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Background:The present study in Sprague-Dawley rats determined the effects of a rapid rise of renal perfusion pressure (RPP) upon the activation of mTOR (mechanistic target of rapamycin), and the effects upon the infiltration of CD68-positive macrophages/monocytes and CD3-positive T lymphocytes into the kidneys.Methods:RPP was elevated by 40 mm Hg for 30 minutes in male Sprague-Dawley rats while measuring renal blood flow and urine flow rate. Sham rats were studied in the same way, but RPP was not changed. Since initial studies found that the acute increase of RPP resulted in activation of mTORC1 (phosphorylation of S6S235/236), the effects of inhibition of mTORC1 with rapamycin pretreatment were then determined.Results:It was found that a 30-minute increase of RPP (≈40 mm Hg) resulted in an 8-fold increase of renal sodium excretion which was blunted by rapamycin treatment. Renal blood flow was not affected by the elevation of RPP. Activation of mTORC1 was observed. Significant increases in CD68-positive macrophages were found in both the cortex (intraglomerular and periglomerular regions) and in the outer medullary interstitial regions of the kidney and prevented by rapamycin treatment. Increases in CD3-positive T lymphocytes were observed exclusively in the periglomerular regions and prevented by rapamycin treatment. Upregulation of several proinflammatory markers was observed.Conclusions:We conclude that elevation of RPP rapidly activates mTORC1 resulting in infiltration of immune cells into the kidney.

Background: Epigenetic marks (eg, DNA methylation) may capture the effect of gene-environment interactions. DNA methylation is involved in blood pressure (BP) regulation and hypertension development; however, no studies have evaluated its relationship with 24-hour BP phenotypes (daytime, nighttime, and 24-hour average BPs). Methods: We examined the association of whole blood DNA methylation with 24-hour BP phenotypes and clinic BPs in a discovery cohort of 281 Black participants using reduced representation bisulfite sequencing. We developed a deep and region-specific methylation sequencing method, Bisulfite ULtrapLEx Targeted Sequencing and utilized it to validate our findings in a separate validation cohort (n=117). Results: Analysis of 38 215 DNA methylation regions (MRs), derived from 1 549 368 CpG sites across the genome, identified up to 72 regions that were significantly associated with 24-hour BP phenotypes. No MR was significantly associated with clinic BP. Two to 3 MRs were significantly associated with various 24-hour BP phenotypes after adjustment for age, sex, and body mass index. Together, these MRs explained up to 16.5% of the variance of 24-hour average BP, while age, sex, and BMI explained up to 11.0% of the variance. Analysis of one of the MRs in an independent cohort using Bisulfite ULtrapLEx Targeted Sequencing confirmed its association with 24-hour average BP phenotype. Conclusions: We identified several MRs that explain a substantial portion of variances in 24-hour BP phenotypes, which might be excellent markers of cumulative effect of factors influencing 24-hour BP levels. The Bisulfite ULtrapLEx Targeted Sequencing workflow has potential to be suitable for clinical testing and population screenings on a large scale.

In the present study, novel methods were developed which allowed continuous (24/7) measurement of blood pressure (BP) and renal blood flow (RBF) in freely moving rats and the intermittent collection of arterial and renal venous blood to estimate kidney metabolic fluxes of O2and metabolites. The study determined the effects of a high salt (HS) diet upon whole kidney O2consumption and the metabolomic profiles of normal Sprague Dawley (SD) rats. A separate group of rats was studied to determine changes in the cortex (Cx) and outer medulla (OM) tissue metabolomic and mRNAseq profiles before and following the switch from a 0.4% to a 4.0% NaCl diet. Significant changes in the metabolomic and transcriptomic profiles occurred with feeding of the HS diet. A progressive increase of kidney O2consumption was found despite a reduction in expression of most of the mRNA encoding enzymes of TCA cycle. Increased glycolysis was evident with the elevation of mRNA expression encoding key glycolytic enzymes and release of pyruvate and lactate from the kidney in the renal venous blood. Glycolytic production of NADH is used in either the production of lactate or oxidized via the malate aspartate shuttle. Aerobic glycolysis (e.g., Warburg-effect) may account for the needed increase in cellular energy. The study provides evidence that kidney metabolism responds to a HS diet enabling enhanced energy production while protecting from oxidate stress and injury.

BACKGROUND: Dietary sodium intake affects blood pressure (BP) and vascular function. Emerging studies in animal models suggest epigenetic changes such as DNA methylation might be involved. We hypothesized that dietary sodium restriction induces DNA methylation changes in T-cells and arterioles in humans. METHODS: Fifty subjects (46% African American, 49% women) were placed on a 1200mg sodium/day diet for 2 weeks. All food was prepared and provided in accordance with subjects’ dietary preferences. BP and flow-mediated dilation (FMD) were evaluated. Methylation sequencing (pre- and post- low-sodium diet) was performed on DNA obtained from T-cells (n=50) and biopsied arterioles (n=10) using reduced representation bisulfite sequencing. RNA-seq was also performed on arterioles (n=11). RESULTS: During the low-sodium diet period, subjects consumed an average of 946mg of sodium/day. The average systolic and diastolic BP reductions following the low-sodium intake were −8±13/−4±9 mmHg (p

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial and smooth muscle cells (iEC and iVSMC). The defined genetic makeup and the ease of genetic engineering in multiple cell types on an isogenic background make these cells potentially powerful tools for studying molecular mechanisms underlying vascular function and disease. While several studies have characterized iEC and iVSMC and compared them with other cultured cells, no studies have analyzed these cells in conjunction with native human cells. In this study, we characterized iEC, iVSMC, and fractions of native human arterioles by examining large panels of marker genes. iEC and iVSMC were differentiated (n=4) from a custom-made iPSC line. Differentiated cells were sorted using VE-cadherin ( CDH5 ) to obtain iEC (positive selection) and iVSMC (negative selection). Arterioles were isolated from adipose tissues obtained from 4 humans, and the endothelial and vessel remainder fractions were separated by scraping. RNA-seq was performed on all samples. We focused the analysis on 10 and 15 marker genes for EC and VSMC, respectively, that we curated from the literature. iEC exhibited robust expression of 9 of the 10 EC markers such as CD34 (FPKM, 137±58), PECAM1 (153±32), and VWF (44±7), while iVSMC had negligible expression of EC marker genes. Conversely, iVSMC exhibited robust expression of 11 of the 15 VSMC marker genes, while the expression of these genes in iEC was depleted by several fold. Eight of the 10 EC marker genes were enriched in the endothelial fraction of native arterioles by several fold compared with intact arterioles, while 12 of the 15 VSMC marker genes were substantially depleted in the endothelial fraction. EC marker genes were significantly depleted and VSMC marker genes enriched in the vessel remainder fraction. In summary, iEC and iVSMC, as well as endothelial and vessel remainder factions from native human arterioles, exhibit robust expression of large panels of EC and VSMC marker genes based on agnostic RNA-seq analysis. These novel findings indicate that iEC, iVSMC, and endothelial and vessel remainder factions of native human arterioles are relevant tools for studying molecular regulation in specific vascular cell types.

Chromatin conformation influences gene transcription by changing the spatial organization and accessibility of DNA regulatory elements. Endothelial cells (ECs) play an important role in the regulation of blood pressure. We have shown that human induced pluripotent stem cell (iPSC)-derived ECs (iECs) exhibit robust expression of a large panel of EC marker genes. In this study, we developed genome-wide maps of chromatin conformation in iECs using Hi-C and Micro-C techniques. With Hi-C, we identified 309 and 1,584 loops at 5,000 base pairs (bp) and 10,000 bp resolution, respectively, from a total of more than 171 million chromatin contacts. We also identified 62 and 582 contact domains at the two levels of resolution. From a total of more than 356 million contacts in the Micro-C map of iECs, we identified 3,259 and 6,497 loops and 1,262 and 3,542 contact domains at 5,000 bp and 10,000 bp resolution, respectively, including many identified by Hi-C. A subsample of the Micro-C data that was similar to the Hi-C map in size identified four fold more loops and six fold more domains at 5,000 bp resolution. Using approaches that we published recently, we identified 54 overlapping genes between 1,278 genes known to be relevant to ECs and 251 genes known to be involved in blood pressure regulation. We searched the 3,259 loops in the Micro-C map of iECs for the 54 genes and 26,585 single nucleotide polymorphisms (SNPs) in 1,071 haplotypes associated with blood pressure. EDNRA (endothelin receptor type A) and SMAD3 (SMAD family member 3) were in the same loops as blood pressure-associated haplotypes, were the closest genes to the haplotype regions, and were expression quantitative trait locus (eQTL) genes for SNPs in the haplotypes. GJA1 (connexin-43) was in the same loop as the rs6925750 haplotype and an eQTL gene for some SNPs in the haplotype, but GJA1 was not the closest gene to the haplotype. In fact, the GJA1 promoter was 498,217 bp away from the haplotype region, indicating possible long-range regulation of GJA1 by SNPs in this blood pressure-associated haplotype. In summary, we have developed a genome-wide chromatin conformation map for human iECs, which may reveal new insights into the mechanisms underlying the regulation of endothelial gene expression and blood pressure.

Background: Although renal mitochondrial dysfunction has been observed in various models of hypertension, the events leading to this dysfunction remain unclear. The present study focused on the role of salt (NaCl) intake upon renal mitochondrial bioenergetic function in Dahl salt-sensitive (SS) rats hypothesizing that a high salt (HS) diet would impair mitochondrial bioenergetics via stimulation of NOX-induced oxidative stress. Method: The HS induced progressive alteration of oxidative phosphorylation (OxPhos) and ATP synthesis in mitochondria obtained from renal cortex and outer medulla (OM) of male SS rats were compared to those of SS rats with dual knockout of NOX4 and p67 phox subunit of NOX2 (NOX DKO). Mitochondria were isolated from the cortex and OM of groups of age matched rats fed a 0.4% NaCl (LS) diet since weaning and at days 3, 7, 14 & 21 of a 4.0% NaCl (HS) diet feeding. ADP-induced (state 3) O 2 consumption rates (OCR) were measured in mitochondria energized with pyruvate and malate as substrates using an Oroboros Oxygraph-2k Instrument. Results: In SS rats, a biphasic pattern in state 3 OCR was observed with progressive uncoupling of OxPhos occurring in both cortex and OM. In the early phase of hypertension (days 3 & 7), state 3 OCR was increased (155 ± 8.2 to 190 ± 12.2 to 203.4 ± 12.1 nmol/min/mg in cortex and 101.4 ± 5.1 to 121.8 ± 7.3 to 112.8 ± 6.8 nmol/min/mg in OM; n = 5). In the established phase of hypertension (days 14 & 21), this increase was not sustained as state 3 OCR was reduced to 153 ± 7.65 and 132 ± 6.6 nmol/min/mg in cortex and 105 ± 7.3 and 63.3 ± 6.5 nmol/min/mg in OM. In contrast, SS NOX DKO rats fed a HS diet showed no significant differences in state 3 OCR in both cortex and OM, as was also observed in a control group of age matched NOX DKO rats maintained on a LS diet throughout the study. Conclusions: In SS rats, a HS diet causes initial increase in the efficiency of OxPhos for ATP production followed by a progressive decrease in the efficiency of OxPhos for ATP production in the established phase of hypertension. This biphasic pattern with ultimate reduction in the efficiency of OxPhos for energy production was not observed in SS NOX DKO rats demonstrating the important causal role of NOX-induced oxidative stress in the renal mitochondrial bioenergetic dysfunction.

Goal: The effects of a high salt diet on renal cortical ( Cx ) and outer medulla ( OM ) tissue metabolism of normal age matched male Sprague-Dawley rats (SD; 15-16 wk age; n-5) was determined with rats fed 0.4% NaCl (LS) and after switching to 4.0% NaCl (HS) diet at day HS14 and HS21. Methods: From snap-frozen Cx and OM tissue, novel analytical approaches were developed and applied at the JAX lab to determine metabolic profiles using 4 modes (C18+/- and HILIC+/-) using a Thermo Q-Exactive Orbitrap coupled to a Vanquish UPLC system. Results: Kidney weight was increased (P Conclusion: Kidney weight increased after 2 wks of HS diet which was associated with increased amino acid metabolism and reduced glycolysis in OM (e.g. anabolic profile). Those metabolites (above) then returned to levels observed on a LS diet as kidney weight stabilized.

Complex phenotypes such as blood pressure regulation involve multiple organ systems, tissues, and specific cell types. Transgenic rodent models with the expression of fluorescent proteins under the control of tissue- and cell-type-specific promoters are powerful tools for identifying and isolating specific cells but have been primarily limited to mice. Our long-term goal is to establish a Cre-driver rat resource for conditional and physiologically predictive rat models of human diseases. We developed two Cre-driver transgenic strains of rats (SS/PodocinCreERT2 and SS/VECadCre) capable of activating a second Cre-sensitive tdTomato reporter transgene integrated into the rat Rosa26 locus in the Dahl salt-sensitive hypertension model background. Cells with reporter activation were identified under fluorescence microscopy or harvested using fluorescence-activated cell sorting (FACS). A tamoxifen-inducible Cre transgene under the control of the Nphs2 (podocin) gene promoter was used to harvest fluorescently labeled podocytes after tamoxifen injection. A constitutive Cre under the control of the Cdh5 (VE-Cadherin) gene promoter was used for the isolation of endothelial cells. Correspondingly, tens to hundreds of thousands of podocytes or endothelial cells were isolated from a single kidney or mesenteric artery. Isolated cells were enriched for specific marker genes and could be used in downstream assays including single-cell transcriptional profiling. These new Cre/reporter dual transgenic rat models provide a much-needed resource for the biomedical research community to study the role of these cell types in a broad range of health and disease modeling studies.

Background: There is evidence that the mTOR signaling pathway plays an important role in salt induced hypertension in the Dahl salt-sensitive (SS) rat via regulation of Na + excretion. PP242 inhibition of mTORC1-2 prevented hypertension in SS rats and intrarenal PP242 produced a 4-fold increase of Na + excretion while selective inhibition of mTORC1 with rapamycin had no effect. The goal of the present study was to identify the renal tubular Na + transporters regulated by the mTORC2 pathway. Methods: Natriuretic effectors of mTORC2 were deduced from the differences of Na + excreted with PP242 treatment beyond that observed by the natriuretic actions of benzamil (B; ENaC inhibitor), furosemide (F; NKCC2 inhibitor), hydrochlorothiazide (H; NCC inhibitor) and in combination (B+F+H). Unanesthetized SS rats were first administered B, F, H or B+F+H (i.p.) and 2 days later with co-administration of PP242 with these same reagents. Rats were studied when fed 0.4% NaCl (LS) and then following 3 weeks of a 4.0% NaCl (HS) diet. The effect of PP242 on ENaC activity in freshly isolated cortical collecting ducts was also examined using cell-attached patch clamp electrophysiology. Results: Urine Na + excretion rates determined at 20 min. intervals over 80 minutes increased nearly 2-fold with administration of PP242 + B, compared to B alone [0.10 + 0.02 vs. 0.04 + 0.01 mmol/100gbw (n=8; P+ 0.02 vs. 0.10 + 0.02 mmol/100gbw (n=8; P+ 0.01 vs. 0.03 + 0.01 mmol/100gbw (n=8; P=0.06)]. PP242 given in the presence of F+B+H did not further enhance the overall natriuretic response. Rats fed HS showed little enhancement of natriuresis when PP242 was administered with any of the drugs. Minimum changes in K + excretion were observed with PP242 in LS or HS studies. Patch clamp studies found that PP242 reduced ENaC activity through reduction of the open channel probability (Po) in both LS and HS fed rats. Western blot data indicated that PP242 reduced pNCC T53 and phosphorylation of SGK1. Conclusion: Together, these studies found evidence that the natriuretic actions of PP242 are a consequence of inhibitory actions on the ENaC and NCC transporters in the distal nephron segments.

Hydrogen peroxide (H2O2) production in the renal outer medulla is an important determinant of renal medullary blood flow and blood pressure (BP) salt-sensitivity in Dahl salt-sensitive (SS) rats. The mechanisms and pathways responsible for these actions are poorly understood. Recently, we have discovered that the mTOR complex 2 (mTORC2) plays a critical role in BP salt-sensitivity of SS rats by regulating Na+ homeostasis. PP242, an inhibitor of mTORC1/2 pathways exhibits potent natriuretic actions and completely prevented salt-induced hypertension in SS rats. In the present study, we have found that chronic infusion of H2O2 into the single remaining kidney of Sprague Dawley (SD) rats (3 days) stimulated the functional marker (pAKTSer473/AKT) of mTORC2 activity measured by Western Blot analysis. No changes in mTORC1 activity in OM were observed as determined by pS6Ser235/236/S6. Using fluorescent microscopy and the Na+ sensitive dye Sodium Green, we have shown that H2O2 (100 µM added in the bath) increased intracellular sodium concentration ([Na+]i) in renal medullary thick ascending limbs (mTALs) isolated from SD rats. These responses were almost completely abolished by pretreatment of mTAL with 10 µM PP242, indicating that mTORC1/2 pathways were involved in the H2O2 induced increase of [Na+]i. mTAL cell volume remained unchanged (± 1%) by H2O2 as determined by 3D reconstruction confocal laser scanning microscopy techniques. Consistent with the microscopy data, Western Blot analysis of proteins obtained from freshly isolated mTAL treated with 100 µM H2O2 exhibited increased activity/phosphorylation of AKT (pAKTSer473/AKT) that was inhibited by PP242. This was associated with increased protein activity of the apical membrane cotransporter Na+-K+-2Cl− (NKCC2) and the Na/H exchanger (NHE-3). Na+-K+-ATPase activity was increased as reflected an increase in the ratio of pNa+-K+-ATPaseSer16 to total Na+-K+-ATPase. Overall, the results indicate that H2O2 mediated activation of mTORC2 plays a key role in transducing the observed increases of cytosolic [Na+]i despite associated increases of basolateral pump activity.

Background ACE2 has been identified as the entry receptor for coronaviruses into human cells, including SARS-COV-2 that causes COVID-19. Since hypertension is a leading comorbidity in non-survivors of COVID-19, we tested for association between ACE2 gene and hypertension in interaction with specific pre-existing conditions known to be associated with COVID-19 severity. Methods Genetic analysis of ACE2 gene was conducted in French-Canadian and British populations. Results In French-Canadian individuals, the T allele of the single nucleotide polymorphism (SNP) rs2074192 of ACE2 gene was a risk factor for hypertension in adult obese males [odds ratio (OR)=1.39, 95% confidence interval (CI) 1.06-1.83)] and even more so in obese males who smoked (OR=1.67, CI: 1.24-2.55), but not in lean males, non-smoker males or females. The T allele was significantly associated with severity of hypertension and with earlier penetrance of hypertension in obese smoking males. Significant interaction between the T allele and obesity was present in both sexes. The association of ACE2 (rs233575) genotype with blood pressure was also seen in adolescents but the interaction with obesity was present only in females. Several variants in ACE2 gene were found to be associated with hypertension in obese, smoking males in British individuals of the UK Biobank. In addition, we observed more severe outcomes to COVID-19 in association with ACE2 risk alleles in obese, smoking males. Conclusion This is the first report that ACE2 variants are associated with earlier penetrance and more severe hypertension and with more severe outcomes of COVID-19 in obese smoking males., Graphical Abstract Graphical Abstract

Although the molecular and functional responses related to renal compensatory hypertrophy after unilateral nephrectomy (UNX) has been well described, many aspects of these events remain unclear. One question is how the remaining kidney senses the absence of the contralateral organ, and another is what the role of the renin-angiotensin system is in these responses. Both acute anesthetized and chronic unanesthetized experiments were performed using the angiotensin II type 1 receptor blocker losartan and the renin inhibitor aliskiren to determine the contribution of the renin-angiotensin system to immediate changes and losartan for chronic changes of renal blood flow (RBF) and the associated hypertrophic events in male Sprague-Dawley rats. Chronic experiments used implanted RBF probes and arterial catheters for continuous data collection, and the glomerular filtration rate was determined by noninvasive transcutaneous FITC-sinistrin measurements. The results of the acute experiments found that RBF increased nearly 25% (4.6 ± 0.5 to 5.6 ± 0.6 mL/min/g kidney wt) during the first 15 min following UNX and that this response was abolished by losartan (6.7 ± 0.7 to 7.0 ± 0.7 mL/min/g kidney wt) or aliskiren (5.8 ± 0.4 to 6.0 ± 0.4 mL/min/g kidney wt) treatment. Thereafter, RBF increased progressively over 7 days, and kidney weight increased by 19% of pre-UNX values. When normalized to kidney weight determined at day 7 after UNX, RBF was not significantly different from pre-UNX levels. Semiquantification of CD31-positive capillaries revealed increases of the glomeruli and peritubular capillaries that paralleled the kidney hypertrophy. None of these chronic changes was inhibited by losartan treatment, indicating that neither the compensatory structural nor the RBF changes were angiotensin II type 1 receptor dependent. NEW & NOTEWORTHY This study found that the immediate increases of renal blood flow (RBF) following unilateral nephrectomy (UNX) are a consequence of reduced angiotensin II type 1 (AT(1)) receptor stimulation. The continuous monitoring of RBF and intermittent measurement of glomerular filtration rate (GFR) in conscious rats during the 1-wk period of rapid hypertrophy following UNX provided unique insights into the regulation of RBF and GFR when faced with increased metabolic loads. It was found that neither kidney hypertrophy nor the associated increase of capillaries was an AT(1)-dependent phenomenon.

The kidneys play a crucial role in maintaining the homeostasis of body fluids. The regulation of renal blood flow (RBF) is essential to the vital functions of filtration and metabolism in kidney function. Many acute studies have been carried out in anesthetized animals to measure RBF under various conditions to determine mechanisms responsible for the regulation of kidney perfusion. However, for technical reasons, it has not been possible to measure RBF continuously (24 h/day) in unrestrained unanesthetized rats over prolonged periods. These methods allow the continuous determination of RBF over many weeks while also simultaneously recording blood pressure (BP) with implanted catheters (fluid-filled or by telemetry). RBF monitoring is carried out with rats placed in a circular servo-controlled rat cage that enables the unrestrained movement of the rat throughout the study. At the same time, the tangling of cables from the flow probe and arterial catheters is prevented. Rats are first instrumented with an ultrasonic flow probe placement on the left renal artery and an arterial catheter implanted in the right femoral artery. These are routed subcutaneously to the nape of the neck, and connected to the flowmeter and pressure transducer, respectively, to measure RBF and BP. Following surgical implantation, rats are immediately placed in the cage to recover for at least one week and stabilize the ultrasonic probe recordings. Urine collection is also feasible in this system. The surgical and post-surgical procedures for continuous monitoring are demonstrated in this protocol.

The present study examined the extent to which leukocyte infiltration into the kidneys in Ang II (angiotensin II)-induced hypertension is determined by elevation of renal perfusion pressure (RPP). Male Sprague-Dawley rats were instrumented with carotid and femoral arterial catheters for continuous monitoring of blood pressure and a femoral venous catheter for infusion. An inflatable aortic occluder cuff placed between the renal arteries with computer-driven servo-controller maintained RPP to the left kidney at control levels during 7 days of intravenous Ang II (50 ng/kg per minute) or vehicle (saline) infusion. Rats were fed a 0.4% NaCl diet throughout the study. Ang II–infused rats exhibited nearly a 50 mm Hg increase of RPP (carotid catheter) to the right kidney while RPP to the left kidney (femoral catheter) was controlled at baseline pressure throughout the study. As determined at the end of the studies by flow cytometry, right kidneys exhibited significantly greater numbers of T cells, B cells, and monocytes/macrophages compared with the servo-controlled left kidneys and compared with vehicle treated rats. No difference was found between Ang II servo-controlled left kidneys and vehicle treated kidneys. Immunostaining found that the density of glomeruli, cortical, and outer medullary capillaries were significantly reduced in the right kidney of Ang II–infused rats compared with servo-controlled left kidney. We conclude that in this model of hypertension the elevation of RPP, not Ang II nor dietary salt, leads to leukocyte infiltration in the kidney and to capillary rarefaction.

NADPH oxidase 4 (NOX4) is the most abundant NOX isoform in the kidney; however, its importance for renal function has only recently emerged. The NOX4-dependent pathway regulates many factors essential for proper sodium handling in the distal nephron. However, the functional significance of this pathway in the control of sodium reabsorption during the initiation of chronic kidney disease is not established. The goal of this study was to test Nox4-dependent ENaC regulation in two models: SS hypertension and STZ-induced type 1 diabetes. First, we showed that genetic ablation of Nox4 in Dahl salt-sensitive (SS) rat attenuated a high-salt (HS)-induced increase in epithelial Na+ channel (ENaC) activity in the cortical collecting duct. We also found that H2 O2 upregulated ENaC activity, and H2 O2 production was reduced in both the renal cortex and medulla in SSNox4-/- rats fed an HS diet. Second, in the streptozotocin model of hyperglycemia-induced renal injury ENaC activity in hyperglycemic animals was elevated in SS but not SSNox4-/- rats. NaCl cotransporter (NCC) expression was increased compared to healthy controls, while expression values between SS and SSNox4-/- groups were similar. These data emphasize a critical contribution of the NOX4-mediated pathway in maladaptive upregulation of ENaC-mediated sodium reabsorption in the distal nephron in the conditions of HS- and hyperglycemia-induced kidney injury.

We have reported that a high-salt (4.0% NaCl) dietary intake activates mTORC1 and inhibition of this pathway with rapamycin blunts the chronic phase of salt-induced hypertension and renal injury in Dahl salt-sensitive (SS) rats. In SS rats, high-salt intake is known to increase the renal production of H 2 O 2 by NOX4, the most abundant NOX isoform in the kidney, and the global knockout of NOX4 blunts salt-sensitivity in these rats. Here, we explored the hypothesis that elevations of H 2 O 2 by NOX4 in high-salt fed SS rat stimulate mTORC1 for the full development of salt-induced hypertension and renal injury. Our in vitro studies found that H 2 O 2 activates mTORC1 independent of PI3K/AKT and AMPK pathways. To determine the in vivo relevance of NOX4/H 2 O 2 /mTORC1 in the salt-induced hypertension, SS- Nox4 knockout (SS Nox4 −/− ) rats were daily administrated with vehicle/rapamycin fed a high-salt diet for 21 days. Rapamycin treatment of SS Nox4−/− rats had shown no augmented effect on the salt-induced hypertension nor upon indices of renal injury. Significant reductions of renal T lymphocyte and macrophage together with inhibition of cell proliferation were observed in rapamycin treated rats suggesting a role of mTORC1 independent of NOX4 in the proliferation of immune cell. Given the direct activation of mTORC1 by H 2 O 2 and absence of any further protection from salt-induced hypertension in rapamycin-treated SS Nox4 −/− rats, we conclude that NOX4-H 2 O 2 is a major upstream activator of mTORC1 that contributes importantly to salt-induced hypertension and renal injury in the SS rat model.

Chromosomal substitution strategies provide a powerful tool to anonymously reveal the relationship between DNA sequence variants and a normal or disease phenotype of interest. Even in this age of CRISPR-Cas9 genome engineering, the knockdown or overexpression of a gene provides relevant information to our understanding of complex disease only when a close association of an allelic variant with the phenotype has first been established. Limitations of genetic linkage approaches led to the development of more efficient breeding strategies to substitute chromosomal segments from one animal strain into the genetic background of a different strain, enabling a direct comparison of the phenotypes of the strains with variant(s) that differ only at a defined locus. This substitution can be a whole chromosome (consomic), a part of a chromosome (congenic), or as small as only a single or several alleles (subcongenics). In contrast to complete knockout of a specific candidate gene of interest, which simply studies the effects of complete elimination of the gene, the substitution of naturally occurring variants can provide special insights into the functional actions of wild-type alleles. Strategies for production of these inbred strains are reviewed, and a number of examples are used to illustrate the utility of these model systems. Consomic/congenic strains provide a number of experimental advantages in the study of functions of genes and their variants, which are emphasized in this article, such as replication of experimental studies; determination of temporal relationships throughout a life; rigorously controlled experiments in which relations between genotype and phenotype can be tested with the confounding effects of heterogeneous genetic backgrounds, both targeted and multilayered; and "omic" studies performed at many levels of functionality, from molecules to organelles, cells to organs, and organs to organismal behavior across the life span. The application of chromosomal substitution strategies and development of consomic/congenic rat and mouse strains have greatly expanded our knowledge of genomic variants and their phenotypic relationship to physiological functions and to complex diseases such as hypertension and cancer. © 2020 American Physiological Society. Compr Physiol 10:365-388, 2020.

The SS (Dahl salt sensitive) rat is an established model of hypertension and renal damage that is accompanied with immune system activation in response to a high-salt diet. Investigations into the effects of sodium-independent and dependent components of the diet were shown to affect the disease phenotype with SS/MCW (JrHsdMcwi) rats maintained on a purified diet (AIN-76A) presenting with a more severe phenotype relative to grain-fed SS/CRL (JrHsdMcwiCrl) rats. Since contributions of the immune system, environment, and diet are documented to alter this phenotype, this present study examined the epigenetic profile of T cells isolated from the periphery and the kidney from these colonies. T cells isolated from kidneys of the 2 colonies revealed that transcriptomic and functional differences may contribute to the susceptibility of hypertension and renal damage. In response to high-salt challenge, the methylome of T cells isolated from the kidney of SS/MCW exhibit a significant increase in differentially methylated regions with a preference for hypermethylation compared with the SS/CRL kidney T cells. Circulating T cells exhibited similar methylation profiles between colonies. Utilizing transcriptomic data from T cells isolated from the same animals upon which the DNA methylation analysis was performed, a predominant negative correlation was observed between gene expression and DNA methylation in all groups. Lastly, inhibition of DNA methyltransferases blunted salt-induced hypertension and renal damage in the SS/MCW rats providing a functional role for methylation. This study demonstrated the influence of epigenetic modifications to immune cell function, highlighting the need for further investigations.

Previous studies utilizing the SSp67phox−/− rat have demonstrated the importance of systemic NADPH oxidase NOX2-derived reactive oxygen species (ROS) production in the pathogenesis of Dahl Salt-Sensitive (SS) hypertension and renal damage. It is established that the immune system contributes to the development of SS hypertension and our laboratory has observed an enrichment of NOX2 subunits in infiltrating T cells. However, the contribution of immune cell-derived ROS in SS hypertension remains unknown. To assess the role of ROS in immune cells, SSp67phox−/− rats underwent total body irradiation and received bone marrow transfer from either SS (+SS) or SSp67phox−/− (+SSp67phox−/−) donor rats. Demonstrated in a respiratory burst assay, response to phorbol 12-myristate 13-acetate stimulus (135 μM) was 10.2-fold greater in peritoneal macrophages isolated from +SS rats compared to nonresponsive + SSp67phox−/− cells, validating that + SS rats were capable of producing NOX2-derived ROS in cells of hematopoietic origin. After 3 weeks of high salt challenge, there was an exacerbated increase in mean arterial pressure in +SS rats compared to + SSp67phox−/− control rats (176.1 ± 4.7 vs 147.9 ± 8.4 mmHg, respectively), which was accompanied by a significant increase in albuminuria (168.3 ± 23.7 vs 107.0 ± 20.4 mg/day) and renal medullary protein cast formation (33.2 ± 4.7 vs 8.1 ± 3.5%). Interestingly, upon analysis of renal immune cells, there was trending increase of CD11b/c + monocytes and macrophages in the kidney of +SS rats (4.7 ± 0.4 vs 3.5 ± 0.5 × 106 cells/kidney, +SS vs + SSp67phox−/−, p = 0.06). These data altogether demonstrate that immune cell production of NOX2-derived ROS is sufficient to exacerbate Dahl SS hypertension, renal damage, and renal inflammation.

Diabetic kidney disease (DKD) is one of the leading pathological causes of decreased renal function and progression to end-stage kidney failure. To explore and characterize age-related changes in DKD and associated glomerular damage, we used a rat model of type 2 diabetic nephropathy (T2DN) at 12 wk and older than 48 wk. We compared their disease progression with control nondiabetic Wistar and diabetic Goto-Kakizaki (GK) rats. During the early stages of DKD, T2DN and GK animals revealed significant increases in blood glucose and kidney-to-body weight ratio. Both diabetic groups had significantly altered renin-angiotensin-aldosterone system function. Thereafter, during the later stages of disease progression, T2DN rats demonstrated a remarkable increase in renal damage compared with GK and Wistar rats, as indicated by renal hypertrophy, polyuria accompanied by a decrease in urine osmolarity, high cholesterol, a significant prevalence of medullary protein casts, and severe forms of glomerular injury. Urinary nephrin shedding indicated loss of the glomerular slit diaphragm, which also correlates with the dramatic elevation in albuminuria and loss of podocin staining in aged T2DN rats. Furthermore, we used scanning ion microscopy topographical analyses to detect and quantify the pathological remodeling in podocyte foot projections of isolated glomeruli from T2DN animals. In summary, T2DN rats developed renal and physiological abnormalities similar to clinical observations in human patients with DKD, including progressive glomerular damage and a significant decrease in renin-angiotensin-aldosterone system plasma levels, indicating these rats are an excellent model for studying the progression of renal damage in type 2 DKD.

DNA methylation, an epigenetic mark, may reflect the interactions between DNA, environment, and lifestyle. It has been implicated in the development and progression of hypertension, a risk factor for cardiovascular disease. We hypothesize that regions of DNA methylation in blood cells can explain 24h BP phenotypes in African Americans. We performed Reduced Representation Bisulfite Sequencing (RRBS) in a discovery cohort of 281 African Americans. Several DNA methylation regions (MRs) were significantly associated with continuously monitored 24-h, daytime, or nighttime SBP, DBP, PP, and MAP after adjustments for covariates age, sex, and body mass index (False Discovery Rate (FDR) = 0.013 - 0.050). Each of these MRs explained a substantial portion of 24h BP variance, ranging from 6.5% - 9.4%. After FDR adjustment, there were no MRs significantly associated with clinic BPs (FDR > 0.1374), calculated by the average of 4 resting measurements (2 per arm) by sphygmomanometer. To interrogate specific regions of DNA methylation, our lab developed a potentially clinically applicable, deep, and targeted methylation sequencing method called Bisulfite-Specific PCR ULtrapLEx Targeted Sequencing (BULLET-Seq), and tested it in two reference samples for three MRs of interest. BULLET-Seq is able to accurately quantify the 10% changes in the dilution series when methylation rates ranged from ~40% to 90% (a chr19 methylation region; R 2 = 0.95 - 0.97) and can modestly measure these changes when rates range from ~2% to 4% (a chr5 region, R 2 = 0.82), and is questionable when methylation rates are below 2% (a chr13 region, R 2 = 0.03 - 0.27). Validation of the chr19 MR in an independent cohort (n=117) was performed in a single BULLET-Seq run. After covariate adjustment, the chr19 region was significantly associated with 24h BPs (SBP, DBP, and MAP; FDR < 0.05), confirming the findings from the discovery cohort. The MR accounted for up to 1.75% of BP variance in the 24h phenotypes. In conclusion, the reported DNA MRs have potential to be excellent markers for the cumulative effect of factors that influence 24h BPs and the BULLET-Seq workflow can be applied in clinical and population settings to screen up to thousands of patients.

It has appeared evident that blood flow to the renal cortex which is required for glomerular filtration far exceeds the delivery of O 2 required for tubular metabolic needs. Yet, the metabolic needs of kidneys are second only to the heart per gram tissue and changes in O 2 extraction and utilization have not been directly measured in face of enhanced workloads such those imposed by high salt (HS) diets. We have developed techniques enabling continuous 24 hr/d monitoring of renal blood flow (RBF; Transonic flow probe) together with blood pressure (BP, 24hr/d) and intermittent sampling of arterial and renal venous blood over 21 days. O 2 content (mL/dL) was determined as 1.31 х Hemoglobin (Hb) (g/dL) х HbO 2 (%) + 0.003 х PaO 2 (mmHg); O 2 consumption (ml/min) was determined as RBF (ml/min) х (arterial-renal venous O 2 content difference). Sprague Dawley rats (SD; n=7; 10 wk age) were compared to Dahl salt-sensitive (SS; n=6; 8 wk age) rats fed 0.4% NaCl and during the 21 days of 4.0% NaCl (HS) diet. Beginning 7 days following surgery, RBF and BP were recorded 24 hr/day, and arterial and renal venous blood sampled for determination of blood gases (ABL800 FLEX radiometer) at 7, 14 and 21 of the HS diet. Average 24 hr mean BP of SD rats increased with the HS diet from 111±2 to 119±5 mmHg and SS rats rose from 121±5 to 155±9 mmHg (p+ 0.6 ml/min (p+ 0.9). RBF when expressed per gram kidney weight (gkw; determined in separate groups of rats) remained unchanged in both SD and SS rats in response to the HS diet. SS rats exhibited lower levels of RBF gkw than SD rats (p2 extraction and consumption was increased significantly in SD rats but not SS rats fed the HS diet. The results of the study indicate that despite high levels of RBF per tissue weight, increased metabolic work imposed by a HS diet resulted in a significant increase of RBF, O 2 extraction and consumption in normal rats. In contrast, SS rats exhibited lower levels of RBF but failed to increase either O 2 delivery or extraction. This failure may contribute importantly to altered metabolic substrate utilization in SS rats fed a HS diet, progression of ischemic related injury and hypertension.

Studies were carried out to determine whether acute elevation of renal perfusion pressure (RPP) activates the mechanistic target of rapamycin complex 1 (mTORC1) and inflammation-related genes which may trigger a rapid infiltration of immune cells. RPP was elevated by 40 mmHg (HP group) for 30 minutes in male SD rats (n=5, 10-12 weeks of age) while measuring renal blood flow (RBF; Transonic ultrasonic probe) and urine flow rate. Sham rats (Sham group) were studied in the same way, but RPP was not changed. Since initial studies found that the acute increase of RPP resulted in activation of mTORC1 (pS6 S235/6 /S6; PT308 /AKT ), the effects of inhibition of mTORC1 with rapamycin (Rapa) pretreatment (1.5 mg/kg; n=10) were then determined (HP+Rapa group). RBF was well autoregulated in both HP and HP+Rapa treated rats averaging 6.9 ± 0.5 vs 7.0 ± 0.8 ml/min/gkw in HP (p=0.72) and 8.1 ± 0.8 vs 8.4 ± 0.5 in HP+Rapa group (p=0.34) with a 40 mmHg elevation of RPP. Pressure-natriuresis was unexpectedly blunted in HP+Rapa treated rats increasing from 0.40 ± 0.21 to 4.0 ± 1.1 in HP rats compared to 0.21 ± 0.07 to 2.3 ± 0.5 μmol/min/gkw; pS235/6 /S6) in renal cortex (2.8 ± 0.4 vs 4.8 ± 0.5 A.U.; p

The kinetics and efficiency of mitochondrial oxidative phosphorylation (OxPhos) can depend on the choice of respiratory substrates. Furthermore, potential differences in this substrate dependency among different tissues are not well-understood. Here, we determined the effects of different substrates on the kinetics and efficiency of OxPhos in isolated mitochondria from the heart and kidney cortex and outer medulla (OM) of Sprague-Dawley rats. The substrates were pyruvate+malate, glutamate+malate, palmitoyl-carnitine+malate, alpha-ketoglutarate+malate, and succinate±rotenone at saturating concentrations. The kinetics of OxPhos were interrogated by measuring mitochondrial bioenergetics under different ADP perturbations. Results show that the kinetics and efficiency of OxPhos are highly dependent on the substrates used, and this dependency is distinctly different between heart and kidney. Heart mitochondria showed higher respiratory rates and OxPhos efficiencies for all substrates in comparison to kidney mitochondria. Cortex mitochondria respiratory rates were higher than OM mitochondria, but OM mitochondria OxPhos efficiencies were higher than cortex mitochondria. State 3 respiration was low in heart mitochondria with succinate but increased significantly in the presence of rotenone, unlike kidney mitochondria. Similar differences were observed in mitochondrial membrane potential. Differences in H(2)O(2) emission in the presence of succinate±rotenone were observed in heart mitochondria and to a lesser extent in OM mitochondria, but not in cortex mitochondria. Bioenergetics and H(2)O(2) emission data with succinate±rotenone indicate that oxaloacetate accumulation and reverse electron transfer may play a more prominent regulatory role in heart mitochondria than kidney mitochondria. These studies provide novel quantitative data demonstrating that the choice of respiratory substrates affects mitochondrial responses in a tissue-specific manner.

In 2015, the American Heart Association awarded 4-year funding for a Strategically Focused Research Network focused on hypertension composed of 4 Centers: Cincinnati Children’s Hospital, Medical College of Wisconsin, University of Alabama at Birmingham, and University of Iowa. Each center proposed 3 integrated (basic, clinical, and population science) projects around a single area of focus relevant to hypertension. Along with scientific progress, the American Heart Association put a significant emphasis on training of next-generation hypertension researchers by sponsoring 3 postdoctoral fellows per center over 4 years. With the center projects being spread across the continuum of basic, clinical, and population sciences, postdoctoral fellows were expected to garner experience in various types of research methodologies. The American Heart Association also provided a number of leadership development opportunities for fellows and investigators in these centers. In addition, collaboration was highly encouraged among the centers (both within and outside the network) with the American Heart Association providing multiple opportunities for meeting and expanding associations. The area of focus for the Cincinnati Children’s Hospital Center was hypertension and target organ damage in children utilizing ambulatory blood pressure measurements. The Medical College of Wisconsin Center focused on epigenetic modifications and their role in pathogenesis of hypertension using human and animal studies. The University of Alabama at Birmingham Center’s areas of research were diurnal blood pressure patterns and clock genes. The University of Iowa Center evaluated copeptin as a possible early biomarker for preeclampsia and vascular endothelial function during pregnancy. In this review, challenges faced and successes achieved by the investigators of each of the centers are presented.