TLR7 activation by imiquimod worsens glycemic control in female FVB/N mice consuming a high-fat diet.

Toll-like receptor-7 (TLR7) activation promotes autoimmunity, and metabolic syndrome (MetS) is a common comorbidity in patients with autoimmune disease. We previously demonstrated hyperinsulinemia in TLR7 agonist imiquimod (IMQ)-treated, high-fat diet (HFD)-fed female C57BL/6 mice. Since mouse strains differ in susceptibility to MetS and target organ damage, this study investigated whether 12 weeks of exposure to HFD and IMQ promoted MetS, autoimmunity, and target organ damage in female FVB/N mice. Supporting early-stage autoimmunity, spleen-to-tibia ratio, and anti-nuclear antibodies (ANA) were significantly increased by IMQ. No significant effect of IMQ on urinary albumin excretion or left ventricular hypertrophy was observed. HFD increased liver-to-tibia ratio, which was further exacerbated by IMQ. HFD increased fasting blood glucose levels at the end of 12 weeks, but there was no significant effect of IMQ treatment on fasting blood glucose levels at 6 or 12 weeks of treatment. However, oral glucose tolerance testing at 12 weeks revealed impaired glucose tolerance in HFD-fed mice compared to control diet mice together with IMQ treatment exacerbating the impairment. Accordingly, these data suggest TLR7 activation also exacerbates HFD-induced dysregulation of glucose handling FVB/N mice, supporting the possibility that endogenous TLR7 activation may contribute to dysglycemia in patients with autoimmune disease.

Cardiac Spinal Afferent Denervation Attenuates Renal Dysfunction in Rats With Cardiorenal Syndrome Type 2.

Cardiorenal syndrome type 2 (CRS2) is defined as a chronic cardiovascular disease, usually chronic heart failure (CHF), resulting in chronic kidney disease. We hypothesized that the cardiac spinal afferent reflex (CSAR) plays a critical role in the development of CRS2. Our data suggest that cardiac afferent ablation by resiniferatoxin not only improves cardiac function but also benefits the kidneys and increases long-term survival in the myocardial infarction model of CHF. We also found that renal denervation has a similar reno-protective effect in CHF rats. We believe this novel work contributes to the development of a unique neuromodulation therapy to treat CHF patients.

Development of High Fat Diet-Induced Hyperinsulinemia in Mice Is Enhanced by Co-treatment With a TLR7 Agonist.

Metabolic syndrome (MetS) is common in Systemic Lupus Erythematosus (SLE) patients and is associated with increased cardio-renal risk. Toll-like receptor 7 (TLR7) stimulation promotes the development of SLE through mechanisms including activating type I Interferon (IFN) and autoreactive B cells. The current study tested whether combined TLR7 agonist treatment and exposure to a high fat, high sucrose "Western diet" intervention affects the early-stage development of SLE or MetS features. Female C57BL/6 mice were untreated or treated with the TLR7 agonist imiquimod (IMQ) and fed a high-fat diet (HFD; fat 42% kcal, sucrose 34% kcal) or control diet (fat 12.6% kcal, sucrose 34% kcal) for 6 weeks. Supporting early-stage induction of autoimmunity, spleen weights were significantly increased and anti-nuclear antibody (ANA) positivity was detected in IMQ-treated mice. Increased body weight, gonadal fat pad mass, and plasma leptin levels were observed between HFD and control animals for both IMQ and untreated mice. However, the increase in these parameters with HFD was slightly but significantly diminished in IMQ-treated mice. Both the HFD and IMQ treatments significantly increased fasting blood glucose levels. Notably, IMQ treatment affected fasting insulin concentrations in a diet-dependent manner, with hyperinsulinemia observed in IMQ-HFD treated mice. Together, this indicates that the IMQ model of SLE is associated with metabolic alterations, impaired glycemic control, and hyperinsulinemia under HFD conditions. This model may be helpful in further investigating the relationship between MetS and SLE, and supports a role of TLR7 signaling in promoting or accelerating the development of dysglycemia and hyperinsulinemia.

Modified chitosan for effective renal delivery of siRNA to treat acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden decrease in renal function and impacts growing number of people worldwide. RNA interference (RNAi) showed potential to treat diseases with no or limited conventional therapies, including AKI. Suitable carriers are needed to protect and selectively deliver RNAi to target cells to fully explore this therapeutic modality. Here, we report on the synthesis of chitosan modified with α-cyclam-p-toluic acid (C-CS) as a novel siRNA carrier for targeted delivery to injured kidneys. We demonstrate that conjugation of the α-cyclam-p-toluic acid to chitosan imparts the C-CS polymer with targeting and antagonistic properties to cells overexpressing chemokine receptor CXCR4. In contrast, the parent α-cyclam-p-toluic acid showed no such properties. Self-assembled C-CS/siRNA nanoparticles rapidly accumulate in the injured kidneys and show long retention in renal tubules. Apoptosis and metabolic and inflammatory pathways induced by p53 are important pathological mechanisms in the development of AKI. Nanoparticles with siRNA against p53 (sip53) were formulated and intravenously injected for attenuation of IRI-AKI. Due to the favorable accumulation in injured kidneys, the treatment with C-CS/sip53 decreased renal injury, extent of renal apoptosis, macrophage and neutrophil infiltration, and improved renal function. Overall, our study suggests that C-CS/siRNA nanoparticles have the potential to effectively accumulate and deliver therapeutic siRNAs to injured kidneys through CXCR4 binding, providing a novel way for AKI therapy.

Autoimmune-mediated renal disease and hypertension.

Hypertension is a major risk factor for cardiovascular disease, chronic kidney disease (CKD), and mortality. Troublingly, hypertension is highly prevalent in patients with autoimmune renal disease and hastens renal functional decline. Although progress has been made over the past two decades in understanding the inflammatory contributions to essential hypertension more broadly, the mechanisms active in autoimmune-mediated renal diseases remain grossly understudied. This Review provides an overview of the pathogenesis of each of the major autoimmune diseases affecting the kidney that are associated with hypertension, and describes the current state of knowledge regarding hypertension in these diseases and their management. Specifically, discussion focuses on Systemic Lupus Erythematosus (SLE) and Lupus Nephritis (LN), Immunoglobulin A (IgA) Nephropathy, Idiopathic Membranous Nephropathy (IMN), Anti-Neutrophil Cytoplasmic Antibody (ANCA)-associated glomerulonephritis, and Thrombotic Thrombocytopenic Purpura (TTP). A summary of disease-specific animal models found to exhibit hypertension is also included to highlight opportunities for much needed further investigation of underlying mechanisms and novel therapeutic approaches.

Evidence of Renal Iron Accumulation in a Male Mouse Model of Lupus.

Lupus nephritis represents a common and serious complication of the autoimmune disease Systemic Lupus Erythematosus (SLE). Clinical studies suggest that several proteins related to iron metabolism, including transferrin, serve as urinary biomarkers of lupus nephritis. We previously reported that in female NZBWF1 mice, a commonly used mouse model of SLE with a female sex bias, increased urinary transferrin excretion and renal iron accumulation occur around the onset of albuminuria. The current study investigated whether similar findings occur in male mice of a different mouse model of SLE, the MRL/lpr mouse. Two different cohorts were studied: MRL/lpr mice at an early, pre-albuminuric age (8 weeks), and after developing albuminuria (>100 mg/dL, confirmed by ELISA); age-matched MRL/MpJ control strain mice served for comparison. Urinary transferrin excretion was dramatically increased in the older, albuminuric MRL/lpr mice compared to the age-matched MRL/MpJ (P < 0.05), but there was no significant difference between strains at 8 weeks of age. Similarly, there were no significant differences between strains in renal cortical or outer medullary non-heme iron concentrations at 8 weeks. In the older, albuminuric MRL/lpr mice, renal cortical and outer medullary non-heme iron concentrations were significantly increased compared with age-matched MRL/MpJ mice, as was the expression of the iron storage protein ferritin (P < 0.01). Together, these data show that increased urinary transferrin excretion and renal tissue iron accumulation also occurs in albuminuric male MRL/lpr mice, suggesting that renal iron accumulation may be a feature of multiple mouse models of SLE.

Sodium 4-phenylbutyrate treatment protects against renal injury in NZBWF1 mice.

Systemic lupus erythematosus (SLE) is an autoimmune disease predominantly affecting women and often leading to lupus nephritis and kidney damage. Endoplasmic reticulum (ER) stress has been implicated in several forms of kidney disease, but whether ER stress contributes to renal injury in SLE is unknown. To investigate this, a small molecule chaperone, sodium 4-phenylbutyrate (4-PBA), was administered to the New Zealand Black x New Zealand White F1 hybrid (NZBWF1) mouse model of SLE. In a prevention study, treatment with 4-PBA from 20 weeks of age (prior to the development of renal injury) delayed the onset of albuminuria and significantly reduced additional indices of renal injury compared with vehicle-treated NZBWF1 mice at 36 weeks of age, including collagen deposition, tubular casts, renal cell apoptosis, and blood urea nitrogen (BUN) concentration. To test whether ER stress contributes to the progression of renal injury once albuminuria has developed, mice were monitored for the onset of albuminuria (3+ or ≥300 mg/dl by dipstick measurement of 24-h urine sample) and once established, were either killed (onset group), or underwent 4-PBA or vehicle treatment for 4 weeks. Treatment with 4-PBA blocked the worsening of glomerular injury, reduced the number of dilated or cast-filled tubules, and reduced the number of apoptotic cells compared with vehicle-treated mice. BUN and left ventricle to bodyweight ratio (LV:BW) were also reduced by 4-PBA treatment. Renal expression of the endogenous chaperones, protein disulphide isomerase (PDI), and 78 kDa glucose-regulated protein (GRP78, also known as binding Ig protein (BiP)), were increased in 4-PBA-treated mice. Together, these results suggest a therapeutic potential for agents like 4-PBA in combating renal injury in SLE.

ETA receptor activation contributes to T cell accumulation in the kidney following ischemia-reperfusion injury.

Renal ischemia-reperfusion (IR) injury and acute kidney injury (AKI) increase the risk of developing hypertension, with T cells suspected as a possible mechanistic link. Endothelin promotes renal T cell infiltration in several diseases, predominantly via the ETA receptor, but its contribution to renal T cell infiltration following renal IR injury is poorly understood. To test whether ETA receptor activation promotes T cell infiltration of the kidney following IR injury, male C57BL/6 mice were treated with the ETA receptor antagonist ABT-627 or vehicle, commencing 2 days prior to unilateral renal IR injury. Mice were sacrificed at 24 h or 10 days post-IR for assessment of the initial renal injury and subsequent infiltration of T cells. Vehicle and ABT-627-treated mice displayed significant upregulation of endothelin-1 (ET-1) in the IR compared to contralateral kidney at both 24 h and 10 days post-IR (P < 0.001). Renal CD3+ T cell numbers were increased in the IR compared to contralateral kidneys at 10 days, but ABT-627-treated mice displayed a 35% reduction in this effect in the outer medulla (P < 0.05 vs. vehicle) and a nonsignificant 23% reduction in the cortex compared to vehicle-treated mice. Whether specific T cell subsets were affected awaits confirmation by flow cytometry, but outer medullary expression of the T helper 17 transcription factor RORγt was reduced by ABT-627 (P = 0.06). These data indicate that ET-1 acting via the ETA receptor contributes to renal T cell infiltration post-IR injury. This may have important implications for immune system-mediated long-term consequences of AKI, an area which awaits further investigation.

Renal iron accumulation occurs in lupus nephritis and iron chelation delays the onset of albuminuria.

Proteins involved in iron homeostasis have been identified as biomarkers for lupus nephritis, a serious complication of systemic lupus erythematosus (SLE). We tested the hypothesis that renal iron accumulation occurs and contributes to renal injury in SLE. Renal non-heme iron levels were increased in the (New Zealand Black x New Zealand White) F1 (NZB/W) mouse model of lupus nephritis compared with healthy New Zealand White (NZW) mice in an age- and strain-dependent manner. Biodistribution studies revealed increased transferrin-bound iron accumulation in the kidneys of albuminuric NZB/W mice, but no difference in the accumulation of non-transferrin bound iron or ferritin. Transferrin excretion was significantly increased in albuminuric NZB/W mice, indicating enhanced tubular exposure and potential for enhanced tubular uptake following filtration. Expression of transferrin receptor and 24p3R were reduced in tubules from NZB/W compared to NZW mice, while ferroportin expression was unchanged and ferritin expression increased, consistent with increased iron accumulation and compensatory downregulation of uptake pathways. Treatment of NZB/W mice with the iron chelator deferiprone significantly delayed the onset of albuminuria and reduced blood urea nitrogen concentrations. Together, these findings suggest that pathological changes in renal iron homeostasis occurs in lupus nephritis, contributing to the development of kidney injury.

EHD4 is a novel regulator of urinary water homeostasis.

The Eps15-homology domain-containing (EHD) protein family comprises 4 members that regulate endocytic recycling. Although the kidney expresses all 4 EHD proteins, their physiologic roles are largely unknown. This study focused on EHD4, which we found to be expressed differentially across nephron segments with the highest expression in the inner medullary collecting duct. Under baseline conditions, Ehd4-/- [EHD4-knockout (KO)] mice on a C57Bl/6 background excreted a higher volume of more dilute urine than control C57Bl/6 wild-type (WT) mice while maintaining a similar plasma osmolality. Urine excretion after an acute intraperitoneal water load was significantly increased in EHD4-KO mice compared to WT mice, and although EHD4-KO mice concentrated their urine during 24-h water restriction, urinary osmolality remained significantly lower than in WT mice, suggesting that EHD4 plays a role in renal water handling. Total aquaporin 2 (AQP2) and phospho-S256-AQP2 (pAQP2) protein expression in the inner medulla was similar in the two groups in baseline conditions. However, localization of both AQP2 and pAQP2 in the renal inner medullary principal cells appeared more dispersed, and the intensity of apical membrane staining for AQP2 was reduced significantly (by ∼20%) in EHD4-KO mice compared to WT mice in baseline conditions, suggesting an important role of EHD4 in trafficking of AQP2. Together, these data indicate that EHD4 play important roles in the regulation of water homeostasis.-Rahman, S. S., Moffitt, A. E. J., Trease, A. J., Foster, K. W., Storck, M. D., Band, H., Boesen, E. I. EHD4 is a novel regulator of urinary water homeostasis.

Interleukin-1ß as a driver of renal NGAL production.

Neutrophil gelatinase-associated lipocalin (NGAL) is increasingly regarded as a biomarker of acute kidney injury, or kidney injury in general, but the stimuli responsible for its production are incompletely understood. This study tested the relationship between the pro-inflammatory cytokine interleukin-1ß (IL-1ß) and both circulating and renal NGAL, using chronic subcutaneous infusion of IL-1ß in mice and tissue culture of renal cell lines. Following a 14-day subcutaneous infusion of vehicle or IL-1ß (10ng/h) in male C57Bl/6 mice, a striking positive correlation (r2=0.94; P<0.01) was observed between plasma IL-1ß and NGAL concentrations. NGAL was markedly increased in the kidneys of IL-1ß-infused mice compared with vehicle-treated mice, both at the protein and mRNA level, indicating increased local as well as systemic production of NGAL. Immunohistochemical staining revealed prominent increases of NGAL in the proximal tubular epithelium of IL-1ß infused mice. These effects occurred in the absence of overt renal injury, with plasma creatinine concentration not significantly different between groups. Further showing that IL-1ß has a direct effect on NGAL production by tubular epithelial cells, exposure of a proximal tubular cell line (HK-2 cells) and a cortical collecting duct principal cell line (mpkCCD cells) to IL-1ß for 24h produced a significant increase of NGAL mRNA levels (>30-fold). These data indicate IL-1ß serves as a powerful stimulus for renal production of NGAL.

Lack of an apparent role for endothelin-1 in the prolonged reduction in renal perfusion following severe unilateral ischemia-reperfusion injury in the mouse.

Therapeutic approaches to block the progression from acute kidney injury to chronic kidney disease are currently lacking. Endothelin-1 (ET-1) is a powerful vasoconstrictor, induced by hypoxia, and previously implicated in renal ischemia-reperfusion (IR) injury. This study tested the hypothesis that blunting the vascular influence of ET-1, either through endothelin ETA receptor blockade (ABT-627) or vascular endothelial cell deletion of ET-1 (VEET KO), would improve recovery of renal perfusion and repair of injury following a severe ischemic insult in mice (45 min unilateral renal ischemia). Male C57Bl/6 mice receiving vehicle or ABT-627 commencing 2 days prior to surgery, and VEET KO mice and wild-type littermates (WT) underwent 45 min unilateral renal IR surgery followed by 28 days recovery. Renal blood velocity was measured by pulsed-wave Doppler ultrasound before and after surgery. Renal blood velocity was not significantly different between pairs of groups before surgery. Unilateral IR induced a marked reduction in renal blood velocity of the IR kidney at 24 h postsurgery in all groups, which partially recovered but remained below baseline at 28 days post-IR. Despite the lack of effect on renal blood velocity, ETA receptor blockade significantly attenuated the atrophy of the post-IR kidney, whereas this was not significantly affected by lack of endothelial ET-1 expression. These data suggest that although blockade of the ETA receptor is mildly beneficial in preserving renal mass following a severe ischemic insult, this protective effect does not appear to involve improved recovery of renal perfusion.

Outside the mainstream: novel collecting duct proteins regulating water balance.

Body water balance is critical to survival and, therefore, very tightly regulated by the hypothalamus and kidney. A key mechanism involved in this process, the arginine vasopressin-mediated phosphorylation and apical membrane insertion of aquaporin 2 in the collecting duct, has been extensively studied; however, with the increased availability of conditional knockout animals, several novel collecting duct proteins have recently been implicated in water homeostasis. In this Mini-Review, we briefly discuss these novel proteins and their roles in the regulation of water homeostasis.

Estradiol regulates AQP2 expression in the collecting duct: a novel inhibitory role for estrogen receptor α.

While there is evidence that sex hormones influence multiple systems involved in salt and water homeostasis, the question of whether sex hormones regulate aquaporin-2 (AQP2) and thus water handling by the collecting duct has been largely ignored. Accordingly, the present study investigated AQP2 expression, localization and renal water handling in intact and ovariectomized (OVX) female rats, with and without estradiol or progesterone replacement. OVX resulted in a significant increase in urine osmolality and increase in p256-AQP2 in the renal cortex at 7 days post-OVX, as well as induced body weight changes. Relative to OVX alone, estradiol repletion produced a significant increase in urine output, normalized urinary osmolality and reduced both total AQP2 (protein and mRNA) and p256-AQP2 expression, whereas progesterone repletion had little effect. Direct effects of estradiol on AQP2 mRNA and protein levels were further tested in vitro using the mpkCCD principal cell line. Estradiol treatment of mpkCCD cells reduced AQP2 at both the mRNA and protein level in the absence of deamino-8-d-AVP (dDAVP) and significantly blunted the dDAVP-induced increase in AQP2 at the protein level only. We determined that mpkCCD and native mouse collecting ducts express both estrogen receptor (ER)α and ERß and that female mice lacking ERα displayed significant increases in AQP2 protein compared with wild-type littermates, implicating ERα in mediating the inhibitory effect of estradiol on AQP2 expression. These findings suggest that changes in estradiol levels, such as during menopause or following reproductive surgeries, may contribute to dysregulation of water homeostasis in women.

Endothelin receptors, renal effects and blood pressure.

The endothelin system has emerged as a key player in the renal control of salt and water homeostasis, exerting profound effects on both the renal vasculature and tubular epithelial cells. Recent advances include new actions of endothelins in the glomerulus, an emerging role for the ETA receptor in chronic kidney disease (CKD) progression and in tubular function, and a more detailed understanding of the tubular response to high salt intake. A large body of evidence also implicates dysfunction of the endothelin system in hypertension, particularly salt-sensitive hypertension, although recent data suggests important sex-differences may exist. Finally, clinical trials indicate that antagonists of endothelin receptors hold great promise in treating resistant hypertension and proteinuric renal disease.

Indoleamine 2,3-dioxygenase inhibition alters the non-coding RNA transcriptome following renal ischemia-reperfusion injury.

BACKGROUND: Indoleamine 2,3 dioxygenase (IDO) degrades the essential amino acid tryptophan and has been shown to minimize rejection in animal models of renal transplantation. Ischemia-reperfusion injury (IRI) is unavoidable in renal transplantation and correlates with shorter graft survival times. Despite its favorable effects on rejection, there is evidence that IDO may facilitate renal IRI. Differentiating the negative impact of IDO on IRI from its pro-tolerant effects in allograft rejection is of clinical relevance. In these studies we hypothesized that constitutive IDO activity may influence renal genes associated with recovery from IRI, and that IDO inhibition may unmask these effects. METHODS: We examined the renal transcriptome in a rat model of IRI with and without IDO inhibition with 1-methyl-d-tryptophan (1-MT), and assessed for alterations in the gene expression signature. RESULTS: These studies demonstrated that during recovery from renal IRI, pre-treatment with 1-MT alleviated alterations in 105 coding sequences associated with IRI, and in turn triggered new changes in 66 non-coding transcripts, the majority of which were represented by small nucleolar RNA. CONCLUSION: These results suggest a biologic role for non-coding, IDO-dependent genes in regulating the early response to IRI.

Renal collecting duct NOS1 maintains fluid-electrolyte homeostasis and blood pressure.

Nitric oxide is a pronatriuretic and prodiuretic factor. The highest renal NO synthase (NOS) activity is found in the inner medullary collecting duct. The collecting duct (CD) is the site of daily fine-tune regulation of sodium balance, and led us to hypothesize that a CD-specific deletion of NOS1 would result in an impaired ability to excrete a sodium load leading to a salt-sensitive blood pressure phenotype. We bred AQP2-CRE mice with NOS1 floxed mice to produce flox control and CD-specific NOS1 knockout (CDNOS1KO) littermates. CDs from CDNOS1KO mice produced 75% less nitrite, and urinary nitrite+nitrate (NOx) excretion was significantly blunted in the knockout genotype. When challenged with high dietary sodium, CDNOS1KO mice showed significantly reduced urine output, sodium, chloride, and NOx excretion, and increased mean arterial pressure relative to flox control mice. In humans, urinary NOx is a newly identified biomarker for the progression of hypertension. These findings reveal that NOS1 in the CD is critical in the regulation of fluid-electrolyte balance, and this new genetic model of CD NOS1 gene deletion will be a valuable tool to study salt-dependent blood pressure mechanisms.

Use of ultrasound to assess renal reperfusion and P-selectin expression following unilateral renal ischemia.

Renal ischemia-reperfusion injury is a major cause of acute kidney injury that carries a high mortality rate and increases the risk of later development of hypertension and chronic kidney disease. Although mouse models have contributed much to our understanding of the mechanisms involved, studying aspects of the injury process in vivo remains technically challenging. This study validates the use of noninvasive ultrasound imaging to assess both renal perfusion and vascular adhesion molecule expression following 1-h unilateral renal ischemia in male and female mice. Pulsed-wave Doppler measurements of renal arterial blood velocity revealed renal perfusion recoveries of 56 ± 9% in male and 69 ± 10% in female mice 1 h after the commencing of reperfusion, which is similar to what we have previously published using conventional invasive methodology. At 24 h postischemia, renal perfusion was 40 ± 8% in male and 46 ± 7% in female mice, representing a further significant reduction of perfusion (P(Time) < 0.001). Using ultrasound imaging of a P-selectin-targeted contrast agent, a significant increase in vascular P-selectin protein expression was observed after 1-h reperfusion in the cortex of the postischemic compared with contralateral kidney in both male and female mice (18 ± 5 vs. 3 ± 3 intensity units in male and 30 ± 6 vs. 0 ± 4 in female mice, P(Ischemia) < 0.01). An approximately sixfold increase in P-selectin mRNA was observed ex vivo in the renal vasculature of male and female mice at this time point (P < 0.01). In conclusion, ultrasound represents an effective and noninvasive method for the measurement of both renal perfusion and vascular adhesion molecule expression in mice.

ETA activation mediates angiotensin II-induced infiltration of renal cortical T cells.

T cells and endothelin (ET-1) both contribute to angiotensin II (AngII)-dependent hypertension. To determine whether ET-1, via the ET(A) receptor, facilitates T cell infiltration in the kidney during AngII-dependent hypertension, we measured T cell infiltration in response to four different treatments: saline, AngII infusion, AngII infusion with an ET(A) receptor antagonist, or AngII infusion with triple-antihypertensive therapy. After 14 days, AngII increased both BP and the numbers of CD3(+) and proliferating cells in the kidney. Mice treated concomitantly with the ET(A) receptor antagonist had lower BP and fewer CD3(+) and proliferating cells in the renal cortex. Mice treated with triple therapy had similar reductions in BP but no change in renal cortical CD3(+) cells compared with kidneys from AngII-infused hypertensive mice. In the outer medulla, both the ET(A) receptor antagonist and triple therapy reduced the number of CD3(+) cells and macrophages. Taken together, these data suggest that ET(A) receptor activation in AngII-mediated hypertension increases CD3(+) cells and proliferation in the renal cortex independent of changes in BP, but changes in the number of inflammatory cells in the renal medulla are BP dependent.