Nascent shifts in renal cellular metabolism, structure, and function due to chronic empagliflozin in prediabetic mice.

Sodium-glucose cotransporter, type 2 inhibitors (SGLT2i) are emerging as the gold standard for treatment of type 2 diabetes (T2D) with renal protective benefits independent of glucose lowering. We took a high-level approach to evaluate the effects of the SGLT2i, empagliflozin (EMPA) on renal metabolism and function in a prediabetic model of metabolic syndrome. Male and female 12-wk-old TallyHo (TH) mice, and their closest genetic lean strain (Swiss-Webster, SW) were treated with a high-milk-fat diet (HMFD) plus/minus EMPA (@0.01%) for 12-wk. Kidney weights and glomerular filtration rate were slightly increased by EMPA in the TH mice. Glomerular feature analysis by unsupervised clustering revealed sexually dimorphic clustering, and one unique cluster relating to EMPA. Periodic acid Schiff (PAS) positive areas, reflecting basement membranes and mesangium were slightly reduced by EMPA. Phasor-fluorescent life-time imaging (FLIM) of free-to-protein bound NADH in cortex showed a marginally greater reliance on oxidative phosphorylation with EMPA. Overall, net urine sodium, glucose, and albumin were slightly increased by EMPA. In TH, EMPA reduced the sodium phosphate cotransporter, type 2 (NaPi-2), but increased sodium hydrogen exchanger, type 3 (NHE3). These changes were absent or blunted in SW. EMPA led to changes in urine exosomal microRNA profile including, in females, enhanced levels of miRs 27a-3p, 190a-5p, and 196b-5p. Network analysis revealed "cancer pathways" and "FOXO signaling" as the major regulated pathways. Overall, EMPA treatment to prediabetic mice with limited renal disease resulted in modifications in renal metabolism, structure, and transport, which may preclude and underlie protection against kidney disease with developing T2D.NEW & NOTEWORTHY Renal protection afforded by sodium glucose transporter, type 2 inhibitors (SGLT2i), e.g., empagliflozin (EMPA) involves complex intertwined mechanisms. Using a novel mouse model of obesity with insulin resistance, the TallyHo/Jng (TH) mouse on a high-milk-fat diet (HMFD), we found subtle changes in metabolism including altered regulation of sodium transporters that line the renal tubule. New potential epigenetic determinants of metabolic changes relating to FOXO and cancer signaling pathways were elucidated from an altered urine exosomal microRNA signature.

Susceptibility of female rats to cardiac arrhythmias following refeeding after severe food restriction.

BACKGROUND: Many studies have shown malnutrition and inadequate caloric consumption have adverse acute effects on cardiovascular structure and function. METHODS: To determine the adverse long term cardiovascular effects, we studied cardiac morphology and function in female (F) and male (M) severe food restricted rats 3 months after refeeding (sFR-Refed). RESULTS: Two weeks of a normal chow diet in which calories were reduced by 60% decreased body weight (BW) by approximately 15% in both sexes. Within 2 weeks of refeeding, no differences in BW were detected between CT and sFR-Refed groups. However, male rats gained almost 3 times more BW than the females over the 3-month refeeding period. Sex differences were also observed in cardiac pathology. Hearts from F-sFR-Refed rats exhibited more atrophy and less hypertrophy, while M-sFR-Refed rats predominantly exhibited hypertrophic remodeling. While there were no differences in the frequency of ventricular arrhythmias induced by ischemia/reperfusion (I/R) in the isolated heart between M-CT and M-sFR-Refed rats, I/R induced twice as many arrhythmias in the F-sFR-Refed rats compared to F-CT. CONCLUSIONS: These findings indicate the female heart is more susceptible to the long term adverse cardiovascular effects of sFR months after refeeding. Thus, this study provides a rationale for studying sex differences in cardiovascular risk in individuals who experience sFR for voluntary (e.g., very low-calorie dieting) or involuntary (e.g., poverty) reasons earlier in life.

Caloric Restriction and Cardiovascular Health: the Good, the Bad, and the Renin-Angiotensin System.

Much excitement exists over the cardioprotective and life-extending effects of caloric restriction (CR). This review integrates population studies with experimental animal research to address the positive and negative impact of mild and severe CR on cardiovascular physiology and pathophysiology, with a particular focus on the renin-angiotensin system (RAS). We also highlight the gaps in knowledge and areas ripe for future physiological research.

Preliminary study of ovariectomy and chronic losartan-induced alterations in brain AT1 receptors.

Women who undergo oophorectomy prior to the age of natural menopause have a higher risk of neurological and psychological impairment. Treatment with the angiotensin receptor blocker (ARB) losartan for 10 weeks following ovariectomy of Long-Evans rats at 3 months of age reduced the ovariectomy-induced cognitive decrements. Following completion of the behavioral experiments, (Campos et al., 2019), the brains were harvested for preliminary receptor autoradiographic studies of AT1 receptor (AT1R) binding in selected brain regions using quantitative densitometric analysis of autoradiograms of 125I-sarcosine1, isoleucine8 angiotensin II binding. Four of the brain regions (amygdala, ventral subiculum, piriform cortex, and cingulate cortex) are associated with cognitive and emotional behavior while one (lateral hypothalamus) is associated with homeostasis. The density of AT1R varied by region: ventral subiculum > amygdala and cingulate cortex, and piriform cortex > cingulate cortex. Losartan treatment decreased AT1R binding in the ventral subiculum of sham and ovariectomized rats by 41.6%, and 46% in the piriform cortex of the sham rats, but tended to increase AT1R binding in the piriform cortex and cingulate cortex 77% and 107%, respectively, in the ovariectomized rats. AT1R binding did not differ significantly between intact male and sham-vehicle female rats among surveyed brain regions. These results suggest that losartan-induced changes in brain AT1R expression may contribute to the reduced anxiety-like behavior and memory impairments seen in ovariectomized rats, but replication of these observations will be needed to determine the extent to which brain AT1R changes mediate the adverse behavioral effects of ovariectomy.

Proteinase-activated receptor-2 (PAR2) on blood pressure and electrolyte handling in the late pregnant rat.

NEW FINDINGS: What is the central question of this study? Pregnancy requires marked renal sodium and potassium retention and cumulative plasma volume expansion, in the setting of reduced blood pressure. Research in male rodents has shown that activation of PAR2 can produce peripheral vasodilatation, stimulate renal sodium chloride reabsorption and inhibit renal potassium secretion. Here, we investigate PAR2 activation in virgin and normal pregnant rats. What is the main finding and its importance? PAR2 expression and sensitivity to activation are increased in pregnancy. This implicates a possible role for PAR2 in supporting the renal/vascular adaptations of pregnancy required for normal maternal plasma volume expansion. ABSTRACT: A healthy pregnancy involves renal and systemic haemodynamic adaptations, which allow renal sodium and potassium retention and cumulative plasma volume expansion, accompanied by a decline in blood pressure attributable to a reduction in the total peripheral vascular resistance. When these adaptations do not occur, pregnancy is compromised. The mechanisms permitting these opposing adaptations are largely unknown. Research in male rodents has shown that activation of PAR2 can produce peripheral vasodilatation, stimulate renal sodium chloride reabsorption and inhibit renal potassium secretion. Here, we investigate PAR2 activation in female virgin and normal late pregnant (LP) rats. We measured the mRNA expression of PAR2 in the renal cortex, outer medulla and inner medulla of virgin and LP rats using quantitative real-time PCR. We also measured in vivo blood pressure, natriuretic and kaliuretic responses to PAR2-activating peptide (SLIGRL-NH2 ) in anaesthetized virgin and LP rats. We found that PAR2 mRNA was increased in the inner medulla of LP rats. We also found that LP rats had larger decreases in blood pressure and increases in net sodium retention compared with virgin rats. These findings suggest that pregnancy enhances sensitivity to the blood pressure-lowering and sodium-retaining effects of PAR2.

Male bias in ACE2 basic science research: missed opportunity for discovery in the time of COVID-19.

Throughout the world, including the United States, men have worse outcomes from COVID-19 than women. SARS-CoV-2, the causative virus of the COVID-19 pandemic, uses angiotensin-converting enzyme 2 (ACE2) to gain cellular entry. ACE2 is a member of the renin-angiotensin system (RAS) and plays an important role in counteracting the harmful effects mediated by the angiotensin type 1 receptor. Therefore, we conducted Ovid MEDLINE and Embase database searches of basic science studies investigating the impact of the biological variable of sex on ACE2 expression and regulation from 2000, the year ACE2 was discovered, through December 31, 2020. Out of 2,131 publications, we identified 853 original research articles on ACE2 conducted in primary cells, tissues, and/or whole mammals excluding humans. The majority (68.7%) of these studies that cited the sex of the animal were conducted in males, while 11.2% were conducted solely in females; 9.26% compared ACE2 between the sexes, while 10.8% did not report the sex of the animals used. General findings are that sex differences are tissue-specific and when present, are dependent upon gonadal state. Renal, cardiac, and adipose ACE2 is increased in both sexes under experimental conditions that model co-morbidities associated with worse COVID-19 outcomes including hypertension, obesity, and renal and cardiovascular diseases; however, ACE2 protein was generally higher in the males. Studies in Ace2 knockout mice indicate ACE2 plays a greater role in protecting the female from developing hypertension than the male. Studying the biological variable of sex in ACE2 research provides an opportunity for discovery in conditions involving RAS dysfunction and will shed light on sex differences in COVID-19 severity.

Persistent Renin-Angiotensin System Sensitization Months After Body Weight Recovery From Severe Food Restriction in Female Fischer Rats.

Background Prior exposure to periods of severe food restriction (sFR) is associated with increased risk of developing hypertension and cardiovascular disease later in life. Methods and Results To investigate the mechanism of these long-term adverse effects of sFR, 4-month-old female Fischer rats were divided in 2 groups and maintained on a normal diet ad libitum (control) or on an sFR diet with 60% reduction in daily food intake for 2 weeks that resulted in a 15% reduction in body weight. After the 2-week sFR period ended, both groups received normal chow ad libitum for 3 months. Within 2 weeks after refeeding was initiated in the sFR group, body weight was restored to control levels; however, plasma angiotensinogen (1.3-fold; P<0.05), Ang-[1-8] (2.0-fold; P<0.05), and angiotensin-converting enzyme activity (1.1-fold; P<0.01) were all elevated 3 months after refeeding. Angiotensin type 1 receptor activity was also increased as evidenced by augmented pressor responses to angiotensin-[1-8] (P<0.01) and depressor responses to the angiotensin type 1 receptor antagonist, losartan (P<0.01) in the sFR group. Conclusions These results indicate that sensitization of the renin-angiotensin system persisted months after the sFR period ended. These findings may have implications for women who voluntarily or involuntarily experience an extended period of sFR and thus may be at increased risk of developing cardiovascular disease through sensitization of the renin-angiotensin system even though their body weight, mean arterial pressure, and heart rate appear normal.

Sex-Specific Modulation of Blood Pressure and the Renin-Angiotensin System by ACE (Angiotensin-Converting Enzyme) 2.

We showed ACE (angiotensin-converting enzyme) 2 is higher in the kidney of male compared with female mice. To further investigate this sex difference, we examined the role of ACE2 in Ang-[1-8] (angiotensin [1-8])-induced hypertension and regulation of the renin-angiotensin system in the kidney of WT (wild type) and Ace2 KO (knockout) mice. Mean arterial pressure rose faster in WT male than WT female mice after Ang-[1-8] infusion. This sex difference was attenuated in ACE2 KO mice. Ang-[1-8] infusion reduced glomerular AT1R (angiotensin type 1 receptor) binding in WT female mice by 30%, and deletion of Ace2 abolished this effect. In contrast, Ang-[1-8] infusion increased glomerular AT1R binding in WT male mice by 1.2-fold, and this effect of Ang-[1-8] persisted in Ace2 KO male mice (1.3-fold). ACE2 also had an effect on renal protein expression of the neutral endopeptidase NEP (neprilysin), the enzyme that catabolizes Ang-[1-10] (angiotensin [1-10]), the precursor of Ang-[1-8]. Ang-[1-8] infusion downregulated NEP protein expression by 20% in WT male, whereas there was a slight increase in NEP expression in WT female mice. Deletion of Ace2 resulted in lowered NEP expression after Ang-[1-8] infusion in both sexes. These findings suggest sex-specific ACE2 regulation of the renin-angiotensin system contributes to female protection from Ang-[1-8]-induced hypertension. These findings have ramifications for the current coronavirus disease 2019 (COVID-19) pandemic, especially in hypertension since ACE2 is the SARS-CoV-2 receptor and hypertension is a major risk factor for poor outcomes.

Short-term very low caloric intake causes endothelial dysfunction and increased susceptibility to cardiac arrhythmias and pathology in male rats.

NEW FINDINGS: What is the central question of this study? What are the effects of a 2 week period of severe food restriction on vascular reactivity of resistance arteries and on cardiac structure and function? What is the main finding and its importance? This study showed, for the first time, that a 2 week period of severe food restriction in adult male Fischer rats caused endothelial dysfunction in mesenteric arteries and increased the susceptibility to ischaemia-reperfusion-induced arrhythmias and cardiac pathology. Our findings might have ramifications for cardiovascular risk in people who experience periods of inadequate caloric intake. ABSTRACT: Severe food restriction (sFR) is a common dieting strategy for rapid weight loss. Male Fischer rats were maintained on a control (CT) or sFR (40% of CT food intake) diet for 14 days to mimic low-calorie crash diets. The sFR diet reduced body weight by 16%. Haematocrits were elevated by 10% in the sFR rats, which was consistent with the reduced plasma volume. Mesenteric arteries from sFR rats had increased sensitivity to vasoconstrictors, including angiotensin II [maximum (%): CT, 1.30 ± 0.46 versus sFR, 11.5 ± 1.6; P < 0.0001; n = 7] and phenylephrine [maximum (%): CT, 78.5 ± 2.8 versus sFR, 94.5 ± 1.7; P < 0.001; n = 7] and reduced sensitivity to the vasodilator acetylcholine [EC50 (nm): CT, 49.2 ± 5.2 versus sFR, 71.6 ± 6.8; P < 0.05; n = 7]. Isolated hearts from sFR rats had a 1.7-fold increase in the rate of cardiac arrhythmias in response to ischaemia-reperfusion and more cardiac pathology, including myofibrillar disarray with contractions and cardiomyocyte lysis, than hearts from CT rats. The sFR dietary regimen is similar to very low-calorie commercial and self-help weight-loss programmes, which provide ∼800-1000 kcal day-1 . Therefore, these findings in rats warrant the study of cardiovascular function in individuals who engage in extreme dieting or are subjected to bouts of very low caloric intake for other reasons, such as socioeconomic factors and natural disasters.

The Angiotensin Type 1 Receptor Antagonist Losartan Prevents Ovariectomy-Induced Cognitive Dysfunction and Anxiety-Like Behavior in Long Evans Rats.

Women who have bilateral oophorectomies prior to the age of natural menopause are at increased risk of developing mild cognitive decline, dementia, anxiety, and depressive type disorders. Clinical and animal studies indicate angiotensin type 1 receptor (AT1R) blockers (ARBs) have blood pressure (BP)-independent neuroprotective effects. To investigate the potential use of ARBs in normotensive women at increased risk of developing neurocognitive problems, we studied a rat model of bilateral oophorectomy. Long Evans rats were sham-operated (Sham) or ovariectomized (Ovx) at 3 months of age and immediately treated continuously with vehicle (Veh) or the ARB losartan (Los) for the duration of the experiment. In contrast to many hypertensive rat models, ovariectomy did not increase mean arterial pressure (MAP) in these normotensive rats. Ovariectomized rats spent less time in the open arms of the elevated plus maze (EPM) [(% total time): Veh, 34.1 ± 5.1 vs. Ovx, 18.7 ± 4.4; p < 0.05] and in the center of the open field (OF) [(s): Veh, 11.1 ± 1.7 vs. Ovx, 6.64 ± 1.1; p < 0.05]. They also had worse performance in the novel object recognition (NOR) test as evidenced by a reduction in the recognition index [Veh, 0.62 ± 0.04 vs. Ovx, 0.45 ± 0.03; p < 0.05]. These adverse effects of ovariectomy were prevented by Los. Losartan also reduced plasma corticosterone in Ovx rats compared to Veh treatment [(ng/mL): Ovx-Veh, 238 ± 20 vs. Ovx-Los, 119 ± 42; p < 0.05]. Ovariectomy increased AT1R mRNA expression in the CA3 region of the hippocampus (Hc) [(copies x 106/µg RNA): Sham-Veh, 7.15 ± 0.87 vs. Ovx-Veh, 9.86 ± 1.7; p < 0.05]. These findings suggest the neuroprotective effects of this ARB in normotensive Ovx rats involve reduction of plasma corticosterone and blockade of increased AT1R activity in the hippocampus. These data suggest ARBs have therapeutic potential for normotensive women at increased risk of developing cognitive and behavioral dysfunction due to bilateral oophorectomy prior to the natural age of menopause.

Adaptive remodeling of renal Na+ and K+ transport during pregnancy.

PURPOSE OF REVIEW: Renal ion transport undergoes dramatic changes during the course of gestation. These adaptations are necessary to meet the dynamic requirements of pregnancy and support fetal development. Pregnancy is characterized by a high demand for both sodium and potassium. Recently there has been work in the field profiling the modifications of the renal tubules in pregnancy to meet these demands. The purpose of this review is to summarize these findings. RECENT FINDINGS: The work to date suggests an important role for the distal nephron in both the renal sodium and potassium reabsorption during pregnancy. There is strong evidence that renal sodium reabsorption is mediated by the epithelial sodium channel (ENaC). Whereas renal potassium reabsorption is mediated by upregulation of potassium retaining transporters (HKA2) and downregulation of potassium secreting channels (ROMK, BK). SUMMARY: Fetal growth restriction and hypertensive disorders of pregnancy including preeclampsia are marked by suboptimal maternal plasma volume expansion, which is determined by renal electrolyte handling. Therefore, understanding the physiologic demand for sodium and potassium in pregnancy and the adaptations required to support these needs is necessary for the effective treatment of diseased states of pregnancy.