Acute kidney injury due to renal ischemia-reperfusion injury (IRI) may lead to chronic or end stage kidney disease. A greater understanding of the cellular mechanisms underlying IRI are required to develop therapeutic options aimed at limiting or reversing damage from IRI. Prior work has shown that deletion of the α subunit of the epithelial Na+ channel (ENaC) in endothelial cells protects from IRI by increasing the availability of nitric oxide. While canonical ENaCs consist of an α, β, and γ subunit, there is evidence of non-canonical ENaC expression in endothelial cells involving the α subunit. We therefore tested whether the deletion of the γ subunit of ENaC also protects mice from IRI to differentiate between these channel configurations. Mice with endothelial-specific deletion of the γ subunit and control littermates were subjected to unilateral renal artery occlusion followed by 48 h of reperfusion. No significant difference was noted in injury between the two groups as assessed by serum creatinine and blood urea nitrogen, levels of specific kidney injury markers, and histological examination. While deletion of the γ subunit did not alter infiltration of immune cells or cytokine message, it was associated with an increase in levels of total and phosphorylated endothelial nitric oxide synthase (eNOS) in the injured kidneys. Our studies demonstrate that even though deletion of the γ subunit of ENaC may allow for greater activation of eNOS, this is not sufficient to prevent IRI, suggesting the protective effects of α subunit deletion may be due, in part, to other mechanisms.

High salt diet is associated with increased risk of adverse cardiovascular events due to its effects on blood pressure, vascular stiffening, and fibrosis. The kidney maintains Na+ homeostasis in times of dietary excess by excreting it in urine. Additionally, the brain’s thirst centers sense excess Na+ and increase water intake. Recent work showed mice given high Na+ loads, provided in chow and water, have changes in glucocorticoid, mineralocorticoid, and urea production that lead to increased free water absorption, conservation of fluid with less water intake, and a switch to a catabolic state. However, mice given a salt load were not followed beyond a few weeks of treatment nor were the same results seen when free access to water was given. Therefore, we were interested in the metabolic changes that occur with long‐term dietary Na+ manipulation with free access to water. Mice were fed an 8% NaCl diet (HSD) beginning at~10 weeks of age and given free access to water. After three months, the HSD mice weighed significantly less than the controls (30.1±2.4g vs 33.3±2.8g), and this trend continued through 16 months of HSD treatment when the HSD animals weighed ~10g less than the controls (33.8±1.7g vs 44.8±7.4g). At 12 months, the HSD group consumed more food (19.9±1.0g vs 11.7±0.5g), therefore the weight loss cannot be accounted for through consumption. The HSD cohort also consumed more water (14.5±3.5mL vs 2.0±0.4mL) and produced more urine (16.4±2.7mL vs 4.0±0.2mL) than their control counterparts. At both 8 months and 16 months, the blood urea nitrogen levels in the HSD treated animals were significantly lower, suggesting these animals did not have increased urea production. Additionally, the HSD mice had 12.8% body fat at 8 months treatment as compared to 25.0% body fat in age‐matched controls. However, lean mass by weight was not significantly different between the HSD and control animals (27.9±0.4g vs 28.7±0.4g), suggesting there was no muscle wasting due to salt consumption. To address the physiological mechanism by which high salt intake reduced body fat and weight, mice were studied in metabolic cages to measure the major determinants of energy balance after just 5 weeks HSD, prior to the divergence in body weight. There was no difference in feeding between groups, however water intake was significantly elevated in the HSD animals. Total activity also did not differ between the HSD and control animals. Energy expenditure was significantly increased in HSD mice during the light cycle and there was a strong trend towards increased 24 hour energy expenditure (P=0.09). Finally, the respiratory quotient was significantly reduced in the HSD cohort during both the light and dark cycles, demonstrating increased whole‐body fat oxidation. Taken together, these data demonstrate that animals given an increased salt load have a higher metabolic demand and increased fatty acid oxidation, which were associated with reduced body weight and adiposity. These results suggest that the effects of sodium extend beyond blood press...

Mercury (Hg) shows the toxic effects in the environment although the etiology is not well characterized and the prevention of toxic effects induced by Hg is an important aspect of metabolic regulation in organisms. Channa punctata, a variety of species of fish was used in this study and the role of calcium carbonate on cholesterol, triglyceride and protein level in liver induced by HgCl2 was adopted. Fish were exposed to 1 and 10 µM of HgCl2 for 1h and cholesterol and triglyceride levels in excised liver were enhanced in response to HgCl2 when compared to respective controls however the effects were more pronounced for 1 µM concentration. Similar stimulatory effects on protein contents were demonstrated whenever they were exposed to HgCl2 (1 and 10 µM) and higher proteins were recorded for 10 µM concentration. The results indicate that HgCl2 causes severe toxic effects enhancing the above parameters. To clarify the role of CaCO3 on prevention of these effects, fish were treated with different concentrations (100 µM and 1 mM) of CaCO3 and CaCO3 + HgCl2. Cholesterol and triglyceride in liver were effectively reduced with CaCO3 (1 mM) + HgCl2 (10 µM) and CaCO3 (100 µM) + HgCl2 (1 µM) while 100 µM of CaCO3 potentially reduced the effects of HgCl2. Although CaCO3 was shown to reduce protein content effectively, however 100 µM concentrations have been found to inhibit the effects of HgCl2 preferentially. Our findings suggest that calcium carbonate might be involved in prevention of the toxic effects of Hg and may contribute to the survival process of this species.

Aldosterone is a steroid hormone that is necessary for sodium and water reabsorption in the distal tubules and collecting ducts of the kidney that is released in response to changes in volume and potassium levels. While aldosterone is typically associated with fluid retention due to its role in promoting sodium reabsorption, previous work has shown that aldosterone further exacerbates urine production in a rat model of diabetes insipidus. Because of this, we aimed to understand the effects of chronic aldosterone administration in mice with no underlying pathology to further elucidate the effects of chronic aldosterone administration on water reabsorption in the kidneys. Aldosterone was administered at a dose of 240 μg/kg/day via subcutaneous minipump to C57Bl/6 male mice housed in metabolic cages. By Day 15, mice given aldosterone had increased urine output compared to control animals (Ctl 4.8 ± 1.6 mL; Aldo 8.4 ± 2.4 mL), and this difference persisted through day 28. The aldosterone treated animals trended toward drinking more water starting at day 7, however significance was not obtained. To understand whether these animals were producing concentrated urine, we measured urine osmolality by freeze‐point depression. By Day 4, there was a significant decrease in daytime urine osmolality in the aldosterone administered mice compared to the control mice (Ctl 1219.8 ± 473.4 mOsm/kg; Aldo 638.8 ± 143.8 mOsm/kg), and by day 17, there was also a significant decrease in nighttime urine osmolality of the aldosterone treated versus control mice (Ctl 905.3 ± 175.1 mOsm/kg; Aldo 522.2 ± 127.3 mOsm/kg). Mice receiving aldosterone had a higher whole blood [Na+] (Ctl 146 ± 1.9mmol/L; Aldo 152 ± 1.3mmol/L) and lower whole blood [K+] (Ctl 4.8 ± 0.56mmol/L; Aldo 3.0 ± 0.34mmol/L) as measured by iSTAT cartridge after terminal cardiac blood draw. At a molecular level, a significant increase (~3.5 fold) in Aqp4 whole kidney mRNA levels were measured in the aldosterone treated mice compared to the control mice at both 2 weeks and 4 weeks of treatment. Additionally, Aqp1 and Aqp3 mRNA levels trended toward being lower in the aldosterone treated animals although significance was not reached. Aqp2 levels were variable, but unchanged at a transcript level. Protein expression and localization of these aquaporins are currently being investigated by Western blot and immunofluorescent staining. Together, these data indicate that chronic aldosterone administration causes a diuresis with decreased concentrating ability. We are currently testing whether correction of the serum [K+] corrects the concentrating defect, and whether vasopressin is involved in these responses.

The steroid hormone aldosterone is an important regulator of fluid balance and electrolyte homeostasis. While acute aldosterone signaling is canonically associated with fluid retention due to its role in promoting sodium reabsorption through ENaC in the distal nephron, chronic aldosterone exposure has been shown to exacerbate urine production in a rat model of diabetes insipidus through its actions on aquaporin 2 (AQP2) localization and expression. In our studies, we aimed to characterize not only the effect of chronic aldosterone administration on AQP2 expression and localization, but also its effect on the basolateral water channels, AQP3 and AQP4. Given the large urinary output we observed in our animals following chronic aldosterone treatments, we hypothesized all three of these channels would show reduced expression.Aldosterone was administered to male C57Bl/6 mice at a dose of 200 μg/kg/day for 28 days via subcutaneous minipumps. Aldosterone treatment caused a significant diuresis as compared to control animals by day 15 (Ctl 4.8 ± 1.6 ml; Aldo 8.4 ± 2.4 ml), and this difference persisted for the remainder of the experiment. Mice given aldosterone also consumed more water as compared to their control counterparts (Ctl 4.0 ± 1.2 ml; Aldo 9.6 ± 2.3 ml). Urine osmolality was measured by freeze-point depression, with a significant decrease in urinary concentration being noted during the daytime by day 4 (Ctl 1219.8 ± 473.4 mOsm/kg; Aldo 638.8 ± 143.8 mOsm/kg) and during the night by day 17 (Ctl 905.3 ± 175.1 mOsm/kg; Aldo 522.2 ± 127.3 mOsm/kg). At the end of the 28 days of aldosterone administration, mice receiving aldosterone had a higher whole blood [Na+] (Ctl 146 ± 1.9mmol/l; Aldo 152 ± 1.3mmol/l) and lower whole blood [K+] (Ctl 4.8 ± 0.56mmol/l; Aldo 3.0 ± 0.34mmol/l) as measured by iSTAT cartridge with terminal cardiac blood draw.Kidneys were examined by Western blot and immunofluorescent staining for expression and localization of the three AQPs. AQP2 expression decreased after both 14 and 28 days of aldosterone administration, with line scan analysis showing a decreased signal present at the apical membrane. AQP3 expression was decreased in the basolateral membranes of principal cells within the cortex of the kidney but increased in cells within the inner medulla after 28 days of aldosterone. AQP4 levels were significantly increased in AQP2-positive cells of the inner stripe of the outer medulla and the inner medulla, suggesting that this channel may be upregulated to compensate for changes in the other two aquaporins. Potassium supplementation to separate the effects of aldosterone administration from hypokalemia did not rescue the expression of these channels.Overall, our studies reveal that chronic aldosterone administration in healthy mice leads to polyuria and polydipsia, with decreased apical expression of AQP2 and variable changes in AQP3 and AQP4 dependent on location within in the kidney. This study was funded by NIH grants R01 DK130901 (to SS), R01 HL147818 and R01 DK129285 (to TRK), and P30 DK079307 (the Pittsburgh Center for Kidney Research). SMM was supported by T32 DK061296, T32 DK007052, and a grant from Relypsa. 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.