Search

SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism inWe used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of thisTrp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Background: SH2B3 (SH2B adaptor protein 3) is an adaptor protein that negatively regulates cytokine signaling and cell proliferation. A common missense single nucleotide polymorphism in SH2B3 (rs3184504) results in substitution of tryptophan (Trp) for arginine (Arg) at amino acid 262 and is a top association signal for hypertension in human genome-wide association studies. Whether this variant is causal for hypertension, and if so, the mechanism by which it impacts pathogenesis is unknown. Methods: We used CRISPR-Cas9 technology to create mice homozygous for the major (Arg/Arg) and minor (Trp/Trp) alleles of this SH2B3 polymorphism. Mice underwent angiotensin II (Ang II) infusion to evaluate differences in blood pressure (BP) elevation and end-organ damage including albuminuria and renal fibrosis. Cytokine production and Stat4 phosphorylation was also assessed in Arg/Arg and Trp/Trp T cells. Results: Trp/Trp mice exhibit 10 mmHg higher systolic BP during chronic Ang II infusion compared to Arg/Arg controls. Renal injury and perivascular fibrosis are exacerbated in Trp/Trp mice compared to Arg/Arg controls following Ang II infusion. Renal and ex vivo stimulated splenic CD8 + T cells from Ang II-infused Trp/Trp mice produce significantly more interferon gamma (IFNg) compared to Arg/Arg controls. Interleukin-12 (IL-12)-induced IFNg production is greater in Trp/Trp compared to Arg/Arg CD8 + T cells. In addition, IL-12 enhances Stat4 phosphorylation to a greater degree in Trp/Trp compared to Arg/Arg CD8 + T cells, suggesting that Trp-encoding SH2B3 exhibits less negative regulation of IL-12 signaling to promote IFNg production. Finally, we demonstrated that a multi-SNP model genetically predicting increased SH2B3 expression in lymphocytes is inversely associated with hypertension and hypertensive chronic kidney disease in humans.. Conclusions: Taken together, these results suggest that the Trp encoding allele of rs3184504 is causal for BP elevation and renal dysfunction, in part through loss of SH2B3-mediated repression of T cell IL-12 signaling leading to enhanced IFNg production.

Previous work in our laboratory has implicated a significant role for the immune system in potentiating the hypertensive and kidney damage phenotypes which occur in the Dahl Salt-Sensitive (SS) rat. Toll-like receptors (TLRs), usually expressed on macrophages, play a key role in the innate immune system. A number of TLRs have been implicated in cardiovascular disease and chronic kidney disease with particular interest in TLR4, which specifically binds gram-negative lipopolysaccharide or endogenous molecules produced as a result of tissue injury. We previously performed a transcriptomic analysis which demonstrated TLR4 upregulation in renal medullary tissue of Dahl SS rats fed a high salt (4.0% NaCl) diet. The present study tests the hypothesis that TLR4 influences high salt diet-induced hypertension and renal damage in the Dahl SS rat. Using CRISPR/Cas9 technology, a mutation was generated in the TLR4 gene on the Dahl SS background, creating a 1-bp frameshift deletion resulting in a premature stop codon. SS TLR4-/- males and parental Dahl SS males (SS TLR4+/+ ), maintained on a 0.4% NaCl chow, underwent carotid artery telemetry implantation at 7 weeks of age. After recovery and baseline mean arterial pressure (MAP) recordings (day 0: 125.6 ± 0.5 vs 124.2 ± 2 mmHg), animals were switched to a 4.0% NaCl chow for 3 weeks. Overnight urine collections occurred once during the low salt period and weekly throughout the high salt challenge. Kidneys were flushed prior to euthanasia. Although SS TLR4-/- MAP was not different from SS TLR4+/+ during the high salt challenge (day 21: 176.8 ± 14 mmHg, n=2 vs 172.3 ± 18 mmHg, n=4), high salt day 21 urinary albumin excretion was significantly reduced in SS TLR4-/- (272.7 ± 65 mg/day, n=5, pTLR4+/+ (427.5 ± 67 mg/day, n=7), indicating reduced renal damage in TLR4 absence. Furthermore, flow cytometric analysis of isolated renal immune cells indicated a 33% reduction in CD45+ total leukocytes (5.0 ± 1.2 vs 3.4 ± 0.9 x10 6 cells) and a 31% reduction in CD11bc+ monocytes/macrophages (3.9 ± 0.9 vs 2.6 ± 0.7 x10 6 cells) in SS TLR4-/- (n=5) vs SS TLR4+/+ (n=7) kidneys. Together, these data indicate that TLR4 may drive the immune response during the induction and progression of salt-sensitive hypertension and renal disease.

Heart failure with preserved ejection fraction (HFpEF) is a clinical syndrome where patients, despite having a normal left ventricular systolic function, present with symptoms of volume overload. Recent data have shown that HFpEF is associated with elevation of inflammatory biomarkers. In a well-known rodent hypertensive model (DOCA-salt), we isolated CD45+ leukocytes from the heart and then performed CITE-Seq, a novel technique to obtain transcriptomic and surface marker expression on single cells, on a total of 4,359 and 7,600 cells from four sham and four DOCA-salt left ventricles, respectively. Analysis showed significant differential gene expression in the myeloid (macrophage/monocytic) population. We then took the top 20 genes that were differentially expressed between DOCA-salt vs sham treated myeloid cells and performed a genetic analysis called PrediXcan in Vanderbilt’s DNA databank, BioVU. Our analysis used gene expression prediction models built from the GTEx Project and tested its association with the HFpEF phenotype, which was derived on ICD-9 and 10 codes and natural language programming. From BioVU, 88,660 subjects were included in the association analysis. Of the 20 genes, 8 did not meet prediction model criteria for PrediXcan. In the remaining 12 genes, genetically predicted expression of only 2 ( Lgals1 and Ctsl ) are associated with a HFpEF phenotype. Of these, the gene encoding galectin 1, Lgals1 , had the lowest p-value (0.02) and highest beta coefficient (0.32) corresponding to an odds ratio for HFpEF of 1.38. Galectin-1 is a well known mediator of inflammation resolution in infection and tumor biology, however its role in heart failure is unknown. We are currently exploring the role of galectin-1 in the pathophysiology of HFpEF through Lgals1 -/- and LysM-Cre x Lgals1 fl/fl mice. In conclusion, using two different approaches in mice and humans, we identified galectin-1 as a new potential mediator in HFpEF development.

Genomic sequence and gene expression association studies in animals and humans have identified genes that may be integral in the pathogenesis of various diseases. CD14 (cluster of differentiation 14)—a cell surface protein involved in innate immune system activation—is one such gene associated with cardiovascular and hypertensive disease. We previously showed that this gene is upregulated in renal macrophages of Dahl salt-sensitive animals fed a high-salt diet; here we test the hypothesis that CD14 contributes to the elevated pressure and renal injury observed in salt-sensitive hypertension. Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9), we created a targeted mutation in the CD14 gene on the Dahl SS (SS/JrHSDMcwi) background and validated the absence of CD14 peptides via mass spectrometry. Radiotelemetry was used to monitor blood pressure in wild-type and CD14 −/ − animals challenged with high salt and identified infiltrating renal immune cells via flow cytometry. Germline knockout of CD14 exacerbated salt-sensitive hypertension and renal injury in female animals but not males. CD14 −/− females demonstrated increased infiltrating macrophages but no difference in infiltrating lymphocytes. Transplant of CD14 +/+ or CD14 −/− bone marrow was used to isolate the effects of CD14 knockout to hematopoietic cells and confirmed that the differential phenotype observed was due to knockout of CD14 in hematopoietic cells. Ovariectomy was used to remove the influence of female sex hormones, which completely abrogated the effect of CD14 knockout. These studies provide a novel treatment target and evidence of a new dichotomy in immune activation between sexes within the context of hypertensive disease where CD14 regulates immune cell activation and renal injury.

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.

Hypertension, or an elevated blood pressure, is the primary modifiable risk factor for cardiovascular disease, the number one cause of mortality worldwide. We previously demonstrated that Th17 activation and interleukin 17A (IL-17A)/IL-21 production is integral for the full development of a hypertensive phenotype as well as the renal and vascular damage associated with hypertension. Rho-associated coiled-coil containing protein Kinase 2 (ROCK2) serves as a molecular switch upregulating Th17 and inhibiting regulatory T cell (Treg) differentiation. We hypothesize that hypertension is characterized by excessive T cell ROCK2 activation leading to increased Th17/Treg ratios and ultimately end-organ damage. We first showed in vitro that KD025, an experimental orally bioavailable ROCK2 inhibitor inhibits Th17 cell proliferation and IL-17A/IL-21 production. To determine if hypertensive stimuli such as endothelial stretch increases T cell ROCK2 expression, we cultured human aortic endothelial cells exposed to 5% (normotensive) or 10% (hypertensive) stretch with circulating human T cells and HLA-DR+ antigen presenting cells. Hypertensive stretch increased T cell ROCK2 expression 2-fold. We then tested the effect of ROCK2 inhibition with KD025 (50mg/kg i.p. daily) in vivo on angiotensin II (Ang II)-induced hypertension. Treatment with KD025 significantly attenuated the hypertensive response within 1 week of Ang II treatment (systolic blood pressure: 139± 8 vs 108±7mmHg) and this persisted for the duration of the 4 week study reaching blood pressures 20 mmHg lower (135±13mmHg) than vehicle treated mice (158±4mmHg p

We have previously reported that depletion Cyclophilin D (CypD), a regulatory subunit of mitochondrial permeability transition pore, improves vascular function and attenuates hypertension, however, specific regulation of CypD in hypertension is not clear. Analysis of human arterioles from hypertensive patients did not reveal alterations in CypD levels but showed 3-fold increase in CypD acetylation. We hypothesized that CypD-K166 acetylation promotes vascular oxidative stress and hypertension, and measures to reduce CypD acetylation can improve vascular function and reduce hypertension. Essential hypertension and animal models of hypertension are linked to inactivation of mitochondrial deacetylase Sirt3 by highly reactive lipid oxidation products, isolevuglandins (isoLGs), and supplementation of mice with mitochondria targeted scavenger of isoLGs, mito2HOBA, improves CypD deacetylation. To test the specific role of CypD-K166 acetylation, we developed CypD-K166R deacetylation mimic mutant mice. Mitochondrial respiration, vascular function and systolic blood pressure in CypD-K166R mice was similar to wild-type C57Bl/6J mice. Meanwhile, angiotensin II-induced hypertension was substantially attenuated in CypD-K166R mice (144 mmHg) compared with wild-type mice (161 mmHg). Angiotensin II infusion in wild-type mice significantly increased mitochondrial superoxide, impaired endothelial dependent relaxation, and reduced the level of endothelial nitric oxide which was prevented in angiotensin II-infused CypD-K166R mice. Hypertension is linked to increased levels of inflammatory cytokines TNFα and IL-17A promoting vascular oxidative stress and end-organ damage. We have tested if CypD-K166R mice are protected from cytokine-induced oxidative stress. Indeed, ex vivo incubation of aorta with the mixture of angiotensin II, TNFα and IL-17A (24 hours) increased mitochondrial superoxide by 2-fold in wild-type aortas which was abrogated in CypD-K166R mice. These data support the pathophysiological role of CypD acetylation in inflammation, oxidative stress and hypertensive end-organ damage. We propose that targeting CypD acetylation may have therapeutic potential in treatment of vascular dysfunction and hypertension.

The Dahl salt-sensitive (SS) rat is an established model of SS hypertension and renal damage. In addition to salt, other dietary components were shown to be important determinants of hypertension in SS rats. With previous work eliminating the involvement of genetic differences, grain-fed SS rats from Charles River Laboratories (SS/CRL; 5L2F/5L79) were less susceptible to salt-induced hypertension and renal damage compared with purified diet-fed SS rats bred at the Medical College of Wisconsin (SS/MCW; 0.4% NaCl, AIN-76A). With the known role of immunity in hypertension, the present study characterized the immune cells infiltrating SS/MCW and SS/CRL kidneys via flow cytometry and RNA sequencing in T-cells isolated from the blood and kidneys of rats maintained on their respective parental diet or on 3 weeks of high salt (4.0% NaCl, AIN-76A). SS/CRL rats were protected from salt-induced hypertension (116.5±1.2 versus 141.9±14.4 mm Hg), albuminuria (21.7±3.5 versus 162.9±22.2 mg/d), and renal immune cell infiltration compared with SS/MCW. RNA-seq revealed >50% of all annotated genes in the entire transcriptome to be significantly differentially expressed in T-cells isolated from blood versus kidney, regardless of colony or chow. Pathway analysis of significantly differentially expressed genes between low and high salt conditions demonstrated changes related to inflammation in SS/MCW renal T-cells compared with metabolism-related pathways in SS/CRL renal T-cells. These functional and transcriptomic T-cell differences between SS/MCW and SS/CRL show that dietary components in addition to salt may influence immunity and the infiltration of immune cells into the kidney, ultimately impacting susceptibility to salt-induced hypertension and renal damage.

Studies from our laboratory have revealed an important role for the maternal diet and the dietary protein source in the development of hypertension and renal injury in Dahl salt-sensitive (SS) rats. The current study sought to compare salt-induced hypertension, renal damage, and immune cell infiltration in the offspring of breeders fed either a casein- or gluten-based diet, with the hypothesis that offspring from gluten-fed breeders would fail to develop these SS phenotypes. When fed identical diets post-weaning, the F1 generation gluten offspring demonstrated lower mean arterial pressure (149.1±3.1 versus 162.5±5.8 mm Hg), albuminuria (166.2±34.6 versus 250.9±27.8 mg/day), and outer medullary protein casting (7.4±0.8% versus 13.1±1.3%) in response to high salt compared with the casein offspring (n=9–11). The gluten offspring also had fewer CD45+ leukocytes, CD11b/c+ monocytes/macrophages, CD3+ T cells, and CD45R+ B cells infiltrating the kidney. Analysis of the F2 generation gluten offspring also exhibited lower mean arterial pressure and renal damage compared with rats born from casein breeders (n=7–9), with no difference in renal immune cell infiltration. CMKLR1 —receptor for the novel prohypertensive adipokine chemerin—was found via polymerase chain reaction array to be significantly upregulated (2.99-fold) in renal T cells isolated from F2 offspring of casein-fed versus gluten-fed parents. Furthermore, CMKLR1 inhibition via α-NETA (2-[α-naphthoyl] ethyltrimethylammonium iodide) treatment significantly attenuated renal immune cell infiltration, hypertension, and renal damage in SS rats fed high salt. Together, these data demonstrate the influence of the parental diet in determining the salt-induced hypertension, renal damage, and inflammatory phenotype of the offspring.

AIM Our previous studies have demonstrated the importance of dietary factors in the determination of hypertension in Dahl salt-sensitive (SS) rats. Since the gut microbiota has been implicated in chronic diseases like hypertension, we hypothesized that dietary alterations shift the microbiota to mediate the development of salt-sensitive hypertension and renal disease. METHODS This study utilized SS rats from the Medical College of Wisconsin (SS/MCW) maintained on a purified, casein-based diet (0.4% NaCl AIN-76A, Dyets) and from Charles River Laboratories (SS/CRL) fed a whole grain diet (0.75% NaCl 5L79, LabDiet). Faecal 16S rDNA sequencing was used to phenotype the gut microbiota. Directly examining the contribution of the gut microbiota, SS/CRL rats were administered faecal microbiota transfer (FMT) experiments with either SS/MCW stool or vehicle (Vehl) in conjunction with the HS AIN-76A diet. RESULTS SS/MCW rats exhibit renal damage and inflammation when fed high salt (HS, 4.0% NaCl AIN-76A), which is significantly attenuated in SS/CRL. Gut microbiota phenotyping revealed distinct profiles that correlate with disease severity. SS/MCW FMT worsened the SS/CRL response to HS, evidenced by increased albuminuria (67.4 ± 6.9 vs 113.7 ± 25.0 mg/day, Vehl vs FMT, P = .007), systolic arterial pressure (158.6 ± 5.8 vs 177.8 ± 8.9 mmHg, Vehl vs FMT, P = .09) and renal T-cell infiltration (1.9-fold). Amplicon sequence variant (ASV)-based analysis of faecal 16S rDNA sequencing data revealed taxa that significantly shifted with FMT: Erysipelotrichaceae_2, Parabacteroides gordonii, Streptococcus alactolyticus, Bacteroidales_1, Desulfovibrionaceae_2, Ruminococcus albus. CONCLUSIONS These data demonstrate that dietary modulation of the gut microbiota directly contributes to the development of Dahl SS hypertension and renal injury.

Preeclampsia (PE) is a disorder of pregnancy, which is categorized by hypertension and proteinuria or signs of end-organ damage. Though PE is the leading cause of maternal and fetal morbidity and mortality, the mechanisms leading to PE remain unclear. The present study examined the contribution of dietary protein source (casein versus wheat gluten) to the risk of developing maternal syndrome utilizing two colonies of Dahl salt-sensitive (SS/JrHsdMcwi) rats. While the only difference between the colonies is the diet, the colonies exhibit profound differences in the pregnancy phenotypes. The SS rats maintained on the wheat gluten (SSWG) chow are protected from developing maternal syndrome; however, approximately half of the SS rats fed a casein-based diet (SSC) exhibit maternal syndrome. Those SSC rats that develop pregnancy-specific increases in blood pressure and proteinuria have no observable differences in renal or placental immune profiles compared to the protected SS rats. A gene profile array of placental tissue revealed a downregulation in Nos3 and Cyp26a1 in the SSC rats that develop maternal syndrome accompanied with increases in uterine artery resistance index suggesting the source of this phenotype could be linked to inadequate remodeling within the placenta. Investigations into the effects of multiple pregnancies on maternal health replicated similar findings. The SSC colony displayed an exacerbation in proteinuria, renal hypertrophy and renal immune cell infiltration associated with an increased mortality rate while the SSWG colony were protected highlighting how dietary protein source could have beneficial effects in PE.

PURPOSE OF REVIEW: To summarize key advances in our understanding of the role of interleukin 17A (IL-17A) in the pathogenesis of hypertension and highlight important areas for future research and clinical translation. RECENT FINDINGS: While T helper 17 (Th17) cells are major producers of IL-17A, there are several additional innate and adaptive immune cell sources including gamma-delta T cells, innate lymphoid cells, and natural killer cells. IL-17A promotes an increase in blood pressure through multiple mechanisms including inhibiting endothelial nitric oxide production, increasing reactive oxygen species formation, promoting vascular fibrosis, and enhancing renal sodium retention and glomerular injury. IL-17A production from Th17 cells is increased by high salt conditions in vitro and in vivo. There is also emerging data linking salt, the gut microbiome, and intestinal T cell IL-17A production. Novel therapeutics targeting IL-17A signaling are approved for the treatment of autoimmune diseases and show promise in both animal models of hypertension and human studies. SUMMARY: Hypertensive stimuli enhance IL-17A production. IL-17A is a key mediator of renal and vascular dysfunction in hypertensive mouse models and correlates with hypertension in humans. Large randomized clinical trials are needed to determine whether targeting IL-17A might be an effective adjunct treatment for hypertension and its associated end-organ dysfunction.

The immune system has a clear role in the development of hypertension and renal damage in male Dahl salt-sensitive (SS) rats, but far less is known about this phenotype in females, especially in regard to the contribution of immune-related mechanisms. The current study examined the hypertensive and kidney injury phenotype in response to a 3 week high salt challenge (HS, 4.0% NaCl, AIN-76A) in female versus male SS rats (n>10/group). Measured via radiotelemetry, there was no difference in low salt (LS, 0.4% NaCl) mean arterial pressure (MAP) between females and males (129±2 vs 124±2 mmHg, respectively). However, after 3 weeks of HS, females had a significant attenuation in the development of hypertension compared to males (161±3 vs 177±7 mmHg). This coincided with significantly less renal damage, evident by markedly reduced albuminuria (105±16 vs 183±16 mg/day) and medullary protein cast formation (7.0±0.7 vs 14.6±1.1%) in the females. Assessed via flow cytometry, there were fewer CD45+ total leukocytes (48% reduction), CD3+ T cells (51%), CD45R+ B cells (73%), and CD11b/c+ monocytes/macrophages (47%) in female versus male kidneys. To interrogate the contribution of adaptive immunity, specifically T cells, to the development of this sex difference, the same parameters were investigated in female and male SS CD247-/- rats which lack CD3+ T cells. The absence of functional T cells significantly reduced blood pressure in both females (147±6 vs 161±3 mmHg; SS CD247-/- vs SS) and males (157±8 vs 177±7 mmHg) after 3 weeks of HS, with no statistical difference between female and male SS CD247-/- rats. While a reduction in albuminuria (females: 37±12 vs 105±16 mg/day; males: 88±13 vs 183±16 mg/day, SS CD247-/- vs SS) and medullary protein cast formation (females: 1.8±0.4 vs 7.0±0.7%; males: 9.1±0.7 vs 14.6±1.1%) was observed in SS CD247-/- versus SS rats regardless of sex, there was greater protection from these measures of kidney damage in SS CD247-/- female versus SS CD247-/- males. Together, our data indicate that female SS rats are significantly protected from high salt-induced hypertension and renal disease compared to males rats. The deletion of T cells normalized high salt blood pressure between male and female rats but sex differences in renal damage still persisted.

Studies examining mechanisms of Dahl salt-sensitive (SS) hypertension have implicated the infiltration of leukocytes in the kidneys, which contribute to renal disease and elevated blood pressure. However, the signaling pathways by which leukocytes traffic to the kidneys remain poorly understood. The present study nominated a signaling pathway by analyzing a kidney RNA sequencing data set from SS rats fed either a low-salt (0.4% NaCl) diet or a high-salt (4.0% NaCl) diet. From this analysis, chemokine (C-C motif) ligand 2 (CCL2) and chemokine (C-C motif) receptor 2 (CCR2) were nominated as a potential pathway modifying renal leukocyte infiltration and contributing to SS hypertension. The functional role of the CCL2/CCR2 pathway was tested by daily administration of CCR2 antagonist (RS-102895 at 5 mg·kg−1·day−1in DMSO) or DMSO vehicle for 3 or 21 days by intraperitoneal injections during the high salt challenge. Blood pressure, renal leukocyte infiltration, and renal damage were evaluated. The results demonstrated that RS-102895 treatment ameliorated renal damage (urinary albumin excretion; 43.4 ± 5.1 vs. 114.7 ± 15.2 mg/day in vehicle, P < 0.001) and hypertension (144.3 ± 2.2 vs. 158.9 ± 4.8 mmHg in vehicle, P < 0.001) after 21 days of high-salt diet. It was determined that renal leukocyte infiltration was blunted by day 3 of the high-salt diet (1.4 ± 0.1 vs. 1.9 ± 0.2 in vehicle × 106CD45+cells/kidney, P = 0.034). An in vitro chemotaxis assay validated the effect of RS-102895 on leukocyte chemotaxis toward CCL2. The results suggest that increased CCL2 in SS kidneys is important in the early recruitment of leukocytes, and blockade of this recruitment by administering RS-102895 subsequently blunted the renal damage and hypertension.

PURPOSE OF REVIEW: This review will provide an in-depth coverage of the epidemiological and pre-clinical literature surrounding the role of dietary protein in hypertension, with a special emphasis on the history of our work on the Dahl salt-sensitive rat. RECENT FINDINGS: Our studies have dedicated much effort into understanding the relationship between dietary protein and its effect on the development of salt-sensitive hypertension and renal injury. Our evidence over the last 15 years have demonstrated that both the source and amount of dietary protein can influence the severity of disease, where we have determined mechanisms related to immunity, the maternal environment during pregnancy, and more recently the gut microbiota, which significantly contribute to these diet-induced effects. SUMMARY: Deeper understanding of these dietary protein-related mechanisms may provide insight on the plausibility of dietary modifications as future therapeutic avenues for hypertension and renal disease.

This review will highlight recent studies that have investigated the relationship between Na+, renal macrophage polarization, and renal damage. A hyperosmotic environment drives the macrophage toward a proinflammatory phenotype and away from an anti-inflammatory phenotype. Animal models of salt-sensitive hypertension demonstrate a characteristic infiltration of macrophages into the kidney that is greatly reduced when blood pressure is lowered. Because general immunosuppression or macrophage depletion leads to a host of adverse side effects, more recent studies have modulated the interaction of specific signaling molecules, including NOD-like receptor family pyrin domain-containing 3, chemokine (C-X-C motif) ligand 16, and VEGF, to prevent the end-organ renal damage that accumulates in salt-sensitive disease.

The present study, performed in Dahl salt-sensitive (SS) and SS- Rag1−/− rats lacking T and B lymphocytes, tested the hypothesis that immune cells amplify salt-sensitive hypertension and kidney damage in response to a high-protein diet. After being weaned, SS and SS- Rag1−/− rats were placed on an isocaloric, 0.4% NaCl diet containing normal (18%) or high (30%) protein. At 9 wk of age, rats were switched to a 4.0% NaCl diet containing the same amount of dietary protein and maintained on the high-salt diet for 3 wk. After being fed the high-salt diet, SS rats fed high protein had amplified hypertension and albumin excretion (158.7 ± 2.6 mmHg and 140.8 ± 16.0 mg/day, respectively, means ± SE) compared with SS rats fed normal protein (139.4 ± 3.6 mmHg and 69.4 ± 11.3 mg/day). When compared with the SS rats, SS- Rag1−/− rats fed high protein were protected from exacerbated hypertension and albuminuria (142.9 ± 5.8 mmHg and 66.2 ± 10.8 mg/day). After 3 wk of the high-salt diet, there was a corresponding increase in total leukocyte infiltration (CD45+) in the kidneys of both strains fed high-protein diet. The SS- Rag1−/− rats fed high-protein diet had 74–86% fewer CD3+ T lymphocytes and CD45R+ B lymphocytes infiltrating the kidney versus SS rats, but there was no difference in the infiltration of CD11b/c+ monocytes and macrophages, suggesting that the protective effects observed in the SS- Rag1−/− rats are specific to the reduction of lymphocytes. With the SS- Rag1−/− rats utilized as a novel tool to explore the effects of lymphocyte deficiency, these results provide evidence that adaptive immune mechanisms contribute to the exacerbation of salt-induced hypertension and renal injury mediated by increased dietary protein intake.

New findings What is the central question of this study? Recruitment of immune cells to the kidney potentiates hypertensive pathology, but more refined methods are needed to assess these cells functionally. Adoptive transfer studies of immune cells have been limited in rat models and especially in the study of salt-sensitive hypertension. We tested the hypothesis that splenocyte transfer into T-cell-deficient rats is sufficient to exacerbate salt-sensitive hypertension. What is the main finding and its importance? We demonstrate that transfer of splenocytes into T-cell-deficient animals exacerbates salt-sensitive hypertension, and an enrichment in the CD4+ compartment specifically induces this phenomenon. Abstract Increasing evidence of immune system activation during the progression of hypertension and renal injury has led to a need for new methods to study individual cell types. Transfer of immune cells serves as a powerful tool to isolate effects of specific subsets. Transfer studies in Rag1-/- mice have demonstrated an important role of T-cell activation in hypertension, but this approach has yielded limited success in rat models. Using the T-cell-deficient Dahl salt-sensitive (SS) rat, SSCD247-/- , we hypothesized that splenocyte transfer from SS wild-type animals into SSCD247-/- animals would populate the T-cell compartment. The Dahl SS background provides a model for studying salt-sensitive hypertension; therefore, we also tested whether the dietary salt content of the donor would confer differential salt sensitivity in the recipient. To test this, donors were maintained on either a low-salt or a high-salt diet, and at postnatal day 5 the recipients received splenocyte transfer from one of these groups before a high-salt diet challenge. We showed that splenocyte transfer elevated blood pressures while rats were fed low salt and exacerbated the salt-sensitive increase in pressure when they were fed fed high salt. Furthermore, transfer of splenocytes conferred exacerbated renal damage. Lastly, we confirmed the presence of T cells in the circulation and in the spleen, and that infiltration of immune cells, including T cells, macrophages and B cells, into the kidney was elevated in those receiving the transfer. Interestingly, the source of the splenocytes, from donors fed either a low-salt or a high-salt diet, did not significantly affect these salt-sensitive phenotypes.

The Dahl Salt-Sensitive rat maintained on a low salt, casein-based diet (SSC) develops maternal syndrome (hypertension and proteinuria) compared to virgin controls. Our group has previously demonstrated that a substitution of the casein protein for a wheat gluten protein in the rat chow (SSWG) protects the Dahl SS rat from the development of maternal syndrome. This protection is observed in both the blood pressure (141±3 vs 114±4 mmHg, p p p< 0.01) suggesting dietary protein source influences pregnancy in Dahl SS rats. While the exact mechanisms responsible for the development of maternal syndrome are unclear, it is thought that poor placentation is the initiating step in the disease process. Since a substitution of the protein source in the rat chow greatly impacted the phenotype in the Dahl SS rat, we tested the hypothesis that there was differential gene expression in the placental tissue between the SSC rats prone to maternal syndrome compared to the protected SSWG rats. To investigate these phenotypic differences, a PCR array approach was utilized to explore gene expression in placental tissue of both SSC and SSWG rats. Placental tissue from both strains at gestational day 20 was homogenized and RNA isolated to investigate 89 genes related to rat pregnancy and preeclampsia. Using a commercially available PCR array approach identified 4 genes that were differentially expressed between the SSC and SSWG placentas. There was a significant downregulation in expression of Atp1b1 (0.0018-fold change, p , Nos3 (0.13-fold change, p , Cdh13 (0.42-fold change, p and Cyp26a1 (0.48-fold change, p

Previous work has implicated the immune system in potentiating salt-sensitive (SS) hypertension and kidney damage in the Dahl SS rat model. The innate immunity protein Cluster of Differentiation 14 (CD14), normally involved in bacterial responses of macrophages, has been associated with hypertension in humans. The present study tests the hypothesis that CD14 influences the extent of high-salt diet induced renal damage and hypertension in the Dahl SS rat, by modulating activation of the innate immune system. CD14 was mutated in the Dahl SS background using CRISPR-Cas9 technology, leading to the loss of CD14 protein in peritoneal macrophages (pMacs) as evidenced by mass spectrometry-based parallel reaction monitoring. pMac stimulation in vitro demonstrated altered IL-1β production in CD14-/- animals. To monitor SS phenotypes in vivo , male and female CD14+/+ and CD14-/- animals maintained on a 0.4% NaCl diet underwent carotid catheter telemetry implantation at 7 weeks of age (n=12/14). After recovery and baseline recordings, animals were switched to a 4.0% NaCl diet for 21 days. There was no significant difference in the SS increase in blood pressure or albuminuria between genotypes of males. However, female CD14-/- animals demonstrated exacerbated salt-sensitivity of blood pressure (+45mmHg) compared to wild types (+31mmHg). To determine what may mediate this difference, immune cells were isolated from the kidneys of these animals. Accordingly, males showed no difference in immune cell infiltration; whereas, female CD14-/- animals showed an elevation in the infiltration of CD11b/c+ macrophages. To determine whether knockout of CD14 in immune cells specifically was responsible for this phenotypic difference, a lethal dose of total body irradiation (11Gy) of CD14+/+ recipient animals followed by either CD14-/- or CD14+/+ bone marrow transfer experiment was done in female animals (n=6/7). Remarkably, even though all host tissue cells were wildtype, transfer of CD14-/- bone marrow recapitulated the exacerbated SS phenotypes, including increased renal infiltration of macrophages. Together, these data demonstrate that CD14 signaling in hematopoietic cells of females modulates the full extent of the immune response and blunts SS hypertension.

Studies of Dahl salt-sensitive (SS) rats have shown that renal CD3+ T cells and ED-1+ macrophages are involved in the development of salt-sensitive hypertension and renal damage. The present study demonstrated that the increase in renal immune cells, which accompanies renal hypertrophy and albuminuria in high-salt diet-fed Dahl SS rats, is absent in Sprague-Dawley and SSBN13 rats that are protected from the SS disease phenotype. Flow cytometric analysis demonstrated that >70% of the immune cells in the SS kidney are M1 macrophages. PCR profiling of renal myeloid cells showed a salt-induced upregulation in 9 of 84 genes related to Toll-like receptor signaling, with notable upregulation of the Toll-like receptor 4/CD14/MD2 complex. Because of the prominent increase in macrophages in the SS kidney, we used liposome-encapsulated clodronate (Clod) to deplete macrophages and assess their contribution to salt-sensitive hypertension and renal damage. Dahl SS animals were administered either Clod-containing liposomes (Clod-Lipo), Clod, or PBS-containing liposomes as a vehicle control. Clod-Lipo treatment depleted circulating and splenic macrophages by ∼50%; however, contrary to our hypothesis, Clod-Lipo-treated animals developed an exacerbated salt-sensitive response with respect to blood pressure and albuminuria, which was accompanied by increased renal T and B cells. Interestingly, those treated with Clod also demonstrated an exacerbated phenotype, but it was less severe than Clod-Lipo-treated animals and independent of changes to the number of renal immune cells. Here, we have shown that renal macrophages in Dahl SS animals sustain a M1 proinflammatory phenotype in response to increased dietary salt and highlighted potential adverse effects of Clod-Lipo macrophage depletion.

Based on previous studies suggesting a role of renal nerves in renal inflammation, the present studies were performed to test the hypothesis that renal nerves mediate renal damage in Dahl salt-sensitive (SS) hypertension by increasing renal leukocyte infiltration. Experiments were performed in Dahl SS rats with bilateral renal denervation (RDN) and bilateral sham operation ( n = 10 or 11 per group) and with unilateral RDN and contralateral sham operation ( n = 10). After denervation, rats were switched from a low-salt 0.4% NaCl (LS) diet to a high-salt 4% NaCl (HS) diet and maintained on HS diet for 21 days. Bilateral RDN reduced the magnitude of hypertension assessed by radiotelemetry in Dahl SS rats compared with sham-operated rats (mean arterial pressure 140.9 ±4.8 mmHg and 159.7 ± 3.5 mmHg, respectively) and reduced proteinuria at day 21 of HS diet. However, assessment of renal leukocyte infiltration demonstrated no significant effect of bilateral RDN on the number of infiltrating leukocytes (RDN 3.6 ± 0.5 × 106 vs. sham operated 4.3 ± 0.3 × 106 CD45+ cells) or any of the subsets examined by flow cytometry. The unilateral RDN experiment showed no effect of RDN on the renal infiltration of leukocytes (RDN 6.5 ± 0.9 × 106 vs. sham operated 6.1 ± 1.1 × 106 CD45+ cells/kidney) or renal damage in RDN vs. sham-operated kidney after 21 days of HS diet. This work investigated the relationship between renal nerves and renal inflammation during Dahl SS hypertension. Contrary to our hypothesis, the results of this work suggest that immune cell infiltration in the kidney of Dahl SS rats is not mediated by the renal nerves.

Studies examining mechanisms of Dahl salt-sensitive (SS) hypertension have implicated the infiltration of leukocytes in the kidneys, which contribute to renal disease and elevated blood pressure. H...

DNA methylation plays a key role in the regulation of gene expression. In the present study, we investigated the impact of changes in DNA methylation in Dahl Salt Sensitive (SS) rats fed a casein-based AIN-76A chow containing low salt (LS) or high salt (HS). In addition to the hypertension and renal damage which occurs following 14 days of HS feeding, we assessed 5-Methylcytosine levels at single-base resolution by reduced representation bisulfite genome sequencing (RRBS) of DNA from the renal outer medulla of SS rats. Several hundred differentially methylated regions (DMRs) were identified between the SS rats on the LS or HS diet. Approximately a quarter of the DMRs were located in transcriptional start site regions, a third in intragenic regions, and the remainder in intergenic regions. To investigate the functional role of methylation in SS hypertension, we administered decitabine (DNA methyltransferase inhibitor, 5mg/kg, ip) or vehicle at Days 0, 3, 5, and 7 during a 14 day HS period. While there was no difference in MAP during the LS period (127±3 vs. 126±6 mmHg, vehicle vs. decitabine, n=9), at the end of 2 weeks of HS there was a significant increase in MAP in the vehicle SS rats compared to the decitabine-treated rats (163±6 vs 141±4 mmHg, p ). This protective effect in MAP in the decitabine-treated group also exhibit a reduction in albumin excretion rate compared to controls (119±19 vs. 182±24 mg/day, p ). Upon analysis of the immune cell infiltration profile, there was a reduction in number of CD45+ total leukocytes, CD11b/c+ monocytes/ macrophages, and CD45R+ B cells in the decitabine-treated animals compared to controls. Decitabine administration also significantly reduced renal histological damage as indicated by the presence of outer medullary protein casts compared to vehicle-treated rats (6.8±1.0% vs. 10.6±1.3%, p

Previous studies utilizing the SS p67 phox–/– 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. Our laboratory has established a key role for renal immune cell infiltration in the development of SS hypertension and observed an enrichment of NOX2 subunits in infiltrating T cells. The contribution of ROS specifically derived from immune cells in SS hypertension remains unknown. To assess the role of ROS in immune cells, SS p67 phox–/– rats underwent total body irradiation and received bone marrow transfer from either SS p67 phox–/– (p67/p67) or SS (p67/SS) donor rats. p67/SS rats would be capable of producing NOX2-derived ROS only in the transferred SS cells of hematopoietic origin while the p67/p67 control group would be unable to produce NOX2-derived ROS globally. Mean arterial pressure (MAP) was not different between the p67/SS and p67/p67 rats when fed a 0.4% NaCl diet (112.6±1.4 vs 110.7±2.4 mmHg, n=4-5). After 3 weeks of high salt, however, there was a significantly greater increase in MAP in the p67/SS rats (176.1±4.7 mmHg) compared to the p67/p67 control rats (147.9±8.4 mmHg), which was accompanied by a significant increase in albuminuria (230.3±13.9 vs 153.1±14.9 mg/day, p67/SS vs p67/p67, n=5-6). Interestingly, upon analysis of the immune cells in the kidney, no differences were detected in the number of CD45+ total leukocytes, CD11b/c+ monocytes/macrophages, CD3+ T cells, and CD45R+ B cells between the p67/SS and p67/p67 (n=4-5), indicating that the recruitment of immune cells into the kidney during hypertension and renal damage is independent of NOX2-derived ROS production from the renal parenchyma. The respiratory burst response to phorbol 12-myristate 13-acetate stimulus (135 μM) in peritoneal macrophages isolated from p67/SS was 4.0-fold greater compared to nonresponsive p67/p67 cells, and was completely inhibited by superoxide dismutase, demonstrating that the reconstituted bone marrow cells in the p67/SS are capable of producing superoxide. In summary, the production of NOX2-derived ROS from immune cells alone is sufficient for the development of Dahl SS hypertension and renal damage.

Hypertension is a worldwide epidemic and global health concern as it is a major risk factor for the development of cardiovascular diseases. A relationship between the immune system and its contributing role to the pathogenesis of hypertension has been long established, but substantial advancements within the last few years have dissected specific causal molecular mechanisms. This review will briefly examine these recent studies exploring the involvement of either innate or adaptive immunity pathways. Such pathways to be discussed include innate immunity factors such as antigen presenting cells and pattern recognition receptors, adaptive immune elements including T and B lymphocytes, and more specifically, the emerging role of T regulatory cells, as well as the potential of cytokines and chemokines to serve as signaling messengers connecting innate and adaptive immunity. Together, we summarize these studies to provide new perspective for what will hopefully lead to more targeted approaches to manipulate the immune system as hypertensive therapy.

Epidemiological data indicates that acute kidney injury (AKI) is an independent risk factor for the development of hypertension and chronic kidney disease in patients. Previous studies demonstrated that rats develop sodium-dependent hypertension and kidney damage following experimental AKI induced by a renal ischemia-reperfusion (IR) insult; furthermore, these high salt deleterious effects could be blunted by administration of immunosuppressive agents. The present study was performed on Dahl SS (SS) rats and SS rats with a null mutation in the CD247 gene (SS-CD247) leading to depletion of T-lymphocytes in order to specifically examine the role of T cells in this response (n=5-6 rats/group). As assessed by serum creatinine (SCr) levels, no difference was observed in the initial response to IR injury between SS and SS-CD247: SCr increased from 0.44±0.03 to 2.16±0.32 mg/dl in SS rats 24 hours after an initial 30 minute period of renal ischemia and returned to control levels after 8 days of recovery. Moreover, no differences were noted in mean arterial pressure (MAP) or albumin excretion rate (UAlb) between SS and SS-CD247 after 43 days of recovery from IR injury while the rats were maintained on a low salt (0.4% NaCl) diet. When the rats were fed a 4.0% NaCl diet for two weeks, MAP and UAlb significantly increased in the sham SS to 178±9 mmHg and 189±25 mg/day, respectively; values significantly greater than observed in the sham SS-CD247 rats (148±2 mmHg and 87±17 mg/day). As expected, the SS rats recovered from IR injury demonstrated an exaggerated increase in MAP (peaking at 183±2 mmHg) and UAlb (275±54 mg/day) in response to high salt. There was no difference in the number of total CD3+ lymphocytes in the kidneys of IR and sham SS after high salt, though the ratio of CD4+/CD8+ T cells was increased in the IR group. Compared to sham CD247, an exaggerated elevation of MAP (157±9 mmHg) and UAlb (210±32 mg/day) was also observed in the SS-CD247 rats recovered from IR injury, demonstrating enhanced responsiveness following IR injury in animals lacking T cells. These data indicate that T lymphocytes amplify salt-sensitive hypertension and renal damage, but other mechanisms also mediate the salt-sensitive hypertension and renal damage that occurs in animals recovered from IR injury.