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The renin-angiotensin system is a powerful regulator of arterial pressure and body fluid volume. Increasing evidence suggests that the angiotensin type 2 receptor (AT(2)R), which mediates the vasodilatory and natriuretic actions of angiotensin peptides, is enhanced in females and may, therefore, represent an innovative therapeutic target. We investigated the therapeutic potential of direct AT(2)R stimulation on renal function in 11- to 12-week-old anesthetized male and female Sprague-Dawley rats. Renal blood flow was examined in response to a graded infusion of the highly selective, nonpeptide AT(2)R agonist, compound 21 (100, 200, and 300 ng/kg per minute), in the presence and absence of AT(2)R blockade (PD123319; 1 mg/kg per hour). Direct AT(2)R stimulation significantly increased renal blood flow in both males and females, without influencing arterial pressure. This was dose dependent in females only and occurred to a greater extent in females at the highest dose of compound 21 administered (males: 13.1±2.4% versus females: 23.0±3.2% change in renal blood flow at 300 ng/kg per minute versus baseline; P<0.01). In addition, AT(2)R stimulation significantly increased sodium and water excretion to a similar extent in males and females (P(Group)=0.05 and 0.005). However, there was no significant change in glomerular filtration rate in either sex, suggesting that altered tubular function may be responsible for AT(2)R-induced natriuresis rather than hemodynamic effects. Taken together, this study provides evidence that direct AT(2)R stimulation produces vasodilatory and natriuretic effects in the male and female kidney. The AT(2)R may, therefore, represent a valuable therapeutic target for the treatment of renal and cardiovascular diseases in both men and women. [ABSTRACT FROM AUTHOR]

Stimulation of the angiotensin II type 2 receptor (AT 2 R) evokes protective effects in various cardiovascular diseases. Thus, this study aimed to investigate the effects of AT 2 R stimulation, with or without AT 1 R blockade, in a model of hypertension with concomitant type 1 diabetes mellitus (T1DM). Spontaneously hypertensive rats (SHRs) were given either citrate or a single dose of streptozotocin (STZ; 55 mg/kg, i.p.) to induce diabetes. After 4 weeks of diabetes, animals were administered either a vehicle (saline), AT 2 R agonist, β-Pro 7 Ang III (0.1 mg/kg/day via osmotic mini-pump), AT 1 R blocker, candesartan (2 mg/kg/day via drinking water), or a combination of both for a further 8 weeks. β-Pro 7 Ang III treatment had no effect on blood pressure, but attenuated the significant increase in cardiac interstitial collagen and protein expression of fibrotic and inflammatory markers, and superoxide levels that was evident in diabetic SHRs. These effects were not observed with candesartan, despite its blood pressure lowering effects. Although β-Pro 7 Ang III had no effect on aortic fibrosis, it significantly attenuated MCP-1 protein expression and superoxide levels when compared to both the non-diabetic and diabetic SHRs, to a similar extent as candesartan. In both the heart and vasculature, the effects of β-Pro 7 Ang III in combination with candesartan were similar to those of β-Pro 7 Ang III alone, and superior to candesartan alone. It was concluded that in hypertension with concomitant diabetes, AT 2 R stimulation with a novel ligand alone, or in combination with AT 1 R blockade, improved the cardiac and vascular structural changes that were strongly associated with inflammation and oxidative stress, independent of blood pressure regulation., Competing Interests: Declaration of Competing Interest Please declare any financial or personal interests that might be potentially viewed to influence the work presented. Interests could include consultancies, honoraria, patent ownership or other. If there are none state ‘there are none’., (Copyright © 2023. Published by Elsevier Masson SAS.)

A solitary functioning kidney (SFK) from birth predisposes to hypertension and kidney dysfunction, and this may be associated with impaired fluid and sodium homeostasis. Brief and early angiotensin-converting enzyme inhibition (ACEi) in a sheep model of SFK delays onset of kidney dysfunction. We hypothesized that modulation of the renin-angiotensin system via brief postnatal ACEi in SFK would reprogram renal sodium and water handling. Here, blood pressure (BP), kidney haemodynamics and kidney excretory function were examined in response to an isotonic saline load (0.13 ml/kg/min, 180 min) at 20 months of age in SFK (fetal unilateral nephrectomy at 100 days gestation; term 150 days), sham and SFK+ACEi sheep (ACEi in SFK 4-8 weeks of age). Basal BP was higher in SFK than sham (∼13 mmHg), and similar between SFK and SFK+ACEi groups. Saline loading caused a small increase in BP (∼3-4 mmHg) the first 2 h in SFK and sham sheep but not SFK+ACEi sheep. Glomerular filtration rate did not change in response to saline loading. Total sodium excretion was similar between groups. Total urine excretion was similar between SFK and sham animals but was ∼40% less in SFK+ACEi animals compared with SFK animals. In conclusion, the present study indicates that water homeostasis in response to a physiological challenge is attenuated at 20 months of age by brief early life ACEi in SFK. Further studies are required to determine if ACEi in early life in children with SFK could compromise fluid homeostasis later in life., (© 2023 The Author(s).)

Atherosclerosis is characterized by the narrowing of the arterial lumen due to subendothelial lipid accumulation, with hypercholesterolemia being a major risk factor. Despite the recent advances in effective lipid-lowering therapies, atherosclerosis remains the leading cause of mortality globally, highlighting the need for additional therapeutic strategies. Accumulating evidence suggests that the sympathetic nervous system plays an important role in atherosclerosis. In this article, we reviewed the sympathetic innervation in the vasculature, norepinephrine synthesis and metabolism, sympathetic activity measurement, and common signaling pathways of sympathetic activation. The focus of this paper was to review the effectiveness of pharmacological antagonists or agonists of adrenoceptors (α1, α2, β1, β2, and β3) and renal denervation on atherosclerosis. All five types of adrenoceptors are present in arterial blood vessels. α1 blockers inhibit atherosclerosis but increase the risk of heart failure while α2 agonism may protect against atherosclerosis and newer generations of β blockers and β3 agonists are promising therapies against atherosclerosis; however, new randomized controlled trials are warranted to investigate the effectiveness of these therapies in atherosclerosis inhibition and cardiovascular risk reduction in the future. The role of renal denervation in atherosclerosis inhibition in humans is yet to be established.

Gut dysfunction has emerged as a contributor to hypertension, the leading risk factor for disease globally, including stroke, heart failure, and kidney disease. This is underpinned by breakdown of the homeostatic relationship connecting intestinal epithelial function, the microbiota and immune responses. Antihypertensive medications have been shown to reverse intestinal dysfunction and gut dysbiosis. However, the mechanisms underlying this restoration of gut structure and function remain largely unknown. In this review, we examine current knowledge supporting a role for impaired intestinal epithelial permeability in hypertension, focusing on electrolyte movement, the renin-angiotensin-aldosterone system, and the restorative effects of orally administered antihypertensive medications and antibiotics. Further work is required to determine if the association between intestinal dysfunction and hypertension is causal. This is a rapidly evolving field, with intestinal dysfunction and dysbiosis representing an area that may be exploited to improve treatment of hypertension and cardiovascular disease., Competing Interests: Disclosures D. Lyras has received funding from MSD (Merck Sharpe and Dohme; Grant number: IIS57443) to investigate bezlotoxumab. MSD had no input into this review. The other authors report no conflicts.

Background: Sex differences play a critical role in the incidence and severity of cardiovascular diseases, whereby men are at a higher risk of developing cardiovascular disease compared to age-matched premenopausal women. Marked sex differences at the cellular and tissue level may contribute to susceptibility to cardiovascular disease and end-organ damage. In this study, we have performed an in-depth histological analysis of sex differences in hypertensive cardiac and renal injury in middle-aged stroke-prone spontaneously hypertensive rats (SHRSPs) to determine the interaction between age, sex and cell senescence., Methods: Kidneys, hearts and urine samples were collected from 6.5- and 8-month-old (Mo) male and female SHRSPs. Urine samples were assayed for albumin and creatinine content. Kidneys and hearts were screened for a suite of cellular senescence markers (senescence-associated β-galactosidase, p16 INK4a , p21, γH2AX). Renal and cardiac fibrosis was quantified using Masson's trichrome staining, and glomerular hypertrophy and sclerosis were quantified using Periodic acid-Schiff staining., Results: Marked renal and cardiac fibrosis, concomitant with albuminuria, were evident in all SHRSPs. These sequelae were differentially affected by age, sex and organ. That is, the level of fibrosis was greater in the kidney than the heart, males had greater levels of fibrosis than females in both the heart and kidney, and even a 6-week increase in age resulted in greater levels of kidney fibrosis in males. The differences in kidney fibrosis were reflected by elevated levels of cellular senescence in the kidney in males but not females. Senescent cell burden was significantly less in cardiac tissue compared to renal tissue and was not affected by age or sex., Conclusions: Our study demonstrates a clear sex pattern in age-related progression of renal and cardiac fibrosis and cellular senescence in SHRSP rats. A 6-week time frame was associated with increased indices of cardiac and renal fibrosis and cellular senescence in male SHRSPs. Female SHRSP rats were protected from renal and cardiac damage compared to age-matched males. Thus, the SHRSP is an ideal model to investigate the effects of sex and aging on organ injury over a short timeframe., (© 2023. The Author(s).)

A child with a congenital solitary functioning kidney (SFK) may develop kidney disease from early in life due to hyperfiltration injury. Previously, we showed in a sheep model of SFK that brief angiotensin-converting enzyme inhibition (ACEi) early in life is reno-protective and increases renal functional reserve (RFR) at 8 months of age. Here we investigated the long-term effects of brief early ACEi in SFK sheep out to 20 months of age. At 100 days gestation (term = 150 days) SFK was induced by fetal unilateral nephrectomy, or sham surgery was performed (controls). SFK lambs received enalapril (SFK+ACEi; 0.5 mg/kg, once daily, orally) or vehicle (SFK) from 4 to 8 weeks of age. At 8, 14 and 20 months of age urinary albumin excretion was measured. At 20 months of age, we examined basal kidney function and RFR via infusion of combined amino acid and dopamine (AA+D). SFK+ACEi resulted in lower albuminuria (∼40%) at 8 months, but not at 14 or 20 months of age compared with vehicle-SFK. At 20 months, basal GFR (∼13%) was lower in SFK+ACEi compared with SFK, but renal blood flow (RBF), renal vascular resistance (RVR) and filtration fraction were similar to SFK. During AA+D, the increase in GFR was similar in SFK+ACEi and SFK animals, but the increase in RBF was greater (∼46%) in SFK+ACEi than SFK animals. Brief ACEi in SFK delayed kidney disease in the short-term but these effects were not sustained long-term., (© 2023 The Author(s).)

This study aimed to investigate the effect of the sympatholytic drug moxonidine on atherosclerosis. The effects of moxonidine on oxidised low-density lipoprotein (LDL) uptake, inflammatory gene expression and cellular migration were investigated in vitro in cultured vascular smooth muscle cells (VSMCs). The effect of moxonidine on atherosclerosis was measured by examining aortic arch Sudan IV staining and quantifying the intima-to-media ratio of the left common carotid artery in apolipoprotein E-deficient (ApoE -/- ) mice infused with angiotensin II. The levels of circulating lipid hydroperoxides in mouse plasma were measured by ferrous oxidation-xylenol orange assay. Moxonidine administration increased oxidised LDL uptake by VSMCs via activation of α2 adrenoceptors. Moxonidine increased the expression of LDL receptors and the lipid efflux transporter ABCG1. Moxonidine inhibited mRNA expression of inflammatory genes and increased VSMC migration. Moxonidine administration to ApoE -/- mice (18 mg/kg/day) decreased atherosclerosis formation in the aortic arch and left common carotid artery, associated with increased plasma lipid hydroperoxide levels. In conclusion, moxonidine inhibited atherosclerosis in ApoE -/- mice, which was accompanied by an increase in oxidised LDL uptake by VSMCs, VSMC migration, ABCG1 expression in VSMCs and lipid hydroperoxide levels in the plasma.

Mesenchymal (human amniotic mesenchymal stem cell [HAMSC]) and epithelial cells (human amnion epithelial cell [HAEC]) derived from human amniotic membranes possess characteristics of pluripotency. However, the pluripotency of HAMSC and HAEC are sustained only for a finite period. This in vitro cell growth can be extended by cell immortalisation. Many well-defined immortalisation systems have been used for artificially overexpressing genes such as human telomerase reverse transcriptase in HAMSC and HAEC leading to controlled and prolonged cell proliferation. In recent years, much progress has been made in our understanding of the cellular machinery that regulates the cell cycle when immortalised. This review summarises the current understanding of molecular mechanisms that contribute to cell immortalisation, the strategies that have been employed to immortalise amnion-derived cell types, and their likely applications in regenerative medicine., (© 2022 The Authors. Cell Biology International published by John Wiley & Sons Ltd on behalf of International Federation of Cell Biology.)

A high salt (HS) diet is associated with an increased risk for cardiovascular diseases (CVDs) and fibrosis is a key contributor to the organ dysfunction involved in CVDs. The activation of the renin angiotensin type 2 receptor (AT 2 R) has been considered as organ protective in many CVDs. However, there are limited AT 2 R-selective agonists available. Our first reported β-substituted angiotensin III peptide, β-Pro 7 -AngIII, showed high selectivity for the AT 2 R. In the current study, we examine the potential anti-fibrotic and anti-inflammatory effects of this novel AT 2 R-selective peptide on HS-induced organ damage. FVB/N mice fed with a 5% HS diet for 8 weeks developed cardiac and renal fibrosis and inflammation, which were associated with increased TGF-β1 levels in heart, kidney and plasma. Four weeks' treatment (from weeks 5-8) with β-Pro 7 -AngIII inhibited the HS-induced cardiac and renal fibrosis and inflammation. These protective effects were accompanied by reduced local and systemic TGF-β1 as well as reduced cardiac myofibroblast differentiation. Importantly, the anti-fibrotic and anti-inflammatory effects caused by β-Pro 7 -AngIII were attenuated by the AT 2 R antagonist PD123319. These results demonstrate, for the first time, the cardio- and reno-protective roles of the AT 2 R-selective β-Pro 7 -AngIII, highlighting it as an important therapeutic that can target the AT 2 R to treat end-organ damage.

The concept of local formation of angiotensin II in the kidney has changed over the last 10-15 years. Local synthesis of angiotensinogen in the proximal tubule has been proposed, combined with prorenin synthesis in the collecting duct. Binding of prorenin via the so-called (pro)renin receptor has been introduced, as well as megalin-mediated uptake of filtered plasma-derived renin-angiotensin system (RAS) components. Moreover, angiotensin metabolites other than angiotensin II [notably angiotensin-(1-7)] exist, and angiotensins exert their effects via three different receptors, of which angiotensin II type 2 and Mas receptors are considered renoprotective, possibly in a sex-specific manner, whereas angiotensin II type 1 (AT 1 ) receptors are believed to be deleterious. Additionally, internalized angiotensin II may stimulate intracellular receptors. Angiotensin-converting enzyme 2 (ACE2) not only generates angiotensin-(1-7) but also acts as coronavirus receptor. Multiple, if not all, cardiovascular diseases involve the kidney RAS, with renal AT 1 receptors often being claimed to exert a crucial role. Urinary RAS component levels, depending on filtration, reabsorption, and local release, are believed to reflect renal RAS activity. Finally, both existing drugs (RAS inhibitors, cyclooxygenase inhibitors) and novel drugs (angiotensin receptor/neprilysin inhibitors, sodium-glucose cotransporter-2 inhibitors, soluble ACE2) affect renal angiotensin formation, thereby displaying cardiovascular efficacy. Particular in the case of the latter three, an important question is to what degree they induce renoprotection (e.g., in a renal RAS-dependent manner). This review provides a unifying view, explaining not only how kidney angiotensin formation occurs and how it is affected by drugs but also why drugs are renoprotective when altering the renal RAS. SIGNIFICANCE STATEMENT: Angiotensin formation in the kidney is widely accepted but little understood, and multiple, often contrasting concepts have been put forward over the last two decades. This paper offers a unifying view, simultaneously explaining how existing and novel drugs exert renoprotection by interfering with kidney angiotensin formation., (Copyright © 2022 by The Author(s).)

Background: Children born with a solitary functioning kidney (SFK) are predisposed to develop hypertension and kidney injury. Glomerular hyperfiltration and hypertrophy contribute to the pathophysiology of kidney injury. Angiotensin-converting enzyme inhibition (ACEi) can mitigate hyperfiltration and may be therapeutically beneficial in reducing progression of kidney injury in those with an SFK., Methods: SFK was induced in male sheep fetuses at 100 days gestation (term=150 days). Between 4 and 8 weeks of age, SFK lambs received enalapril (SFK+ACEi; 0.5mg/kg per day, once daily, orally) or vehicle (SFK). At 8 months, we examined BP, basal kidney function, renal functional reserve (RFR; GFR response to combined amino acid and dopamine infusion), GFR response to nitric oxide synthase (NOS) inhibition, and basal nitric oxide (NO) bioavailability (basal urinary total nitrate and nitrite [NOx])., Results: SFK+ACEi prevented albuminuria and resulted in lower basal GFR (16%), higher renal blood flow (approximately 22%), and lower filtration fraction (approximately 35%), but similar BP, compared with vehicle-treated SFK sheep. Together with greater recruitment of RFR (approximately 14%) in SFK+ACEi than SFK animals, this indicates a reduction in glomerular hyperfiltration-mediated kidney dysfunction. During NOS inhibition, the decrease in GFR (approximately 14%) was greater among SFK+ACEi than among SFK animals. Increased (approximately 85%) basal urinary total NOx in SFK+ACEi compared with SFK animals indicates elevated NO bioavailability likely contributed to improvements in kidney function and prevention of albuminuria., Conclusions: Brief and early ACEi in SFK is associated with reduced glomerular hyperfiltration-mediated kidney disease up to 8 months of age in a sheep model., (Copyright © 2022 by the American Society of Nephrology.)

The associations between high-density lipoprotein cholesterol (HDL-C) and blood pressure (BP) or hypertension are inconsistent in previous studies. This study aimed to assess these associations in a large cohort of Chinese adults and across different age groups. This cross-sectional association study included 22,081 Chinese adults. Associations of HDL-C with BP and hypertension were analyzed using linear or logistic regression, with or without adjustment for confounding factors. HDL-C was inversely associated with BP and hypertension. These associations were still apparent after adjustment for age, sex, fasting plasma glucose, and low-density lipoprotein cholesterol. Sub-analyses revealed: (1) in the whole cohort and females alone, HDL-C was inversely associated with BP and hypertension in young and middle-aged but not older participants; (2) in males alone, HDL-C was not associated with systolic BP or hypertension. However, HDL-C was either inversely, or not, or positively associated with BP in young, middle-aged, and older males, respectively. After further adjustment for body mass index (BMI), the negative associations of HDL-C with BP and hypertension in the whole cohort became positive ones, and the positive associations only presented in males. These findings suggest that further adjustment for BMI changes inverse associations of HDL-cholesterol with BP and hypertension to positive associations in a cohort of Chinese adults., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Clostridioides difficile is the most common cause of infectious antibiotic-associated diarrhea, with disease mediated by two major toxins TcdA and TcdB. In severe cases, systemic disease complications may arise, resulting in fatal disease. Systemic disease in animal models has been described, with thymic damage an observable consequence of severe disease in mice. Using a mouse model of C. difficile infection, we examined this disease phenotype, focussing on the thymus and serum markers of systemic disease. The efficacy of bezlotoxumab, a monoclonal TcdB therapeutic, to prevent toxin mediated systemic disease complications was also examined. C. difficile infection causes toxin-dependent thymic damage and CD4 + CD8 + thymocyte depletion in mice. These systemic complications coincide with changes in biochemical markers of liver and kidney function, including increased serum urea and creatinine, and hypoglycemia. Administration of bezlotoxumab during C. difficile infection prevents systemic disease and thymic atrophy, without blocking gut damage, suggesting the leakage of gut contents into circulation may influence systemic disease. As the thymus has such a crucial role in T cell production and immune system development, these findings may have important implications in relapse of C. difficile disease and impaired immunity during C. difficile infection. The prevention of thymic atrophy and reduced systemic response following bezlotoxumab treatment, without altering colonic damage, highlights the importance of systemic disease in C. difficile infection, and provides new insights into the mechanism of action for this therapeutic. Abbreviations : Acute kidney injury (AKI); Alanine Transaminase (ALT); Aspartate Aminotransferase (AST); C. difficile infection (CDI); chronic kidney disease (CKD); combined repetitive oligo-peptides (CROPS); cardiovascular disease (CVD); Double positive (DP); hematoxylin and eosin (H&E); immunohistochemical (IHC); multiple organ dysfunction syndrome (MODS); phosphate buffered saline (PBS); standard error of the mean (SEM); surface layer proteins (SLP); Single positive (SP); wild-type (WT).

Majority of patients with hypertension and chronic kidney disease (CKD) undergoing renal denervation (RDN) are maintained on antihypertensive medication. However, RDN may impair compensatory responses to hypotension induced by blood loss. Therefore, continuation of antihypertensive medications in denervated patients may exacerbate hypotensive episodes. This study examined whether antihypertensive medication compromised hemodynamic responses to blood loss in normotensive (control) sheep and in sheep with hypertensive CKD at 30 months after RDN (control-RDN, CKD-RDN) or sham (control-intact, CKD-intact) procedure. CKD-RDN sheep had lower basal blood pressure (BP; ≈9 mm Hg) and higher basal renal blood flow (≈38%) than CKD-intact. Candesartan lowered BP and increased renal blood flow in all groups. 10% loss of blood volume alone caused a modest fall in BP (≈6-8 mm Hg) in all groups but did not affect the recovery of BP. 10% loss of blood volume in the presence of candesartan prolonged the time at trough BP by 9 minutes and attenuated the fall in renal blood flow in the CKD-RDN group compared with CKD-intact. Candesartan in combination with RDN prolonged trough BP and attenuated renal hemodynamic responses to blood loss. To minimize the risk of hypotension-mediated organ damage, patients with RDN maintained on antihypertensive medications may require closer monitoring when undergoing surgery or experiencing traumatic blood loss.

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Background: The antifibrotic effects of recombinant human relaxin (RLX) in the kidney are dependent on an interaction between its cognate receptor (RXFP1) and the angiotensin type 2 receptor (AT 2 R) in male models of disease. Whether RLX has therapeutic effects, which are also mediated via AT 2 R, in hypertensive adult and aged/reproductively senescent females is unknown. Thus, we determined whether treatment with RLX provides cardiorenal protection via an AT 2 R-dependent mechanism in adult and aged female stroke-prone spontaneously hypertensive rats (SHRSPs)., Methods: In 6-month-old (6MO) and 15-month-old ([15MO]; reproductively senescent) female SHRSP, systolic BP (SBP), GFR, and proteinuria were measured before and after 4 weeks of treatment with vehicle (Veh), RLX (0.5 mg/kg per day s.c.), or RLX+PD123319 (AT 2 R antagonist; 3 mg/kg per day s.c.). Aortic endothelium-dependent relaxation and fibrosis of the kidney, heart, and aorta were assessed., Results: In 6MO SHRSP, RLX significantly enhanced GFR by approximately 25% ( P =0.001) and reduced cardiac fibrosis ( P =0.01) as compared with vehicle-treated counterparts. These effects were abolished or blunted by PD123319 coadministration. In 15MO females, RLX reduced interstitial renal ( P =0.02) and aortic ( P =0.003) fibrosis and lowered SBP (13±3 mm Hg; P =0.04) relative to controls. These effects were also blocked by PD123319 cotreatment (all P =0.05 versus RLX treatment alone). RLX also markedly improved vascular function by approximately 40% ( P <0.001) in 15MO SHRSP, but this was not modulated by PD123319 cotreatment., Conclusions: The antifibrotic and organ-protective effects of RLX, when administered to a severe model of hypertension, conferred cardiorenal protection in adult and reproductively senescent female rats to a great extent via an AT 2 R-mediated mechanism., Competing Interests: K.M. Denton reports scientific advisor or membership with American Journal of Physiology–Renal Physiology, Experimental Physiology, Frontiers of Science–Integrative Physiology, Hypertension, and Journal of Hypertension. C.S. Samuel reports scientific advisor or membership as a senior editor of Clinical and Experimental Pharmacology and Physiology, an associate editor of Frontiers in Pharmacology–Cardiovascular and Smooth Muscle Pharmacology, a subject editor of Nephrology, and a scientific advisory board member of Relaxera Pharmazeutische Gesellschaft GmbH & Co. KG, Bensheim, Germany. All remaining authors have nothing to disclose., (Copyright © 2021 by the American Society of Nephrology.)

Renal sympathetic nerves contribute to renal excretory function during volume expansion. We hypothesized that intact renal innervation is required for excretion of a fluid/electrolyte load in hypertensive chronic kidney disease (CKD) and normotensive healthy settings. Blood pressure, kidney hemodynamic and excretory response to 180 min of isotonic saline loading (0.13 ml/kg/min) were examined in female normotensive (control) and hypertensive CKD sheep at 2 and 11 months after sham (control-intact, CKD-intact) or radiofrequency catheter-based RDN (control-RDN, CKD-RDN) procedure. Basal blood pressure was ~ 7 to 9 mmHg lower at 2, and 11 months in CKD-RDN compared with CKD-intact sheep. Saline loading did not alter glomerular filtration rate in any group. At 2 months, in response to saline loading, total urine and sodium excretion were ~ 40 to 50% less, in control-RDN and CKD-RDN than intact groups. At 11 months, the natriuretic and diuretic response to saline loading were similar between control-intact, control-RDN and CKD-intact groups but sodium excretion was ~ 42% less in CKD-RDN compared with CKD-intact at this time-point. These findings indicate that chronic withdrawal of basal renal sympathetic activity impairs fluid/electrolyte excretion during volume expansion. Clinically, a reduced ability to excrete a saline load following RDN may contribute to disturbances in body fluid balance in hypertensive CKD., (© 2021. The Author(s).)

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In May 2014, the National Institutes of Health (NIH) stated its intent to "require applicants to consider sex as a biological variable (SABV) in the design and analysis of NIH-funded research involving animals and cells." Since then, proposed research plans that include animals routinely state that both sexes/genders will be used; however, in many instances, researchers and reviewers are at a loss about the issue of sex differences. Moreover, the terms sex and gender are used interchangeably by many researchers, further complicating the issue. In addition, the sex or gender of the researcher might influence study outcomes, especially those concerning behavioral studies, in both animals and humans. The act of observation may change the outcome (the "observer effect") and any experimental manipulation, no matter how well-controlled, is subject to it. This is nowhere more applicable than in physiology and behavior. The sex of established cultured cell lines is another issue, in addition to aneuploidy; chromosomal numbers can change as cells are passaged. Additionally, culture medium contains steroids, growth hormone, and insulin that might influence expression of various genes. These issues often are not taken into account, determined, or even considered. Issues pertaining to the "sex" of cultured cells are beyond the scope of this Statement. However, we will discuss the factors that influence sex and gender in both basic research (that using animal models) and clinical research (that involving human subjects), as well as in some areas of science where sex differences are routinely studied. Sex differences in baseline physiology and associated mechanisms form the foundation for understanding sex differences in diseases pathology, treatments, and outcomes. The purpose of this Statement is to highlight lessons learned, caveats, and what to consider when evaluating data pertaining to sex differences, using 3 areas of research as examples; it is not intended to serve as a guideline for research design., (© 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.)

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Insulin-regulated aminopeptidase (IRAP), an enzyme that cleaves vasoactive peptides including oxytocin and vasopressin, is suggested to play a role in pregnancy and the onset of preeclampsia. Our aim was to examine the contribution of IRAP to arterial pressure regulation and placental development during pregnancy in mice. Mean arterial pressure and heart rate were measured via radiotelemetry in 12-week-old female wild-type and IRAP knockout mice. Females were time-mated with males of the same genotype. Placentae were collected at embryonic day 18.5 for histological analysis. Basal heart rate was ∼40 bpm lower in IRAP knockout females compared with wild-type females. The increase in heart rate across gestation was greater in IRAP knockout females than wild-type females. Neither basal nor gestational mean arterial pressure was different between wildtype and IRAP knockout females. Urine output and water intake of IRAP knockout mice were ∼45% less than wild-type mice at late gestation. IRAP deficiency had no effect on fetal weight. Morphological assessment of placentae revealed that IRAP deficiency was associated with reduced labyrinth surface area and accumulation of glycogen in the junctional zone. Our data demonstrate that IRAP deficiency alters maternal fluid handling and impairs placental labyrinth expansion at late gestation, indicating that IRAP contributes to the normal adaptions to pregnancy., (© 2020 The Author(s).)

Overactivity of the sympathetic nervous system and high blood pressure are implicated in the development and progression of chronic kidney disease (CKD) and independently predict cardiovascular events in end-stage renal disease. To assess the role of renal nerves, we determined whether renal denervation (RDN) altered the hypertension and sympathoexcitation associated with a rabbit model of CKD. The model involves glomerular layer lesioning and uninephrectomy, resulting in renal function reduced by one-third and diuresis. After 3-week CKD, blood pressure was 13±2 mm Hg higher than at baseline ( P <0.001), and compared with sham control rabbits, renal sympathetic nerve activity was 1.2±0.5 normalized units greater ( P =0.01). The depressor response to ganglion blockade was also +8.0±3 mm Hg greater, but total norepinephrine spillover was 8.7±3.7 ng/min lower (both P <0.05). RDN CKD rabbits only increased blood pressure by 8.0±1.5 mm Hg. Renal sympathetic activity, the response to ganglion blockade and diuresis were similar to sham denervated rabbits (non-CKD). CKD rabbits had intact renal sympathetic baroreflex gain and range, as well as normal sympathetic responses to airjet stress. However, hypoxia-induced sympathoexcitation was reduced by -9±0.4 normalized units. RDN did not alter the sympathetic response to hypoxia or airjet stress. CKD increased oxidative stress markers Nox5 and MCP-1 (monocyte chemoattractant protein-1) in the kidney, but RDN had no effect on these measures. Thus, RDN is an effective treatment for hypertension in this model of CKD without further impairing renal function or altering the normal sympathetic reflex responses to various environmental stimuli.

Chronic kidney disease (CKD) is associated with greater sympathetic nerve activity but it is unclear if this is a kidney-specific response or due to generalized stimulation of sympathetic nervous system activity. To determine this, we used a rabbit model of CKD in which quantitative comparisons with control rabbits could be made of kidney sympathetic nerve activity and whole-body norepinephrine spillover. Rabbits either had surgery to lesion 5/6 th of the cortex of one kidney by electro-lesioning and two weeks later removal of the contralateral kidney, or sham lesioning and sham nephrectomy. After three weeks, the blood pressure was statistically significantly 20% higher in conscious rabbits with CKD compared to rabbits with a sham operation, but their heart rate was similar. Strikingly, kidney nerve activity was 37% greater than in controls, with greater burst height and frequency. Total norepinephrine spillover was statistically significantly lower by 34%, and kidney baroreflex curves were shifted to the right in rabbits with CKD. Plasma creatinine and urine output were elevated by 38% and 131%, respectively, and the glomerular filtration rate was 37% lower than in sham-operated animals (all statistically significant). Kidney gene expression of fibronectin, transforming growth factor-β, monocyte chemotactic protein1, Nox4 and Nox5 was two- to eight-fold greater in rabbits with CKD than in control rabbits. Overall, the glomerular layer lesioning model in conscious rabbits produced a moderate, stable degree of CKD characterized by elevated blood pressure and increased kidney sympathetic nerve activity. Thus, our findings, together with that of a reduction in total norepinephrine spillover, suggest that kidney denervation, rather than generalized sympatholytic treatments, may represent a preferable management for CKD associated hypertension., (Copyright © 2020 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)

Obtaining growth and physiologic data in the postnatal laboratory animal is common. However, monitoring growth in utero is far more difficult, with little data available except upon termination of pregnancy. High-resolution ultrasound was used to monitor growth, morphology, and fetal well-being in normotensive and hypertensive rabbits (21 fetuses) at day 16, 20, and 26 of the 32 day gestational period. Set protocols, comparable to those routinely assessed in humans, were devised and followed for each examination. Birth weight was greater in offspring of hypertensive as compared to normotensive mothers (p < 0.001); however, litter size was reduced. The greater birth weight was reflected in growth parameters measured throughout gestation indicating the predictive value of ultrasound. High-resolution ultrasound was a reliable and sensitive method for biometric and morphologic assessment of the fetal rabbit, demonstrating that growth trajectory of offspring of hypertensive mothers may be altered early in gestation., (© 2020 American Association for Anatomy.)

Children born with a solitary functioning kidney (SFK) have an increased risk of hypertension and kidney disease from early in adulthood. In response to a reduction in kidney mass, the remaining kidney undergoes compensatory kidney growth. This is associated with both an increase in size of the kidney tubules and the glomeruli and an increase in single nephron glomerular filtration rate (SNGFR). The compensatory hypertrophy and increase in filtration at the level of the individual nephron results in normalization of total glomerular filtration rate (GFR). However, over time these same compensatory mechanisms may contribute to kidney injury and hypertension. Indeed, approximately 50% of children born with a SFK develop hypertension by the age of 18 and 20-40% require dialysis by the age of 30. The mechanisms that result in kidney injury are only partly understood, and early biomarkers that distinguish those at an elevated risk of kidney injury are needed. This review will outline the compensatory adaptations to a SFK, and outline how these adaptations may contribute to kidney injury and hypertension later in life. These will be based largely on the mechanisms we have identified from our studies in an ovine model of SFK, that implicate the renal nitric oxide system, the renin angiotensin system and the renal nerves to kidney disease and hypertension associated with SFK. This discussion will also evaluate current, and speculate on next generation, prognostic factors that may predict those children at a higher risk of future kidney disease and hypertension., (Copyright © 2020 McArdle, Schreuder, Moritz, Denton and Singh.)

Fibrosis is a hallmark of several cardiovascular diseases. The relaxin family peptide receptor 1 (RXFP1) agonist, relaxin, has rapidly occurring anti-fibrotic actions which are mediated through RXFP1 and angiotensin II receptor crosstalk on renal and cardiac myofibroblasts. Here, we investigated whether this would allow relaxin to indirectly activate angiotensin II type 2 receptor (AT 2 R)-specific signal transduction in primary human cardiac myofibroblasts (HCMFs). The anti-fibrotic effects of recombinant human relaxin (RLX; 16.8 nM) or the AT 2 R-agonist, Compound 21 (C21; 1 μM), were evaluated in TGF-β1-stimulated HCMFs, in the absence or presence of an RXFP1 antagonist (1 μM) or AT 2 R antagonist (0.1 μM) to confirm RXFP1-AT 2 R crosstalk. Competition binding for RXFP1 was determined. Western blotting was performed to determine which AT 2 R-specific protein phosphatases were expressed by HCMFs; then, the anti-fibrotic effects of RLX and/or C21 were evaluated in the absence or presence of pharmacological inhibition (NSC95397 (1 μM) for MKP-1; okadaic acid (10 nM) for PP2A) or siRNA-knockdown of these phosphatases after 72 hours. The RLX- or C21-induced increase in ERK1/2 and nNOS phosphorylation, and decrease in α-SMA (myofibroblast differentiation) and collagen-I expression by HCMFs was abrogated by pharmacological blockade of RXFP1 or the AT 2 R, confirming RXFP1-AT 2 R crosstalk in these cells. HCMFs were found to express AT 2 R-dependent MKP-1 and PP2A phosphatases, while pharmacological blockade or siRNA-knockdown of either phosphatase also abolished RLX and/or C21 signal transduction in HCMFs (all P < .05 vs RLX or C21 alone). These findings demonstrated that RLX can indirectly activate AT 2 R-dependent phosphatase activity in HCMFs by signaling through RXFP1-AT 2 R crosstalk, which have important therapeutic implications for its anti-fibrotic actions., (© 2020 Federation of American Societies for Experimental Biology.)

Recently, we designed a group of peptides by sequential substitution of the naturally occurring α-amino acid throughout the Ang III peptide sequence with the corresponding β-amino acid. β-Amino acid substitution at the proline residue of Ang III (β-Pro7-Ang III) resulted in a highly selective AT2R ligand, demonstrating remarkable selectivity for the AT2R in both binding and functional studies. To provide additional functional evidence for the suitability of β-Pro7 Ang III as a novel AT2R agonist, we tested effects of acute systemic administration of β-Pro7-Ang III on renal hemodynamic and excretory function in anesthetized normotensive male and female rats. We also compared the natriuretic effects of acute intrarenal administration of native Ang III and β-Pro7-Ang III in the presence of systemic AT1R blockade in anesthetized female rats to allow for the differentiation of systemic versus direct intrarenal natriuretic actions of β-Pro7-Ang III. In both male and female rats, acute systemic administration of β-Pro7-Ang III elicited renal vasodilatation and natriuresis. Notably, greater renal vasodilatory effects were observed in female versus male rats at the highest dose of β-Pro7-Ang III administered. Moreover, intra-renal administration of β-Pro7-Ang III produced significant natriuretic effects in female rats and, like Ang III, evoked AT2R translocation to the apical plasma membrane in renal proximal tubular cells. Taken together, our findings support the use of β-Pro7-Ang III as a novel AT2R agonist and experimental tool for exploring AT2R function and its potential as a therapeutic target. Furthermore, our findings provide further evidence of a sex-specific influence of AT2R stimulation on renal function., (© 2020 The Author(s).)

Fibrosis is involved in the majority of cardiovascular diseases and is a key contributor to end-organ dysfunction. In the current study, the antifibrotic effects of recombinant human relaxin-2 (serelaxin; RLX) and/or the AT 2 R agonist CGP42112 (CGP) were compared with those of the established AT 1 R antagonist, candesartan cilexetil (CAND), in a high salt-induced cardiac fibrosis model. High salt (HS; 5%) for 8 weeks did not increase systolic blood pressure in male FVB/N mice, but CAND treatment alone significantly reduced systolic blood pressure from HS-induced levels. HS significantly increased cardiac interstitial fibrosis, which was reduced by either RLX and/or CGP, which were not additive under the current experimental conditions, while CAND failed to reduce HS-induced cardiac fibrosis. The antifibrotic effects induced by RLX and/or CGP were associated with reduced myofibroblast differentiation. Additionally, all treatments inhibited the HS-induced elevation in tissue inhibitor of matrix metalloproteinases-1, together with trends for increased MMP-13 expression, that collectively would favor collagen degradation. Furthermore, these antifibrotic effects were associated with reduced cardiac inflammation. Collectively, these results highlight that either RXFP1 or AT 2 R stimulation represents novel therapeutic strategies to target fibrotic conditions, particularly in HS states that may be refractory to AT 1 R blockade., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)