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Aim: Among multi-ethnic Asians, type 2 diabetes (T2D) clustered in three subtypes; mild obesity-related diabetes (MOD), mild age-related diabetes with insulin insufficiency (MARD-II) and severe insulin-resistant diabetes with relative insulin insufficiency (SIRD-RII) had differential cardio-renal complication risk. We assessed the proteomic profiles to identify subtype specific biomarkers and its association with diabetes complications., Methods: 1448 plasma proteins at baseline were measured and compared across the T2D subtypes. Multivariable cox regression was used to assess associations between significant proteomics features and cardio-renal complications., Results: Among 645 T2D participants (SIRD-RII [19%], MOD [45%], MARD-II [36%]), 295 proteins expression differed significantly across the groups. These proteins were enriched in cell adhesion, neurogenesis and inflammatory response processes. In SIRD-RII group, ADH4, ACY1, THOP1, IGFBP2, NEFL, ENTPD2, CALB1, HAO1, CTSV, ITGAV, SCLY, EDA2R, ERBB2 proteins significantly associated with progressive CKD and LILRA5 protein with incident heart failure (HF). In MOD group, TAFA5, RSPO3, EDA2R proteins significantly associated with incident HF. In MARD-II group, FABP4 protein significantly associated with progressive CKD and PTPRN2 protein with major adverse cardiovascular events. Genetically determined NEFL and CALB1 were associated with kidney function decline., Conclusions: Each T2D subtype has unique proteomics signature and association with clinical outcomes and underlying mechanisms., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The macula densa (MD) is a distinct cluster of approximately 20 specialized kidney epithelial cells that constitute a key component of the juxtaglomerular apparatus. Unlike other renal tubular epithelial cell populations with functions relating to reclamation or secretion of electrolytes and solutes, the MD acts as a cell sensor, exerting homeostatic actions in response to sodium and chloride changes within the tubular fluid. Electrolyte flux through apical sodium transporters in MD cells triggers release of paracrine mediators, affecting blood pressure and glomerular hemodynamics. In this issue of the JCI, Gyarmati and authors explored a program of MD that resulted in activation of regeneration pathways. Notably, regeneration was triggered by feeding mice a low-salt diet. Furthermore, the MD cells showed neuron-like properties that may contribute to their regulation of glomerular structure and function. These findings suggest that dietary sodium restriction and/or targeting MD signaling might attenuate glomerular injury.

Renin was discovered more than a century ago. Since then, the functions of the renin-angiotensin system in the kidney have been the focus of intensive research revealing its importance in regulation of renal physiology and in the pathogenesis of heart, vascular, and kidney diseases. Inhibitors of renin-angiotensin system components are now foundational therapies for a range of kidney and cardiovascular diseases from hypertension to heart failure to diabetic nephropathy. Despite years of voluminous research, emerging studies continue to reveal new complexities of the regulation of the renin-angiotensin system within the kidney and identification of nonclassical components of the system like the prorenin receptor (PRR) and ACE2 (angiotensin-converting enzyme 2), with powerful renal effects that ultimately impact the broader cardiovascular system. With the emergence of a range of novel therapies for cardiovascular and kidney diseases, the importance of a detailed understanding of the renin-angiotensin system in the kidney will allow for the development of informed complementary approaches for combinations of treatments that will optimally promote health and longevity over the century ahead., Competing Interests: None.

Context: Metabolites in tricarboxylic acid (TCA) pathway have pleiotropic functions., Objective: To study the association between urine TCA cycle metabolites and the risk for chronic kidney disease (CKD) progression in individuals with type 2 diabetes., Design, Setting and Participants: A prospective study in a discovery (n = 1826) and a validation (n = 1235) cohort of type 2 diabetes in a regional hospital and a primary care facility., Exposure and Outcome: Urine lactate, pyruvate, citrate, alpha-ketoglutarate, succinate, fumarate and malate were measured by mass spectrometry. CKD progression was defined as a composite of sustained eGFR below 15 ml/min/1.73 m2 , dialysis, renal death or doubling of serum creatinine., Results: During a median of 9.2 (IQR 8.1-9.7) and 4.0 (3.2-5.1) years of follow-up, 213 and 107 renal events were identified. Cox regression suggested that urine lactate, fumarate and malate were associated with an increased risk (adjusted hazard ratio, aHR [95% CI] 1.63 [1.16-2.28], 1.82 [1.17-2.82] and 1.49 [1.05-2.11], per SD), while citrate was associated with a low risk (aHR 0.83 [0.72-0.96] per SD) for the renal outcome after adjustment for cardio-renal risk factors. These findings were reproducible in the validation cohort. Noteworthy, fumarate and citrate were independently associated with the renal outcome after additional adjustment for other metabolites., Conclusion: Urine fumarate and citrate predict the risk for progression to ESKD independent of clinical risk factors and other urine metabolites. These two metabolites in TCA cycle pathway may play important roles in the pathophysiological network underpinning progressive loss of kidney function in patients with type 2 diabetes., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

In lung, thromboxane A2 (TXA2) activates the TP receptor to induce proinflammatory and bronchoconstrictor effects. Thus, TP receptor antagonists and TXA2 synthase inhibitors have been tested as potential asthma therapeutics in humans. Th9 cells play key roles in asthma and regulate the lung immune response to allergens. Herein, we found that TXA2 reduces Th9 cell differentiation during allergic lung inflammation. Th9 cells were decreased approximately 2-fold and airway hyperresponsiveness was attenuated in lungs of allergic mice treated with TXA2. Naive CD4+ T cell differentiation to Th9 cells and IL-9 production were inhibited dose-dependently by TXA2 in vitro. TP receptor-deficient mice had an approximately 2-fold increase in numbers of Th9 cells in lungs in vivo after OVA exposure compared with wild-type mice. Naive CD4+ T cells from TP-deficient mice exhibited increased Th9 cell differentiation and IL-9 production in vitro compared with CD4+ T cells from wild-type mice. TXA2 also suppressed Th2 and enhanced Treg differentiation both in vitro and in vivo. Thus, in contrast to its acute, proinflammatory effects, TXA2 also has longer-lasting immunosuppressive effects that attenuate the Th9 differentiation that drives asthma progression. These findings may explain the paradoxical failure of anti-thromboxane therapies in the treatment of asthma.

Background: We aimed to explore the three-way interaction among age, gender, and kidney function on the risk of all-cause mortality and cardiovascular mortality among patients with type 2 diabetes (T2D)., Methods: In a retrospective cohort study, patients aged > 40 years with T2D with serum creatinine and urine albumin measured from 2013 to 2019 were included from a multi-institutional diabetes registry. The exposure was estimated glomerular filtration rate (eGFR), outcomes were all-cause mortality (primary outcome) and cardiovascular disease (CVD) mortality (secondary outcome). We applied multivariable cox proportional hazards regression analysis to compute the association between eGFR and mortality., Results: A total of 36,556 patients were followed for up to 6 years during which 2492 (6.82%) died from all causes, and 690 (1.9%) died from CVD. We observed a significant three-way interaction (p = 0.021) among age (younger, < 65; older, ≥65 years), gender and eGFR for the risk of all-cause mortality. Using age- and gender-specific eGFR of 90 ml/min/1.73m 2 as the reference point, the adjusted hazard rate (HR) (95% CI) for all-cause mortality at eGFR of 40 ml/min/1.73m 2 was 3.70 (2.29 to 5.99) in younger women and 1.86 (1.08 to 3.19) in younger men. The corresponding adjusted HRs in older women and older men were 2.38 (2.02 to 2.82) and 2.18 (1.85 to 2.57), respectively. Similar results were observed for CVD deaths, although the three-way interaction was not statistically significant. Sensitivity analysis yielded similar results., Conclusions: In this T2D population, younger women with reduced kidney function might be more susceptible to higher risks of all-cause mortality and CVD mortality than younger men., (© 2024. The Author(s).)

Diabetic nephropathy (DN) is characterized by abnormal kidney energy metabolism, but its causes and contributions to DN pathogenesis are not clear. To examine this issue, we carried out targeted metabolomics profiling in a mouse model of DN that develops kidney disease resembling the human disorder. We found a distinct profile of increased lactate levels and impaired energy metabolism in kidneys of mice with DN, and treatment with an angiotensin-receptor blocker (ARB) reduced albuminuria, attenuated kidney pathology and corrected many metabolic abnormalities, restoring levels of lactate toward normal while increasing kidney ATP content. We also found enhanced expression of lactate dehydrogenase isoforms in DN. Expression of both the LdhA and LdhB isoforms were significantly increased in kidneys of mice, and treatment with ARB significantly reduced their expression. Single-cell sequencing studies showed specific up-regulation of LdhA in the proximal tubule, along with enhanced expression of oxidative stress pathways. There was a significant correlation between albuminuria and lactate in mice, and also in a Southeast Asian patient cohort consisting of individuals with type 2 diabetes and impaired kidney function. In the individuals with diabetes, this association was independent of ARB and angiotensin-converting enzyme inhibitor use. Furthermore, urinary lactate levels predicted the clinical outcomes of doubling of serum creatinine or development of kidney failure, and there was a significant correlation between urinary lactate levels and biomarkers of tubular injury and epithelial stress. Thus, we suggest that kidney metabolic disruptions leading to enhanced generation of lactate contribute to the pathogenesis of DN and increased urinary lactate levels may be a potential biomarker for risk of kidney disease progression., (Copyright © 2023 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Diabetic corneal neuropathy (DCN) is a common complication of diabetes. However, there are very limited therapeutic options. We investigated the effects of a peroxisome proliferator-activated receptor-α (PPAR-α) agonist, fenofibrate, on 30 patients (60 eyes) with type 2 diabetes. On in vivo confocal microscopy evaluation, there was significant stimulation of corneal nerve regeneration and a reduction in nerve edema after 30 days of oral fenofibrate treatment, as evidenced by significant improvement in corneal nerve fiber density (CNFD) and corneal nerve fiber width, respectively. Corneal epithelial cell morphology also significantly improved in cell circularity. Upon clinical examination, fenofibrate significantly improved patients' neuropathic ocular surface status by increasing tear breakup time along with a reduction of corneal and conjunctival punctate keratopathy. Tear substance P (SP) concentrations significantly increased after treatment, suggesting an amelioration of ocular surface neuroinflammation. The changes in tear SP concentrations was also significantly associated with improvement in CNFD. Quantitative proteomic analysis demonstrated that fenofibrate significantly upregulated and modulated the neurotrophin signaling pathway and linolenic acid, cholesterol, and fat metabolism. Complement cascades, neutrophil reactions, and platelet activation were also significantly suppressed. Our results showed that fenofibrate could potentially be a novel treatment for patients with DCN., (© 2023 by the American Diabetes Association.)

Background: Ang II (angiotensin II) type 1 (AT 1 ) receptors play a critical role in cardiovascular diseases such as hypertension. Rodents have 2 types of AT 1 receptor (AT 1A and AT 1B ) of which knock-in Tagln -mediated smooth muscle AT 1A silencing attenuated Ang II-induced hypertension. Although vascular remodeling, a significant contributor to organ damage, occurs concurrently with hypertension in Ang II-infused mice, the contribution of smooth muscle AT 1A in this process remains unexplored. Accordingly, it is hypothesized that smooth muscle AT 1A receptors exclusively contribute to both medial thickening and adventitial fibrosis regardless of the presence of hypertension., Methods: About 1 µg/kg per minute Ang II was infused for 2 weeks in 2 distinct AT 1A receptor silenced mice, knock-in Tagln -mediated constitutive smooth muscle AT 1A receptor silenced mice, and Myh11 -mediated inducible smooth muscle AT 1A together with global AT 1B silenced mice for evaluation of hypertensive cardiovascular remodeling., Results: Medial thickness, adventitial collagen deposition, and immune cell infiltration in aorta were increased in control mice but not in both smooth muscle AT 1A receptor silenced mice. Coronary arterial perivascular fibrosis in response to Ang II infusion was also attenuated in both AT 1A receptor silenced mice. Ang II-induced cardiac hypertrophy was attenuated in constitutive smooth muscle AT 1A receptor silenced mice. However, Ang II-induced cardiac hypertrophy and hypertension were not altered in inducible smooth muscle AT 1A receptor silenced mice., Conclusions: Smooth muscle AT 1A receptors mediate Ang II-induced vascular remodeling including medial hypertrophy and inflammatory perivascular fibrosis regardless of the presence of hypertension. Our data suggest an independent etiology of blood pressure elevation and hypertensive vascular remodeling in response to Ang II.

The kidney has large regenerative capacity, but this is compromised when kidney damage is excessive and renal tubular epithelial cells (TECs) undergo SNAI1-driven growth arrest. Here we investigate the role of IL11 in TECs, kidney injury and renal repair. IL11 stimulation of TECs induces ERK- and p90RSK-mediated GSK3β inactivation, SNAI1 upregulation and pro-inflammatory gene expression. Mice with acute kidney injury upregulate IL11 in TECs leading to SNAI1 expression and kidney dysfunction, which is not seen in Il11 deleted mice or in mice administered a neutralizing IL11 antibody in either preemptive or treatment modes. In acute kidney injury, anti-TGFβ reduces renal fibrosis but exacerbates inflammation and tubule damage whereas anti-IL11 reduces all pathologies. Mice with TEC-specific deletion of Il11ra1 have reduced pathogenic signaling and are protected from renal injury-induced inflammation, fibrosis, and failure. In a model of chronic kidney disease, anti-IL11 therapy promotes TEC proliferation and parenchymal regeneration, reverses fibroinflammation and restores renal mass and function. These data highlight IL11-induced mesenchymal transition of injured TECs as an important renal pathology and suggest IL11 as a therapeutic target for restoring stalled endogenous regeneration in the diseased kidney., (© 2022. The Author(s).)

Background A beneficial role for prostanoids in hypertension is suggested by clinical studies showing nonsteroidal anti-inflammatory drugs, which block the production of all prostanoids, cause sodium retention and exacerbate hypertension. Among prostanoids, prostaglandin E2 and its E-prostanoid receptor 4 receptor (EP4R) have been implicated in blood pressure control. Our previous study found that conditional deletion of EP4R from all tissues in adult mice exacerbates angiotensin II-dependent hypertension, suggesting a powerful effect of EP4R to resist blood pressure elevation. We also found that elimination of EP4R from vascular smooth muscle cells did not affect the severity of hypertension, suggesting nonvascular targets of prostaglandin E mediate this antihypertensive effect. Methods and Results Here we generated mice with cell-specific deletion of EP4R from macrophage-specific EP4 receptor knockouts or kidney epithelial cells (KEKO) to assess the contributions of EP4R in these cells to hypertension pathogenesis. Macrophage-specific EP4 receptor knockouts showed similar blood pressure responses to alterations in dietary sodium or chronic angiotensin II infusion as Controls. By contrast, angiotensin II-dependent hypertension was significantly augmented in KEKOs (mean arterial pressure: 146±3 mm Hg) compared with Controls (137±4 mm Hg; P =0.02), which was accompanied by impaired natriuresis in KEKOs. Because EP4R expression in the kidney is enriched in the collecting duct, we compared responses to amiloride in angiotensin II-infused KEKOs and Controls. Blockade of the epithelial sodium channel with amiloride caused exaggerated natriuresis in KEKOs compared with Controls (0.21±0.01 versus 0.15±0.02 mmol/24 hour per 20 g; P =0.015). Conclusions Our data suggest EP4R in kidney epithelia attenuates hypertension. This antihypertension effect of EP4R may be mediated by reducing the activity of the epithelial sodium channel, thereby promoting natriuresis.

Background: ACE2 is a key enzyme in the renin-angiotensin system (RAS) capable of balancing the RAS by metabolizing angiotensin II (AngII). First described in cardiac tissue, abundance of ACE2 is highest in the kidney, and it is also expressed in several extrarenal tissues. Previously, we reported an association between enhanced susceptibility to hypertension and elevated renal AngII levels in global ACE2-knockout mice., Methods: To examine the effect of ACE2 expressed in the kidney, relative to extrarenal expression, on the development of hypertension, we used a kidney crosstransplantation strategy with ACE2-KO and WT mice. In this model, both native kidneys are removed and renal function is provided entirely by the transplanted kidney, such that four experimental groups with restricted ACE2 expression are generated: WT→WT (WT), KO→WT (KidneyKO), WT→KO (SystemicKO), and KO→KO (TotalKO). Additionally, we used nanoscale mass spectrometry-based proteomics to identify ACE2 fragments in early glomerular filtrate of mice., Results: Although significant differences in BP were not detected, a major finding of our study is that shed or soluble ACE2 (sACE2) was present in urine of KidneyKO mice that lack renal ACE2 expression. Detection of sACE2 in the urine of KidneyKO mice during AngII-mediated hypertension suggests that sACE2 originating from extrarenal tissues can reach the kidney and be excreted in urine. To confirm glomerular filtration of ACE2, we used micropuncture and nanoscale proteomics to detect peptides derived from ACE2 in the Bowman's space., Conclusions: Our findings suggest that both systemic and renal tissues may contribute to sACE2 in urine, identifying the kidney as a major site for ACE2 actions. Moreover, filtration of sACE2 into the lumen of the nephron may contribute to the pathophysiology of kidney diseases characterized by disruption of the glomerular filtration barrier., Competing Interests: T.M. Coffman reports serving in an advisory or leadership role for Cell Metabolism and The Journal of Clinical Investigation (editorial boards), Kidney Research Institute University of Washington, Singapore Eye Research Institute, and the Singapore Health Services (board of directors). S.B. Gurley serving as Associate Editor for the Diabetes and a family member being employed by, and having ownership interest in, United Therapeutics. M.P. Hutchens reports having ownership interest in ATT, Exxon, Frontier, and Verizon; and having patents or royalties with ProjectLite, LLC. P.D. Piehowski reports receiving research funding from Jacobs (worldwide research principal investigator) and Pfizer; and being employed by, and having patents or royalties with, Pacific Northwest National Laboratory. R. Wakasaki reports having ownership interest in Fuji Film Holdings, KDDI, Lion, and Toyota. All remaining authors have nothing to disclose., (Copyright © 2022 by the American Society of Nephrology.)

The functional interpretation of genome-wide association studies (GWAS) is challenging due to the cell-type-dependent influences of genetic variants. Here, we generated comprehensive maps of expression quantitative trait loci (eQTLs) for 659 microdissected human kidney samples and identified cell-type-eQTLs by mapping interactions between cell type abundances and genotypes. By partitioning heritability using stratified linkage disequilibrium score regression to integrate GWAS with single-cell RNA sequencing and single-nucleus assay for transposase-accessible chromatin with high-throughput sequencing data, we prioritized proximal tubules for kidney function and endothelial cells and distal tubule segments for blood pressure pathogenesis. Bayesian colocalization analysis nominated more than 200 genes for kidney function and hypertension. Our study clarifies the mechanism of commonly used antihypertensive and renal-protective drugs and identifies drug repurposing opportunities for kidney disease., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

A major pathway in hypertension pathogenesis involves direct activation of ANG II type 1 (AT 1 ) receptors in the kidney, stimulating Na + reabsorption. AT 1 receptors in tubular epithelia control expression and stimulation of Na + transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT 1 receptors in smooth muscle (SMKO mice). Although impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary Na + excretion in SMKO mice, we considered whether alterations in Na + transporter expression might also play a role; therefore, we carried out proteomic analysis of key Na + transporters and associated proteins. Here, we show that levels of Na + -K + -2Cl - cotransporter isoform 2 (NKCC2) and Na + /H + exchanger isoform 3 (NHE3) are reduced at baseline in SMKO mice, accompanied by attenuated natriuretic and diuretic responses to furosemide. During ANG II hypertension, we found widespread remodeling of transporter expression in wild-type mice with significant increases in the levels of total NaCl cotransporter, phosphorylated NaCl cotransporter (Ser 71 ), and phosphorylated NKCC2, along with the cleaved, activated forms of the α- and γ-epithelial Na + channel. However, the increases in α- and γ-epithelial Na + channel with ANG II were substantially attenuated in SMKO mice. This was accompanied by a reduced natriuretic response to amiloride. Thus, enhanced urinary Na + excretion observed after cell-specific deletion of AT 1 receptors from smooth muscle cells is associated with altered Na + transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial in the kidney, modulating the expression of Na + transporters and contributing to the regulation of pressure natriuresis. NEW & NOTEWORTHY The use of drugs to block the renin-angiotensin system to reduce blood pressure is common. However, the precise mechanism for how these medications control blood pressure is incompletely understood. Here, we show that mice lacking angiotensin receptors specifically in smooth muscle cells lead to alternation in tubular transporter amount and function. Thus, demonstrating the importance of vascular-tubular cross talk in the control of blood pressure.

Dr Irvine Page proposed the Mosaic Theory of Hypertension in the 1940s advocating that hypertension is the result of many factors that interact to raise blood pressure and cause end-organ damage. Over the years, Dr Page modified his paradigm, and new concepts regarding oxidative stress, inflammation, genetics, sodium homeostasis, and the microbiome have arisen that allow further refinements of the Mosaic Theory. A constant feature of this approach to understanding hypertension is that the various nodes are interdependent and that these almost certainly vary between experimental models and between individuals with hypertension. This review discusses these new concepts and provides an introduction to other reviews in this compendium of Circulation Research .

Activation of AT 1 (type 1 Ang) receptors stimulates cardiomyocyte hypertrophy in vitro. Accordingly, it has been suggested that regression of cardiac hypertrophy associated with renin-Ang system blockade is due to inhibition of cellular actions of Ang II in the heart, above and beyond their effects to reduce pressure overload. We generated 2 distinct mouse lines with cell-specific deletion of AT 1A receptors, from cardiomyocytes. In the first line (C-SMKO), elimination of AT 1A receptors was achieved using a heterologous Cre recombinase transgene under control of the Sm22 promoter, which expresses in cells of smooth muscle lineage including cardiomyocytes and vascular smooth muscle cells of conduit but not resistance vessels. The second line (R-SMKO) utilized a Cre transgene knocked-in to the Sm22 locus, which drives expression in cardiac myocytes and vascular smooth muscle cells in both conduit and resistance arteries. Thus, although both groups lack AT 1 receptors in the cardiomyocytes, they are distinguished by presence (C-SMKO) or absence (R-SMKO) of peripheral vascular responses to Ang II. Similar to wild-types, chronic Ang II infusion caused hypertension and cardiac hypertrophy in C-SMKO mice, whereas both hypertension and cardiac hypertrophy were reduced in R-SMKOs. Thus, despite the absence of AT 1A receptors in cardiomyocytes, C-SMKOs develop robust cardiac hypertrophy. By contrast, R-SMKOs developed identical levels of hypertrophy in response to pressure overload-induced by transverse aortic banding. Our findings suggest that direct activation of AT 1 receptors in cardiac myocytes has minimal influence on cardiac hypertrophy induced by renin-Ang system activation or pressure overload.

Hypertension is the most common chronic disease in the world, yet the precise cause of elevated blood pressure often cannot be determined. Animal models have been useful for unraveling the pathogenesis of hypertension and for testing novel therapeutic strategies. The utility of animal models for improving the understanding of the pathogenesis, prevention, and treatment of hypertension and its comorbidities depends on their validity for representing human forms of hypertension, including responses to therapy, and on the quality of studies in those models (such as reproducibility and experimental design). Important unmet needs in this field include the development of models that mimic the discrete hypertensive syndromes that now populate the clinic, resolution of ongoing controversies in the pathogenesis of hypertension, and the development of new avenues for preventing and treating hypertension and its complications. Animal models may indeed be useful for addressing these unmet needs.

Background Prostaglandin E 2 ( PGE 2 ) is a major prostanoid with multiple actions that potentially affect blood pressure ( BP ). PGE 2 acts through 4 distinct E-prostanoid ( EP ) receptor isoforms: EP 1 to EP 4. The EP 4 receptor ( EP 4R) promotes PGE 2 -dependent vasodilation, but its role in the pathogenesis of hypertension is not clear. Methods and Results To address this issue, we studied mice after temporal- and cell-specific deletion of EP 4R. First, using a mouse line with loss of EP 4 expression induced universally after birth, we confirm that EP 4R mediates a major portion of the acute vasodilatory effects of infused PGE 2 . In addition, EP 4 contributes to control of resting BP , which was increased by 5±1 mm Hg in animals with generalized deficiency of this receptor. We also show that EP 4 is critical for limiting elevations in BP caused by high salt feeding and long-term infusion of angiotensin II . To more precisely identify the mechanism for these actions, we generated mice in which EP 4R loss is induced after birth and is limited to smooth muscle. In these mice, acute PGE 2 -dependent vasodilation was attenuated, indicating that this response is mediated by EP 4R in vascular smooth muscle cells. However, absence of EP 4R only in this vascular compartment had a paradoxical effect of lowering resting BP , whereas the protective effect of EP 4R on limiting angiotensin II-dependent hypertension was unaffected. Conclusions Taken together, our findings support a complex role for EP 4R in regulation of BP and in hypertension, which appears to involve actions of the EP 4R in tissues beyond vascular smooth muscle cells.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease worldwide, but its molecular pathogenesis is not well defined, and there are no specific treatments. In humans, there is a strong genetic component determining susceptibility to DN. However, specific genes controlling DN susceptibility in humans have not been identified. In this study, we describe a mouse model combining type 1 diabetes with activation of the renin-angiotensin system (RAS), which develops robust kidney disease with features resembling human DN: heavy albuminuria, hypertension, and glomerulosclerosis. Additionally, there is a powerful effect of genetic background regulating susceptibility to nephropathy; the 129 strain is susceptible to kidney disease, whereas the C57BL/6 strain is resistant. To examine the molecular basis of this differential susceptibility, we analyzed the glomerular transcriptome of young mice early in the course of their disease. We find dramatic differences in regulation of immune and inflammatory pathways, with upregulation of proinflammatory pathways in the susceptible (129) strain and coordinate downregulation in the resistant (C57BL/6) strain. Many of these pathways are also upregulated in rat models and in humans with DN. Our studies suggest that genes controlling inflammatory responses, triggered by hyperglycemia and RAS activation, may be critical early determinants of susceptibility to DN., (© 2018 by the American Diabetes Association.)

The renin-angiotensin-aldosterone system plays crucial roles in cardiovascular physiology and pathophysiology. However, many of the signaling mechanisms have been unclear. The angiotensin II (ANG II) type 1 receptor (AT 1 R) is believed to mediate most functions of ANG II in the system. AT 1 R utilizes various signal transduction cascades causing hypertension, cardiovascular remodeling, and end organ damage. Moreover, functional cross-talk between AT 1 R signaling pathways and other signaling pathways have been recognized. Accumulating evidence reveals the complexity of ANG II signal transduction in pathophysiology of the vasculature, heart, kidney, and brain, as well as several pathophysiological features, including inflammation, metabolic dysfunction, and aging. In this review, we provide a comprehensive update of the ANG II receptor signaling events and their functional significances for potential translation into therapeutic strategies. AT 1 R remains central to the system in mediating physiological and pathophysiological functions of ANG II, and participation of specific signaling pathways becomes much clearer. There are still certain limitations and many controversies, and several noteworthy new concepts require further support. However, it is expected that rigorous translational research of the ANG II signaling pathways including those in large animals and humans will contribute to establishing effective new therapies against various diseases.

The activity of the renin-angiotensin-aldosterone system is triggered by the release of the protease renin from the kidneys, which in turn is controlled in the sense of negative feedback loops. It is widely assumed that Ang II (angiotensin II) directly inhibits renin expression and secretion via a short-loop feedback by an effect on renin-producing cells (RPCs) mediated by AT 1 (Ang II type 1) receptors. Because the concept of such a direct short-loop negative feedback control, which originates mostly from in vitro experiments, has not yet been systematically proven in vivo, we aimed to test the validity of this concept by studying the regulation of renin synthesis and secretion in mice lacking Ang II-AT 1 receptors on RPCs. We found that RPCs of the kidney express Ang II-AT 1 receptors. Mice with conditional deletion of Ang II-AT 1 receptors in RPCs were normal with regard to the number of renin cells, renal renin mRNA, and plasma renin concentrations. Renin expression and secretion of these mice responded to Ang I (angiotensin I)-converting enzyme inhibition and to Ang II infusion like in wild-type (WT) controls. In summary, we did not obtain evidence that Ang II-AT 1 receptors on RPCs are of major relevance for the normal regulation of renin expression and secretion in mice. Therefore, we doubt the existence of a direct negative feedback function of Ang II on RPCs., (© 2018 American Heart Association, Inc.)

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease in the developed world. Accordingly, an urgent need exists for new, curative treatments as well as for biomarkers to stratify risk of DN among individuals with diabetes mellitus. A barrier to progress in these areas has been a lack of animal models that faithfully replicate the main features of human DN. Such models could be used to define the pathogenesis, identify drug targets and test new therapies. Owing to their tractability for genetic manipulation, mice are widely used to model human diseases, including DN. Questions have been raised, however, about the general utility of mouse models in human drug discovery. Standard mouse models of diabetes typically manifest only modest kidney abnormalities, whereas accelerated models, induced by superimposing genetic stressors, recapitulate key features of human DN. Incorporation of systems biology approaches and emerging data from genomics and metabolomics studies should enable further model refinement. Here, we discuss the current status of mouse models for DN, their limitations and opportunities for improvement. We emphasize that future efforts should focus on generating robust models that reproduce the major clinical and molecular phenotypes of human DN.

The renal collecting duct (CD), as the terminal segment of the nephron, is responsible for the final adjustments to the amount of sodium excreted in urine. While angiotensin II modulates reabsorptive functions of the CD, the contribution of these actions to physiological homeostasis is not clear. To examine this question, we generated mice with cell-specific deletion of AT 1A receptors from the CD. Elimination of AT 1A receptors from both principal and intercalated cells (CDKO mice) had no effect on blood pressures at baseline or during successive feeding of low- or high-salt diets. In contrast, the severity of hypertension caused by chronic infusion of angiotensin II was paradoxically exaggerated in CDKO mice compared with controls. In wild-type mice, angiotensin II induced robust expression of cyclooxygenase-2 (COX-2) in renal medulla, primarily localized to intercalated cells. Upregulation of COX-2 was diminished in CDKO mice, resulting in reduced generation of vasodilator prostanoids. This impaired expression of COX-2 has physiological consequences, since administration of a specific COX-2 inhibitor to CDKO and control mice during angiotensin II infusion equalized their blood pressures. Stimulation of COX-2 was also triggered by exposure of isolated preparations of medullary CDs to angiotensin II. Deletion of AT 1A receptors from principal cells alone did not affect angiotensin II-dependent COX2 stimulation, implicating intercalated cells as the main source of COX2 in this setting. These findings suggest a novel paracrine role for the intercalated cell to attenuate the severity of hypertension. Strategies for preserving or augmenting this pathway may have value for improving the management of hypertension.

Systolic Blood Pressure Intervention Trial (SPRINT) was a multicenter randomized controlled trial showing the significant benefit of intensive reduction of blood pressure to a target of 120 mm Hg in individuals with hypertension and elevated cardiovascular risk. Because SPRINT includes the largest cohort of adults with chronic kidney disease (CKD) to be prospectively studied in a hypertension intervention trial, it has particular relevance to the field of nephrology. Here, we review the findings of SPRINT and assess their potential impact on guidelines for treatment of hypertension in patients with CKD. We believe that the data from SPRINT will support a recommendation for lowering blood pressure targets to 120 mm Hg in a substantial segment of adults with CKD., (© 2017 S. Karger AG, Basel.)

Postnatal inhibition or deletion of angiotensin II (ANG II) AT1 receptors impairs renal medullary mircrovascular development through a mechanism that may include vascular endothelial growth factor (VEGF). The present study was designed to test if VEGF/VEGF receptor signaling is necessary for the development of the renal medullary microcirculation. Endothelial cell-specific immunolabeling of kidney sections from rats showed immature vascular bundles at postnatal day (P) 10 with subsequent expansion of bundles until P21. Medullary VEGF protein abundance coincided with vasa recta bundle formation. In human fetal kidney tissue, immature vascular bundles appeared early in the third trimester (GA27-28) and expanded in size until term. Rat pups treated with the VEGF receptor-2 (VEGFR2) inhibitor vandetanib (100 mg·kg(-1)·day(-1)) from P7 to P12 or P10 to P16 displayed growth retardation and proteinuria. Stereological quantification showed a significant reduction in total length (386 ± 13 vs. 219 ± 16 m), surface area, and volume of medullary microvessels. Vascular bundle architecture was unaffected. ANG II-AT1A/1B (-/-) mice kidneys displayed poorly defined vasa recta bundles whereas mice with collecting duct principal cell-specific AT1A deletion displayed no medullary microvascular phenotype. In conclusion, VEGFR2 signaling during postnatal development is necessary for expansion of the renal medullary microcirculation but not structural patterning of the vasa recta bundles, which occurs through an AT1-mediated mechanism.

The main actions of the renin-angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT1Rs). The major murine AT1R isoform, AT1AR, is expressed throughout the nephron, including the collecting duct in both principal and intercalated cells. Principal cells play the major role in sodium and water reabsorption. Although aldosterone is considered to be the dominant regulator of sodium reabsorption by principal cells, recent studies suggest a role for direct actions of AT1R. To specifically examine the contributions of AT1AR in principal cells to BP regulation and the development of hypertension in vivo, we generated inbred 129/SvEv mice with deletion of AT1AR from principal cells (PCKO). At baseline, we found that BPs measured by radiotelemetry were similar between PCKOs and controls. During 1-week of low-salt diet (<0.02% NaCl), BPs fell significantly (P<0.05) and to a similar extent in both groups. On a high-salt (6% NaCl) diet, BP increased but was not different between groups. During the initial phase of angiotensin II-dependent hypertension, there was a modest but significant attenuation of hypertension in PCKOs (163±6 mm Hg) compared with controls (178±2 mm Hg; P<0.05) that was associated with enhanced natriuresis and decreased alpha epithelial sodium channel activation in the medulla of PCKOs. However, from day 9 onward, BPs were indistinguishable between groups. Although effects of AT1AR on baseline BP and adaptation to changes in dietary salt are negligible, our studies suggest that direct actions of AT1AR contribute to the initiation of hypertension and epithelial sodium channel activation., (© 2016 American Heart Association, Inc.)

We investigated the role of angiotensin type 1a receptors (AGTR1a) in vascular injury induced by aldosterone activation of mineralocorticoid receptors in Agtr1a(-/-) and wild-type (WT) mice infused with aldosterone for 14 days while receiving 1% NaCl in drinking water. Aldosterone increased systolic blood pressure (BP) by ≈30 mm Hg in WT mice and ≈50 mm Hg in Agtr1a(-/-) mice. Aldosterone induced aortic and small artery remodeling, impaired endothelium-dependent relaxation in WT mice, and enhanced fibronectin and collagen deposition and vascular inflammation. None of these vascular effects were observed in Agtr1a(-/-) mice. Aldosterone effects were prevented by the AGTR1 antagonist losartan in WT mice. In contrast to aldosterone, norepinephrine caused similar BP increase and mesenteric artery remodeling in WT and Agtr1a(-/-) mice. Agtr1a(-/-) mice infused with aldosterone did not increase sodium excretion in response to a sodium chloride challenge, suggesting that sodium retention could contribute to the exaggerated BP rise induced by aldosterone. Agtr1a(-/-) mice had decreased mesenteric artery expression of the calcium-activated potassium channel Kcnmb1, which may enhance myogenic tone and together with sodium retention, exacerbate BP responses to aldosterone/salt in Agtr1a(-/-) mice. We conclude that although aldosterone activation of mineralocorticoid receptors raises BP more in Agtr1a(-/-) mice, AGTR1a is required for mineralocorticoid receptor stimulation to induce vascular remodeling and inflammation and endothelial dysfunction., (© 2016 American Heart Association, Inc.)

Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)-dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II-dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%-10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II-dependent vascular responses in the kidney to effect natriuresis and BP control., (Copyright © 2015 by the American Society of Nephrology.)

The relationship between dietary salt intake and the associated risk of hypertension and cardiovascular disease is an important public health concern. In this issue of the JCI, a study by Zhang and associates shows that consumption of a high-sodium diet induces expression of cyclooxygenase-2 (COX-2) in macrophages, resulting in enhanced levels of prostaglandin E2 (PGE2), autocrine activation of the macrophage E-prostanoid 4 (EP4) receptor, and subsequent triggering of parallel pathways in the kidney and in skin that help dispose of excess sodium. The authors found that blockade or genetic elimination of the COX-2/PGE2/EP4 receptor pathway in hematopoietic cells causes salt-sensitive hypertension in mice. These studies illuminate an unexpected central role for the macrophage in coordinating homeostatic responses to dietary salt intake and suggest a complex pathophysiology for hypertension associated with NSAID use.

Vascular endothelial growth factor A (VEGFA) production by podocytes is critical for glomerular endothelial health. VEGFA is also expressed in tubular epithelial cells in kidney; however, its physiologic role in the tubule has not been established. Using targeted transgenic mouse models, we found that Vegfa is expressed by specific epithelial cells along the nephron, whereas expression of its receptor (Kdr/Vegfr2) is largely restricted to adjacent peritubular capillaries. Embryonic deletion of tubular Vegfa did not affect systemic Vegfa levels, whereas renal Vegfa abundance was markedly decreased. Excision of Vegfa from renal tubules resulted in the formation of a smaller kidney, with a striking reduction in the density of peritubular capillaries. Consequently, elimination of tubular Vegfa caused pronounced polycythemia because of increased renal erythropoietin (Epo) production. Reducing hematocrit to normal levels in tubular Vegfa-deficient mice resulted in a markedly augmented renal Epo production, comparable with that observed in anemic wild-type mice. Here, we show that tubulovascular cross-talk by Vegfa is essential for maintenance of peritubular capillary networks in kidney. Disruption of this communication leads to increased renal Epo production and resulting polycythemia, presumably to counterbalance microvascular losses., (Copyright © 2015 by the American Society of Nephrology.)

Angiotensin type 1 (AT1) receptors are key effector elements of the renin-angiotensin system, mediating virtually all of the classical physiological actions of angiotensin II. Pharmacological blockade of the AT1 receptor effectively lowers blood pressure in a substantial proportion of patients with hypertension, indicating the pivotal role of these receptors in human hypertension. AT1 receptors are expressed in many different organ systems where they have myriad cellular actions. However, several lines of evidence have suggested that direct actions of AT1 receptors in kidney have a major role in regulation of blood pressure and in the pathogenesis of hypertension. Here we review recent studies suggesting that renal epithelium and vasculature may be key cellular targets, where AT1 receptor activation has powerful physiological impact. We will also examine novel regulatory mechanisms by peptides associated with the C-terminus of the AT1 receptor., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Objective: Elevations in brain angiotensin-II cause increased energy expenditure and a lean phenotype. Interestingly, the metabolic effects of increased brain angiotensin-II mimic the actions of leptin, suggesting an interaction between the two systems. Here we demonstrate that angiotensin-type 1a receptors (AT1aR) in the subfornical organ (SFO), a forebrain structure emerging as an integrative metabolic center, play a key role in the body weight-reducing effects of leptin via brown adipose tissue (BAT) thermogenesis., Methods: Cre/LoxP technology coupled with targeted viral delivery to the SFO in a mouse line bearing a conditional allele of the Agtr1a gene was utilized to determine the interaction between leptin and SFO AT1aR in metabolic regulation., Results: Selective deletion of AT1aR in the SFO attenuated leptin-induced weight loss independent of changes in food intake or locomotor activity. This was associated with diminished leptin-induced increases in core body temperature, blunted upregulation of BAT thermogenic markers, and abolishment of leptin-mediated sympathetic activation to BAT., Conclusions: These data identify a novel interaction between angiotensin-II and leptin in the control of BAT thermogenesis and body weight, and highlight a previously unrecognized role for the forebrain SFO in metabolic regulation.

Aims: Diabetes is associated with both dysfunction of the lower urinary tract (LUT) and overactivity of the renin-angiotensin system (RAS). Although it is well known that the RAS affects normal LUT function, very little is known about RAS effects on the diabetic LUT. Accordingly, we investigated the effects of chronic angiotensin II (AngII) treatment on the LUT in a model of type 1 diabetes., Methods: Ins2(Akita) diabetic mice (20 weeks old) and their age-matched background controls underwent conscious cystometric evaluation after 4 weeks of chronic AngII treatment (700 ng/kg/min by osmotic pump) or vehicle (saline)., Results: Diabetic mice had compensated LUT function with bladder hypertrophy. Specifically, micturition volume, residual volume, and bladder capacity were all increased, while voiding efficiency and pressure generation were unchanged as bladder mass, contraction duration, and phasic urethral function were increased. AngII significantly increased voiding efficiency and peak voiding pressure and decreased phasic frequency irrespective of diabetic state and, in diabetic but not normoglycemic control mice, significantly decreased residual volume and increased contraction duration and nonphasic contraction duration., Conclusions: The Ins2(Akita) diabetic mice had compensated LUT function at 20 weeks of age. Even under these conditions, AngII had beneficial effects on LUT function, resulting in increased voiding efficiency. Future studies should therefore be conducted to determine whether AngII can rescue the decompensated LUT function occurring in end-stage diabetic uropathy., (© 2013 Wiley Periodicals, Inc.)

The renin-angiotensin system has powerful effects in control of the blood pressure and sodium homeostasis. These actions are coordinated through integrated actions in the kidney, cardiovascular system and the central nervous system. Along with its impact on blood pressure, the renin-angiotensin system also influences a range of processes from inflammation and immune responses to longevity. Here, we review the actions of the "classical" renin-angiotensin system, whereby the substrate protein angiotensinogen is processed in a two-step reaction by renin and angiotensin converting enzyme, resulting in the sequential generation of angiotensin I and angiotensin II, the major biologically active renin-angiotensin system peptide, which exerts its actions via type 1 and type 2 angiotensin receptors. In recent years, several new enzymes, peptides, and receptors related to the renin-angiotensin system have been identified, manifesting a complexity that was previously unappreciated. While the functions of these alternative pathways will be reviewed elsewhere in this journal, our focus here is on the physiological role of components of the "classical" renin-angiotensin system, with an emphasis on new developments and modern concepts., (© 2014 American Physiological Society.)

An essential link between the kidney and blood pressure control has long been known. Here, we review evidence supporting the premise that an impaired capacity of the kidney to excrete sodium in response to elevated blood pressure is a major contributor to hypertension, irrespective of the initiating cause. In this regard, recent work suggests that novel pathways controlling key sodium transporters in kidney epithelia have a critical impact on hypertension pathogenesis, supporting a model in which impaired renal sodium excretion is a final common pathway through which vascular, neural, and inflammatory responses raise blood pressure. We also address recent findings calling into question long-standing notions regarding the relationship between sodium intake and changes in body fluid volume. Expanded understanding of the role of the kidney as both a cause and target of hypertension highlights key aspects of pathophysiology and may lead to identification of new strategies for prevention and treatment.

Disruption of the regulatory role of the kidneys leads to diverse renal pathologies; one major hallmark is inflammation and fibrosis. Conventional magnitude MRI has been used to study renal pathologies; however, the quantification or even detection of focal lesions caused by inflammation and fibrosis is challenging. We propose that quantitative susceptibility mapping (QSM) may be particularly sensitive for the identification of inflammation and fibrosis. In this study, we applied QSM in a mouse model deficient for angiotensin receptor type 1 (AT1). This model is known for graded pathologies, including focal interstitial fibrosis, cortical inflammation, glomerulocysts and inner medullary hypoplasia. We acquired high-resolution MRI on kidneys from AT1-deficient mice that were perfusion fixed with contrast agent. Two MR sequences were used (three-dimensional spin echo and gradient echo) to produce three image contrasts: T1, T2* (magnitude) and QSM. T1 and T2* (magnitude) images were acquired to segment major renal structures and to provide landmarks for the focal lesions of inflammation and fibrosis in the three-dimensional space. The volumes of major renal structures were measured to determine the relationship of the volumes to the degree of renal abnormalities and magnetic susceptibility values. Focal lesions were segmented from QSM images and were found to be closely associated with the major vessels. Susceptibilities were relatively more paramagnetic in wild-type mice: 1.46 ± 0.36 in the cortex, 2.14 ± 0.94 in the outer medulla and 2.10 ± 2.80 in the inner medulla (10(-2) ppm). Susceptibilities were more diamagnetic in knockout mice: -7.68 ± 4.22 in the cortex, -11.46 ± 2.13 in the outer medulla and -7.57 ± 5.58 in the inner medulla (10(-2) ppm). This result was consistent with the increase in diamagnetic content, e.g. proteins and lipids, associated with inflammation and fibrosis. Focal lesions were validated with conventional histology. QSM was very sensitive in detecting pathology caused by small focal inflammation and fibrosis. QSM offers a new MR contrast mechanism to study this common disease marker in the kidney., (Copyright © 2013 John Wiley & Sons, Ltd.)

The National Institute of Diabetes and Digestive and Kidney Diseases-supported Kidney Research National Dialogue (KRND) asked the scientific community to formulate and prioritize research objectives that would improve our understanding of kidney function and disease. Several high-priority objectives for diabetic nephropathy were identified in data and sample collection, hypothesis generation, hypothesis testing, and translation promotion. The lack of readily available human samples linked to comprehensive phenotypic, clinical, and demographic data remains a significant obstacle. With data and biological samples in place, several possibilities exist for using new technologies to develop hypotheses. Testing novel disease mechanisms with state-of-the-art tools should continue to be the foundation of the investigative community. Research must be translated to improve diagnosis and treatment of people. The objectives identified by the KRND provide the research community with future opportunities for improving the prevention, diagnosis, and treatment of diabetic nephropathy.

Glomerular injury leads to podocyte loss, a process directly underlying progressive glomerular scarring and decline of kidney function. The inherent repair process is limited by the inability of podocytes to regenerate. Cells of renin lineage residing alongside glomerular capillaries are reported to have progenitor capacity. We investigated whether cells of renin lineage can repopulate the glomerulus after podocyte injury and serve as glomerular epithelial cell progenitors. Kidney cells expressing renin were genetically fate-mapped in adult Ren1cCreER×Rs-tdTomato-R, Ren1cCre×Rs-ZsGreen-R, and Ren1dCre×Z/EG reporter mice. Podocyte depletion was induced in all three cell-specific reporter mice by cytotoxic anti-podocyte antibodies. After a decrease in podocyte number, a significant increase in the number of labeled cells of renin lineage was observed in glomeruli in a focal distribution along Bowman's capsule, within the glomerular tuft, or in both locations. A subset of cells lining Bowman's capsule activated expression of the glomerular parietal epithelial cell markers paired box protein PAX2 and claudin-1. A subset of labeled cells within the glomerular tuft expressed the podocyte markers Wilms tumor protein 1, nephrin, podocin, and synaptopodin. Neither renin mRNA nor renin protein was detected de novo in diseased glomeruli. These findings provide initial evidence that cells of renin lineage may enhance glomerular regeneration by serving as progenitors for glomerular epithelial cells in glomerular disease characterized by podocyte depletion., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The epidemiological association between high salt intake and hypertension is well established. However, in most patients, the specific defect causing salt-dependent hypertension cannot be discerned. In this issue of Cell Metabolism, Rakova and associates use an unprecedented study design to characterize long-term salt balance in humans (Rakova et al., 2012)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The angiotensin II type 1 receptor (AT(1)R) mediates most hypertensive actions of angiotensin II. To understand the molecular regulation of the AT(1)R in normal physiology and pathophysiology, methods for sensitive and specific detection of AT(1)R protein are required. Here, we examined the specificity of a panel of putative AT(1)R antibodies that are commonly used by investigators in the field. For these studies, we carried out Western blotting and immunohistochemistry with kidney tissue from wild-type mice and genetically modified mice lacking the major murine AT(1)R isoform, AT(1A) (AT(1A)KO), or with combined deficiency of both the AT(1A) and AT(1B) isoforms (AT(1AB)KO). For the 3 antibodies tested, Western blots of protein homogenates from wild-type kidneys yielded distinct bands with the expected size range for AT(1)R. In addition, these bands appeared identical in samples from mice lacking 1 or both murine AT(1)R isoforms. Additionally, the pattern of immunohistochemical staining in kidneys, liver, and adrenal glands of wild-type mice was very similar to that of AT(1AB)KO mice completely lacking all AT(1)R. We verified the absence of AT(1)R subtypes in each mouse line by the following: (1) quantitative polymerase chain reaction documenting the absence of mRNA species, and (2) functionally by assessing angiotensin II-dependent vasoconstriction, which was substantially blunted in both AT(1A)KOs and AT(1AB)KOs. Finally, these antibodies failed to detect epitope-tagged AT(1A)R protein overexpressed in human embryonic kidney cells. We conclude that anti-AT(1)R antibodies available from commercial sources and commonly used in published studies exhibit nonspecific binding in mouse tissue that may lead to erroneous results.

The prostanoid thromboxane A2 has been implicated to contribute to the pathogenesis of many cardiovascular diseases, including hypertension. To study the role of vascular thromboxane-prostanoid (TP) receptors in blood pressure regulation, we generated mice with cell-specific deletion of TP receptors in smooth muscle using Cre/Loxp technology. We crossed the KISM22α-Cre transgenic mouse line expressing Cre recombinase in smooth muscle cells with a mouse line bearing a conditional allele of the Tbxa2r gene (Tp(flox)). In KISM22α-Cre(+)Tp(flox/flox) (TP-SMKO) mice, TP receptors were efficiently deleted from vascular smooth muscle cells. In TP-SMKOs, acute vasoconstrictor responses to the TP agonist U46619 were attenuated to a similar extent in both the peripheral and renal circulations. Yet, acute vascular responses to angiotensin II were unaffected at baseline and after chronic angiotensin II administration. Infusion of high-dose U46619 caused circulatory collapse and death in a majority of control mice but had negligible hemodynamic effects in TP-SMKOs, which were completely protected from U46619-induced sudden death. Baseline blood pressures were normal in TP-SMKOs. However, the absence of TP receptors in vascular smooth muscle cells was associated with significant attenuation of angiotensin II-induced hypertension and diminished vascular remodeling. This was also associated with reduced urinary thromboxane production after chronic angiotensin II. Thus, TP receptors in vascular smooth muscle cells play a major role in mediating the actions of thromboxane A(2) in TP agonist-induced shock, hypertension, and vascular remodeling of the aorta.