Microbiota-associated mechanisms underlying sexual dimorphism in hypertension.

Consistent research over the last 20 years has shown that there are clear sex differences in the pathogenesis of hypertension, the leading risk factor for the development of cardiovascular diseases. More recently, there is evidence in both humans and experimental animal models that causally implicates the gut microbiota in hypertension. It therefore follows that sex differences in the gut microbiota may mediate the extent of disease between sexes. This new field is rapidly changing and advancing, and the purpose of this review is to cover the most up-to-date evidence regarding the sexual dimorphism of the gut microbiota and its potential influence on the differential manifestation of hypertension in males versus females. Emphasis will be placed on the mechanisms thought to contribute to these sex differences in both the gut microbiota and hypertension, including sex steroid hormones, gut-derived metabolites, the immune system, and pregnancy.

Dietary Protein, Chronic Salt-Sensitive Hypertension, and Kidney Damage.

It has been estimated that over a fifth of deaths worldwide can be attributed to dietary risk factors. A particularly serious condition is salt-sensitive (SS) hypertension and renal damage, participants of which demonstrate increased morbidity and mortality. Notably, a large amount of evidence from humans and animals has demonstrated that other components of the diet can also modulate hypertension and associated end-organ damage. Evidence presented in this review provides support for the view that immunity and inflammation serve to amplify the development of SS hypertension and leads to malignant disease accompanied by tissue damage. Interestingly, SS hypertension is modulated by changes in dietary protein intake, which also influences immune mechanisms. Together, the evidence presented in this review from animal and human studies indicates that changes in dietary protein source have profound effects on the gut microbiota, microbiota-derived metabolites, gene expression, immune cell activation, the production of cytokines and other factors, and the development of SS hypertension and kidney damage.

Functional NADPH oxidase 2 in T cells amplifies salt-sensitive hypertension and associated renal damage.

Infiltrating T cells in the kidney amplify salt-sensitive (SS) hypertension and renal damage, but the mechanisms are not known. Genetic deletion of T cells (SSCD247-/-) or of the p67phox subunit of NADPH oxidase 2 (NOX2; SSp67phox-/-) attenuates SS hypertension in the Dahl SS rat. We hypothesized that reactive oxygen species produced by NOX2 in T cells drive the SS phenotype and renal damage. T cells were reconstituted by adoptively transferring splenocytes (∼10 million) from the Dahl SS (SS→CD247) rat, the SSp67phox-/- rat (p67phox→CD247), or only PBS (PBS→CD247) into the SSCD247-/- rat on postnatal day 5. Animals were instrumented with radiotelemeters and studied at 8 wk of age. There were no detectable differences in mean arterial pressure (MAP) or albuminuria between groups when rats were maintained on a low-salt (0.4% NaCl) diet. After 21 days of high-salt diet (4.0% NaCl), MAP and albuminuria were significantly greater in SS→CD247 rats compared with p67phox→CD247 and PBS→CD247 rats. Interestingly, there was no difference between p67phox→CD247 and PBS→CD247 rats in albuminuria or MAP after 21 days. The lack of CD3+ cells in PBS→CD247 rats and the presence of CD3+ cells in rats that received the T cell transfer demonstrated the effectiveness of the adoptive transfer. No differences in the number of CD3+, CD4+, or CD8+ cells were observed in the kidneys of SS→CD247 and p67phox→CD247 rats. These results indicate that reactive oxygen species produced by NOX2 in T cells participates in the amplification of SS hypertension and renal damage.NEW & NOTEWORTHY Our current work used the adoptive transfer of T cells that lack functional NADPH oxidase 2 into a genetically T cell-deficient Dahl salt-sensitive (SS) rat model. The results demonstrated that reactive oxygen species produced by NADPH oxidase 2 in T cells participate in the amplification of SS hypertension and associated renal damage and identifies a potential mechanism that exacerbates the salt-sensitive phenotype.

Impact of bedding on Dahl salt-sensitive hypertension and renal damage.

Salt-sensitive hypertension, increases in blood pressure in response to increased salt intake, is associated with an increased risk of morbidity, mortality, and end-organ damage compared with salt-resistant hypertension. The Dahl salt-sensitive (SS) rat mimics the phenotypic characteristics observed in human hypertension when rats are challenged with a high-salt diet. Our previous work demonstrated that environmental factors, such as dietary protein, alter the severity of salt sensitivity in Dahl SS rats and should be an important consideration in experimental design. The present study investigated how the bedding on which animals were maintained (wood vs. corncob) could impact the SS phenotype in the Dahl SS rat. Animals that were maintained on corncob bedding exhibited a significant attenuation in blood pressure and renal end-organ damage in response to a high-salt diet compared with animals maintained on wood bedding. This attenuation was associated with an improvement in renal function and reduction in immune cell infiltration into the kidneys of Dahl SS rats maintained on corncob bedding. These results indicate that the type of bedding impacts the SS phenotype in the Dahl SS rat and that the bedding used in experiments can be a confounding factor to consider during data interpretation and experimental design.NEW & NOTEWORTHY Results from our present study demonstrate the profound effect of animal bedding on the severity of salt-sensitive hypertension, renal damage, and inflammation in Dahl salt-sensitive rats. This study highlights the important consideration that should be given to environmental factors, namely, the type of bedding in animal facilities, in experimental design and data interpretation.

Gut-Immune-Kidney Axis: Influence of Dietary Protein in Salt-Sensitive Hypertension.

Humans with salt-sensitive hypertension demonstrate increased morbidity, increased mortality, and renal end-organ damage when compared with normotensive subjects or those with salt-resistant hypertension. Substantial evidence from humans and animals has also demonstrated the role of dietary components other than salt to modulate hypertension. Evidence presented in this review provides support for the view that immunity and inflammation serve to amplify the development of salt-sensitive hypertension and leads to malignant disease accompanied by end-organ damage. Interestingly, salt-sensitive disease is modulated by changes in dietary protein intake, which also influences immune mechanisms. Together, the evidence presented in this review from animal and human studies indicates that changes in dietary protein source have profound effects on the gut microbiota, microbiota-derived metabolites, DNA methylation, gene expression, immune cell activation, the production of cytokines and other factors, and the development of salt-sensitive hypertension and related disease phenotypes.

T Cell Immunometabolism and Redox Signaling in Hypertension.

PURPOSE OF REVIEW: In this article, we summarize the current literature supporting metabolic and redox signaling pathways as important mechanisms underlying T cell activation in the context of hypertension. RECENT FINDINGS: T cell immunometabolism undergoes dramatic remodeling in order to meet the demands of T cell activation, differentiation, and proliferation. Recent evidence demonstrates that the T cell oxidation-reduction (redox) system also undergoes significant changes upon activation, which can itself modulate metabolic processes and T cell function. Dysregulation of these signaling pathways can lead to aberrant T cell activation and inappropriate ROS production, both of which are linked to pathological conditions like hypertension. While the contribution of T cells to the progression of hypertension has been thoroughly investigated, how T cell metabolism and redox signaling changes, both separately and together, is an area of study that remains largely untouched. This review presents evidence from our own laboratory as well as others to highlight the importance of these two mechanisms in the study of hypertension.

Dietary protein source contributes to the risk of developing maternal syndrome in the Dahl salt-sensitive rat.

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.

Dietary influences on the Dahl SS rat gut microbiota and its effects on salt-sensitive hypertension and renal damage.

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.

Contribution of Th17 cells to tissue injury in hypertension.

PURPOSE OF REVIEW: Hypertension has been demonstrated to be a chief contributor to morbidity and mortality throughout the world. Although the cause of hypertension is multifactorial, emerging evidence, obtained in experimental studies, as well as observational studies in humans, points to the role of inflammation and immunity. Many aspects of immune function have now been implicated in hypertension and end-organ injury; this review will focus upon the recently-described role of Th17 cells in this pathophysiological response. RECENT FINDINGS: Studies in animal models and human genetic studies point to a role in the adaptive immune system as playing a contributory role in hypertension and renal tissue damage. Th17 cells, which produce the cytokine IL17, are strongly pro-inflammatory cells, which may contribute to tissue damage if expressed in chronic disease conditions. The activity of these cells may be enhanced by physiological factors associated with hypertension such as dietary salt or Ang II. This activity may culminate in the increased sodium retaining activity and exacerbation of inflammation and renal fibrosis via multiple cellular mechanisms. SUMMARY: Th17 cells are a distinct component of the adaptive immune system that may strongly enhance pathways leading to increased sodium reabsorption, elevated vascular tone and end-organ damage. Moreover, this pathway may lend itself towards specific targeting for treatment of kidney disease and hypertension.

Amplification of Salt-Sensitive Hypertension and Kidney Damage by Immune Mechanisms.

Humans with salt-sensitive (SS) hypertension demonstrate increased morbidity, increased mortality, and renal end-organ damage when compared with normotensive subjects or those with salt-resistant hypertension. Increasing evidence indicates that immune mechanisms play an important role in the full development of SS hypertension and associated renal damage. Recent experimental advances and studies in animal models have permitted a greater understanding of the mechanisms of activation and action of immunity in this disease process. Evidence favors a role of both innate and adaptive immune mechanisms that are triggered by initial, immune-independent alterations in blood pressure, sympathetic activity, or tissue damage. Activation of immunity, which can be enhanced by a high-salt intake or by alterations in other components of the diet, leads to the release of cytokines, free radicals, or other factors that amplify renal damage and hypertension and mediate malignant disease.

Sexual Dimorphic Role of CD14 (Cluster of Differentiation 14) in Salt-Sensitive Hypertension and Renal Injury.

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.

Renal Perfusion Pressure Determines Infiltration of Leukocytes in the Kidney of Rats With Angiotensin II-Induced Hypertension.

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.

Influences of environmental factors during preeclampsia.

Preeclampsia is a pregnancy-specific disorder that impacts 5-8% of pregnancies and has long-term cardiovascular and metabolic implications for both mother and fetus. The mechanisms are unclear; however, it is believed that preeclampsia is characterized by abnormal vascularization during placentation resulting in the manifestation of clinical signs such as hypertension, proteinuria, and endothelial dysfunction. Although there is no current cure to alleviate the clinical signs, an emerging area of interest in the field is the influence of environmental factors including diet on the risk of preeclampsia. Because preeclampsia has serious cardiovascular implications to both the mother and fetus and most antihypertensive medications are contraindicated in pregnancy, it is important to investigate other potential therapeutic options such as dietary manipulation. The emerging field of nutrigenomics links diet with the gene expression of known pathways such as oxidative stress and inflammation via microbiome-mediated metabolites and could serve as one potential avenue of therapeutic targets for preeclampsia. Although the exact role of nutrition in the pathogenesis of preeclampsia is unknown, this review will focus on known pathways involved in the development of preeclampsia and how dietary intake modulates the microbiome, oxidative stress, and inflammation with an emphasis on nutrigenomics as a potential avenue of further investigation to better understand this pathology.

CCL2 mediates early renal leukocyte infiltration during salt-sensitive hypertension.

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-1 in 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 × 106 CD45+ 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.

Dietary Protein: Mechanisms Influencing Hypertension and Renal Disease.

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. 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.

Epigenetic Modifications in T Cells: The Role of DNA Methylation in Salt-Sensitive Hypertension.

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.

NOX2-derived reactive oxygen species in immune cells exacerbates salt-sensitive hypertension.

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.

Dietary Effects on Dahl Salt-Sensitive Hypertension, Renal Damage, and the T Lymphocyte Transcriptome.

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.

Salt-sensitive increase in macrophages in the kidneys of Dahl SS rats.

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.