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ATP6AP2 knockout in the renal nephron impairs receptor-mediated endocytosis, increasing urinary albumin and glucose excretion and impairing weight gain. Nonesterified fatty acids (NEFA) in urine are bound to albumin and reabsorbed in the proximal tubule through receptor-mediated endocytosis by the megalin-cubilin complex. We hypothesized that ATP6AP2 knockout increases urinary NEFA excretion through a reduction in megalin. Ten-week-old male C57BL/6 mice with nephron specific inducible ATP6AP2 knockout and noninduced controls were fed either normal diet (ND 12% fat) or high fat diet (HFD 45% fat) for 6 months. ATP6AP2 knockout significantly increased urine albumin:creatinine ratio in both ND and HFD fed mice while normalized urine NEFA concentration increased 489% and 259% in ND and HFD knockout mice compared to respective controls. Knockout decreased renal cortical megalin mRNA by 47% on ND and 49% on HFD while megalin protein expression decreased by 36% and 44% respectively. At the same time, markers of mTOR activity were increased while autophagy was impaired. Our results indicate that nephron specific ATP6AP2 knockout increases urinary NEFA excretion in the setting of impaired receptor-mediated endocytosis. Further investigation should determine whether ATP6AP2 contributes to obesity related ectopic lipid deposition in the proximal tubule., (© 2024. The Author(s).)

The majority of disorders that cause renal potassium wasting present with abnormalities in adrenal hormone secretion. While these findings frequently lead patients to seek endocrine evaluation, clinicians often struggle to accurately diagnose these conditions, delaying treatment and adversely impacting patient care. At the same time, growing insight into the genetic and molecular basis of these disorders continues to improve their diagnosis and management. In this review, we outline a practical integrated approach to the evaluation of renal hypokalemia syndromes that are seen in endocrine practice while highlighting recent advances in understanding of the genetics and pathophysiology behind them., (© 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.)

Objective: Morning total cortisol (TC) levels have been shown to predict adrenal dysfunction (AD) in the general population, but their utility in cirrhosis is unknown., Methods: A retrospective cohort study was performed including all noncritically ill patients at our institution between 2011 and 2022 admitted with acute decompensated cirrhosis who underwent standard-dose adrenocorticotropic hormone (ACTH) stimulation testing. Adrenal dysfunction was defined as an increase in TC (delta TC) level <9 µg/dl 60 minutes after ACTH dosing. Spearman correlation was utilized to assess the relationship between binding globulins and cortisol levels. Multivariate regression analysis was performed to determine if basal TC level or common clinical parameters were predictive of AD., Results: One hundred and nineteen patients were included, with a median model for end-stage liver disease score of 18. Albumin levels did not correlate with basal TC levels (ρ = 0.127; P = 0.169); basal TC did not correlate with delta TC (ρ = 0.050; P = 0.591). The degree of hypoalbuminemia did not alter these relationships. On multivariate regression, only albumin level [odds ratio (OR) = 0.418; 95% confidence interval (CI), 0.196-0.890; P = 0.024] and MELD score (OR, 1.094; 95% CI, 1.019-1.174; P = 0.014) were predictive of AD. Basal TC levels were not predictive of AD (OR = 0.991; 95% CI, 0.903-1.088; P = 0.855) or delta TC (β = 0.000; 95% CI -0.147 to 0.147; P = 0.999)., Conclusion: Baseline TC levels do not predict ACTH stimulation testing response in patients with acute decompensated cirrhosis. Clinicians should avoid utilizing an isolated morning cortisol result as a screening method for AD in this population., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background The renin-angiotensin system plays a crucial role in human physiology, and its main hormone, angiotensin, activates 2 G-protein-coupled receptors, the angiotensin type-1 and type-2 receptors, in almost every organ. However, controversy exists about the location, distribution, and expression levels of these receptors. Concerns have been raised over the low sensitivity, low specificity, and large variability between lots of commercially available antibodies for angiotensin type-1 and type-2 receptors, which makes it difficult to reconciliate results of different studies. Here, we describe the first non-antibody-based sensitive and specific targeted quantitative mass spectrometry assay for angiotensin receptors. Methods and Results Using a technique that allows targeted analysis of multiple peptides across multiple samples in a single mass spectrometry analysis, known as TOMAHAQ (triggered by offset, multiplexed, accurate mass, high resolution, and absolute quantification), we have identified and validated specific human tryptic peptides that permit identification and quantification of angiotensin type-1 and type-2 receptors in biological samples. Several peptide sequences are conserved in rodents, making these mass spectrometry assays amenable to both preclinical and clinical studies. We have used this method to quantify angiotensin type-1 and type-2 receptors in postmortem frontal cortex samples of older adults (n=28) with Alzheimer dementia. We correlated levels of angiotensin receptors to biomarkers classically linked to renin-angiotensin system activation, including oxidative stress, inflammation, amyloid-β load, and paired helical filament-tau tangle burden. Conclusions These robust high-throughput assays will not only catalyze novel mechanistic studies in the angiotensin research field but may also help to identify patients with an unbalanced angiotensin receptor distribution who would benefit from angiotensin receptor blocker treatment.

Background & Aims: Characterization of relative adrenal insufficiency (RAI) in cirrhosis is heterogeneous with regard to studied patient populations and diagnostic methodology. We aimed to describe the prevalence and prognostic importance of RAI in non-critically ill patients with cirrhosis., Methods: A systematic review and meta-analysis was performed using MeSH terms and Boolean operators to search five large databases (Ovid-MEDLINE, ScienceDirect, Web of Science, Cochrane Library and ClinicalTrials.gov). The population of interest was patients with cirrhosis and without critical illness. The primary outcome was the pooled prevalence of RAI as defined by a peak total cortisol level <18 μg/dl, delta total cortisol <9 μg/dl or composite of the two thresholds in response either a standard-dose or low-dose short synacthen test. Odds ratios and standardized mean differences from random-effects models estimated important clinical outcomes and patient characteristics by adrenal functional status., Results: Twenty-two studies were included in final analysis, comprising 1991 patients with cirrhosis. The pooled prevalence of RAI was 37% (95% CI 33-42%). The prevalence of RAI varied by Child-Pugh classification, type of stimulation test used, specific diagnostic threshold and by severity of illness. Ninety-day mortality was significantly higher in patients with RAI (OR 2.88, 95% CI 1.69-4.92, I2 = 15%, p < 0.001)., Conclusions: Relative adrenal insufficiency is highly prevalent in non-critically ill patients with cirrhosis and associated with increased mortality. Despite the proposed multifactorial pathogenesis, no studies to date have investigated therapeutic interventions in this specific population., (© 2022 The Authors. Liver International published by John Wiley & Sons Ltd.)

Supine hypertension-orthostatic hypotension disease poses a management challenge to clinicians. Data on short term outcomes of patients with orthostatic hypotension (OH) who are hospitalized with hypertensive (HTN) crises is lacking. The Nationwide Readmission Database 2016-2019 was queried for all hospitalizations of HTN crises. Hospitalizations were stratified according to whether OH was present or not. We employed propensity score to match hospitalizations for patients with OH to those without, at 1:1 ratio. Outcomes evaluated were 30-days readmission with HTN crises or falls, as well as hospital outcomes of in-hospital mortality, acute kidney injury, acute congestive heart failure, acute coronary syndrome, type 2 myocardial infarction, aortic dissection, stroke, length of stay (LOS), discharge to nursing home and hospitalization costs. We included a total of 9451 hospitalization (4735 in the OH group vs 4716 in the control group). OH group was more likely to be readmitted with falls (Odds ratio [OR]:3.27, P < 0.01) but not with HTN crises (P = 0.05). Both groups had similar likelihood of developing acute kidney injury (P = 0.08), stroke/transient ischemic attack (P = 0.52), and aortic dissection (P = 0.66). Alternatively, OH group were less likely to develop acute heart failure (OR:0.54, P < 0.01) or acute coronary syndrome (OR:0.39, P < 0.01) in the setting of HTN crises than non-OH group. OH group were more likely to have longer LOS and have higher hospitalization costs. Patients with OH who are admitted with HTN crises tend to have similar or lower HTN-related complications to non-OH group while having higher likelihood of readmission with falls, LOS and hospitalization costs. Further studies are needed to confirm such findings., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Losartan is an oral antihypertensive agent that is rapidly metabolized to EXP3174 (angiotensin-subtype-1-receptor blocker) and EXP3179 (peroxisome proliferator-activated receptor gamma [PPARγ] agonist), which was shown in animal studies to reduce inflammation, enhance mitochondrial energetics, and improve muscle repair and physical performance. We conducted an exploratory pilot study evaluating losartan treatment in prefrail older adults (age 70-90 years, N = 25). Participants were randomized to control (placebo) or treatment (daily oral losartan beginning at 25 mg per day and increasing every 8 weeks) for a total of 6 months. Fatigue, hyperkalemia, and hypotension were the most observed side effects of losartan treatment. Participants in the losartan group had an estimated 89% lower odds of frailty (95% confidence interval [CI]: 18% to 99% lower odds, p = .03), with a 0.3-point lower frailty score than the placebo group (95% CI: 0.01-0.5 lower odds, p = .04). Frailty score was also negatively associated with serum losartan and EXP3179 concentrations. For every one standard deviation increase in EXP3179 (ie, 0.0011 ng/μL, based on sample values above detection limit) and EXP3174 (ie, 0.27 ng/μL, based on sample values above detection limit), there was a 0.0035 N (95% CI: 0.0019-0.0051, p < .001) and a 0.0027 N (95% CI: 0.00054-0.0043, p = .007) increase in average knee strength, respectively., (© The Author(s) 2022. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hypothalamus-pituitary-adrenal axis assessment in patients with cirrhosis is challenging. The phenotype of fatigue, hypotension, electrolyte disarray, and abdominal pain characterizing primary adrenal insufficiency (AI) overlaps significantly with decompensated liver disease. Reliance on total cortisol assays in hypoproteinemic states is problematic, yet abnormal stimulated levels in cirrhosis are associated with poor clinical outcomes. Alternative measures including free plasma or salivary cortisol levels have theoretical merit but are limited by unclear prognostic significance and undefined cirrhosis-specific reference ranges. Further complicating matters is that AI in cirrhosis represents a spectrum of impairment. Although absolute cortisol deficiency can occur, this represents a minority of cases. Instead, there is an emerging concept that cirrhosis, with or without critical illness, may induce a "relative" cortisol deficiency during times of stress. In addition, the limitations posed by decreased synthesis of binding globulins in cirrhosis necessitate re-evaluation of traditional AI diagnostic thresholds., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society.)

Angiotensin II (Ang II) type-2 receptors (AT 2 R) are expressed in the adult kidney, prominently in renal proximal tubule cells (RPTCs), and play an important role in opposing renal sodium (Na + ) retention induced by Ang II stimulation of Ang II type-1 receptor (AT 1 R). Natriuresis induced by AT 1 R blockade is due at least in part to AT 2 R activation and whole body deletion of AT 2 Rs reduces the natriuretic response to increased blood pressure (BP). The major endogenous AT 2 R agonist mediating the natriuretic response is Ang III, the Ang II heptapeptide metabolite generated by aminopeptidase A, and the principal nephron site mediating inhibition of Na + reabsorption by the AT 2 R is the renal proximal tubule (RPT). AT 2 Rs induce natriuresis via a bradykinin, nitric oxide and cyclic GMP (cGMP) signaling cascade. Recent studies demonstrated a key role for protein phosphatase 2A (PP2A) in the AT 2 R-mediated natriuretic response upstream of cGMP. By inducing natriuresis, AT 2 Rs lower BP in the Ang II-infusion model of hypertension. PP2A activation and the natriuretic response to AT 2 R stimulation are defective in spontaneously hypertensive rats, a model of primary hypertension in humans. AT 2 R agonists are candidates for proximal tubule natriuretic agents in Na + and fluid retention disorders.

Type 2 myocardial infarction (T2MI) is an ischemic injury that occurs due to a mismatch between myocardial oxygen supply and demand. T2MI can occur with hypertensive crisis. Nevertheless, the impact of T2MI on hypertensive crisis outcome is poorly understood due to limited data. This study was a retrospective analysis of the National Readmission Database year 2018. Patients were included if the primary diagnosis was hypertensive crisis, hypertensive urgency, or hypertensive emergency. Patients were excluded if they had type 1 myocardial infarction (T1MI), severe sepsis, septic shock, gastrointestinal bleeding, or hemorrhagic anemia at index admission. The primary outcome was 90-day readmission with T1MI. Secondary outcomes were in-hospital mortality, length of stay, resource utilization, and all-cause 90-day readmission. Subgroup analysis was done according to urgency and emergency presentation. A total of 101,211 index hospitalizations were included in our cohort, of whom 3,644 (3.6%) received a diagnosis of T2MI. A total of 912 patients were readmitted within 90 days with T1MI. T2MI was an independent predictor of 90-day readmission with T1MI (adjusted odds ratio [aOR] 2.64, 95% confidence interval [CI] 1.90 to 3.66, p <0.01). Subgroup analysis including only hypertensive urgency and hypertensive emergency yielded similar results (aOR 2.80, 95% CI 1.56 to 5.01, p <0.01 and aOR 2.28, 95% CI 1.59 to 3.27, p <0.01, respectively). In conclusion, T2MI was an independent predictor of poor outcome in patients presenting with hypertensive crisis. Further studies are needed to guide the management of T2MI in this population., Competing Interests: Disclosures Dr. Elgendy has disclosures unrelated to this manuscript content including receiving research grants from Caladrius Biosciences, Inc. The other authors have no conflicts of interest to declare., (Copyright © 2021 Elsevier Inc. All rights reserved.)

ATP6AP2 expression is increased in the nephron during high-fat diet (HFD) and its knockout (ATP6AP2 KO) reduces body weight (WT) in mice. We evaluated the contribution of ATP6AP2 to urinary glucose (UG) and albumin (Ualb) handling during HFD. We hypothesized that nephron ATP6AP2 KO increases UG and Ualb and minimizes HFD-induced obesity. Eight-week-old male C57BL/6J mice with inducible nephron-specific ATP6AP2 KO and noninduced controls were fed either normal diet (ND, 12% kcal fat) or HFD (45% kcal fat) for 6 months. ATP6AP2 KO mice on ND had 20% (P < 0.01) lower WT compared with controls. HFD-fed mice had 41% (P < 0.05) greater WT than ND-fed control mice. In contrast, ATP6AP2 KO abrogated the increase in WT induced by HFD by 40% (P < 0.05). Mice on HFD had less caloric intake compared with ND controls (P < 0.01). There were no significant differences in metabolic rate between all groups. UG and Ualb was significantly increased in ATP6AP2 KO mice on both ND and HFD. ATP6AP2 KO showed greater levels of proximal tubule apoptosis and histologic evidence of proximal tubule injury. In conclusion, our results demonstrate that nephron-specific ATP6AP2 KO is associated with glucosuria and albuminuria, most likely secondary to renal proximal tubule injury and/or dysfunction. Urinary loss of nutrients may have contributed to the reduced WT of knockout mice on ND and lack of WT gain in response to HFD. Future investigation should elucidate the mechanisms by which loss of renal ATP6AP2 causes proximal tubule injury and dysfunction., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Recent studies suggested that renal gluconeogenesis is substantially stimulated in the kidney in presence of obesity. However, the mechanisms responsible for such stimulation are not well understood. Recently, our laboratory demonstrated that mice fed high fat diet (HFD) exhibited increase in renal Atp6ap2 [also known as (Pro)renin receptor] expression. We hypothesized that HFD upregulates renal gluconeogenesis via Atp6ap2-PGC-1α and AKT pathway. Using real-time polymerase chain reaction, western blot analysis and immunostaining, we evaluated renal expression of the Atp6ap2 and renal gluconeogenic enzymes, PEPCK and G6Pase, in wild type and inducible nephron specific Atp6ap2 knockout mice fed normal diet (ND, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 8 weeks. Compared with ND, HFD mice had significantly higher body weight (23%) (P < 0.05), renal mRNA and protein expression of Atp6ap2 (39 and 35%), PEPCK (44 and 125%) and G6Pase (39 and 44%) respectively. In addition, compared to ND, HFD mice had increased renal protein expression of PGC-1α by 32% (P < 0.05) and downregulated AKT by 33% (P < 0.05) respectively in renal cortex. Atp6ap2-KO abrogated these changes in the mice fed HFD. In conclusion, we identified novel regulation of renal gluconeogenesis by Atp6ap2 in response to high fat diet via PGC1-α/AKT-1 pathway.

Abnormal mitochondrial biogenesis and function has been linked to multiple diseases including diabetes. Recently, we demonstrated the role of renal (Pro)renin receptor (PRR) in the dysregulation of mitochondria. We hypothesized that PRR contributes to the reduction of mitochondrial biogenesis and function in diabetic kidney via PGC-1α/AMPK/SIRT-1 signaling pathway. In vivo and in vitro studies were conducted in C57BL/6 mouse and mouse renal mesangial cells (mRMCs). Control and streptozotocin-induced diabetic mice were injected with scramble or PRR shRNA and followed for a period of eight weeks. PRR mRNA and protein expression increased by 44% and 39% respectively (P<0.05) in kidneys of diabetic mice, and in mRMCs exposed to high glucose by 43 and 61% respectively compared to their respective controls. These results were accompanied by reduced mRNA and protein expressions of PGC-1α (67% and 75%), nuclear respiratory factors (NRF-1, 48% and 53%), mitochondrial transcriptional factor A (mtTFA, 56% and 40%), mitochondrial DNA copy number by 75% (all, P<0.05), and ATP production by 54%, respectively in diabetic kidneys and in mRMCs exposed to high glucose. Compared to non-diabetic control mice, PRR knockdown in diabetic mice and in mRMCs, not only attenuated the PRR mRNA and protein expression but also normalized mRNA and protein expressions of PGC-1α, NRF-1, mtTFA, mitochondrial DNA copy number, and ATP production. Treatment with AMPK inhibitor, Compound C, or SIRT-1 inhibitor, EX-527, alone, or combined with PRR siRNA caused marked reduction of mRNA expression of PGC-1α, NRF-1 and mtTFA, and ATP production in mRMCs exposed to high glucose. In conclusion, our study demonstrated the contribution of the PRR to the reduction of mitochondrial biogenesis and function in diabetic kidney disease via decreasing AMPK/SIRT-1/ PGC-1α signaling pathway., Competing Interests: The authors have declared that no competing interests exist.

Recently we demonstrated that increased renal (Pro)renin receptor (PRR) expression in diabetes contributes to development of diabetic kidney disease. However, the exact mechanisms involving PRR activity and diabetic kidney dysfunction are unknown. We hypothesized that PRR is localized in renal mitochondria and contributes to renal fibrosis and apoptosis through oxidative stress-induced mitochondria dysfunction. Controls and streptozotocin-induced diabetic C57BL/6 mice were injected with scramble shRNA and PRR shRNA and followed for a period of eight weeks. At the end of study, diabetic mice showed increased expressions of PRR and NOX4 in both total kidney tissue and renal mitochondria fraction. In addition, renal mitochondria of diabetic mice showed reduced protein expression and activity of SOD2 and ATP production and increased UCP2 expression. In diabetic kidney, there was upregulation in the expressions of caspase3, phos-Foxo3a, phos-NF-κB, fibronectin, and collagen IV and reduced expressions of Sirt1 and total-FOXO3a. Renal immunostaining revealed increased deposition of PRR, collagen and fibronectin in diabetic kidney. In diabetic mice, PRR knockdown decreased urine albumin to creatinine ratio and the renal expressions of PRR, NOX4, UCP2, caspase3, phos-FOXO3a, phos-NF-κB, collagen, and fibronectin, while increased the renal mitochondria expression and activity of SOD2, ATP production, and the renal expressions of Sirt1 and total-FOXO3a. In conclusion, increased expression of PRR localized in renal mitochondria and diabetic kidney induced mitochondria dysfunction, and enhanced renal apoptosis and fibrosis in diabetes by upregulation of mitochondria NOX4/SOD2/UCP2 signaling pathway.

Recent studies have demonstrated that the renal (pro)renin receptor (PRR) regulates expression of the alpha subunit of the epithelial sodium channel (α-ENaC). In this study we hypothesized that the renal PRR mediates high fat diet (HFD)-induced sodium retention and elevated systolic blood pressure (SBP) by enhancing expression of the epithelial sodium channel (α-ENaC). In our study we used a recently developed inducible nephron specific PRR knockout mouse. Mice (n = 6 each group) were allocated to receive regular diet (RD, 12 kcal% fat) or a high-fat diet (HFD, 45 kcal% fat) for 10 weeks. Body weight (BW), SBP, urine volume (UV) and urine sodium (UNaV), as well as renal interstitial Angiotensin II (Ang II), and renal medullary expression of PRR, p-SGK-1, α-ENaC were monitored in RD and HFD mice with or without PRR knockout. At baseline, there were no significant differences in BW, BP, UV or UNaV between different animal groups. At the end of the study, HFD mice had significant increases in SBP, BW, and significant reductions in UV and UNaV. Compared to RD, HFD significantly increased mRNA and protein expression of PRR, α-ENaC, p-SGK-1, and Ang II. Compared to HFD alone, PRR knockout mice on HFD had reduced mRNA and protein expression of PRR, p-SGK-1, and α-ENaC, as well as increased UV, UNaV and significantly reduced SBP. RIF Ang II was significantly increased by HFD and did not change in response to PRR knockout. We conclude that obesity induced sodium retention and elevated SBP are mediated by the PRR-SGK-1- α-ENaC pathway independent of Ang II., Competing Interests: The authors have declared that no competing interests exist.

Objective: Mice with global null mutation of Ceacam1 (Cc1 -/- ), display impairment of insulin clearance that causes hyperinsulinemia followed by insulin resistance, elevated hepatic de novo lipogenesis, and visceral obesity. In addition, they manifest abnormal vascular permeability and elevated blood pressure. Liver-specific rescuing of Ceacam1 reversed all of the metabolic abnormalities in Cc1 -/-liver+ mice. The current study examined whether Cc1 -/- male mice develop endothelial and cardiac dysfunction and whether this relates to the metabolic abnormalities caused by defective insulin extraction., Methods and Results: Myography studies showed reduction of agonist-stimulated nitric oxide production in resistance arterioles in Cc1 -/- , but not Cc1 -/-liver+ mice. Liver-based rescuing of CEACAM1 also attenuated the abnormal endothelial adhesiveness to circulating leukocytes in parallel to reducing plasma endothelin-1 and recovering plasma nitric oxide levels. Echocardiography studies revealed increased septal wall thickness, cardiac hypertrophy and reduced cardiac performance in Cc1 -/- , but not Cc1 -/-xliver+ mice. Insulin signaling experiments indicated compromised IRS1/Akt/eNOS pathway leading to lower nitric oxide level, and activated Shc/MAPK pathway leading to more endothelin-1 production in the aortae and hearts of Cc1 -/- , but not Cc1 -/-xliver+ mice. The increase in the ratio of endothelin-1 receptor A/B indicated an imbalance in the vasomotor activity of Cc1 -/- mice, which was normalized in Cc1 -/-xliver+ mice., Conclusions: The data underscore a critical role for impaired CEACAM1-dependent hepatic insulin clearance pathways and resulting hyperinsulinemia and lipid accumulation in aortae and heart in regulating the cardiovascular function., (Copyright © 2018 The Authors. Published by Elsevier GmbH.. All rights reserved.)

We hypothesized that PRR contributes to renal inflammation in the 2-kidney, 1-clip (2K1C) renal ischaemia model. Male Sprague-Dawley rats were fed normal sodium diet. Blood pressure (BP) was obtained on days 0 and 28 after left renal artery clipping that reduced renal blood flow by 40%. Renal expression of TNF-α, COX-2, NF-κB, IL-1β, MCP-1 and collagen type I were assessed in sham and 2K1C rats with or without left renal administration of scramble or PRR shRNA. At baseline, there were no differences in BP. Compared to sham, MAP significantly increased in clipped animals (sham 102 ± 1.9 vs 2K1C 131.8 ± 3.09 mmHg, P < .05) and was not influenced by scramble or PRR shRNA treatment. Compared to sham and contra lateral (non-clipped) kidney, there was upregulation in mRNA and protein expression of PRR (99% and 45%, P < .01), TNF-α (72% and 50%, P < .05), COX-2 (72% and 39%, P < .05), p-NF-κB (92%, P < .05), MCP-1 (87%, P < .05) and immunostaining of collagen type I in the clipped kidney. These increases were not influenced by scramble shRNA. Compared to 2K1C and scramble shRNA, PRR shRNA treatment in the clipped kidney significantly reduced the expression of PRR (62% and 57%, P < .01), TNF-α (51% and 50%, P < .05), COX-2 (50% and 56%, P < .05), p-NF-κB by 68% (P < .05), MCP-1 by 73% (P < .05) and collagen type I respectively. Ang II was increased in both kidneys and did not change in response to scramble or PRR shRNA treatments. We conclude that PRR mediates renal inflammation in renal ischaemia independent of blood pressure and Ang II., (© 2017 John Wiley & Sons Australia, Ltd.)

Purpose of Review: The intrarenal renin-angiotensin-aldosterone system (RAS) is an independent paracrine hormonal system with an increasingly prominent role in hypertension and renal disease. Two enzyme components of this system are angiotensin-converting enzyme (ACE) and more recently discovered ACE2. The purpose of this review is to describe recent discoveries regarding the roles of intrarenal ACE and ACE2 and their interaction., Recent Findings: Renal tubular ACE contributes to salt-sensitive hypertension. Additionally, the relative expression and activity of intrarenal ACE and ACE2 are central to promoting or inhibiting different renal pathologies including renovascular hypertension, diabetic nephropathy, and renal fibrosis. Renal ACE and ACE2 represent two opposing axes within the intrarenal RAS system whose interaction determines the progression of several common disease processes. While this relationship remains complex and incompletely understood, further investigations hold the potential for creating novel approaches to treating hypertension and kidney disease.

The specific aims section of National Institutes of Health and other grants is the most important component, as it summarizes the scientific premise, gap in current knowledge, hypotheses, methods, and expected results of the project proposed. The reviewer usually reads this section first and forms an immediate opinion, usually confirmed on reading the entire grant. This treatise reviews the philosophical background underlying generation of hypotheses, emphasizes the important characteristics of the specific aims section, and offers a point-by-point roadmap for writing. This perspective arose out of a new Endocrine Society initiative in which senior investigators review the specific aims of next-generation members.

Autophagy plays a major role in podocytes health and disease. P62, also known as sequestosome-1 (SQSTM1), is a marker for autophagic activity and is required for the formation and degradation of ubiquitnated protein by autophagy. Knockout of p62 enhanced extracellular signal-regulated kinases (ERK1/2) activity. (pro)renin receptor (PRR) is expressed in podocytes where it contributes to the homeostasis of these cells. The influence of autophagy on PRR expression is unknown. We hypothesized that in podocytes, upregulation of autophagic activity increases PRR expression via reduction of p62 and stimulation of ERK1/2 signaling pathway. Cultured mouse podocytes were treated with the autophagy activators, rapamycin or Earle's balanced salt solution (EBSS), for 48 h. Both rapamycin and EBSS significantly decreased p62 protein levels, increased ERK1/2 activation by phosphorylating pTpY185/187, and increased mRNA and protein expressions of PRR. Utilizing confocal microscopy demonstrated that rapamycin and EBSS significantly decreased p62/SQSTM1 and increased PRR protein expressions. Similarly, by enhancing autophagic activity by transfection with autophagy-related 5 (ATG5) cDNA or ATG7 cDNA, results similar to those observed with rapamycin and EBSS treatments were produced. Inhibition of autophagic flux with bafilomycin A1 reversed the effects of rapamycin. ERK1/2 inhibitor U0126 significantly attenuated mRNA and protein expressions of PRR in podocytes treated with rapamycin. In conclusion, upregulation of autophagy enhanced PRR expression through reduction of p62 and stimulation of ERK1/2 activity signaling pathway., (Copyright © 2017 the American Physiological Society.)

Cyclooxygenase-2 (COX-2) plays an important role in mediating actions of the renin-angiotensin system (RAS). This review sheds light on the recent developments regarding the complex interactions between components of RAS and COX-2; and their implications on renal function and disease. COX-2 is believed to counter regulate the effects of RAS activation and therefore counter balance the vasoconstriction effect of Ang II. In kidney, under normal conditions, these systems are essential for maintaining a balance between vasodilation and vasoconstriction. However, recent studies suggested a pivotal role for this interplay in pathology. COX-2 increases the renin release and Ang II formation leading to increase in blood pressure. COX-2 is also associated with diabetic nephropathy, where its upregulation in the kidney contributes to glomerular injury and albuminuria. Selective inhibition of COX-2 retards the progression of renal injury. COX-2 also mediates the pathologic effects of the (Pro)renin receptor (PRR) in the kidney. In summary, this review discusses the interaction between the RAS and COX-2 in health and disease.

Background: Recent studies reported increased (Pro)renin receptor (PRR) expression during low-salt intake. We hypothesized that PRR plays a role in regulation of renal epithelial sodium channel (ENaC) through serum and glucocorticoid-inducible kinase isoform 1 (SGK-1)-neural precursor cell expressed, developmentally downregulated 4-2 (Nedd4-2) signaling pathway., Method: Male Sprague-Dawley rats on normal-sodium diet and mouse renal inner medullary collecting duct cells treated with NaCl at 130  mmol/l (normal salt), or 63  mmol/l (low salt) were studied. PRR and α-ENaC expressions were evaluated 1 week after right uninephrectomy and left renal interstitial administration of 5% dextrose, scramble shRNA, or PRR shRNA (n = 6 each treatment)., Results: In-vivo PRR shRNA significantly reduced expressions of PRR throughout the kidney and α-ENaC subunits in the renal medulla. In inner medullary collecting duct cells, low salt or angiotensin II (Ang II) augmented the mRNA and protein expressions of PRR (P < 0.05), SGK-1 (P < 0.05), and α-ENaC (P < 0.05). Low salt or Ang II increased the phosphorylation of Nedd4-2. In cells treated with low salt or Ang II, PRR siRNA significantly downregulated the mRNA and protein expressions of PRR (P < 0.05), SGK-1 (P < 0.05), and α-ENaC expression (P < 0.05)., Conclusion: We conclude that PRR contributes to the regulation of α-ENaC via SGK-1-Nedd4-2 signaling pathway.

Introduction: Previous studies demonstrated that stimulation of angiotensin subtype 1 receptor (AT1R) led to increased renal generation of prostaglandins E2 (PGE2) and renal inflammation. In turn, PGE2 increases AT1R activity. The conversion of PGE2 to the less active metabolite prostaglandin F2α (PGF2α) via 9-ketoreductase interrupts this feedback loop. The effects of diabetes on the interface between AT1R, PGE2 and PGF2α are not well established. We hypothesized that in diabetes, an aberrant AT1R activity enhances the biosynthesis of PGE2 and impairs the activity of PGE 9-ketoreductase, leading to accumulation of PGE2., Materials and Methods: Using microdialysis technique, we monitored renal interstitial fluid levels of angiotensin II (Ang II), PGE2 and PGF2α in control and AT1R blocker, valsartan, treated diabetic rats (N=8 each). We utilized the PGF2α to PGE2 ratio as indirect measure of PGE 9-ketoreductase activity., Results: Diabetes increased renal interstitial fluid levels of Ang II, PGE2 and PGF2α. PGF2α/PGE2 ratio increased by the third week, but declined by the sixth week of diabetes. Valsartan reduced PGE2 and PGF2α levels and increased Ang II and the conversion of PGE2 to PGF2α., Conclusion: Our results suggest that in diabetes, AT1R increases PGE2 generation and reduces conversion of PGE2 to PGF2α with the progression of diabetes., (© The Author(s) 2015.)

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAMl), a substrate of the insulin receptor tyrosine kinase, regulates insulin action by promoting insulin clearance. Global null mutation of Ceacam1 gene (Cc1(-/-)) results in features of the metabolic syndrome, including insulin resistance, hyperinsulinemia, visceral adiposity, elevated blood pressure, and albuminuria. It also causes activation of the renal renin-angiotensin system (RAS). In the current study, we tested the hypothesis that high-fat diet enhances the expression of RAS components. Three-month-old wild-type (Cc1(+/+)) and Cc1(-/-) mice were fed either a regular or a high-fat diet for 8 wk. At baseline under regular feeding conditions, Cc1(-/-) mice exhibited higher blood pressure, urine albumin-to-creatinine ratio (UACR), and renal expression of angiotensinogen, renin/prorenin, angiotensin-converting enzyme, (pro)renin receptor, angiotensin subtype AT1 receptor, angiotensin II, and elevated PI3K phosphorylation, as detected by p85α (Tyr(508)) immunostaining, inflammatory response, and the expression of collagen I and collagen III. In Cc1(+/+) mice, high-fat diet increased blood pressure, UACR, the expression of angiotensin-converting enzyme and angiotensin II, PI3K phosphorylation, inflammatory response, and the expression of collagen I and collagen III. In Cc1(-/-) mice, high-fat intake further amplified these parameters. Immunohistochemical staining showed increased p-PI3K p85α (Tyr(508)) expression in renal glomeruli, proximal, distal, and collecting tubules of Cc1(-/-) mice fed a high-fat diet. Together, this demonstrates that high-fat diet amplifies the permissive effect of Ceacam1 deletion on renal expression of all RAS components, PI3K phosphorylation, inflammation, and fibrosis., (Copyright © 2015 the American Physiological Society.)

High glucose reduces autophagy and enhances apoptosis of podocytes. Previously, we reported that high glucose induced podocyte injury through upregulation of the (pro)renin receptor (PRR). We hypothesized that increasing PRR reduces autophagy and increases apoptosis of mouse podocytes exposed to high glucose via activation of the PI3K/Akt/mTOR signaling pathway. Mouse podocytes were cultured in normal (5 mmol/l) or high (25 mmol/l) d-glucose for 48 h. High glucose significantly increased mRNA and protein levels of PRR, phosphorylation of PI3K/Akt/mTOR, and p62. In contrast, high glucose decreased activation of UNC-51-like kinase-1 (ULK1) by phosphorylating Ser⁷⁵⁷ and protein levels of microtubule-associated protein-1 light chain 3B (LC3B)-II and Lamp-2. Bafilomycin A1 increased LC3BII and p62 accumulation in high-glucose-treated cells. High glucose reduced the autophagic flux. Confocal microscopy studies showed significant reduction in the protein level of LC3B in response to high glucose. Cyto-ID autophagy staining showed a significant decrease in autophagosome formation with high glucose. In the absence of PRR, activation of Akt with sc-79 or mTOR with MHY-1485 increased p62 accumulation. Caspase-3/7 activity and apoptosis monitored by TUNEL assay were significantly increased in podocytes treated with high glucose. PRR siRNA significantly reversed the effects of high glucose. Based on these data, we conclude that high glucose decreases autophagy and increases apoptosis in mouse podocytes through the PRR/PI3K/Akt/mTOR signaling pathway., (Copyright © 2015 the American Physiological Society.)

The prorenin receptor (PRR), a recently discovered component of the renin-angiotensin system, is expressed in the nephron in general and the collecting duct in particular. However, the physiological significance of nephron PRR remains unclear, partly due to developmental abnormalities associated with global or renal-specific PRR gene knockout (KO). Therefore, we developed mice with inducible nephron-wide PRR deletion using Pax8-reverse tetracycline transactivator and LC-1 transgenes and loxP flanked PRR alleles such that ablation of PRR occurs in adulthood, after induction with doxycycline. Nephron-specific PRR KO mice have normal survival to ∼1 yr of age and no renal histological defects. Compared with control mice, PRR KO mice had 65% lower medullary PRR mRNA and protein levels and markedly diminished renal PRR immunofluorescence. During both normal water intake and mild water restriction, PRR KO mice had significantly lower urine osmolality, higher water intake, and higher urine volume compared with control mice. No differences were seen in urine vasopressin excretion, urine Na(+) and K(+) excretion, plasma Na(+), or plasma osmolality between the two groups. However, PRR KO mice had reduced medullary aquaporin-2 levels and arginine vasopressin-stimulated cAMP accumulation in the isolated renal medulla compared with control mice. Taken together, these results suggest nephron PRR can potentially modulate renal water excretion., (Copyright © 2015 the American Physiological Society.)

We hypothesized that direct AT2R stimulation improves albuminuria in diabetes by preventing renal inflammation and improving oxidative stress. Normoglycemic controls (NCs) and streptozotocin-induced diabetes Sprague-Dawley rats (DM) were treated for 4 weeks with vehicle (V) or the AT2R agonist Compound 21 (C21). At the end of study, we evaluated blood pressure, urinary albumin to creatinine ratio (UACR), renal interstitial fluid (RIF) levels of tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), nitric oxide (NO), cGMP, and 8-isoprostane, and renal expression of TNF-α, IL-6, and AT2R. There were no significant differences in blood pressure between different treatments. DM rats demonstrated increased UACR, RIF TNF-α, IL-6 and 8-isoprostane, and messenger RNA (mRNA) for TNF-α and IL-6. DM rats also had reduced RIF NO and cGMP. C21 treatment of DM rats limited the increase in UACR, normalized RIF TNF-α, IL-6 and 8-isoprostane, and in mRNA for TNF-α and IL-6, and increased RIF NO and cGMP. In NC rats, C21 treatment did not change these parameters. AT2R mRNA and protein expressions increased in DM rats compared with NC but were not influenced by C21 treatment. We conclude that direct AT2R stimulation in diabetic rats improves diabetic albuminuria through the prevention of renal inflammation and improved production of NO and cGMP.

Although angiotensin II subtype-2 receptor (AT2R) was discovered over 2 decades ago, its contribution to physiology and pathophysiology is not fully elucidated. Current knowledge suggests that under normal physiologic conditions, AT2R counterbalances the effects of angiotensin II subtype-1 receptor (AT1R). A major obstacle for AT2R investigations was the lack of specific agonists. Most of the earlier AT2R studies were performed using the peptidic agonist, CG42112A, or the nonpeptidic antagonist PD123319. CGP42112A is nonspecific for AT2R and in higher concentrations can bind to AT1R. Recently, the development of specific nonpeptidic AT2R agonists boosted the efforts in identifying the therapeutic potentials for AT2R stimulation. Unlike AT1R, AT2R is involved in vasodilation by the release of bradykinin and nitric oxide, anti-inflammation, and healing from injury. Interestingly, the vasodilatory effects of AT2R stimulation were not associated with significant reduction in blood pressure. In the kidney, AT2R stimulation produced natriuresis, increased renal blood flow, and reduced tissue inflammation. In animal studies, enhanced AT2R function led to reduction of cardiac inflammation and fibrosis, and reduced the size of the infarcted area. Similarly, AT2R stimulation demonstrated protective effects in vasculature and brain.

The localization and regulation of (pro)renin receptor (PRR) expression in kidney collecting duct cells are not well established. We hypothesized that low salt (LS) contributes to the regulation of PRR expression in these cells via the GSK-3β-NFAT5-sirtuin1 (SIRT-1) signaling pathway. Mouse inner medullary collecting duct (mIMCD) cells were treated with NaCl at 130 (normal salt; NS), 63 (LS), or 209 mM (high salt; HS) alone or in combination with NFAT5 scrambled small interfering (si) RNA, NFAT5 siRNA, or the SIRT-1 inhibitor EX-527. Compared with NS, LS increased the mRNA and protein expression of PRR by 71% and 69% (P < 0.05), and reduced phosphorylation of GSK-3β by 62% (P < 0.01), mRNA and protein expressions of NFAT5 by 65% and 45% (P < 0.05), and SIRT-1 by 44% and 50% (P < 0.01), respectively. LS also enhanced p65 NF-κB by 102% (P < 0.01). Treatment with HS significantly reduced the mRNA and protein expression of PRR by 32% and 23% (P < 0.05), and increased the mRNA and protein expression of NFAT5 by 39% and 45% (P < 0.05) and SIRT-1 by 51% and 56% (P < 0.05), respectively. HS+NFAT5 siRNA reduced the mRNA and protein expression of NFAT5 by 51% and 35% (P < 0.01) and increased the mRNA and protein expression of PRR by 148% and 70% (P < 0.01), respectively. HS+EX-527 significantly increased the mRNA and protein expression of PRR by 96% and 58% (P < 0.05), respectively. We conclude that expression of PRR in mIMCD cells is regulated by the GSK-3β-NFAT5- SIRT-1 signaling pathway., (Copyright © 2014 the American Physiological Society.)

(Pro)renin receptor (PRR) expression is upregulated in diabetes. We hypothesized that PRR contributes to podocyte injury via activation of Wnt-β-catenin-snail signaling pathway. Mouse podocytes were cultured in normal (5 mM) or high (25 mM) D-glucose for 3 days. Compared to normal glucose, high glucose significantly decreased mRNA and protein expressions of podocin and nephrin, and increased mRNA and protein expressions of PRR, Wnt3a, β-catenin, and snail, respectively. Confocal microscopy studies showed significant reduction in expression and reorganization of podocyte cytoskeleton protein, F-actin, in response to high glucose. Transwell functional permeability studies demonstrated significant increase in albumin flux through podocytes monolayer with high glucose. Cells treated with high glucose and PRR siRNA demonstrated significantly attenuated mRNA and protein expressions of PRR, Wnt3a, β-catenin, and snail; enhanced expressions of podocin mRNA and protein, improved expression and reorganization of F-actin, and reduced transwell albumin flux. We conclude that high glucose induces podocyte injury via PRR-Wnt-β-catenin-snail signaling pathway.

The carcinoembryonic antigen-related cell adhesion molecule 1 regulates insulin sensitivity by promoting hepatic insulin clearance. Mice bearing a null mutation of Ceacam1 gene (Cc1(-/-)) develop impaired insulin clearance followed by hyperinsulinemia and insulin resistance, in addition to visceral obesity and increased plasma fatty acids. Because insulin resistance is associated with increased blood pressure, we investigated whether they develop higher blood pressure with activated renal renin-angiotensin system and whether this is mediated, in part, by the upregulation of renal (pro)renin receptor (PRR) expression. Compared with age-matched wild-type littermates, Cc1(-/-) mice exhibited increased blood pressure with increased activation of renal renin-angiotensin systems and renal PRR expression. Cytoplasmic and nuclear immunostaining of phospho-PI3K p85α and phospho-Akt was enhanced in the kidney of Cc1(-/-) mice. In murine renal inner medullary collecting duct epithelial cells with lentiviral-mediated small hairpin RNA knockdown of carcinoembryonic antigen-related cell adhesion molecule 1, PRR expression was upregulated and phosphorylation of PI3K (Tyr508), Akt (Ser473), NF-κB p65 (Ser276), cAMP response element-binding protein/activated transcription factor (ATF)-1 (Ser133), and ATF-2 (Thr71) was enhanced. Inhibiting PI3K with LY294002 or Akt with Akt inhibitor VIII attenuated PRR expression. In conclusion, global null deletion of Ceacam1 caused an increase in blood pressure with increased renin-angiotensin system activation together with upregulation of PRR via PI3K-Akt activation of cAMP response element-binding protein 1, ATF-1, ATF-2, and NF-κB p65 transcription factors.

Aldosterone is increased in diabetes and contributes to the development of diabetic nephropathy. The authors hypothesized that reduction in aldosterone production in diabetes by amlodipine or aliskiren improves diabetic kidney disease by attenuating renal oxidative stress and fibrosis. Normoglycemic and streptozotocin-induced diabetic Sprague-Dawley rats were given vehicle, amlodipine, or aliskiren alone and combined for 6 weeks. At the end of study, we evaluated blood pressure (BP), 24-hour urinary sodium (UNaV) and aldosterone excretion rates, renal interstitial fluid (RIF) levels of nitric oxide (NO), cyclic guanosine 3',5'-monophosphate (cGMP), and 8-isoprostane, and renal morphology. BP was not significantly different between any of experimental groups. UNaV increased in diabetic animals and was not affected by different treatments. Urinary aldosterone excretion increased in diabetic rats receiving vehicle and decreased with amlodipine and aliskiren alone or combined. RIF NO and cGMP levels were reduced in vehicle-treated diabetic rats and increased with amlodipine or aliskiren given alone and combined. RIF 8-isoprostane levels and renal immunostaining for periodic acid-Schiff and fibronectin were increased in vehicle-treated diabetic rats and decreased with aliskiren alone or combined with amlodipine. The authors conclude that inhibition of aldosterone by amlodipine or aliskiren ameliorates diabetes induced renal injury via improvement of NO-cGMP pathway and reduction in oxidative stress and fibrosis, independent of BP changes.

Effects of low salt (LS) on (pro)renin receptor (PRR) expression are not well established. We hypothesized that LS enhances renal PRR expression via the cGMP-protein kinase G (PKG) signaling pathway. Sprague-Dawley rats were fed a normal-salt (NS) or LS diet associated with intrarenal cortical administration of vehicle (V), the nitric oxide (NO) synthase inhibitor nitro-l-arginine methyl ester (l-NAME), the NO donor S-nitroso-N-acetyl-dl-penicillamine (SNAP), the cGMP analog 8-bromoguanosine (8-Br)-cGMP, the guanylyl cyclase inhibitor 1H-[1, 2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), or a PKG inhibitor (PKGi) for 6 days via osmotic minipump. We evaluated the effects of each treatment on renal interstitial fluid (RIF) levels of nitrate/nitrite and cGMP and renal PRR expression. There were no significant changes in blood pressure with any of the treatments. Urinary sodium excretion was significantly lower in rats given a LS diet. Compared with NS + V, RIF nitrate/nitrite and cGMP levels increased in LS + V rats. In NS groups, RIF nitrate/nitrite and cGMP levels did not change with l-NAME, ODQ, or PKGi and increased in response to SNAP. 8-Br-cGMP increased RIF cGMP but not RIF nitrate/nitrite. In LS groups, RIF nitrate/nitrite decreased with l-NAME and did not change with ODQ or PKGi whereas RIF cGMP decreased with l-NAME, ODQ, and PKGi. PRR mRNA and protein increased in LS + V. In NS rats, PRR mRNA and protein increased in response to 8-Br-GMP and were not affected by any of other treatments. In LS rats, PRR mRNA and protein decreased significantly in response to l-NAME, ODQ, and PKGi. We conclude that LS intake enhances renal expression of PRR via cGMP-PKG signaling pathway.

We hypothesized that compared with hydrochlorothiazide (HCTZ), the renin inhibitor aliskiren (ALISK) or amlodipine (AMLO) and their combination reduce albuminuria via reduction in renal inflammation, independent of blood pressure (BP) changes. We studied normal and streptozotocin-induced diabetic (DM) Sprague-Dawley rats treated for 6 weeks with vehicle, ALISK, HCTZ, or AMLO individually and combined and evaluated the effects of treatments on BP, urine albumin to creatinine ratio, renal interstitial fluid levels of angiotensin II, tumor necrosis factor alpha (TNF-α), and interleukin 6 (IL-6) and renal expression of TNF-α, IL-6, transforming growth factor beta 1, and nuclear factor kappa B. There were no differences in BP between treatments. Only ALISK and its combinations reduced renal interstitial fluid angiotensin II. Urine albumin to creatinine ratio increased in DM rats and decreased with ALISK alone or combined with HCTZ or AMLO. HCTZ or AMLO individually and combined did not influence urine albumin to creatinine ratio. Renal interstitial fluid TNF-α and IL-6, and the renal expression of TNF-α, IL-6, transforming growth factor beta 1, and nuclear factor kappa B were increased in DM rats. These renal inflammatory markers were reduced only with ALISK or AMLO individually or combined with other treatments. We conclude that ALISK alone and combined with HCTZ or AMLO reduced albuminuria in diabetes via reduction in renal inflammation, independent of BP changes.

(Pro)renin receptor (PRR) is expressed in renal vasculature, glomeruli, and tubules. The physiological regulation of this receptor is not well established. We hypothesized that sodium depletion increases PRR expression through cGMP- protein kinase G (PKG) signaling pathway. Renal PRR expressions were evaluated in Sprague-Dawley rats on normal sodium or low-sodium diet (LS) and in cultured rat proximal tubular cells and mouse renal inner medullary collecting duct cells exposed to LS concentration. LS augmented PRR expression in renal glomeruli, proximal tubules, distal tubules, and collecting ducts. LS also increased cGMP production and PKG activity. In cells exposed to normal sodium, cGMP analog increased PKG activity and upregulated PRR expression. In cells exposed to LS, blockade of guanylyl cyclase with 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one decreased PKG activity and downregulated PRR expression. PKG inhibition decreased phosphatase protein phosphatase 2A activity; suppressed LS-mediated phosphorylation of extracellular signal-regulated kinase, c-Jun N-terminal kinase, c-Jun, and nuclear factor-κB p65; and attenuated LS-mediated PRR upregulation. LS also enhanced DNA binding of cAMP response element binding protein 1 to cAMP response elements, nuclear factor-κB p65 to nuclear factor-κB elements, and c-Jun to activator protein 1 elements in PRR promoter in proximal tubular cells. We conclude that sodium depletion upregulates renal PRR expression via the cGMP-PKG signaling pathway by enhancing binding of cAMP response element binding protein 1, nuclear factor-κB p65, and c-Jun to PRR promotor.

Angiotensin subtype-1 receptor (AT(1)R) influences inflammatory processes through enhancing signal transducer and activator of transcription proteins 3 (STAT3) signal transduction, resulting in increased tumor necrosis factor-α (TNF-α) production. Although angiotensin subtype-2 receptor (AT(2)R), in general, antagonizes AT(1)R-stimulated activity, it is not known if AT(2)R has any anti-inflammatory effects. In this study, we tested the hypothesis that AT(2)R activation plays an anti-inflammatory role by reducing STAT3 phosphorylation and TNF-α production. Changes in AT(2)R expression, TNF-α production, and STAT3 phosphorylation were quantified by Western blotting, Bio-Plex cytokine, and phosphoprotein cellular signaling assays in PC12W cells that express AT(2)R but not AT(1)R, in response to the AT(2)R agonist, CGP-42112 (CGP, 100 nm), or AT(2)R antagonist PD-123319 (PD, 1 μm). A 100% increase in AT(2)R expression in response to stimulation with its agonist CGP was observed. Further, AT(2)R activation reduced TNF-α production by 39% and STAT3 phosphorylation by 83%. In contrast, PD decreased AT(2)R expression by 76%, increased TNF-α production by 84%, and increased STAT3 phosphorylation by 67%. These findings suggest that increased AT(2)R expression may play a role in the observed decrease in inflammatory pathway activation through decreased TNF-α production and STAT3 signaling. Restoration of AT(2)R expression and/or its activation constitute a potentially novel therapeutic target for the management of inflammatory processes.

1. Transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) are expressed in renal glomeruli, and contribute to the development of diabetic nephropathy. Recently, we showed that (pro)renin receptor (PRR) is upregulated in the kidneys of the streptozocin (STZ)-induced diabetes rat model. We hypothesized that in the presence of hyperglycaemia, increased renal PRR expression contributes to enhanced TGF-β1-CTGF signalling activity, leading to the development of diabetic kidney disease. 2. In vivo and in vitro studies were carried out in Sprague-Dawley rats and rat mesangial cells (RMC). PRR blockade was achieved in vivo by treating STZ induced diabetes rats with the handle region peptide (HRP) of prorenin and in vitro by HRP or PRR siRNA in RMC. Angiotensin AT1 receptor blockade was achieved by valsartan treatment. 3. Results showed that expression of PRR, TGF-β1 and CTGF were upregulated in diabetic kidneys and RMC exposed to high glucose. Glucose exposure also induced PRR phosphorylation, a process that was inhibited by HRP, valsartan or PRR siRNA. HRP and valsartan significantly attenuated renal TGF-β1 and CTGF expression in diabetic animals and high glucose treated RMC. Similar results were observed in high glucose exposed RMC in response to PRR siRNA. TGF-β receptor blockade decreased CTGF expression in RMC. Combined administration of valsartan and PRR siRNA showed further reduction of TGF-β1 and CTGF expression in RMC. 4. In conclusion, PRR contributes to kidney disease in diabetes through an enhanced TGF-β1-CTGF signalling cascade., (© 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.)

Inhibition of the renin-angiotensin system has been a highly successful therapeutic approach for the prevention of hypertension-related end organ damage. Angiotensin converting enzyme inhibitors and angiotensin II receptor blockers lower blood pressure and reduce morbidity and mortality in patients with cardiovascular and kidney disease. However, progression to end-stage disease remains common in these patient populations. A compensatory increase in plasma renin activity occurs with the use of either angiotensin converting enzyme inhibitors or angiotensin II receptor blockers, thus causing increased levels of angiotensin II, which may limit the therapeutic effectiveness of these agents. The direct renin inhibitor, aliskiren, suppresses the renin-angiotensin system by inhibiting its first and rate-limiting step. This early inhibition reduces the production of all downstream components of the system. In this review, recent clinically relevant advances in the understanding of renin-angiotensin system biology are explored as a rationale for combining aliskiren with other blockers of the renin-angiotensin system. These combinations more fully inhibit the renin-angiotensin system, with the goal of providing additional therapeutic benefits in diseases associated with chronic activation of the renin-angiotensin system.

Angiotensin II type 2 receptor (AT₂R) counteracts most effects of angiotensin II type 1 receptor (AT(1)R). We hypothesized that direct AT₂R stimulation reduces renal production of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and transforming growth factor-β1 (TGF-β1) and enhances the production of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) in the clipped kidney of 2-kidney, 1-clip (2K1C) hypertension rat model. We used Sprague-Dawley rats to evaluate changes in renal interstitial fluid recovery levels of TNF-α, IL-6, NO, and cGMP; renal expression of AT₁R, AT₂R, TGF-β1, TNF-α, and IL-6 in sham and 2K1C rats treated for 4 days with vehicle, AT₂R agonist compound 21 (C21), or AT₂R antagonist PD123319 (PD), alone and combined (n=6, each group). Systolic blood pressure increased significantly in 2K1C and was not influenced by any treatment. Clipped kidneys showed significant increases in renal expression of AT₁R, AT₂R, TNF-α, IL-6, TGF-β1 and decreases in NO and cGMP levels. These factors were not influenced by PD treatment. In contrast, C21 caused significant decrease in renal TNF-α, IL-6, TGF-β1 and an increase in NO and cGMP levels. Combined C21 and PD treatment partially reversed the observed C21 effects. Compared to sham, there were no significant changes in TNF-α, IL-6, TGF-β1, NO, or cGMP in the nonclipped kidneys of 2K1C animals. We conclude that direct AT₂R stimulation reduces early renal inflammatory responses and improves production of NO and cGMP in renovascular hypertension independent of blood pressure reduction.

The safety of angiotensin II receptor blockers (ARBs) for the treatment of hypertension and cardiovascular and renal diseases has been well documented in numerous randomized clinical trials involving thousands of patients. However, recent concerns have surfaced about possible links between ARBs and increased risks of myocardial infarction and cancer. Less is known about the safety of the direct renin inhibitor aliskiren, which was approved as an antihypertensive in 2007. This article provides a detailed review of the safety of ARBs and aliskiren, with an emphasis on the risks of cancer and myocardial infarction associated with ARBs. Safety data were identified by searching PubMed and Food and Drug Administration (FDA) Web sites through April 2011. ARBs are generally well tolerated, with no known class-specific adverse events. The possibility of an increased risk of myocardial infarction associated with ARBs was suggested predominantly because the Valsartan Antihypertensive Long-Term Use Evaluation (VALUE) trial reported a statistically significant increase in the incidence of myocardial infarction with valsartan compared with amlodipine. However, no large-scale, randomized clinical trials published after the VALUE study have shown a statistically significant increase in the incidence of myocardial infarction associated with ARBs compared with placebo or non-ARBs. Meta-analyses examining the risk of cancer associated with ARBs have produced conflicting results, most likely due to the inherent limitations of analyzing heterogeneous data and a lack of published cancer data. An ongoing safety investigation by the FDA has not concluded that ARBs increase the risk of cancer. Pooled safety results from clinical trials indicate that aliskiren is well tolerated, with a safety profile similar to that of placebo. ARBs and aliskiren are well tolerated in patients with hypertension and certain cardiovascular and renal conditions; their benefits outweigh possible safety concerns.

Renal (pro)renin receptor (PRR) expression is increased in diabetes. The exact mechanisms involved in this process are not well established. We hypothesized that high glucose up-regulates PRR through protein kinase C (PKC)-Raf-ERK and PKC-c-Jun N-terminal kinase (JNK)-c-Jun signaling pathways. Rat mesangial cells exposed to 30 mm d-glucose demonstrated significant increase in PRR mRNA and protein expression, intracellular phosphorylation of Raf-1 (Y340/341), ERK, JNK, nuclear factor-kappaB (NF-kappaB) p65 (S536) and c-Jun (S63). By chromatin immunoprecipitation assay and EMSA, high glucose induced more functional NF-kappaB and activator protein (AP)-1 dimers bound to corresponding cis-regulatory elements in the predicted PRR promoter to up-regulate PRR transcription. Conventional and novel PKC inhibitors Chelerythrine and Rottlerin, Raf-1 inhibitor GW5074, MEK1/2 inhibitor U0126, JNK inhibitor SP600125, NF-kappaB inhibitor Quinazoline, and AP-1 inhibitor Curcumin, respectively, attenuated glucose-induced PRR up-regulation. Chelerythrine and Rottlerin also inhibited glucose-induced phosphorylation of Raf-1 (Y340/341), ERK1/2, JNK, NF-kappaB p65 (S536), and c-Jun (S63). GW5074 and U0126 inhibited the phosphorylation of ERK1/2 and NF-kappaB p65 (S536). SP600125 inhibited phosphorylation of NF-kappaB p65 (S536) and c-Jun (S63). We conclude that high glucose up-regulates the expression of PRR through mechanisms dependent on both PKC-Raf-ERK and PKC-JNK-c-Jun signaling pathways. NF-kappaB and AP-1 are involved in high-glucose-induced PRR up-regulation in rat mesangial cells.