Hemodynamic phenotyping of transgenic rats with ubiquitous expression of an angiotensin-(1-7)-producing fusion protein

Activation of the angiotensin (Ang) converting enzyme 2/Ang-(1-7)/MAS receptor pathway of the renin-angiotensin system induces protective mechanisms in different diseases. Herein, we describe the cardiovascular phenotype of a new transgenic rat line (TG7371) that expresses an Ang-(1-7)-producing fusion protein. The transgene-specific mRNA and the corresponding protein were shown to be present in all evaluated tissues of TG7371 with the highest expression in aorta and brain. Plasma Ang-(1-7) levels, measured by radioimmunoassay were similar to control SD rats, however high Ang-(1-7) levels were found in the hypothalamus. TG7371 showed lower baseline mean arterial pressure, assessed in conscious or anesthetized rats by telemetry or short-term recordings, associated with increased plasma ANP and higher urinary sodium concentration. Evaluation of regional blood flow and hemodynamic parameters with fluorescent microspheres showed a significant increase in blood flow in different tissues (kidneys, mesentery, muscle, spleen, brown fat, heart and skin), with a resulting decreased total peripheral resistance. TG7371 rats also presented increased cardiac and global sympathetic tone, increased plasma AVP levels and decreased free water clearance. Altogether, our data show that expression of an Ang-(1-7)-producing fusion protein induced a hypotensive phenotype due to widespread vasodilation and consequent fall in peripheral resistance. This phenotype was associated with an increase in ANP together with an increase in AVP and sympathetic drive, which did not fully compensate the lower BP. Here we present the hemodynamic impact of long-term increase in tissue expression of an Ang-(1-7)-fusion protein and provide a new tool to investigate this peptide in different pathophysiological conditions.

PECULIARITIES OF IONOREGULATORY RENAL FUNCTION OF RATS IN THE DYNAMICS OF EXPERIMENTAL DIABETES MELLITUS DEVELOPMENT WITH UNDERLYING PHARMACOLOGICAL BLOCADE OF RENIN-ANGIOTENSINALDOSTERONE SYSTEM

The aim of the study – to explore the role of the renin-angiotensin-aldosteronesystem (RAAS) in the disturbance of ionoregulatory renal function in alloxan-inducedexperimental diabetes mellitus (EDM).Material and methods. The experiments were carried out on 78 white non-linearmature male rats with 11-, 26- and 46-day long alloxan-induced EDM with underlyingpharmacological blockade of RAAS by administration of kaptopril. The study ofionoregulating function of the kidneys was provided by the clearance method under thecondition of water 2-hour diuresis.Results. Pharmacological blockade of RAAS in rats with alloxan-induced EDM causedan intensification of natriuresis at all stages of the experiment: increased urinaryconcentration of sodium ions, its excretion and clearance. On the 11th day of EDM, thesodium filtration charge increased with the development of hyponatremia, proximal anddistal sodium reabsorption standardized in volume of glomerular filtrate (GF) decreased,kaliuresis was suppressed, and sodium-free water clearance elevated. In case of 26-daylong EDM, the sodium filtration charge decreased, its absolute and relative reabsorption,the distal sodium reabsorption standardized by GF increased. Kaliuresis increased. In46-day long EDM, the sodium filtration charge decreased, and hyponatremia enhanced.Absolute and relative sodium reabsorption reduced due to both – proximal and distal.Kaliuresis augmented, the clearance of sodium-free water declined.Conclusions. The increase in urinary sodium loss during the 11-day EDM is stipulatedby glomerular hyperfiltration, causing a functional weakening of the tubulotubularbalance and relative dysfunction of the distal segment of the nephron, emphasizing therenoprotective effect of RAAS on ionoregulatory function of the kidneys. The decrease inthe total reabsorption potential of the tubular segment of the nephron in the dynamics ofEDM development reflects on the proximal tubules, and preserved tubulotubular balancecertifies functional intactness of the distal tubules in 26-day long EDM. RAAS pathologicalactivation and attenuation of the renal blood flow autoregulation by tubuloglomerularfeedback may serve as an initiating factor in the development of tubular disorders in 26-day long alloxan diabetes with following progression in 46-day long EDM.

Is Water-Only Fasting Safe?

Background Water-only fasting (WF) is a practice used to improve and maintain health. Objective The aim of the study was to show whether WF performed for 8 days may be a threat to the health and/or life of people undergoing this practice. Methods Twelve middle-aged men participated in the study. During the 8-day WF, the subjects ate no food except for drinking mineral water. Before and after WF, all subjects had a series of tests performed, beginning with the level of perceived stress and somatic measurements. The concentrations of creatinine, sodium (Na+), potassium (K+), total calcium (Ca), magnesium (Mg++), urea (U), uric acid (UA) and total protein were determined in this urine and in the serum. For these substances, the values ​​of clearance, renal filtration and fractional excretion were calculated. The osmotic clearance and free water clearance as well as the amount of daily urinary excretion of creatinine, Na+, K+, Ca, Mg++, U and UA were also calculated. Moreover, the concentration of glucose in the serum and the concentration of β-hydroxybutyrate in the plasma were determined. In urine, specific gravity, pH and osmolality were also measured. Results After 8 days of WF, the study showed a significant reduction in the level of perceived stress, weight loss, changes in body composition, dehydration, increased ketogenesis, hyperuricemia, decreased serum glucose concentration, and hyponatremia. These changes were accompanied by Na+, K+ and protein sparing, decreased serum Ca and Mg++ concentrations, and reduced daily volume of more acidic urine with elevated specific gravity. Conclusions After 8 days of WF, all subjects were found to remain safe and feel the sense of well-being. However, the appearance of the above-mentioned adverse metabolic effects, despite partially effective renal compensations, suggests that the further continuation of fasting intervention by the subjects would be detrimental to their body.

The renal and cardiovascular effects of SGLT2 inhibition in combination with loop diuretics in patients with type 2 diabetes and chronic heart failure (RECEDE-CHF) trial

Introduction The SGLT2 inhibitors have been shown to reduce heart failure (HF) hospitalisations in patients with Type 2 diabetes (T2D), and to improve outcomes in those with HF irrespective of T2D status. Given this, it is likely that they will be co-prescribed with a loop diuretic. The combined diuretic effect of SGLT2 inhibitors and loop diuretic has not been well defined. Purpose The aim of this study was to assess the diuretic and natriuretic effect of empagliflozin in patients with T2D and chronic HF in combination with loop diuretics. Methods and analysis RECEDE-CHF (Renal and Cardiovascular Effects of SGLT2 inhibition in combination with loop Diuretics in patients with T2D and Chronic HF) was a randomised, double-blind, placebo-controlled, cross-over trial. Patients were randomised to empagliflozin 25 mg once a day or placebo for 6 weeks with study assessments at day 3 and week 6. Following a 2 week washout period, patients then entered the second treatment arm. The primary outcome was change in 24-hour urinary volume, when compared to placebo at day 3 and week 6. Results 23 participants mean age 69.8 years, (male 73.9%) with T2D and chronic HF (all ejection fraction <50%) on a mean furosemide dose of 49.6 mg/day were recruited. In comparison to placebo, empagliflozin caused a significant increase in 24-hour urinary volume at both time points (day 3, mean difference of 549.3 ml, 95% confidence interval 151.4 to 947.2, p=0.004 and week 6, mean difference of 542.8 ml, 95% CI 134.9 to 950.7, p=0.005), adjusted for treatment order and baseline 24-hour urine volume. Empagliflozin did not cause a significant increase in the 24-hour urinary sodium as measured in mmol/L at both time points (day 3, mean difference compared to placebo −2.96 mmol/L, 95% CI: −21.55 to 15.64, p=1.00, week 6 mean difference compared to placebo −8.41 mmol/L, 95% CI: −27.00 to 10.18, p=0.81). Empagliflozin caused a significant increase in electrolyte-free water clearance (cH20e) at day 3 (mean difference compared to placebo 287.36 ml/min, (95% CI 31.79 to 542.93, p=0.022) and at week 6 (mean difference when compared to placebo 255.69 ml/min (95% CI: −284.12 to 220.76, p=0.048) when corrected for furosemide dose. 21.7% (n=5) participants had to have their furosemide dose reduced by 50% whilst on the active treatment arm of empagliflozin by day 3. On discontinuation of the active treatment arm with empagliflozin, two participants experienced hospital admissions with decompensated cardiac congestion. Conclusions Empagliflozin caused a significant increase in 24-hour urine volume when used in combination with loop diuretic when compared to placebo. There was no increase in 24-hour urinary sodium excretion. The greater electrolyte-free water clearance induced by empagliflozin observed in this study may support a proposed hypothesis that this could result in a greater fluid clearance from the interstitial space than from the circulating volume. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation (BHF)

Renal function is a major determinant of ICU-acquired hypernatremia: A balance study on sodium handling

Background and ObjectivesThe development of ICU-acquired hypernatremia (IAH) is almost exclusively attributed to ‘too much salt and too little water’. However, intrinsic mechanisms also have been suggested to play a role. To identify the determinants of IAH, we designed a prospective controlled study.MethodsPatients with an anticipated length of stay ICU > 48 hours were included. Patients with hypernatremia on admission and/or on renal replacement therapy were excluded. Patients without IAH were compared with patients with borderline hypernatremia (≥ 143 mmol/L, IAH 143) and more severe hypernatremia (≥ 145 mmol/L, IAH 145).ResultsWe included 89 patients, of which 51% developed IAH 143 and 29% IAH 145. Sodium intake was high in all patients. Fluid balances were slightly positive and comparable between the groups. Patients with IAH 145 were more severely ill on admission, and during admission, their sodium intake, cumulative sodium balances, serum creatinine and copeptin levels were higher. According to the free water clearance, all the patients conserved water. On multivariate analysis, the baseline serum creatinine was an independent risk factor for the development of IAH 143 and IAH 145. Also, the copeptin levels remained significant for IAH 143 and IAH 145. Sodium intake remained only significant for patients with IAH 145.ConclusionsOur data support the hypothesis that IAH is due to the combination of higher sodium intake and a urinary concentration deficit, as a manifestation of the renal impairment elicited by severe illness.

Influence of electrolyte balance on the prognosis of long-term cardiovascular events after acute coronary syndrome

Aim. To assess the effect of electrolyte changes on the prognosis of long-term cardiovascular events after acute coronary syndrome (ACS).Material and methods. The study included 105 patients with ACS who underwent coronary angiography (CA) with coronary stenting. At the study inclusion (before CA with coronary stenting), we collected data on traditional risk factors, analyzed levels of urinary sodium and potassium, kaliuresis and natriuresis. Free water clearance (FWC) and electrolyte free water clearance (EFWC), as well as fluid balance using bioelectrical impedance analysis were determined. Study endpoints (fatal and nonfatal cardiovascular events) were determined 6,2±0,2 months after CA with coronary stenting.Results. It was found that a decrease in urinary sodium (χ2=5,64, p=0,02, Constanta B0 =-0,62, Estimate =-16,5) and natriuresis (χ2=4,1, р=0,044, Constanta B0 =-1,38, Estimate =-5,2) increase the death risk. Urinary sodium of 0,2 mol/L and natriuresis of 0,5 mol are threshold levels of increased risk of death. Urinary potassium decrease was associated with an increase in death risk (threshold level — 0,5 mol/L, χ2=4,99, р=0,025, Constanta B0 =-0,63, Estimate =-70,4) and acute myocardial infarction (threshold level — 0,06 mol/L, χ2=3,93, р=0,04, Constanta B0 =-0,99, Estimate =-58,0) in the long-term period. Increase in EFWC increased the likelihood of long-term transient ischemic attack after ACS (χ2=4,61, р=0,03, Constanta B0 =-2,95, Estimate =-1,0). There were no significant relationships in the matter of FWC (p>0,05). However, with a decrease in intracellular fluid volume compared to normal values and a decrease in FWC or an increase in EFWC, the likelihood of longterm composite endpoints after ACS increases.Conclusion. As a result of the study, risk markers for long-term fatal and non-fatal cardiovascular events after ACS were established: decrease in urinary sodium <0,2 mol/l and potassium <0,5 mol/l; decrease in FWC and increase in EFWC with or without cellular dehydration. The established markers can complement the current cardiovascular risk score methods in patients with ACS.

Racial Differences in Risk Factors for Kidney Stone Formation

Background and objectivesIncidence of kidney stone disease is rising. It is not known whether mechanisms of stone formation differ across racial groups. Our objective was to identify differing lithogenic risk factors across racial groups in idiopathic nephrolithiasis.Design, setting, participants, & measurementsWe conducted a retrospective cohort study evaluating metabolic risk factors in black and age-matched white idiopathic stone formers at our tertiary referral center. We compared serum and urine metabolic risk factors pre- and post-treatment across racial groups using analysis of covariance. Generalized linear modeling was used to build regression models for risk of stone formation in both groups.ResultsAmong 117 black and 172 white stone formers, urine volume was lower in black stone formers (1.4±0.8 versus 2.0±0.8 L/d, P<0.001). Urine calcium was lower in black stone formers (116±70 versus 217±115 mg/d, P<0.001). Supersaturations for calcium oxalate were similar among the groups, whereas calcium phosphate supersaturation was higher in white stone formers, and uric acid supersaturation was higher in black stone formers. Electrolyte free water clearance was significantly lower in black stone formers (207±780 versus 435±759 ml/d, P=0.02). In the subgroup of 77 black patients and 107 white patients with post-treatment evaluations, urine volume rose significantly and similarly in both groups. Urine sodium was unchanged in whites but increased in blacks by 40 mmol/d (95% confidence interval, 32 to 48 mmol/d). Electrolyte free water clearance remained lower in black stone formers (385±891 versus 706±893 ml/d, P=0.02). Post-treatment supersaturations were similar across the groups except for calcium phosphate, which improved with treatment in whites.ConclusionsBlack stone formers have lower 24-hour urine calcium excretion and urine volume. Increases in urine volume with treatment were associated with increased solute, but not free water, excretion in black stone formers.

0823 Age-Related Changes in Nocturnal Urine Composition

Introduction In humans sleeping nocturnally, nocturnal polyuria (NP) refers to high rate of overnight urine production. NP is a heterogeneous condition that may reflect both free water and/or sodium diuresis, but the influence of age on differential fluid handling remains poorly understood. This study examined diuresis rate, sodium clearance, and free water clearance (FWC) by age, time of day (nighttime vs. daytime) and NP status (positive/negative) in subjects under entrained conditions sleeping nocturnally. Methods Convenience samples (age range 18-91; 82 men, 148 women) recruited from a urology ambulatory care unit (n=135) or continence clinic (n=95) collected 8 urine samples at 3-hour intervals over a single 24-hr period. Three separate mixed linear models were constructed for diuresis rate, sodium clearance, and FWC using four predictors: NP status (present [&gt;90mL/h] vs. absent), time of day (night = 0100, 0400, 0700), age (as a continuous measure), and study source. Results Subjects with NP experienced both higher nighttime vs. daytime diuresis rate (1.89 vs. 1.44 mL/min, p&lt;0.001), sodium clearance (0.91 vs. 0.74 mL/min, p&lt;0.001), and FWC (-0.38 vs. -0.71 mL/min, p&lt;0.001), whereas subjects without NP demonstrated lower nighttime vs. daytime diuresis rate (0.94 vs. 1.06, p=0.004) and no difference in sodium clearance (0.59 vs. 0.64, p=0.120) or FWC (-0.80 vs. -0.86, p=0.268). Regardless of NP status, FWC increased with age (p=0.039), and older age (&gt;70) was accompanied by an increase in the ratio of nighttime/daytime diuresis rate and both nighttime and daytime sodium clearance. Conclusion Irrespective of NP, older adults experience proportionally greater nocturnal sodium clearance, as well as a complex surge in both daytime and nighttime FWC. The data imply that both nocturnal sodium clearance and FWC may reflect the relevant substrate underlying excess nocturnal urine production in elderly persons. Support N/A

0822 Frail Older Men With Nocturia are Disproportionately Affected by Excess Nocturnal Urine Production

Introduction Nocturia is a risk factor for falls and hip fractures in older adults. We determined whether the Frailty Index (FI), incorporating comorbidities, functional performance, and physical signs, was associated with nocturia frequency and/or overnight urine production. Methods We examined nightly (24-hour) voiding diaries (men ≥65 years) in an outpatient urologic clinic demonstrating ≥2 nocturnal voids (n=158). FI calculations followed Rockwood (CMAJ 2005;173:489-95). A total of 39 conditions were assessed. Three FI groups were established: Low (≤0.077) (n=59), Intermediate (&gt;0.077 and &lt;0.179) (n=58), and High (≥0.179) (n=41). We compared number of nocturnal voids (NV), nocturnal urine volume (NUV) (in mL), and 24-hr total urine volume (24-hr TUV) (in mL) across groups. Results NV did not differ by group (p=0.333) (median for all groups=3). However, NUV (916 [671-1419] vs. 690 [505-942] vs. 630 [500-1050] mL) differentiated the High, Medium and Low FI groups (p&lt;0.001 via Kruskal-Wallis with Bonferroni pairwise adjustments), respectively. Similarly, 24-hr TUV differentiated the 3 groups (2200 [1800-2550] vs. 1620 [1259-2119] vs. 1650 [1390-2517] mL, p=0.005). Differences in NUV remained significant (p=0.006) after eliminating Diabetes Mellitus cases (n=44). However, differences did not persist for 24-hr TUV (p=0.180). Conclusion Higher NUV, but not 24-hr TUV, was a robust correlate of frailty in these older men. Accounting for diabetes did not diminish the effect. Although undiagnosed sleep apnea remains a possible cause, recent chronobiologic data (Monaghan et al, Age Aging, 2020, in press) suggest that nocturia in the aged is characterized by excess free water clearance early in the sleep period. This argues against solute-driven urine production (as might be expected in sleep apnea) in accounting for the effect. Nocturia may represent a conspicuous and important change in circadian rhythm of urine production occurring in old age. Support N/A