role during the progression of CKD. However, the role of NLRP3 in diabetes-induced tubular adaptation is unknown. Method scRNAseq analysis showed previously that the TECs do not express NLRP3 or inflammasome components. However, a group of patients diagnosed with Cryopyrin-Associated Autoinflammatory Syndromes, have a mutation that overexpress NLRP3, which is associated with chronic and systemic inflammation. To investigate the role of NLRP3 on tubular cells under diabetes conditions, we generated a mouse line by crossing mice bearing LoxP-target A350V allele with Pax8-driven Cre transgenic mice. Animals carrying the A350V allele have overexpression of NLRP3 in tubular cells. The combination of unilateral nephrectomy and treatment with multiple low doses of streptozotocin (60 mg/Kg) accelerate the kidney injury caused by hyperglycaemia. Kidney function, histology and gene expression were evaluated at the end of 16 weeks of diabetes. In vitro experiments carried out using TECs isolated from Pax8-A350V, and stimulated with LPS (1µg/ml) and/or ATP (20 mM). Results In silico analysis showed TECs do not express NLRP3 or inflammasome components. A350V-TECs under inflammasome activation did not produce IL-1β or IL-18. However, the Nlrp3 overexpression upregulated kidney injury markers, as well IL-6 and TNF. Moreover, the increase in kidney inflammation was associated with significative upregulation of fibrosis markers (Tgfb1, Asma, and Fn1) and extracellular molecules (Ctgf, AnxaII, and Chd2). Diabetic mice carrying the A350V mutation did not have significative differences in blood glucose, albuminuria, or body weight when compared with diabetic control mice. The overexpression of NLRP3 in tubular cells was associated with significative reduction in GFR and BUN when compared with controls mice, even though no differences in tubular injury markers observed. Furthermore, Pax8-A350V diabetic mice expressed significantly more Nlrp3 and Il1b than control group. Although, no differences observed in cryopyrin staining. Conclusion Our data suggest the NLRP3-associated damage in tubular epithelial cells occurs in a non-canonical manner.

INVESTIGATING NEW THERAPEUTIC OPPORTUNITIES FOR HYPERTENSION THROUGH TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS OF THE KIDNEY RENIN-ANGIOTENSIN SYSTEM

Objective: We systematically characterised the expression of all renin-angiotensin system (RAS) genes in the human kidney and searched in silico for new pharmacological therapeutic opportunities to modify RAS. Design and method: We compiled data from open access resources to characterise all components of the RAS pathway. We used transcriptomic profiles of 73 human kidney tissues from the Genotype-Tissue Expression Project, integrated genome-wide DNA genotypes and RNA-sequencing-derived transcriptome profiles of 478 human kidneys from Human Kidney Tissue Resource and, kidney tissue proteomics from 72 samples recruited by Clinical Proteomic Tumour Analysis Consortium. Additionally, we used an independent single-cell RNA sequencing data and gene expression datasets curated from the Kidney Precision Medicine Project (KPMP) and Enrichr (web-based enrichment analysis tool). Lastly, we integrated the Connectivity Map and Drug Gene Interaction Database to identify drug repurposing opportunities for treatment of hypertension targeting the RAS genes. Results: We identified 37 genes encoding components of the RAS pathway with measurable expression in human kidney tissue. 15% of these gene-pairs demonstrated a positive (r>0.5) correlation in kidney expression most consistent with a functionally integrated network of interacting molecules. Angiotensinogen (AGT), the key component of the pathway - showed the highest level of mRNA expression in the liver, and we demonstrated that its hepatic mRNA expression correlates most strongly with its mRNA expression in the kidney (r = 0.71, P = 2.60e-06). Expression levels of mRNA for 92% of RAS genes correlated positively with their protein abundance in the kidney, where renin had the strongest positive correlation (r = 0.64, P<0.01). Enrichr and KPMP revealed that RAS gene expression was enriched in mesangial and epithelial cell types. Existing genetic evidence for correlation between glutamyl aminopeptidase (ENPEP) and systolic blood pressure might relate to its capacity to generate angiotensin III acting on dilator angiotensin II type 2 receptors. This raises potential repurposing opportunities for ENPEP inhibitors like firibastat and tosedostat for the treatment of hypertension. Conclusions: Collectively, this study confirmed synchronized expression of RAS genes in the kidney at both mRNA and protein levels and identified new therapeutic opportunities for treatment of hypertension, through drug repurposing.

Upregulation of Beta 1 and Arachidonic Acid Metabolizing Enzymes in the Mouse Hearts and Kidneys after Sub Chronic Administration of Rofecoxib.

An imbalance in the levels of arachidonic acid (ARA) metabolites in cardiovascular disorders and drug-induced cardiotoxicity have been previously described. This study aimed to investigate the influence of cyclooxygenase-2 (COX-2) selective inhibitors on the gene expression of ARA-metabolizing genes and beta1 gene in the hearts and kidneys of experimental mice. Thirty-five balb/c mice were divided into five groups with seven mice per group. The groups were then given two distinct types of COX-2 selective inhibitors, rofecoxib and celecoxib, in two different doses equivalent to those used in human treatment for 30 days. The mRNA expression of beta1, ace2, and ARA-metabolizing genes, coxs, lipoxygenases (aloxs), and cytochrome p450 (cyp450s) in mice heart and kidneys were assessed. Genes were analyzed using real-time polymerase chain reaction analysis. In addition, rofecoxib-induced histological alterations were examined. It was found that only the high dose of rofecoxib (5 mg/kg) caused toxicological alterations, a finding that was indicated by a significant increase (P < 0.05) in the relative weight of the mouse hearts and increase in the ventricle wall thickness as observed through pathohistological examination. This increase was associated with a significant increase in the mRNA expression level of the beta1 receptor in both the heart and kidneys of the mice (53- and 12-fold, respectively). The expression of both cox1 and 2 genes was increased 4-fold in the kidneys. In addition, the expression of the alox12 gene increased significantly (by 67-fold in the heart and by 21-fold in the kidney), while alox15 gene expression was upregulated in the heart by 8-fold and 5-fold in the kidney. The genes responsible for synthesizing 20- Hydroxyeicosatetraenoic acid (cyp4a12 and cyp1a1) were significantly upregulated (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 7- and 17 -fold, respectively. In addition, the expression of epoxyeicosatrienoic acid-synthesizing genes, cyp2c29 and cyp2j5, was increased significantly (P < 0.05) in the hearts of high-dose rofecoxib-treated mice by 4- and 16-fold, respectively. Rofecoxib caused upregulation of the mRNA expression of the beta 1 gene in association with increased expression of ARA-metabolizing genes in mouse hearts and kidneys. These findings may help us understand the molecular cardiotoxic mechanism of rofecoxib.

The association of low birthweight and prematurity on outcomes in children and adults with nephrotic syndrome-a NEPTUNE cohort study.

In single-center studies, both preterm birth and low birth weight (LBW) are associated with worse outcomes in childhood nephrotic syndrome. Using the Nephrotic Syndrome Study Network (NEPTUNE) observational cohort, we tested the hypothesis that in patients with nephrotic syndrome, hypertension, proteinuria status, and disease progression would be more prevalent and more severe in subjects with LBW and prematurity singly or in combination (LBW/prematurity). Three hundred fifty-nine adults and children with focal segmental glomerulosclerosis (FSGS) or minimal change disease (MCD) and available birth history were included. Estimated glomerular filtration rate (eGFR) decline and remission status were primary outcomes, and secondary outcomes were kidney histopathology, kidney gene expression, and urinary biomarkers. Logistic regression was used to identify associations with LBW/prematurity and these outcomes. We did not find an association between LBW/prematurity and remission of proteinuria. However, LBW/prematurity was associated with greater decline in eGFR. This decline in eGFR was partially explained by the association of LBW/prematurity with APOL1 high-risk alleles, but the association remained after adjustment. There were no differences in kidney histopathology or gene expression in the LBW/prematurity group compared to normal birth weight/term birth. LBW and premature babies who develop nephrotic syndrome have a more rapid decline in kidney function. We did not identify clinical or laboratory features that distinguished the groups. Additional studies in larger groups are needed to fully ascertain the effects of (LBW) and prematurity alone or in combination on kidney function in the setting of nephrotic syndrome.

Leptin-dependent gene expression and function in mouse kidney

Objective: Leptin regulates energy balance via leptin receptor expressed in central and peripheral tissues. Little is known about leptin receptor (Lepr)-mediated kidney gene expression and function. Here, we determined kidney gene expression of Lepr splice variants, leptin-sensitive kidney gene expression, and the role of Lepr in renal tubular cells for the response to a high fat diet. Methods: A) Four groups of male mice were used for urine/plasma analysis, and kidney cortex RNA-seq and quantitative RT-PCR (qPCR) analysis: BTBR wild-type, BTBR ob/ob-vehicle (BTBR background in ob/ob promotes diabetic kidney disease), BTBR ob/ob-chronic leptin replacement [6 days via osmotic mini-pump, 4.7 μg/day], and BTBR ob/ob-acute leptin replacement [single dose of 5 mg/kg body weight i.p. and analysis after 7 hours]; all 12-19 weeks old; n=4~15/group. B) Tubular Lepr knockdown (Pax8-Lepr KO) mice were generated by the Pax8-Cre/loxP system, and 5-week old male Pax8-Lepr KO and littermate control mice (n=8-12/group) were fed either control diet or high fat diet (60 kcal% fat) for 35 weeks. Lepr in situ hybridization and qPCR were performed in kidney tissue, and physiological parameters and urinary albumin to creatinine ratio (UACR) were determined. Results: qPCR analysis of Lepr splice variants A, B and C in BTBR mouse kidney revealed a ratio of ~100:10:1 in cortex and medulla with medullary levels being ~10x higher vs cortex with little effects of leptin deficiency (ob/ob)/or replacement. Chronic leptin replacement in ob/ob mice reduced hyperphagia, hyperglycemia and albuminuria, associated with normalization of kidney mRNA expression of molecular markers of glycolysis, gluconeogenesis, amino acid synthesis and megalin. Acute leptin replacement was ineffective implicating indirect metabolic effects for albuminuria in leptin deficiency. Tubular knockdown of Lepr and in situ hybridization indicated that a minor fraction of kidney Lepr mRNA is expressed by tubular cells compared with interstitial or endothelial cells. Nonetheless, Pax8-Lepr KO mice had 13% lower kidney weight (P=0.038). Moreover, they presented similar high fat diet-induced increases in plasma leptin levels, body and kidney weight, and GFR vs controls but a 50% reduced rise in UACR (P=0.0005). Two additional leptin or tubular Lepr-sensitive genes were identified: Leptin deficiency and tubular Lepr knockdown reduced renal expression of acetoacetyl-CoA synthetase (AACS) and increased expression of gremlin 1 (Grem1), while leptin replacement in ob/ob induced opposite effects. Conclusions: Leptin deficiency may increase albuminuria via systemic metabolic effects that impinge on kidney megalin expression. Hyperleptinemia-induced albuminuria may involve direct effects via tubular Lepr. Implications of the novel tubular Lepr/AACS/Grem1 axis and of Lepr variants remain to be determined. This study was supported by Janssen Pharmaceuticals, Inc. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Western diet exacerbates aristolochic acid-induced nephropathy in mice

Aristolochic acid (AA) ingestion causes Balkan nephropathy, characterized by tubular injury and progression to chronic kidney disease (CKD). AA is taken up by proximal tubule cells via organic anion transport (OAT3) and induces p53-mediated DNA damage, but little is known about modulating factors. The Western diet (WD) is rich in saturated fats, sugars, and salt and contributes to the development of metabolic disorders such as obesity and diabetes, but also to the progression of CKD. The aim of this study was to elucidate the impact of WD on AA-induced kidney injury. 5-week-old male C57BL6 mice were fed with WD or normal chow (NC) for 8 weeks, followed by administration of AA every 3 days at a dose of 3 mg/kg for 3 weeks. Samples were collected after a 3-week recovery period (n=4/group for vehicle; n=7-8/group for AA). The AA-induced increase in plasma creatinine and the reduction of hematocrit were significantly greater in WD vs NC. This was associated with increased kidney gene expression in WD vs NC of markers of DNA damage ( p53), injury ( Kim1 and Ngal), and inflammation ( Tnfa). The WD group had a greater body weight and, therefore, received a higher total dose of AA (0.73 and 0.6 mg), which may have worsened kidney injury. Therefore, in a second series, the same total dose of AA (0.9 mg) was administered to WD and NC groups. WD similarly increased the AA-induced fall in hematocrit and rise in plasma creatinine and renal p53, Kim1, Ngal, and Tnfa mRNA expression compared to the NC group. Moreover, WD increased renal mRNA expression of Oat3. These findings suggest that WD increases the susceptibility to AA nephrotoxicity, possibly through upregulation of OAT3. This model may be useful for studying the mechanistic impact of Western diet on the development and progression of CKD caused by nephrotoxic agents. Supported by National Institutes of Health (NIH) Grants RF1AG061296 (PM, VV), R01DK132690 (VV), and P30 DK079337 (VV), the Department of Veterans Affairs, and a fellowship of the Manpei Suzuki Diabetes Foundation (YO). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Expression of leptin receptor in renal tubules is sparse but implicated in leptin-dependent kidney gene expression and function.

Leptin regulates energy balance via leptin receptors expressed in central and peripheral tissues, but little is known about leptin-sensitive kidney genes and the role of the tubular leptin receptor (Lepr) in response to a high-fat diet (HFD). Quantitative RT-PCR analysis of Lepr splice variants A, B, and C revealed a ratio of ∼100:10:1 in the mouse kidney cortex and medulla, with medullary levels being ∼10 times higher. Leptin replacement in ob/ob mice for 6 days reduced hyperphagia, hyperglycemia, and albuminuria, associated with normalization of kidney mRNA expression of molecular markers of glycolysis, gluconeogenesis, amino acid synthesis, and megalin. Normalization of leptin for 7 h in ob/ob mice did not normalize hyperglycemia or albuminuria. Tubular knockdown of Lepr [Pax8-Lepr knockout (KO)] and in situ hybridization revealed a minor fraction of Lepr mRNA in tubular cells compared with endothelial cells. Nevertheless, Pax8-Lepr KO mice had lower kidney weight. Moreover, while HFD-induced hyperleptinemia, increases in kidney weight and glomerular filtration rate, and a modest blood pressure lowering effect were similar compared with controls, they showed a blunted rise in albuminuria. Use of Pax8-Lepr KO and leptin replacement in ob/ob mice identified acetoacetyl-CoA synthetase and gremlin 1 as tubular Lepr-sensitive genes that are increased and reduced by leptin, respectively. In conclusion, leptin deficiency may increase albuminuria via systemic metabolic effects that impinge on kidney megalin expression, whereas hyperleptinemia may induce albuminuria by direct tubular Lepr effects. Implications of Lepr variants and the novel tubular Lepr/acetoacetyl-CoA synthetase/gremlin 1 axis remain to be determined.NEW & NOTEWORTHY This study provides new insights into kidney gene expression of leptin receptor splice variants, leptin-sensitive kidney gene expression, and the role of the leptin receptor in renal tubular cells for the response to diet-induced hyperleptinemia and obesity including albuminuria.

Assessing Kidney Injury Induced by Mercuric Chloride in Guinea Pigs with In Vivo and In Vitro Experiments.

Acute kidney injury, which is associated with high levels of morbidity and mortality, affects a significant number of individuals, and can be triggered by multiple factors, such as medications, exposure to toxic chemicals or other substances, disease, and trauma. Because the kidney is a critical organ, understanding and identifying early cellular or gene-level changes can provide a foundation for designing medical interventions. In our earlier work, we identified gene modules anchored to histopathology phenotypes associated with toxicant-induced liver and kidney injuries. Here, using in vivo and in vitro experiments, we assessed and validated these kidney injury-associated modules by analyzing gene expression data from the kidneys of male Hartley guinea pigs exposed to mercuric chloride. Using plasma creatinine levels and cell-viability assays as measures of the extent of renal dysfunction under in vivo and in vitro conditions, we performed an initial range-finding study to identify the appropriate doses and exposure times associated with mild and severe kidney injuries. We then monitored changes in kidney gene expression at the selected doses and time points post-toxicant exposure to characterize the mechanisms of kidney injury. Our injury module-based analysis revealed a dose-dependent activation of several phenotypic cellular processes associated with dilatation, necrosis, and fibrogenesis that were common across the experimental platforms and indicative of processes that initiate kidney damage. Furthermore, a comparison of activated injury modules between guinea pigs and rats indicated a strong correlation between the modules, highlighting their potential for cross-species translational studies.

The circadian clock remains intact, but with dampened hormonal output in heart failure

Circadian (24-h) rhythms are important regulators in physiology and disease, but systemic disease may disrupt circadian rhythmicity. Heart failure (HF) is a systemic disease affecting hormonal regulation. We investigate whether HF affects the rhythmic expression of melatonin and cortisol, main endocrine products of the central clock, and cardiac-specific troponin in patients. We corroborate the functionality of the peripheral clock directly in the organs of translational models, inaccessible in human participants. We included 46 HF patients (71.7% male, median age of 60 years, NYHA class II (32.6%) or III (67.4%), ischemic cardiomyopathy (43.5%), comorbidities: diabetes 21.7%, atrial fibrillation 30.4%), and 24 matched controls. Blood was collected at seven time-points during a 24-h period (totalling 320 HF and 167 control samples) for melatonin, cortisol, and cardiac troponin T (cTnT) measurements after which circadian rhythms were assessed through cosinor analyses, both on the individual and the group level. Next, we analysed peripheral circadian clock functionality using cosinor analysis in male animal HF models: nocturnal mice and diurnal zebrafish, based on expression of core clock genes in heart, kidneys, and liver, every 4h during a 24-h period in a light/darkness synchronised environment. Melatonin and cortisol concentrations followed a physiological 24-h pattern in both patients and controls. For melatonin, acrophase occurred during the night for both groups, with significantly decreased amplitude (median 5.2 vs 8.8, P=0.0001) and circadian variation ([maximum]/[minimum]) in heart failure patients. For cortisol, mesor showed a significant increase for HF patients (mean 331.9 vs 275.1, P=0.017) with a difference of 56.8 (95% CI 10.3-103.3) again resulting in a relatively lower variation: median 3.9 vs 6.3 (P=0.0058). A nocturnal blood pressure dip was absent in 77.8% of HF patients. Clock gene expression profiles (Bmal, Clock, Per, Cry) were similar and with expected phase relations in animal HF models and controls, demonstrating preserved peripheral clock functionality in HF. Furthermore, oscillations in diurnal zebrafish were expectedly in opposite phases to those of nocturnal mice. Concordantly, cTnT concentrations in HF patients revealed significant circadian oscillations. Central clock output is dampened in HF patients while the molecular peripheral clock, as confirmed in animal models, remains intact. This emphasises the importance of taking timing into account in research and therapy for HF, setting the stage for another dimension of diagnostic, prognostic and therapeutic approaches. Hartstichting.

Up-regulation of PRKDC was associated with poor renal dysfunction after renal transplantation: A multi-centre analysis.

Renal transplantation is the only efficacious treatment for end-stage kidney disease. However, some people have developed renal insufficiency after transplantation, the mechanisms of which have not been well clarified. Previous studies have focused on patient factors, while the effect of gene expression in the donor kidney on post-transplant renal function has been less studied. Donor kidney clinical data and mRNA expression status were extracted from the GEO database (GSE147451). Weight gene co-expression network analysis (WGCNA) and differential gene enrichment analysis were performed. For external validation, we collected data from 122 patients who accepted renal transplantation at several hospitals and measured the level of target genes by qPCR. This study included 192 patients from the GEO data set, and 13 co-expressed genes were confirmed by WGCNA and differential gene enrichment analysis. Then, the PPI network contained 17 edges as well as 12 nodes, and four central genes (PRKDC, RFC5, RFC3 and RBM14) were identified. We found by collecting data from 122 patients who underwent renal transplantation in several hospitals and by multivariate logistic regression that acute graft-versus-host disease postoperative infection, PRKDC [Hazard Ratio (HR) = 4.44; 95% CI = [1.60, 13.68]; p = 0.006] mRNA level correlated with the renal function after transplantation. The prediction model constructed had good predictive accuracy (C-index = 0.886). Elevated levels of donor kidney PRKDC are associated with renal dysfunction after transplantation. The prediction model of renal function status for post-transplant recipients based on PRKDC has good predictive accuracy and clinical application.

Lipidome modulation by dietary omega-3 polyunsaturated fatty acid supplementation or selective soluble epoxide hydrolase inhibition suppresses rough LPS-accelerated glomerulonephritis in lupus-prone mice.

Lipopolysaccharide (LPS)-accelerated autoimmune glomerulonephritis (GN) in NZBWF1 mice is a preclinical model potentially applicable for investigating lipidome-modulating interventions against lupus. LPS can be expressed as one of two chemotypes: smooth LPS (S-LPS) or rough LPS (R-LPS) which is devoid of O-antigen polysaccharide sidechain. Since these chemotypes differentially affect toll-like receptor 4 (TLR4)-mediated immune cell responses, these differences may influence GN induction. We initially compared the effects of subchronic intraperitoneal (i.p.) injection for 5 wk with 1) Salmonella S-LPS, 2) Salmonella R-LPS, or 3) saline vehicle (VEH) (Study 1) in female NZBWF1 mice. Based on the efficacy of R-LPS in inducing GN, we next used it to compare the impact of two lipidome-modulating interventions, ω-3 polyunsaturated fatty acid (PUFA) supplementation and soluble epoxide hydrolase (sEH) inhibition, on GN (Study 2). Specifically, effects of consuming ω-3 docosahexaenoic acid (DHA) (10 g/kg diet) and/or the sEH inhibitor 1-(4-trifluoro-methoxy-phenyl)-3-(1-propionylpiperidin-4-yl) urea (TPPU) (22.5 mg/kg diet ≈ 3 mg/kg/day) on R-LPS triggering were compared. In Study 1, R-LPS induced robust elevations in blood urea nitrogen, proteinuria, and hematuria that were not evident in VEH- or S-LPS-treated mice. R-LPS-treated mice further exhibited kidney histopathology including robust hypertrophy, hyperplasia, thickened membranes, lymphocytic accumulation containing B and T cells, and glomerular IgG deposition consistent with GN that was not evident in VEH- or SLPS-treated groups. R-LPS but not S-LPS induced spleen enlargement with lymphoid hyperplasia and inflammatory cell recruitment in the liver. In Study 2, resultant blood fatty acid profiles and epoxy fatty acid concentrations reflected the anticipated DHA- and TPPU-mediated lipidome changes, respectively. The relative rank order of R-LPS-induced GN severity among groups fed experimental diets based on proteinuria, hematuria, histopathologic scoring, and glomerular IgG deposition was: VEH/CON< R-LPS/DHA ≈ R-LPS/TPPU<<< R-LPS/TPPU+DHA ≈ R-LPS/CON. In contrast, these interventions had modest-to- negligible effects on R-LPS-induced splenomegaly, plasma antibody responses, liver inflammation, and inflammation-associated kidney gene expression. We show for the first time that absence of O-antigenic polysaccharide in R-LPS is critical to accelerated GN in lupus-prone mice. Furthermore, intervention by lipidome modulation through DHA feeding or sEH inhibition suppressed R-LPS-induced GN; however, these ameliorative effects were greatly diminished upon combining the treatments.

Autophagy activation, histopathological damage, and altered renal epithelial sodium channel and Na+,K+‐ATPase gene expression in offspring kidney after in utero exposure to allethrin

The goal of the current study was to determine the effects of the allethrin, as a pyrethroid-based insecticide, on kidney function in rat offspring. Sixty-day-old Wistar albino female rats were randomly allocated to three groups: control group and two allethrin-treated groups that were intragastrically administered with 34.2 or 68.5 mg/kg b.w. of allethrin starting from the day 6 of pregnancy until delivery. The results revealed that the high dose of allethrin (68.5 mg/kg) reduced the number of glomeruli. Results showed that the glomeruli diameter in animals treated with 34.2 mg/kg of allethrin significantly decreased when compared to the controls (p < 0.001) whereas at the dose of 68.5 mg/kg the glomeruli diameter was significantly higher than those of the control group (p < 0.05). Interestingly, the expression of the autophagic markers Beclin-1 and LC-3 was increased when compared to control indicating an induction of the autophagic mechanism. Furthermore, allethrin administration downregulated in a dose-dependent manner the expression of the epithelial sodium channel (ENaC) gene. The mRNA levels of Na+/K+- ATPase gene were significantly higher in the group treated with the low dose and significantly lower with the high dose, when compared to control. However, there was no effect of allethrin on the gene expression of NHE3 in both treated groups. Taken together, the findings of this study suggested that the exposure to allethrin might contribute to the nephrotoxicity by affecting the number and the diameter of glomeruli, causing histopathological alterations and inducing autophagic-related markers and affecting the mRNA levels of ENaC and Na+/ K + ATPase genes.

In Vivo Toxicity of Oral Administrated Nano-SiO2: Can Food Additives Increase Apoptosis?

Nano-silicon dioxide (nano-SiO2) has a great deal of application in food packaging, as antibacterial food additives, and in drug delivery systems but this nanoparticle, despite its wide range of utilizations, can generate destructive effects on organs such as the liver, kidney, and lungs. This study is aimed at investigating the toxicological effects of nano-SiO2 through apoptotic factors. For this purpose, 40 female rats in 4 groups (n = 10) received 300, 600, and 900mg/kg/day of nano-SiO2 at 20-30nm size orally for 20days. Relative expression of Caspase3, Bcl-2, and BAX genes in kidney and liver was evaluated in real time-PCR. The results indicated the overexpression of BAX and Caspase3 genes in the liver and kidney in groups receiving 300 and 900mg/kg/day of nano-SiO2. Bcl-2 gene was up-regulated in the liver and kidney at 600mg/kg/day compared to the control group. Overexpression of the Bcl-2 gene in the kidney in 300 and 900mg/kg/day recipient groups was observed (P ≤ 0.05). Histopathological examination demonstrated 600mg/kg/day hyperemia in the kidney and lungs. In addition, at 900mg/kg/day were distinguished scattered necrosis and hyperemia in the liver. The rate of epithelialization in the lungs increased. The nano-SiO2 at 300 and 900mg/kg/day can induce more cytotoxicity in the liver and lung after oral exposure. However, cytotoxicity of nano-SiO2 at 600mg/kg/day in the kidney and lung was noticed. Hence, the using of nano-SiO2 as an additive and food packaging should be more considered due to their deleterious effects.

PS-B09-2: ENOS-NO PATHWAY MODULATES AKI TO CKD TRANSITION IN IRI MODEL MICE VIA REGULATION OF B;CATENIN TRANSLOCATION

Background: The incidence of acute kidney injury (AKI) has increased over the past few decades. AKI is also currently recognized as an inducer of chronic kidney disease (CKD) through a process known as the AKI to CKD transition. It has been shown that aging and hypertension are risk factor for the AKI to CKD transition. Because these conditions are related to endothelial dysfunction, we hypothesized that the AKI to CKD transition is promoted by deficiency in the eNOS Nitric Oxide (NO) pathway as an endothelial dysfunction, and we used eNOS deficient mice (eNOSKO) and wild-type (WT) mice (C57B6/J: WT) to determine the mechanism of the AKI to CKD transition. Methods: WT and eNOSKO mice were used to determine the role of the eNOS NO pathway. Ischemic reperfusion was performed two weeks after uni-nephrectomization. The ischemic time was 20 min. The WT and eNOSKO mice were divided into four groups: WT sham, WT ischemic reperfusion injury (IRI), eNOSKO-sham and eNOSKO IRI. The first theses mice were sacrificed on Day 28 (D28) after the IRI (WT IRI D28 and eNOS IRI D28). Pathological analysis was conducted using Masson staining and KIM1 staining. Inflammation was assessed using quantitative PCR (qPCR) (IL1β;, IL6, and F4/80) and flow cytometry (FACS) analysis. Results: There was no evidence of kidney damage in the WT IRI D28 group. However, KIM1 staining detected tubular cell damage in the eNOSKO IRI D28 group. Fibrosis, which was assessed using the Masson staining, was also exacerbated in the eNOSKO IRI D28 group compared with the WT IRI D28 group. These results suggested that deficient eNOS NO signaling promoted the AKI to CKD transition. Next, the WT-IRI and eNOS IRI mice were sacrificed on Day 1 (D1) after the IRI (WT IRI D1 and eNOSKO IRI D1) to evaluate inflammation and kidney damage in the acute phase after IRI. There were no differences in kidney damage and inflammation between the WT-IRI-D1 and eNOSKO-IRI-D1 groups. In addition, at each time point (D7, Day 14 (D14), and D28) after IRI, qPCR was used to detect Wnt β;catenin related gene expression in the entire kidney. Expression of these genes was increased in the eNOSKO IRI D7 group compared with the WT IRI D7 group. Conclusions: Our results showed that after ischemic reperfusion, deficiency in the eNOS NO pathway promotes the AKI to CKD transition via acceleration of Wnt β;catenin. Therefore, activation of the eNOS NO pathway could be a potential therapeutic target in AKI.

Single-Cell Transcriptome Sequencing Reveals Molecular Mechanisms of Renal Injury in Essential Hypertension

Introduction: Hypertensive nephropathy is characterized by glomerular and tubulointerstitial damage, but we know little about changes in cell-specific gene expression in the early stages of hypertensive kidney injury, which usually has no obvious pathological changes. Methods: We performed unbiased single-cell RNA sequencing of rat kidney samples from hypertensive kidney injury to generate 10,602 single-cell transcriptomes from 2 control and 2 early stage hypertensive kidney injury samples. Results: All major cell types of the kidney were represented in the final dataset. Side-by-side comparisons showed that cell type-specific changes in gene expression are critical for functional impairment of glomeruli and tubules and activation of immune cells. In particular, we found a significantly reduced gene expression profile of maintaining vascular integrity in glomerular cells of hypertensive kidney injury. Meanwhile, the expression of genes associated with oxidative stress injury and fibrosis in the renal tubules and collecting ducts was elevated, but the degree of tubular cells response to injury differed between parts. We also found a signature of immune cell infiltration in hypertensive kidney injury. Conclusion: Exploring the changes of gene expression in hypertension-injured kidneys may be helpful to identify the early biomarkers and signal pathways of this disease. Our data provide rich resources for understanding the pathogenesis of hypertensive renal injury and formulating effective treatment strategies.

Hypovitaminosis A Drives the Progression of Tubulointerstitial Lupus Nephritis through Potentiating Predisease Cellular Autoreactivity.

Vitamin A (VA) deficiency (VAD) is observed in both humans and mice with lupus nephritis. However, whether VAD is a driving factor for accelerated progression of lupus nephritis is unclear. In this study, we investigated the effect of VAD on the progression of lupus nephritis in a lupus-prone mouse model, MRL/lpr. We initiated VAD either during gestation or after weaning to reveal a potential time-dependent effect. We found exacerbated lupus nephritis at ∼15 wk of age with both types of VAD that provoked tubulointerstitial nephritis leading to renal failure. This was concomitant with significantly higher mortality in all VAD mice. Importantly, restoration of VA levels after weaning reversed VAD-induced mortality. These results suggest VAD-driven acceleration of tubulointerstitial lupus nephritis. Mechanistically, at the earlier time point of 7 wk of age and before the onset of clinical lupus nephritis, continued VAD (from gestation until postweaning) enhanced plasma cell activation and augmented their autoantibody production, while also increasing the expansion of T lymphocytes that could promote plasma cell autoreactivity. Moreover, continued VAD increased the renal infiltration of plasmacytoid dendritic cells. VAD initiated after weaning, in contrast, showed modest effects on autoantibodies and renal plasmacytoid dendritic cells that were not statistically significant. Remarkably, analysis of gene expression in human kidney revealed that the retinoic acid pathway was decreased in the tubulointerstitial region of lupus nephritis, supporting our findings in MRL/lpr mice. Future studies will elucidate the underlying mechanisms of how VAD modulates cellular functions to exacerbate tubulointerstitial lupus nephritis.

PS-BPB06-5: URINARY CELL TRANSCRIPTOMICS: A NON-INVASIVE EXPRESSION READOUT OF KIDNEY GENES OF RELEVANCE TO HYPERTENSION

Objective: Hypertension affects 35% of the global adult population and is the leading preventable risk factor for premature death globally. The kidney is the key organ of blood pressure regulation in the body. Here, we compare the gene expression profile from renal cells shed naturally into the urine and kidney tissue to determine if urine can provide a non-invasive readout of kidney gene expression and whether hypertension-associated genes can be quantified in urine. Design and Method: Urinary cell and kidney tissue samples were collected from 33 human participants and were both profiled by poly-A RNA-sequencing, generating an average of 30 million paired reads per sample. These were quantified using the standard Genotype Tissue Expression (GTEx) project pipeline and were compared against 43 different human tissues and other bodily fluids using transcriptomic correlation. RNA-sequencing quality metrics were calculated by RNA-SeQC software. Gene set overrepresentation analysis for Gene Ontology (GO) terms and Kyoto Encyclopaedia of Genes and Genomes (KEGG) employed Fishers exact test. Enriched and enhanced kidney genes were collected from the Human Protein Atlas (HPA). Results: Our RNA-sequencing metric analysis revealed that urinary cells can generate robust data of comparable or superior quality to that of saliva at similar read coverage. The top one hundred most highly expressed urinary cell genes show an enrichment for biological themes shared with the renal transcriptome, including immunity (P = 1.2x10–7), glucose metabolism (P = 1.3x10–5) and renal mineral reabsorption (P = 9.8x10–4). In an analysis all protein-coding genes across 43 human tissues/cell-types, urinary cells showed the highest level of transcriptomic correlation with kidney cortex (r2 = 0.65) and kidney medulla (r2 = 0.64). This correlation between urinary cells and kidney tissue was particularly strong (r2 = 0.72) in an analysis restricted to highly specific kidney genes (including uromodulin and the Na-K-Cl cotransporter NKCC2 [loop diuretic target]). 98% (176 out of 179) of kidney genes with a known causal association to blood pressure were expressed in urinary cells. Their urinary expression demonstrated strong correlation with their abundance in kidney cortex (r2 = 0.68) and medulla (r2 = 0.64). Conclusions: Standard poly-A RNA-sequencing of cells harvested from urinary sediments produces robust gene expression profiles. These profiles provide a non-invasive insight into transcriptome of the kidney and permit measuring expression of kidney genes of relevance to BP regulation and hypertension.

Available phosphorus levels modulate gene expression related to intestinal calcium and phosphorus absorption and bone parameters differently in gilts and barrows.

Dietary phytase increases bioavailability of phytate-bound phosphorus (P) in pig nutrition affecting dietary calcium (Ca) to P ratio, intestinal uptake, and systemic utilization of both minerals, which may contribute to improper bone mineralization. We used phytase to assess long-term effects of two dietary available P (aP) levels using a one-phase feeding system on gene expression related to Ca and P homeostasis along the intestinal tract and in the kidney, short-chain fatty acids in stomach, cecum, and colon, serum, and bone parameters in growing gilts and barrows. Growing pigs (37.9±6.2 kg) had either free access to a diet without (Con; 75 gilts and 69 barrows) or with phytase (650 phytase units; n = 72/diet) for 56 days. Samples of blood, duodenal, jejunal, ileal, cecal, and colonic mucosa and digesta, kidney, and metacarpal bones were collected from 24 pigs (6 gilts and 6 barrows per diet). Phytase decreased daily feed intake and average daily gain, whereas aP intake increased with phytase versus Con diet (p<0.05). Gilts had higher colonic expression of TRPV5, CDH1, CLDN4, ZO1, and OCLN and renal expression of TRPV5 and SLC34A3 compared to barrows (p<0.05). Phytase increased duodenal expression of TRPV5, TRPV6, CALB1, PMCA1b, CDH1, CLDN4, ZO1, and OCLN compared to Con diet (p<0.05). Furthermore, phytase increased expression of SCL34A2 in cecum and of FGF23 and CLDN4 in colon compared to Con diet (p<0.05). Alongside, phytase decreased gastric propionate, cecal valerate, and colonic caproate versus Con diet (p<0.05). Phytase reduced cortical wall thickness and index of metacarpal bones (p<0.05). Gene expression results suggested an intestinal adaptation to increased dietary aP amount by increasing duodenal trans- and paracellular Ca absorption to balance the systemically available Ca and P levels, whereas no adaption of relevant gene expression in kidney occurred. Greater average daily gain in barrows related to higher feed intake.

Steroidogenic and innate immune responses in Atlantic salmon are influenced by dietary total lipid, long chain polyunsaturated fatty acids and dissolved oxygen

Recent studies have found that feeding high levels of long chain polyunsaturated fatty acids (LC-PUFA) influenced the steroid biosynthesis pathway of Atlantic salmon on a transcriptomic level, although research observed on a metabolic level is lacking. Dietary lipid and chronic stress have also been suggested to play a role in steroidogenesis. A study was performed on Atlantic salmon post-smolts (184 g) fed with diets of high and low levels of lipid with and without high levels of LC-PUFA under hypoxic (stressed) or normoxic (unstressed) conditions. Liver, head kidney and blood samples were collected after 35 and 116 days to determine short- and long-term effects. On day 35, dissolved oxygen affected the levels of the steroid hormones 11-deoxycortisol, cortisone and testosterone as well as the relative expression of cyp11a involved in steroidogenesis and ud2a2 involved in sex steroid metabolism. On day 116, an interaction between lipid x LC-PUFA x oxygen was found for cyp11a, hsd3b (steroidogenesis) and ud2a2 genes, whereas LC-PUFA and/or oxygen affected the steroid levels of 11-dehydrocorticosterone, corticosterone, cortisol, dehydroepiandrosterone, pregnenolone and testosterone. The observation that both LC-PUFA and oxygen affected cyp11a expression and the levels of pregnenolone, provides evidence for the influence of both parameters on the rate-limiting point of steroid production, which can take >35 days to establish. The 3-way interaction between lipid, LC-PUFA and dissolved oxygen on the expression of steroid genes suggests that the chronic stressor of hypoxia may be over-riding dietary influences of the lipid and LC-PUFA, though the relative proportion of LC-PUFA within the lipid may play a role as well. In terms of fatty acid composition, low dietary lipid and high LC-PUFA increased the levels of total n-6 and n-3 PUFA in the liver, particularly EPA and DHA, and the head kidney to a lesser extent. In terms of immune pathways, hypoxia influenced cytokine gene expression (tgfb, il10), cellular repair gene expression (gr, hsp70 and hsp90) and eicosanoid levels (PGE2 and LTB5) in the head kidney, while dietary lipid and LC-PUFA influenced cytokines (ifnγ and il1β) and eicosanoids (PGE2 and LTB5). These findings demonstrate that feeding different levels of LC-PUFA significantly alters steroidogenesis and innate immune response in Atlantic salmon post-smolts with additional interactions from dietary lipid and hypoxia stress. These results will significantly impact the aquaculture industry since it demonstrates salmon fed high levels of LC-PUFA are likely more resilient to cope with chronic stressors (e.g. prolonged hypoxia) due to lower corticosteroid levels and upregulated immune response.