Objective This study examined vascular actions of angiotensin 1–7 (ANG 1–7) in human atrial and adipose arterioles. Approach and Results The endothelial-derived hyperpolarizing factor of flow mediated dilation (FMD) switches from anti-proliferative nitric oxide (NO) to pro-atherosclerotic hydrogen peroxide (H2O2) in arterioles from humans with coronary artery disease (CAD). Given the known vasoprotective properties of ANG 1–7, we tested the hypothesis that overnight ANG 1–7 treatment restores the NO-component of FMD in arterioles from CAD patients. Endothelial telomerase activity is essential for preserving the NO-component of vasodilation in the human microcirculation, thus we also tested whether telomerase activity was necessary for ANG 1–7 mediated vasoprotection by treating separate arterioles with ANG 1–7 ± the telomerase inhibitor BIBR-1532. ANG 1–7 dilated arterioles from patients without CAD, whereas dilation was significantly reduced in arterioles from CAD patients. In atrial arterioles from CAD patients incubated with ANG 1–7 overnight, the NO synthase inhibitor L-NAME abolished FMD while the H2O2 scavenger PEG catalase had no effect. Conversely, in vessels incubated with ANG 1–7 + BIBR-1532, L-NAME had no effect on FMD but PEG catalase abolished dilation. In cultured human coronary artery endothelial cells, ANG 1–7 significantly increased telomerase activity. These results indicate that ANG 1–7 dilates human microvessels, and dilation is abrogated in the presence of CAD. Further, ANG 1–7 treatment is sufficient to restore the NO component of FMD in arterioles from CAD patients in a telomerase-dependent fashion. Conclusion ANG 1–7 exerts vasoprotection in the human microvasculature via modulation of telomerase activity.

Introduction The present study explored the mechanism of mTORC2 activation in Sprague Dawley (SD) rats and the protective effect of mTORC2 inhibition in salt‐induced hypertension and kidney injury in Dahl salt‐sensitive (SS) rats. Although there are evidences that the mTORC2 pathway contributes to renal podocyte homeostasis and tubular epithelial Na+ and K+ transport, this pathway has remained unexplored in hypertension. Since excess production of H2O2 in the kidney is the hallmark of salt‐induced hypertension in the SS rat, we hypothesized that H2O2 may stimulate mTORC2 and contributes to this salt‐sensitive form of hypertension. Methods H2O2 (347 nmol/Kg/min) was chronically infused for 3 days into renal medullary interstitium of unilaterally nephrectomized (SD) rats to determine the in vivo consequence of H2O2 upon mTORC2 activity. To test the protective effect of PP242 (ATP competitive inhibitor of both mTORC1 and mTORC2), SS rats were divided in to four groups. Groups 1 and 2: vehicle (group 1) or PP242 (group 2) was administrated daily (i.p., 15 mg/Kg/day) for 4 days to SS rats fed a 0.4% NaCl diet (control), then switched to a 4.0% NaCl diet for 21 days. Groups 3 and 4: rats were fed 4.0 % NaCl for 7 days before being treated with vehicle (group 3) or PP242 (group 4) for next 14 days while maintained on the 4.0 % NaCl diet. Kidneys were harvested to measure the protein levels of pAKT S473 and AKT by Western blots. mTORC2 activity was determined by assessing the ratio pAKTS473 and AKT. Microalbuminuria was quantified and kidney sections were immunostained with antibody against anti‐CD3 to determine the renal T lymphocytes infiltration. Results A significant increase of mTORC2 (pAKT S473/AKT) activity was observed in the renal cortex of SD rats infused with H2O2 for 3 days compared to saline infused rats. PP242 treatment significantly reduced H2O2 stimulated mTORC2 activity in nephron segments isolated from SS rat kidneys in vitro. Daily adminsiration of PP242 significantly reduced salt‐induced blood pressure in SS rats which averaged 119 ± 2 mmHg in group 2 rats (n=7) compared to 168 ± 3 mmHg in group 1 rats (n=7). Importantly, PP242 reversed the hypertension from 143 ± 3 mmHg to the base line 126 ± 5 mmHg in 2 days and maintained it at similar levels over the next 12 days in the group 4 rats (n=7) compared with group 3 rats (n=6). Albuminuria was greatly reduced with urine albumin excretion (mg/day) averaging 32.8 ± 3 in group 2 rats compared to 256 ± 37 in group 1 rats. PP242 treatment notably reduced infiltration of T lymphocytes in the kidneys of SS rats fed a 4.0% NaCl diet. CD3+ cells/mm2 averaging 36.0 ± 11.0 compared to 157.0 ± 40.0 in the cortex and 24.0 ± 9.0 compared to 218.0 ± 24.0 in the outer medulla in group 2 versus group 1 rats. Conclusion These data show that mTORC2 is required for the initiation of salt‐induced hypertension and therapeutic suppression of this pathway prevents and reverses the salt‐induced hypertension and and kidney injury in SS rats. This abstract is from the Experimenta...

Cardiovascular-related pathologies are the single leading cause of death in patients with chronic kidney disease (CKD). Previously, we found that a 5/6th nephrectomy model of CKD leads to an upregulation of miR-21-5p in the left ventricle, targeting peroxisome proliferator-activated receptor-α and altering the expression of numerous transcripts involved with fatty acid oxidation and glycolysis. In the present study, we evaluated the potential for knockdown or overexpression of miR-21-5p to regulate lipid content, lipid peroxidation, and mitochondrial respiration in H9C2 cells. Cells were transfected with anti-miR-21-5p (40 nM), pre-miR-21-5p (20 nM), or the appropriate scrambled oligonucleotide controls before lipid treatment in culture or as part of the Agilent Seahorse XF fatty acid oxidation assay. Overexpression of miR-21-5p attenuated the lipid-induced increase in cellular lipid content, whereas suppression of miR-21-5p augmented it. The abundance of malondialdehyde, a product of lipid peroxidation, was significantly increased with lipid treatment in control cells but attenuated in pre-miR-21-5p-transfected cells. This suggests that miR-21-5p reduces oxidative stress. The cellular oxygen consumption rate (OCR) was increased in both pre-miR-21-5p- and anti-miR-21-5p-transfected cells. Levels of intracellular ATP were significantly higher in anti-mR-21-5p-transfected cells. Pre-miR-21-5p blocked additional increases in OCR in response to etomoxir and palmitic acid. Conversely, anti-miR-21-5p-transfected cells exhibited reduced OCR with both etomoxir and palmitic acid, and the glycolytic capacity was concomitantly reduced. Together, these results indicate that overexpression of miR-21-5p attenuates both lipid content and lipid peroxidation in H9C2 cells. This likely occurs by reducing cellular lipid uptake and utilization, shifting cellular metabolism toward reliance on the glycolytic pathway. NEW & NOTEWORTHY Both overexpression and suppression of miR-21-5p augment basal and maximal mitochondrial respiration. Our data suggest that reliance on glycolytic and fatty acid oxidation pathways can be modulated by the abundance of miR-21-5p within the cell. miR-21-5p regulation of mitochondrial respiration can be modulated by extracellular lipids.

Cardiovascular (CV) and renal diseases are increasingly prevalent in the United States and globally. CV-related mortality is the leading cause of death in the United States, while renal-related mortality is the 8th. Despite advanced therapeutics, both diseases persist, warranting continued exploration of disease mechanisms to develop novel therapeutics and advance clinical outcomes for cardio-renal health. CV and renal diseases increase with age, and there are sex differences evident in both the prevalence and progression of CV and renal disease. These age and sex differences seen in cardio-renal health implicate sex hormones as potentially important regulators to be studied. One such regulator is G protein-coupled estrogen receptor 1 (GPER1). GPER1 has been implicated in estrogen signaling and is expressed in a variety of tissues including the heart, vasculature, and kidney. GPER1 has been shown to be protective against CV and renal diseases in different experimental animal models. GPER1 actions involve multiple signaling pathways: interaction with aldosterone and endothelin-1 signaling, stimulation of the release of nitric oxide, and reduction in oxidative stress, inflammation, and immune infiltration. This review will discuss the current literature regarding GPER1 and cardio-renal health, particularly in the context of aging. Improving our understanding of GPER1-evoked mechanisms may reveal novel therapeutics aimed at improving cardio-renal health and clinical outcomes in the elderly.

The mechanisms that regulate renal survival and atrophy under pathological stress remain incompletely understood. This knowledge is essential for developing new strategies to preserve renal function in patients with various forms of renal disease. We have utilized the 5/6 nephrectomy (5/6Nx) model to study the effects of renal insufficiency in 10 week old male Sprague Dawley rats. In this model excision of 2/3 of the left kidney and the entire right kidney induced substantial hypertrophy of the remnant kidney within 7 weeks of surgery. Conversely, excision of 2/3 of the left kidney, while leaving the right kidney intact (1/3 nephrectomy; 1/3Nx), resulted in atrophy of the remnant kidney within the same period. This lead us to hypothesize that removal of the right kidney 7 weeks after 1/3 surgery could induce hypertrophic remodeling of the left remnant kidney even after atrophic remodeling has occurred. To test this, we performed sham surgeries consisting of laparotomy and renal vessel isolation, right uninephrectomy (1Nx), 1/3Nx, or 5/6Nx surgery, as described above (n= 4–11/group). After 7 weeks we performed ultrasound analysis to evaluate the transverse renal cross‐sectional area (CSA) of the left kidney in each model. Sham surgery was then performed on all animals except for a subset of the 1/3Nx group which had their right kidneys excised. The animals were allowed to recover for another 3 weeks before the left kidney remodeling was again evaluated by ultrasound, acute anesthetized blood pressure was measured, and blood and tissues were collected for analysis. Statistical analysis of ultrasound data was completed using two‐way RM ANOVA, and all other by one‐way ANOVA. Ultrasound analysis of left kidney CSA revealed significant hypertrophy in the 1Nx and 5/6Nx models by week 7, when compared to sham‐operated controls, with no further increase in size evident by week 10. We observed the anticipated reduction in CSA in the 1/3Nx group by week 7 (1.06 ± 0.02, sham vs. 0.58 ± 0.01 cm2, 1/3Nx; p <0.05). The 1/3Nx rats subjected to removal of the right kidney experienced a significant hypertrophy of the remnant kidney (1.31 ± 0.04 cm2) between weeks 7 and 10, however this hypertrophy failed to reach the level observed in the 5/6Nx model (2.06 ± 0.05 cm2). Those 1/3Nx rats that retained their right kidney had no apparent change in remnant kidney size by week 10 (0.56 ± 0.03 cm2). These changes in renal dimensions were consistent with wet weights of the remnant kidney weights. Systolic and diastolic blood pressures measured at the end of the study were not statistically different from that of sham operated controls in any surgical group, however pressures tended to be higher in the 1/3Nx rats that had their right kidneys removed at week 7. We determined that removal of the right kidney could stimulate an atrophied left kidney to hypertrophy. In conclusion, the changes observed in this study suggest that these surgical models will allow us to study the mechanisms that regulate hypertrophy and atrophy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The current understanding of sodium (Na + ) handling, which is critical for blood pressure regulation, is based on studies done primarily in male (M) subjects. Our recent studies showed that female (F) Sprague Dawley (SD) rats reach a steady state in urinary Na + excretion after 1 day of increasing dietary Na + intake while M rats take 3-5 days. RNA sequencing data suggests that renal endothelin-1 (ET-1) signaling plays an important role in this difference, however, the role of ET receptors in sex-differences in acclimation to a high salt (HS) diet is not clear. We hypothesized that ET receptors mediate the advanced natriuretic capacity of F rats, compared to M rats, during acclimation to a HS diet. To test our hypothesis, M and F SD rats were implanted with telemeters and randomly assigned to treatment with A-182086, a dual antagonist of ET receptor A and B, or vehicle. After 2-3 days of treatment, rats were placed into metabolic cages and 24-hour urine samples were collected on a normal salt (NS, 0.3% NaCl) diet and after challenging rats with a HS (4% NaCl) diet for 1 day. Treatment with the ET receptor antagonist increased serum ET-1 and mean arterial pressure (MAP) to a comparable extent in both sexes (ET-1: M; 4.17 ± 0.65 vs 0.32 ± 0.02 pg/ml P=0.0004, F; 4.94 ± 1.01 vs 0.30 ± 0.01 pg/ml P<0.0001; MAP: M; 126 ± 2 vs 116 ± 3 mmHg P<0.0001, F; 114 ± 3 vs 107 ± 3 mmHg P<0.0043). On a NS and HS diet, F rats treated with A-182086 consumed more food than corresponding M rats (NS; P=0.0377, HS; P<0.0001), whereas vehicle-treated rats have no sex-difference in food intake. In vehicle-treated rats, urinary Na + excretion was higher in F compared to M rats on either NS or HS diet. Interestingly, ET receptor antagonism eliminates these sex differences in natriuresis during both dietary phases. In particular, A-182086 promotes urinary Na + excretion in M rats fed a HS diet (65.0 ± 2.3 vs 28.2 ± 4.6 % of Na + intake, P=0.0004). In conclusion, ET receptor antagonism eliminates sex-differences in natriuresis on NS and day 1 of HS. In opposition to our hypothesis, ET receptor antagonism promotes HS-induced natriuresis in M rats rather than attenuating the natriuretic capacity of F rats. A better mechanistic understanding of M-F differences in natriuresis could reveal sex-specific targets for hypertension treatment.

Progression of chronic kidney disease (CKD) involves compensatory hypertrophy of remaining nephrons leading to additional nephron loss. Understanding the precise mechanisms responsible for hypertrophy may help develop novel strategies to preserve renal function in people living with CKD. Our laboratory utilized the 5/6 nephrectomy (5/6Nx) rat model of renal insufficiency, where 2/3 of the left kidney and the entire right kidney was surgically excised in 10-week-old male Sprague Dawley rats. The remnant kidney underwent substantial compensatory hypertrophy between 5- and 7-weeks post-surgery. We then performed transcriptomic analysis of kidneys collected 6 weeks after 5/6Nx or sham surgery (n=6/group) to gain an unbiased perspective of molecular pathways involved with hypertrophic remodeling. Through transcriptomic analysis, we found that transcript expression of glial cell derived Neurotrophic factor (GDNF) was 7.64-fold higher in 5/6Nx remnant kidneys compared to sham-operated control. GDNF is an essential kidney morphogen in the developmental process that had not been implicated in renal compensatory hypertrophic remodeling. Immunohistochemistry (IHC) showed increased GDNF protein expression in the 5/6Nx remnant kidney. In the kidney GDNF elicits biological function through binding of its co-receptor GDNF family receptor α (GFRα) which then forms a complex with the tyrosine kinase RET resulting in downstream signaling. We hypothesized that GDNF signaling promotes tubular hypertrophic growth response in the 5/6Nx. To test this, we pharmacologically inhibited the GDNF/GFRα/RET complex using pralsetinib (3mg/kg/bw in corn oil; 1x daily oral gavage) from 5- to 7-weeks post-5/6Nx or sham surgery. Treatment control animals received vehicle. During this time we collected blood, urine, and conducted ultrasound analysis of the remnant kidney transverse cross sectional-area. Analysis of left kidney CSA revealed a decrease in kidney size between weeks 5 and 7 (1.58 ± 0.89 cm2, treatment vs 2.06 ± 0.071 cm2, vehicle; p=0.02; a two-way RM ANOVA), kidney wet weight and kidney/body weight were lower in pralsetinib vs. vehicle treated animals (66.85 ± 4.47% and 67.19 ± 2.30%, respectively; mean ± SEM, p < 0.05, student’s t-test). IHC analysis of renal cortex showed pralestinib treatment decreased proximal tubule diameter compared to vehicle (61.51 ± 3.45 μm, treatment vs 71.33 ± 9.82 μm, vehicle; p=0.01, student’s t-test). Additionally, plasma creatinine and blood urea nitrogen levels were decreased when compared to vehicle treated animals (0.88 ± 0.51 mg/dL, treatment vs 1.4 ± 0.21 mg/dL, vehicle; mean ± SEM, p < 0.03, student’s t-test and 37.63 ± 10.62 mg/dL, treatment vs 71.13 ± 33.50 mg/dL, vehicle; mean ±SEM, p<0.001, student’s t-test, respectively). Taken together, our data suggests inhibition of RET signaling prevented whole kidney hypertrophic growth and proximal tubule remodeling while preserving kidney function following 5/6Nx in rats. NIH-T32 Training Grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Remnant kidney models of chronic kidney disease include the 1/3 nephrectomy (1/3Nx), and 5/6 nephrectomy rats. The 1/3Nx remnant kidney spontaneously atrophies with time, and the 5/6Nx hypertrophies. This disparate remodeling must occur with commensurate vascular changes, examination of which may reveal important molecular pathways and mechanisms underlying changes in renal function. The first step toward realizing this, and the aim of this study, was to characterize the renal vasculature from 1/3Nx, 5/6Nx, and sham Nx rats. Methods: The vasculature was examined in rats 10 weeks following surgery. To-date, these renal vasculatures have been quantified by (1) assessment of vascular density by analysis of bisected, renal vascular casts, and (2) renal blood flow doppler ultrasound. Data: Avg±SEM. Results: 1/3Nx remnant kidney atrophied and 5/6Nx hypertrophied (cross-sectional area: sham n=9; 241±7. 5/6Nx, n=9; 247±8. 1/3Nx, n=12; 132±11: mm 2 ). Despite this, kidney height (intact kidney long axis) to width (short axis) ratio was similar between the remnant models (5/6Nx; 0.8±0.04. 1/3Nx; 0.9±0.04). Sham kidney vascular density was significantly greater than that of 1/3Nx remnant kidneys, which exceeded that of 5/6Nx rats. Cortex vascular density was significantly different between all groups (sham; 95±2%. 1/3Nx; 57±9%. 5/6Nx; 25±11%). The statistical mode of the arterial blood flow distribution was not different between the groups, but mode venous flow was (sham; 100±6. 1/3Nx; 118±9. 5/6Nx; 136±8. Relative units.). Homogeneity of flow was quantified by expressing flow-direction boundaries as a percent of flow area. Sham flow was significantly more homogenous (1.8±0.2) than 1/3Nx (3.7±0.5), and 5/6Nx rats (3.9±0.3). Conclusion: These data demonstrate changes in vascular anatomy and function in both remnant kidney models. Interrogation of anatomical changes and molecular pathways may reveal novel treatment approaches.