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Hypertension and diabetes are the greatest factors influencing the progression of chronic kidney disease (CKD). Investigation into the role of nephron number in CKD alone or with hypertension has revealed a strong inverse relationship between the two; however, not much is known about the connection between nephron number and diabetic kidney disease. The heterogeneous stock-derived model of unilateral renal agenesis (HSRA) rat, a novel model of nephron deficiency, provides a unique opportunity to study the association between nephron number and hypertension and diabetes on CKD. HSRA rats exhibit failure of one kidney to develop in 50-75% of offspring, whereas the remaining offspring are born with two kidneys. Rats born with one kidney (HSRA-S) develop significant renal injury with age compared with two-kidney littermates (HSRA-C). The induction of hypertension as a secondary stressor leads to significantly more renal injury in HSRA-S compared with HSRA-C rats and nephrectomized HSRA-C (HSRA-UNX) rats. The present study sought to address the hypothesis that nephron deficiency in the HSRA rat would hasten renal injury in the presence of a secondary stressor of hyperglycemia. HSRA animals did not exhibit diabetes-related traits at any age; thus, streptozotocin (STZ) was used to induce hyperglycemia in HSRA-S, HSRA-C, and HSRA-UNX rats. STZand vehicle-treated animals were followed for 15 wk. STZ-treated animals developed robust hyperglycemia, but in contrast to the response to hypertension, neither HSRA-S nor HSRA-UNX animals developed proteinuria compared with vehicle treatment. In total, our data indicate that hyperglycemia from STZ alone does not have a significant impact on the onset or progression of injury in young one-kidney HSRA animals. [ABSTRACT FROM AUTHOR]

Preeclampsia is a progressive hypertensive disorder of pregnancy affecting 2%-8% of pregnancies globally. Preexisting chronic hypertension is a major risk factor associated with developing preeclampsia, and growing evidence suggests a role for the gut microbiome in the development of preeclampsia. However, neither alterations in the gut microbiome associated with preeclampsia nor the mechanisms involved are fully understood. In this study, we tested the hypothesis that normal gestational maternal gut microbiome remodeling is impaired in the Dahl salt-sensitive (Dahl S) rat model of superimposed preeclampsia. Gut microbiome profiles of pregnant Dahl S, normal pregnant Sprague-Dawley (SD), and matched virgin controls were assessed by 16S rRNA gene sequencing at baseline; during early, middle, and late pregnancy; and 1-wk postpartum. Dahl S rats had significantly higher abundance in Proteobacteria, and multiple genera were significantly different from SD rats at baseline. The pregnant SD displayed a significant increase in Proteobacteria and genera such as Helicobacter, but these were not different between pregnant and virgin Dahl S rats. By late pregnancy, Dahl S rats had significantly lower a-diversity and Firmicutes compared with their virgin Dahl S controls. b-diversity was significantly different among groups (P < 0.001). KEGG metabolic pathways including those associated with short-chain fatty acids were different in Dahl S pregnancy but not in SD pregnancy. These results reveal an association between chronic hypertension and gut microbiome dysbiosis which may hinder pregnancy-specific remodeling in the gut microbial composition during superimposed preeclampsia. [ABSTRACT FROM AUTHOR]

The burden of hypertension in the United States is increasing and yields significant morbidity and mortality, and sex differences in hypertension are widely recognized. Reduced nitric oxide (NO) bioavailability and increased oxidative stress are known to contribute to the pathogenesis of hypertensive renal injury, and but their contributions to sex differences in injury progression of are undefined. Our purpose was to test the hypothesis that male hypertensive rats have accelerated renal injury compared to females and to determine the contributions of the nitric oxide pathway and oxidative stress in these differences. Male and female Dahl SS/Jr rats, a model that spontaneously develops hypertension with age, were allowed to age on a 0.3% NaCl diet until 3 or 6 months of age, at which points blood pressure was measured and plasma, tissue, and urine were collected. While no significant sex differences in blood pressure were present at either time point, renal injury measured by urine protein excretion was more severe (male = 44.9 ± 6; female = 15±3 mg/day/100 g bw, p =.0001), and renal function was reduced (male = 0.48 ± 0.02; female = 0.7 ± 0.03 ml min‐1 g‐1 kw, p =.001) in males compared to females with age. Both male and female rats exhibited reduced nitric oxide metabolites (3 months: male = 0.65 ± 0.1; female = 0.74 ± 0.3; 6 months: male = 0.16 ± 0.1; female = 0.41 ± 0.1 ml min‐1 g‐1 kw, p, age = 0.02, p, sex = 0.3). Levels of urinary TBARS were similar (3 months: male = 20±1.5; female = 23±1.8; 6 months: male = 26±4.8; female = 23±4.7µM day g‐1 kw, p, age = 0.4, p, sex = 0.9), extracellular superoxide dismutase (EC SOD) mRNA was greater in females (3 months: male = 0.35 ± 0.03; female = 1.4 ± 0.2; 6 months: male = 0.4 ± 0.05; female = 1.3 ± 0.1 normalized counts, p, age = 0.7, p, sex < 0.0001), but EC SOD protein expression was not different (3 months: male = 0.01 ± 0.002; female = 0.01 ± 0.002; 6 months: male = 0.02 ± 0.004; female = 0.01 ± 0.002 relative density, p, age = 0.2, p, sex = 0.8). These data support the presence of significant sex differences in renal injury and function in the Dahl S rat and identify a need for further study into the mechanisms involved. [ABSTRACT FROM AUTHOR]

Arhgef11 is a Rho-guanine nucleotide exchange factor that was previously implicated in kidney injury in the Dahl salt-sensitive (SS) rat, a model of hypertension-related chronic kidney disease. Reduced Arhgef11 expression in an SS-Arhgef11SHR-minimal congenic strain (spontaneously hypertensive rat allele substituted for S allele) significantly decreased proteinuria, fibrosis, and improved renal hemodynamics, without impacting blood pressure compared with the control SS (SS-wild type). Here, SS-Arhgef11-/- and SS-wild type rats were placed on either low or elevated salt (0.3% or 2% NaCl) from 4 to 12 weeks of age. On low salt, starting at week 6 and through week 12, SS-Arhgef11-/- animals demonstrated a 3-fold decrease in proteinuria compared with SS-wild type. On high salt, beginning at week 6, SS-Arhgef11-/- animals demonstrated >2-fold lower proteinuria from weeks 8 to 12 and 30 mm Hg lower BP compared with SS-wild type. To better understand the molecular mechanisms of the renal protection from loss of Arhgef11, both RNA sequencing and discovery proteomics were performed on kidneys from week 4 (before onset of renal injury/proteinuria between groups) and at week 12 (low salt). The omics data sets revealed loss of Arhgef11 (SS-Arhgef11-/-) initiates early transcriptome/protein changes in the cytoskeleton starting as early as week 4 that impact a number of cellular functions, including actin cytoskeletal regulation, mitochondrial metabolism, and solute carrier transporters. In summary, in vivo phenotyping coupled with a multi-omics approach provides strong evidence that increased Arhgef11 expression in the Dahl SS rat leads to actin cytoskeleton-mediated changes in cell morphology and cell function that promote kidney injury, hypertension, and decline in kidney function. [ABSTRACT FROM AUTHOR]

The present study examined whether development of renal injury in the nondiabetic obese Dahl salt-sensitive leptin receptor mutant (SSLepRmutant) strain is associated with elevations in glomerular filtration rate and renal lipid accumulation. Baseline mean arterial pressure at 6 wk of age was similar between Dahl salt-sensitive wild-type (SSWT) and SSLepRmutant rats. However, by 18 wk of age, the SSLepRmutant strain developed hypertension, while the elevation in mean arterial pressure was not as severe in SSWT rats (192 ± 4 and 149 ± 6 mmHg, respectively). At baseline, proteinuria was fourfold higher in SSLepRmutant than SSWT rats and remained elevated throughout the study. The early development of progressive proteinuria was associated with renal hyperfiltration followed by a decline in renal function over the course of study in the SSLepRmutant compared with SSWT rats. Kidneys from the SSLepRmutant strain displayed more glomerulosclerosis and glomerular lipid accumulation than SSWT rats. Glomeruli were isolated from the renal cortex of both strains at 6 and 18 wk of age, and RNA sequencing was performed to identify genes and pathways driving glomerular injury. We observed significant increases in expression of the influx lipid transporters, chemokine (C-X-C motif) ligand 16 (Cxcl16) and scavenger receptor and fatty acid translocase (Cd36), respectively, and a significant decrease in expression of the efflux lipid transporter, ATP-binding cassette subfamily A member 2 (Abca2; cholesterol efflux regulatory protein 2), in SSLepRmutant compared with SSWT rats at 6 and 18 wk of age, which were validated by RT-PCR analysis. These data suggest an association between glomerular hyperfiltration and glomerular lipid accumulation during the early development of proteinuria associated with obesity. [ABSTRACT FROM AUTHOR]

The HSRA rat is a model of congenital abnormalities of the kidney and urogenital tract (CAKUT). Our laboratory has used this model to investigate the role of nephron number (functional unit of the kidney) in susceptibility to develop kidney disease as 50-75% offspring are born with a single kidney (HSRA-S), while 25-50% are born with two kidneys (HSRA-C). HSRA-S rats develop increased kidney injury and hypertension with age compared with nephrectomized two-kidney animals (HSRA-UNX), suggesting that even slight differences in nephron number can be an important driver in decline in kidney function. The HSRA rat was selected and inbred from a family of outbred heterogeneous stock (NIH-HS) rats that exhibited a high incidence of CAKUT. The HS model was originally developed from eight inbred strains (ACI, BN, BUF, F344, M520, MR, WKY, and WN). The genetic make-up of the HSRA is therefore a mosaic of these eight inbred strains. Interestingly, the ACI progenitor of the HS model exhibits CAKUT in 10-15% of offspring with the genetic cause being attributed to the presence of a long-term repeat (LTR) within exon 1 of the c-Kit gene. Our hypothesis is that the HSRA and ACI share this common genetic cause, but other alleles in the HSRA genome contribute to the increased penetrance of CAKUT (75% HSRA vs. 15% in ACI). To facilitate genetic studies and better characterize the model, we sequenced the whole genome of the HSRA to a depth of ~50×. A genome-wide variant analysis of high-impact variants identified a number of novel genes that could be linked to CAKUT in the HSRA model. In summary, the identification of new genes/modifiers that lead to CAKUT/loss of one kidney in the HSRA model will provide greater insight into association between kidney development and susceptibility to develop cardiovascular disease later in life. [ABSTRACT FROM AUTHOR]

Preeclampsia (PE), a multifactorial pregnancy-specific syndrome accounting for up to 8% of pregnancy complications, is a leading cause of maternal and fetal morbidity and mortality. PE is also associated with long-term risk of hypertension and stroke for both mother and fetus. Currently, the only "cure" is delivery of the baby and placenta, largely because the pathogenesis of PE is not yet fully understood. PE is associated with impaired vascular remodeling at the maternal-fetal interface and placental insufficiency; however, specific factors contributing to this impairment have not been identified. To identify molecular pathways involved in PE, we examined temporal transcriptomic changes occurring within the uterus, uterine implantation sites, and placentae from the Dahl salt-sensitive (Dahl S) rat model of superimposed PE compared with Sprague Dawley (SD) rats. We hypothesized that targeted gene analysis and whole transcriptome analysis would identify genetic factors that contribute to development of the preeclamptic phenotype in the Dahl S rat and unveil novel biomarkers, therapeutic targets, and mechanistic pathways in PE. Quantitative real-time PCR (qRT-PCR) and whole genome microarray analysis were performed on isolated total RNA from uterus (day 0), uterine implantation sites (days 7 and 10), and placenta (days 14 and 20). We found 624, 332, 185, and 366 genes to be differentially expressed between Dahl S (PE) and SD (normal pregnancy) on days 0, 7, 10, and 14, respectively. Our data revealed numerous pathways that may play a role in the pathophysiology of spontaneous superimposed PE and allow for further investigation of novel therapeutic targets and biomarker development. [ABSTRACT FROM AUTHOR]

The current study examined the effect of obesity on the development of renal injury within the genetic background of the Dahl salt-sensitive rat with a dysfunctional leptin receptor derived from zinc-finger nucleases (SSLepRmutant strain). At 6 wk of age, body weight was 35% higher in the SSLepRmutant strain compared with SSWT rats and remained elevated throughout the entire study. The SSLepRmutant strain exhibited impaired glucose tolerance and increased plasma insulin levels at 6 wk of age, suggesting insulin resistance while SSWT rats did not. However, blood glucose levels were normal throughout the course of the study. Systolic arterial pressure (SAP) was similar between the two strains from 6 to 10 wk of age. However, by 18 wk of age, the development of hypertension was more severe in the SSLepRmutant strain compared with SSWT rats (201 ± 10 vs. 155 ± 3 mmHg, respectively). Interestingly, proteinuria was substantially higher at 6 wk of age in the SSLepRmutant strain vs. SSWT rats (241 ± 27 vs. 24 ± 2 mg/day, respectively) and remained elevated until the end of the study. The kidneys from the SSLepRmutant strain displayed significant glomerular injury, including podocyte foot process effacement and lipid droplets compared with SSWT rats as early as 6 wk of age. By 18 wk of age, plasma creatinine levels were twofold higher in the SSLepRmutant strain vs. SSWT rats, suggesting the presence of chronic kidney disease (CKD). Overall, these results indicate that the SSLepRmutant strain develops podocyte injury and proteinuria independently of hyperglycemia and elevated arterial pressure that later progresses to CKD. [ABSTRACT FROM AUTHOR]

There is little clinical data of how hypertension may influence individuals with nephron deficiency in the context of being born with a single kidney. We recently developed a new rat model (the heterogeneous stockderived model of unilateral renal agenesis rat) that is born with a single kidney and exhibits progressive kidney injury and decline in kidney function with age. We hypothesized that DOCA-salt would induce a greater increase in blood pressure and therefore accelerate the progression of kidney injury in rats born with a solitary kidney compared with rats that have undergone unilateral nephrectomy. Time course evaluation of blood pressure, kidney injury, and renal hemodynamics was performed in the following six groups of animals from weeks 13 to 18: 1) DOCA-treated rats with a solitary kidney (DOCA+S group), 2) placebo-treated rats with a solitary kidney, 3) DOCA-treated control rats with two kidneys (DOCA+C group), 4) placebo-treated control rats with two kidneys, 5) DOCA-treated rats with two kidneys that underwent uninephrectomy (DOCA+UNX8 group), and 6) placebo-treated rats with two kidneys that underwent uninephrectomy. DOCA+S rats demonstrated a significant rise (P < 0.05) in blood pressure (192 ± 4 mmHg), proteinuria (205 ± 31 mg/24 h), and a decline in glomerular filtration rate (600 ± 42 μl·min-1·g kidney weight-1) relative to the DOCA+UNX8 (173 ± 3 mmHg, 76 ± 26 mg/24 h, and 963 ± 36 μl·min-1·g kidney weight-1) and DOCA+C (154 ± 2 mmHg, 7 ± 1 mg/24 h, and 1,484 ± 121 μl·min-1·g kidney weight-1) groups. Placebo-treated groups showed no significant change among the three groups. An assessment of renal injury markers via real-time PCR/Western blot analysis and histological analysis was concordant with the measured physiological parameters. In summary, congenital solitary kidney rats are highly susceptible to the induction of hypertension compared with uninephrectomized rats, suggesting that low nephron endowment is an important driver of elevated blood pressure, hastening nephron injury through the transmission of elevated systemic blood pressure and thereby accelerating decline in kidney function. [ABSTRACT FROM AUTHOR]

Previously, genetic analyses identified that variants in Arhgef11 may influence kidney injury in the Dahl salt-sensitive (S) rat, a model of hypertensive chronic kidney disease. To understand the potential mechanism by which altered expression and/or protein differences in Arhgef11 could play a role in kidney injury, stably transduced Arhgef11 knockdown cell lines as well as primary cultures of proximal tubule cells were studied. Genetic knockdown of Arhgef11 in HEK293 and NRK resulted in reduced RhoA activity, decreased activation of Rho-ROCK pathway, and less stress fiber formation versus control, similar to what was observed by pharmacological inhibition (fasudil). Primary proximal tubule cells (PTC) cultured from the S exhibited increased expression of Arhgef11, increased RhoA activity, and up regulation of Rho-ROCK signaling compared to control (small congenic). The cells were also more prone (versus control) to TGFβ-1 induced epithelial-mesenchymal transition (EMT), a hallmark feature of the development of renal interstitial fibrosis, and characterized by development of spindle shape morphology, gene expression changes in EMT markers (Col1a3, Mmp9, Bmp7, and Ocln) and increased expression of N-Cadherin and Vimentin. S derived PTC demonstrated a decreased ability to uptake FITC-albumin compared to the small congenic in vitro, which was confirmed by assessment of albumin re-uptake in vivo by infusion of FITC-albumin and immunofluorescence imaging. In summary, these studies suggest that genetic variants in the S form of Arhgef11 via increased expression and/or protein activity play a role in promoting kidney injury in the S rat through changes in cell morphology (Rho-Rock and/or EMT) that impact the function of tubule cells. [ABSTRACT FROM AUTHOR]

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The article evaluates blood pressure, proteinuria and renal hemodynamics in congenic strains with small segments of the protective spontaneously hypertensive rat genome on the salt-sensitive (S) background. Proteinuria and renal function were significantly improved in the congenic strains compared with the S. Findings demonstrate that allelic variants in Arhgef11 acting through the Rho-ROCK pathway could influence kidney injury in the S as well as provide insight into human kidney disease.

The Dahl salt-sensitive (S) rat is a widely studied model of saltsensitive hypertension and develops proteinuria, glomerulosclerosis, and renal interstitial fibrosis. An earlier genetic analysis using a population derived from the S and spontaneously hypertensive rat (SHR) identified eight genomic regions linked to renal injury in the S rat and one protective locus on chromosome 11. The "protective" locus in the S rat was replaced with the SHR genomic segment conferring "susceptibility" to kidney injury. The progression of kidney injury in the S.SHR(11) congenic strain was characterized in the present study. Groups of S and S.SHR(11) rats were followed for 12 wk on either a low-salt (0.3% NaCl) or high-salt (2% NaCl) diet. By week 12 (low-salt), S.SHR(11) demonstrated a significant decline in kidney function compared with the S. Blood pressure was significantly elevated in both strains on high salt. Despite similar blood pressure, the S.SHR(11) exhibited a more significant decline in kidney function compared with the S. The decline in S.SHR(11) kidney function was associated with more severe kidney injury including tubular loss, immune cell infiltration, and tubulointerstitial fibrosis compared with the S. Most prominently, the S.SHR(11) exhibited a high degree of medullary fibrosis and a significant increase in renal vascular medial hypertrophy. In summary, genetic modification of the S rat generated a model of accelerated renal disease that may provide a better system to study progression to renal failure as well as lead to the identification of genetic variants involved in kidney injury. [ABSTRACT FROM AUTHOR]

We have previously implicated genetic variants in Arhgef11, a Rho guanine nucleotide exchange factor, in kidney injury exhibited in the Dahl salt-sensitive (S) rat, a widely studied model of hypertension and chronic kidney disease. We have demonstrated that genetic knockdown (lentiviral shRNA) of Arhgef11 in HEK293 cells results in reduced RhoA activity (~20%), decreased activation of Rho-ROCK pathway (40-60%), and less stress fiber formation vs. scrambled control. S rat derived primary proximal tubule cells (p-PTC) exhibit increased expression of Arhgef11, activation of Rho-ROCK, and when stimulated with the profibrotic cytokine, TGFβ1 are also more prone (vs. control) to epithelial-mesenchymal transition (EMT). These data (along with animal studies) suggest that dysregulation of Rho-ROCK in tubular cells likely play an important role in EMT and promote fibrosis; however, chronic stimulation of this pathway in other kidney cell types including vascular smooth muscle cells (VSMC) may also play a role. Accordingly, S rat primary cultures of VSMC isolated from kidney microvessels (k-VSMC) were studied for differences in the RhoA-ROCK, proliferation, migration, apoptosis, and response to cyclical stretch compared to control k-VSMC (minimal congenic). S rat k-VSMC demonstrated higher expression of Arhgef11 and activation of Rho-ROCK pathway, similar to p-PTC studies. Cell migration was significantly enhanced (scratch assay) in k-VSMC from S rats (485.0±26.2 nm) compared to 296.2±37.6 nm in cells from control. Apoptosis, evaluated by flow cytometry, was significantly increased in k-VSMC from S rats stimulated with TGFβ1 (10ng/ml, 24hrs), but not Ang II, at both early (9.9%±0.9%) and late apoptosis (22.0%±3.5%) compared to control (7.71%±0.75% and 15.5%±1.3%, respectively). S rat derived k-VSMC also demonstrated up-regulation and activation of Rho-ROCK pathway after cyclical stretch, which models in vivo stimuli such as shear stress. In summary, increased expression of Arhgef11 (RhoA-ROCK) exhibited by PTC and VSMC cell types in S rat kidney appear to promote epithelial-mesenchymal transition and enhance proliferation, migration, and apoptosis under baseline and profibrotic conditions, which likely contributes to kidney injury. [ABSTRACT FROM AUTHOR]

It is well known that hypertension can promote decline in renal function, but there is a paucity of information on how hypertension influences single kidney individuals. The HSRA model exhibits a high incidence of congenital single kidney (50-75% offspring) that is characterized by low nephron number (~20% less in solitary kidney vs. control kidney), glomerular and kidney hypertrophy, elevated single nephron glomerular filtration rate (GFR), which culminates in kidney injury and decline in kidney function. We hypothesized that renal hemodynamic stressors, such as DOCA induced hypertension, would accelerate onset and progression of kidney injury in rats born with a solitary kidney (HSRA-S) compared to two-kidney littermates (HSRA-C) or rats that have undergone unilateral nephrectomy (HSRA-UNX). Time course evaluation of blood pressure, renal hemodynamics, and kidney injury was performed in 6 animal groups: (1-2) HSRA-S+DOCA or placebo; (3-4) HSRA-C+DOCA or placebo; and (5-6) HSRA-UNX+DOCA or placebo. At the conclusion of the study (5 weeks), HSRA-S+DOCA treated rats demonstrated a significant (ANOVA; p<0.05) rise in blood pressure (192±4 mmHg), proteinuria (205±31 mg/24hr) and decline in GFR (600+42 μl/min/gKW) relative to HSRA-UNX+DOCA (173±3 mmHg, 76±26 mg/24hr, 963+36 μl/min/gKW) and HSRA-C+DOCA (154±2 mmHg, 7±1 mg/24hr, 1484+121 μl/min/gKW). Animals in placebo groups showed no significant change in blood pressure, proteinuria or GFR from week 13-18 between all three groups. To elucidate the molecular mechanism of nephron deficiency in the HSRA model and better understand how elevated blood pressure promotes rapid decline in renal function, whole transcriptome analysis (RNA sequencing) of embryonic kidney isolated at key stages in nephrogenesis (E14.5, 15.5, and 16.5) was performed which identified a number of novel genes involved in nephrogenesis and kidney development. We are currently working to understand the functional role of these genes and pathways using an in vitro whole organ kidney culture system. In summary, nephron deficient rats are highly susceptible to hypertension induced kidney injury and several novel genes/pathways involved in nephrogenesis likely program the model to develop significant kidney disease with age. [ABSTRACT FROM AUTHOR]

Chronic kidney disease is a major health issue, with hypertension playing a significant role in progression of kidney injury and decline in kidney function. Previously, genetic studies utilized the Dahl salt-sensitive (S) rat, a model of hypertensive related kidney disease and the spontaneously hypertensive rat (hypertensive, but resistant to kidney injury) to identify genomic regions associated with kidney injury. To confirm identified genomic intervals, congenic strains were developed for several chromosomes (RNO) by transfer of SHR genome to the genetic background of the S, including RNO2, 6, 8, 9, 10, 11, 13 and 19. Several of these congenic strains, with the exception of RNO6 and 19 have been extensively investigated. Here, we report that S.SHR(6) and (19) demonstrate significantly improved proteinuria (53±2.5 and 54±2.5 mg/24hr, respectively) compared to control S (116±7.2) and improved measures of kidney function (BUN= 24.3±0.6 and 24.6±0.6 vs. 28±1.3 mg/dl). To facilitate the identification of genetic variants that underlie each genomic region, especially on RNO6 and RNO19, comparative genome hybridization (CGH) was performed (NimbleGen and Agilent) to identify copy number variation (CNV) between the S and SHR. CNV was identified on RNO6 and RNO19 (as well as other chromosomes) which could be linked to the development of kidney injury. In addition, recent sequencing of the entire genomes of the S and SHR has identified essentially all genetic variation (SNP and INDEL) between the two inbred models. The 95% confidence interval of each causative locus spans ~10 Mb. The region on RNO6 contains 54 annotated genes (24 nonsynonymous SNP) and RNO19 contains 101 genes and/or miRNA (68 nonsynonymous SNP). The majority of SNP (>95%) are within intergenic or intronic regions, regardless of the genomic locus. A modest number of SNP (~5%) fall within the putative promoter region of genes which could impact gene expression. In summary, CGH and high quality genome sequence analysis will help guide upcoming congenic substrain analysis to expedite identification of the causative genetic variation(s) that underlie each genomic locus. [ABSTRACT FROM AUTHOR]

The Dahl salt-sensitive (S) rat is a widely studied model of hypertension that develops progressive kidney injury characterized by proteinuria, glomerulosclerosis and tubulointerstitial fibrosis, culminating in a significant decline in kidney function. Through positional cloning approaches, Arhgef11, a Rho guanine nucleotide exchange factor for the Rho-ROCK pathway was identified as a strong candidate gene. The most likely pathophysiological mechanism (based on animal studies) relates to the development of tubulointerstitial fibrosis, suggesting that chronic activation of Rho-ROCK in tubular cells may contribute to kidney injury observed in the S rat. Arhgef11 knockdown cell lines (up to 60% knockdown) as well as primary cultures of proximal tubule cells (PTC) were studied. Knockdown of Arhgef11 resulted in reduced RhoA activity (30% decrease, 0.57 ± 0.05 vs 0.82 ± 0.06 ng/mg protein), decreased activation of Rho-ROCK pathway (40-60%) and less stress fiber formation (2-fold reduction, 2.28 ± 0.18 vs 4.39 ± 0.64 in length/width ratio) versus control. S rat PTCs exhibited increased expression of Arhgef11, activation of Rho-ROCK, and decreased uptake of FITC-albumin (5.85 ± 0.71 vs 8.25 ± 0.64 mg/g protein) compared to control (minimal congenic). Both Arhgef11 knockdown cell-lines and PTCs stimulated with the profibrotic cytokine TGFβ1 were more prone (versus control) to epithelial-mesenchymal transition as demonstrated by increased expression of N-Cadherin, Vimentin, Col1a1 and spindle shape morphology. While dysregulation of the Rho-ROCK pathway in tubular cells appear to play an important role in EMT and promote fibrosis, chronic stimulation in other kidney cell types including vascular smooth muscle cells (VSMC) and glomerular epithelial cell (GEC) could also contribute to S rat kidney injury. Thus, we are currently investigating Arhgef11 knockdown in VSMC and GEC cell-lines as well as primary cells derived from the S rat. In summary, these studies suggest that the functional significance of genetic variants in Arhgef11 may be of major importance in promoting changes in cell morphology (Rho-Rock and/or EMT), function of tubule cells, and/or act in concert with dysregulation in VMSC and GEC to promote kidney injury. [ABSTRACT FROM AUTHOR]

Nuclear hormone receptors of the NR4A subgroup have been implicated in cancer, atherosclerosis, and most prominently in metabolic disease. However, little is known about the role of NR4A1 in kidney health or disease. Fawn-hooded hypertensive (FHH) Nr4a1-/- deficient animals were evaluated for blood pressure, proteinuria, renal function and metabolic parameters from 4-24 weeks of age. At week 24, Nr4a1-/- rats exhibited 4-fold higher proteinuria (81±11.5 mg/24hrs/100gBW) compared to FHH (21±1.0). No difference in telemetry measured blood pressure was observed at any time point, but glomerular filtration rate was significantly decreased in the Nr4a1-/- (542±78.9 ul/min/gKW) vs FHH (937±75.2). Moderate increases in glomerulosclerosis were seen, but were not different between strains. The severity of tubular atrophy, casts, and fibrosis was significantly increased in Nr4a1-/- compared to FHH (19±1.86% vs 12±0.8%, respectively). Tubulointerstitial macrophage infiltration was also significantly increased in kidneys from Nr4a1-/- (245±62 foci per field) compared to FHH (50±13.4) at week 24. Microarray analysis of kidney from week 8-24 found significant up-regulation of important immune mediators, including TGF-β1-2, CCL2 (MCP-1), CCL9 (MIP-1γ), and other chemokines and interleukins. The specific injury (tubulointerstitial), immune cell infiltration, and immune gene expression profile led to the hypothesis that loss of Nr4a1 contributes to the observed injury by an immune-mediated mechanism. To test this hypothesis, bone marrow (bm) cross transplantation studies were performed in 3 groups of irradiated (irr) animals: (1) bmFHH into irrFHH (FHH control); (2) bmFHH into irrNr4a1-/-; and (3) bmNr4a1-/- into irrNr4a1-/- (Nr4a1 control). The bmFHH into irrNr4a1-/- animals showed a significant attenuation of proteinuria starting as early as week 12 and continuing through week 24 (35±4.6 mg/24hrs/100gBW) compared to the Nr4a1-/- control (60±7.5). These data strongly suggest that the mechanism of Nr4a1-/- renal injury is mediated via an immune mechanism, likely due to the predisposition of the FHH to develop renal injury. Further studies are now underway to better understand the precise immune mechanism that promotes injury in this model. [ABSTRACT FROM AUTHOR]

Hypertension plays an important role in the development of chronic kidney disease. The Dahl salt-sensitive (S) rat is a widely studied model of salt-sensitive hypertension that develops concurrent kidney injury and progressive decline in kidney function. Genetic studies using the S rat, along with the kidney injury resistant, but hypertensive SHR (spontaneously hypertensive rat) have identified many genomic regions associated with kidney injury. Through positional cloning approaches, Arhgef11, a Rho guanine nucleotide exchange factor has been identified as a strong candidate gene involved in the kidney injury. The S rat exhibits increased expression of Arhgef11, up-regulation of Rho-ROCK pathway, and coding sequence variants that may alter protein function compared to the SHR. Inhibition of the Rho-ROCK pathway (using fasudil) in the S resulted in an attenuation of proteinuria, providing evidence the pathway could influence kidney injury. To better understand the role of Arhgef11 in the Rho-ROCK pathway, genetic knockdown in cell culture was achieved in HEK293 and NRK cell lines using lentiviral shRNA-Arhfgef11 constructs. Transduced cells achieved ∼70% knockdown of Arhgef11 protein. RhoA activity was significantly decreased ∼40% (p<0.01) in both transduced cell lines compared to scramble control. Gene expression and protein levels of Arhgef11, RhoA, ROCK1, MLC, Cofilin were all significantly down-regulated 40-65% in transduced HEK293 and NRK (p<0.01). Reduced expression of Arhgef11 in transduced cells showed a different cellular distribution and less stress fiber formation and elongation compared to control (2-fold reduction in length/width ratio, p<0.01) by immunofluorescence. Genetic knockdown demonstrated a similar effect as fasudil for both stable cell-lines. Microarray studies have also identified other novel pathways influenced by genetic knockdown of Arhgef11. Current studies are investigating the specific effect of the S or SHR Arhgef11 allele (using lentiviral overexpression) on the Rho-ROCK pathway and stress fiber formation. In summary, our promising data suggests that the biological and functional significance of genetic variants in Arhgef11 could be of major importance in development of kidney injury in the S rat. [ABSTRACT FROM AUTHOR]

Hypertension, diabetes and obesity, along with genetic predisposition, contribute to the growing number of chronic kidney disease patients. Our novel congenic model [S.SHR(11)] was developed through genetic modification of the Dahl salt-sensitive (S) rat, a model of hypertension related renal disease. The S.SHR(11) strain exhibits accelerated kidney injury compared to the already highly susceptible S rat. On either a low or high-salt diet, the S.SHR(11) model predominately exhibited more tubulointerstitial fibrosis compared to the S rat (17.1±1.29% vs. 12.9±1.22%). Increased α-SMA and macrophage infiltration was also observed. The S and S.SHR(11) had similar blood pressure (week 12), despite an early reduction in renal function in the S.SHR(11); however at an advanced age the S.SHR(11) demonstrated significantly higher blood pressure than the S (215±6.6 mm Hg vs. 183±5.9, respectively). This suggests that increased kidney injury is driving the development of hypertension later in life. Since these two animal models are identical with exception of chromosome 11, the causative genetic variants contributing to decreased renal function must reside within this region. The Dahl S and SHR genomes have been sequenced; this data provides a catalog of all the genetic variants between the two models. The 95% confidence interval of the genomic locus contains 28 non-synonymous SNP, with 15 of these SNP occurring within only three genes: Retnlg, Trat1 and Myh15. Two of these genes, Retnlg and Trat1, are known to play a role in immune response leading to our hypothesis that genetic variants in these genes alter protein function and lead to an increased immune response. Bone marrow transplant studies have been initiated to test our hypothesis and preliminary data shows that S rats who receive S.SHR(11) bone marrow have kidney function measurements similar to the S.SHR(11). The sequencing information has also lead to the development of nine new, more refined congenic strains. Through functional analysis of these new congenic animals, identification of the causative genetic variations will be expedited. In summary, we are employing a model of accelerated kidney disease to identify genes or genetic variants responsible for reduced kidney function and hypertension. [ABSTRACT FROM AUTHOR]

Background: Preeclampsia is a disorder of pregnancy with accompanying high disease and economic burdens in the United States. Evidence supporting longstanding effects of preeclampsia on the offspring of affected pregnancies is high, but the effects of current antihypertensive therapies for preeclampsia on cardio-renal outcomes are largely unknown. The purpose of this study was to test the hypothesis that sildenafil citrate, a phosphodiesterase-5 inhibitor, reprograms the risk of hypertension and kidney disease in offspring of preeclamptic pregnancies by altering responses to secondary stressors., Methods: Dahl SS/Jr rats on a 0.3% NaCl diet were mated. At gestational day 10, pregnant dams were randomized to vehicle diet or diet with sildenafil (50 mg/kg per day), which was continued until birth. Pups were weaned at 4 weeks of age and allowed to age on a 0.3% NaCl diet until 3 months of age. At this point, pups were randomized into three groups: baseline or no intervention, 2% NaCl diet challenge for 4 weeks, or a subpressor infusion of angiotensin II (200 ng/kg per minute) for 2 weeks., Results: There were no differences among maternal treatment groups at baseline. Upon introduction of 2% NaCl diet, male offspring of sildenafil-treated dams exhibited an attenuated rise in BP; however, this protection was not observed during angiotensin II infusion., Conclusions: Our findings indicate that intrapartum sildenafil does not reprogram the risk of hypertension and kidney disease in offspring of preeclamptic pregnancies., Competing Interests: All authors have nothing to disclose., (Copyright © 2020 by the American Society of Nephrology.)

Nephron endowment refers to the total number of nephrons an individual is born with, as nephrogenesis in humans is completed by 36 weeks of gestation and no new nephrons are formed post-birth. Nephron number refers to the total number of nephrons measured at any point in time post-birth. Both genetic and environmental factors influence both nephron endowment and number. Understanding how specific genes or factors influence the process of nephrogenesis and nephron loss or demise is important as individuals with lower nephron endowment or number are thought to be at a higher risk of developing renal or cardiovascular disease. Understanding how environmental exposures over the course of a person's lifetime affects nephron number will also be vital in determining future disease risk. Thus, the ability to assess whole kidney nephron number quickly and reliably is a basic experimental requirement to better understand mechanisms that contribute to or promote nephrogenesis or nephron loss. Here, we describe the acid maceration method for the estimation of whole kidney nephron number based on the procedure described by Damadian, Shawayri, and Bricker, with slight modifications. The acid maceration method provides fast and reliable estimates of nephron number (as assessed by counting glomeruli) that are within 5% of those determined using more advanced, albeit expensive, methods such as magnetic resonance imaging. Moreover, the acid maceration method is an excellent high-throughput method to assess nephron number in large numbers of samples or experimental conditions.

Reduced levels of the cardiac human (h)ERG ion channel protein and the corresponding repolarizing current I can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native Kr can cause arrhythmia and sudden cardiac death, but the underlying cellular mechanisms controlling hERG surface expression are not well understood. Here, we identified TRIOBP-1, an F-actin-binding protein previously associated with actin polymerization, as a putative hERG-interacting protein in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas reducing TRIOBP-1 expression via shRNA knockdown resulted in increased hERG protein levels. Immunolabeling in rat cardiomyocytes showed that native TRIOBP-1 colocalized predominantly with myosin-binding protein C and secondarily with rat ERG. In human stem cell-derived cardiomyocytes, TRIOBP-1 overexpression caused intracellular co-sequestration of hERG signal, reduced native I Kr and disrupted action potential repolarization. Ca 2+ currents were also somewhat reduced and cell capacitance was increased. These findings establish that TRIOBP-1 interacts directly with hERG and can affect protein levels, I Kr magnitude and cardiac membrane excitability., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

There is little clinical data of how hypertension may influence individuals with nephron deficiency in the context of being born with a single kidney. We recently developed a new rat model (the heterogeneous stock-derived model of unilateral renal agenesis rat) that is born with a single kidney and exhibits progressive kidney injury and decline in kidney function with age. We hypothesized that DOCA-salt would induce a greater increase in blood pressure and therefore accelerate the progression of kidney injury in rats born with a solitary kidney compared with rats that have undergone unilateral nephrectomy. Time course evaluation of blood pressure, kidney injury, and renal hemodynamics was performed in the following six groups of animals from weeks 13 to 18: 1) DOCA-treated rats with a solitary kidney (DOCA+S group), 2) placebo-treated rats with a solitary kidney, 3) DOCA-treated control rats with two kidneys (DOCA+C group), 4) placebo-treated control rats with two kidneys, 5) DOCA-treated rats with two kidneys that underwent uninephrectomy (DOCA+UNX8 group), and 6) placebo-treated rats with two kidneys that underwent uninephrectomy. DOCA+S rats demonstrated a significant rise (P < 0.05) in blood pressure (192 ± 4 mmHg), proteinuria (205 ± 31 mg/24 h), and a decline in glomerular filtration rate (600 ± 42 μl·min(-1)·g kidney weight(-1)) relative to the DOCA+UNX8 (173 ± 3 mmHg, 76 ± 26 mg/24 h, and 963 ± 36 μl·min(-1)·g kidney weight(-1)) and DOCA+C (154 ± 2 mmHg, 7 ± 1 mg/24 h, and 1,484 ± 121 μl·min(-1)·g kidney weight(-1)) groups. Placebo-treated groups showed no significant change among the three groups. An assessment of renal injury markers via real-time PCR/Western blot analysis and histological analysis was concordant with the measured physiological parameters. In summary, congenital solitary kidney rats are highly susceptible to the induction of hypertension compared with uninephrectomized rats, suggesting that low nephron endowment is an important driver of elevated blood pressure, hastening nephron injury through the transmission of elevated systemic blood pressure and thereby accelerating decline in kidney function., (Copyright © 2016 the American Physiological Society.)

Some studies have reported up to 40% of patients born with a single kidney develop hypertension, proteinuria, and in some cases renal failure. The increased susceptibility to renal injury may be due, in part, to reduced nephron numbers. Notably, children who undergo nephrectomy or adults who serve as kidney donors exhibit little difference in renal function compared with persons who have two kidneys. However, the difference in risk between being born with a single kidney versus being born with two kidneys and then undergoing nephrectomy are unclear. Animal models used previously to investigate this question are not ideal because they require invasive methods to model congenital solitary kidney. In this study, we describe a new genetic animal model, the heterogeneous stock-derived model of unilateral renal agenesis (HSRA) rat, which demonstrates 50%-75% spontaneous incidence of a single kidney. The HSRA model is characterized by reduced nephron number (more than would be expected by loss of one kidney), early kidney/glomerular hypertrophy, and progressive renal injury, which culminates in reduced renal function. Long-term studies of temporal relationships among BP, renal hemodynamics, and renal function demonstrate that spontaneous single-kidney HSRA rats are more likely than uninephrectomized normal littermates to exhibit renal impairment because of the combination of reduced nephron numbers and prolonged exposure to renal compensatory mechanisms (i.e., hyperfiltration). Future studies with this novel animal model may provide additional insight into the genetic contributions to kidney development and agenesis and the factors influencing susceptibility to renal injury in individuals with congenital solitary kidney., (Copyright © 2015 by the American Society of Nephrology.)

Nuclear hormone receptors of the NR4A subgroup have been implicated in cancer, atherosclerosis, and metabolic disease. However, little is known about the role of these receptors in kidney health or disease. Nr4a1-deficient rats (Nr4a1(-/-)) developed on a genetic background susceptible to kidney injury (fawn-hooded hypertensive rat [FHH]) were evaluated for BP, proteinuria, renal function, and metabolic parameters from 4 to 24 weeks-of-age. By week 24, Nr4a1(-/-) rats exhibited significantly higher proteinuria (approximately 4-fold) and decreased GFR compared with FHH controls. The severity of tubular atrophy, tubular casts, and interstitial fibrosis increased significantly in Nr4a1(-/-) rats and was accompanied by a large increase in immune cell infiltration, predominantly macrophages and to a lesser extent T cells and B cells. Global transcriptome and network analyses at weeks 8, 16, and 24 identified several proinflammatory genes and pathways differentially regulated between strains. Bone marrow crosstransplantation studies demonstrated that kidney injury in Nr4a1(-/-) rats was almost completely rescued by bone marrow transplanted from FHH controls. In vitro, macrophages isolated from Nr4a1(-/-) rats demonstrated increased immune activation compared with FHH-derived macrophages. In summary, the loss of Nr4a1 in immune cells appears to cause the increased kidney injury and reduced renal function observed in the Nr4a1(-/-) model., (Copyright © 2014 by the American Society of Nephrology.)