Role of Alström syndrome 1 in the regulation of glomerular hemodynamics.

Inactivating mutations in the ALMS1 gene in humans cause Alström syndrome, characterized by the early onset of obesity, insulin resistance, and renal dysfunction. However, the role of ALMS1 in renal function and hemodynamics is unclear. We previously found that ALMS1 is expressed in thick ascending limbs, where it binds and decreases Na+-K+-2Cl- cotransporter activity. We hypothesized that ALMS1 is expressed in macula densa cells and that its deletion enhances tubuloglomerular feedback (TGF) and reduces glomerular filtration rate (GFR) in rats. To test this, homozygous ALMS1 knockout (KO) and littermate wild-type Dahl salt-sensitive rats were studied. TGF sensitivity was higher in ALMS1 KO rats as measured by in vivo renal micropuncture. Using confocal microscopy, we confirmed immunolabeling of ALMS1 in macula densa cells (nitric oxide synthase 1 positive), supporting a role for ALMS1 in TGF regulation. Baseline glomerular capillary pressure was higher in ALMS1 KO rats, as was mean arterial pressure. Renal interstitial hydrostatic pressure was lower in ALMS1 KO rats, which is linked to increased Na+ reabsorption and hypertension. GFR was reduced in ALMS1 KO rats. Seven-week-old ALMS1 KO rats were not proteinuric, but proteinuria was present in 18- to 22-wk-old ALMS1 KO rats. The glomerulosclerosis index was higher in 18-wk-old ALMS1 KO rats. In conclusion, ALMS1 is involved in the control of glomerular hemodynamics in part by enhancing TGF sensitivity, and this may contribute to decreased GFR. Increased TGF sensitivity, enhanced glomerular capillary pressure, and hypertension may lead to glomerular damage in ALMS1 KO rats. These are the first data supporting the role of ALMS1 in TGF and glomerular hemodynamics.NEW & NOTEWORTHY ALMS1 is a novel protein involved in regulating tubuloglomerular feedback (TGF) sensitivity, glomerular capillary pressure, and blood pressure, and its dysfunction may reduce renal function and cause glomerular damage.

Decreased tubuloglomerular feedback response in high-fat diet-induced obesity.

Obesity increases the risk of renal damage, but the mechanisms are not clear. Normally, kidneys autoregulate to keep the glomerular capillary pressure (PGC), renal blood flow, and glomerular filtration rate in a steady state. However, in obesity, higher PGC, renal blood flow, and glomerular filtration rate are noted. Together, these may lead to glomerular damage. PGC is controlled mainly by afferent arteriole resistance, which, in turn, is regulated by tubuloglomerular feedback (TGF), a vasoconstrictor mechanism. High fat-induced obesity causes renal damage, and this may be related to increased PGC. However, there are no studies as to whether high-fat diet (HFD)-induced obesity affects TGF. We hypothesized that TGF would be attenuated in obesity caused by HFD feeding (60% fat) in Sprague-Dawley rats. Sprague-Dawley rats fed a normal-fat diet (NFD; 12% fat) served as the control. We studied 4 and 16 wk of HFD feeding using in vivo renal micropuncture of individual rat nephrons. We did not observe significant differences in body weight, TGF response, and mean arterial pressure at 4 wk of HFD feeding, but after 16 wk of HFD, rats were heavier and hypertensive. The maximal TGF response was smaller in HFD-fed rats than in NFD-fed rats, indicating an attenuation of TGF in HFD-induced obesity. Baseline PGC was higher in HFD-fed rats than in NFD-fed rats and was associated with higher glomerulosclerosis. We conclude that attenuated TGF and higher PGC along with hypertension in HFD-fed obese Sprague-Dawley rats could explain the higher propensity of glomerular damage observed in obesity.NEW & NOTEWORTHY Reduced tubuloglomerular feedback, higher glomerular capillary pressure, and hypertension in combination may explain the higher glomerular damage observed in high-fat diet-induced obesity.

High SARS-CoV-2 Viral Load in Urine Sediment Correlates with Acute Kidney Injury and Poor COVID-19 Outcome.

BACKGROUND: AKI is a complication of coronavirus disease 2019 (COVID-19) that is associated with high mortality. Despite documented kidney tropism of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there are no consistent reports of viral detection in urine or correlation with AKI or COVID-19 severity. Here, we hypothesize that quantification of the viral load of SARS-CoV-2 in urine sediment from patients with COVID-19 correlates with occurrence of AKI and mortality. METHODS: The viral load of SARS-CoV-2 in urine sediments (U-viral load) was quantified by qRT-PCR in 52 patients with PCR-confirmed COVID-19 diagnosis, who were hospitalized between March 15 and June 8, 2020. Immunolabeling of SARS-CoV-2 proteins Spike and Nucleocapsid was performed in two COVID-19 kidney biopsy specimens and urine sediments. Viral infectivity assays were performed from 32 urine sediments. RESULTS: A total of 20 patients with COVID-19 (39%) had detectable SARS-CoV-2 U-viral load, of which 17 (85%) developed AKI with an average U-viral load four-times higher than patients with COVID-19 who did not have AKI. U-viral load was highest (7.7-fold) within 2 weeks after AKI diagnosis. A higher U-viral load correlated with mortality but not with albuminuria or AKI stage. SARS-CoV-2 proteins partially colocalized with the viral receptor ACE2 in kidney biopsy specimens in tubules and parietal cells, and in urine sediment cells. Infective SARS-CoV-2 was not detected in urine sediments. CONCLUSION: Our results further support SARS-CoV-2 kidney tropism. A higher SARS-CoV-2 viral load in urine sediments from patients with COVID-19 correlated with increased incidence of AKI and mortality. Urinary viral detection could inform the medical care of patients with COVID-19 and kidney injury to improve prognosis.

Aldehyde Dehydrogenase 2 Activator Augments the Beneficial Effects of Empagliflozin in Mice with Diabetes-Associated HFpEF.

To ameliorate diabetes mellitus-associated heart failure with preserved ejection fraction (HFpEF), we plan to lower diabetes-mediated oxidative stress-induced 4-hydroxy-2-nonenal (4HNE) accumulation by pharmacological agents that either decrease 4HNE generation or increase its detoxification.A cellular reactive carbonyl species (RCS), 4HNE, was significantly increased in diabetic hearts due to a diabetes-induced decrease in 4HNE detoxification by aldehyde dehydrogenase (ALDH) 2, a cardiac mitochondrial enzyme that metabolizes 4HNE. Therefore, hyperglycemia-induced 4HNE is critical for diabetes-mediated cardiotoxicity and we hypothesize that lowering 4HNE ameliorates diabetes-associated HFpEF. We fed a high-fat diet to ALDH2*2 mice, which have intrinsically low ALDH2 activity, to induce type-2 diabetes. After 4 months of diabetes, the mice exhibited features of HFpEF along with increased 4HNE adducts, and we treated them with vehicle, empagliflozin (EMP) (3 mg/kg/d) to reduce 4HNE and Alda-1 (10 mg/kg/d), and ALDH2 activator to enhance ALDH2 activity as well as a combination of EMP + Alda-1 (E + A), via subcutaneous osmotic pumps. After 2 months of treatments, cardiac function was assessed by conscious echocardiography before and after exercise stress. EMP + Alda-1 improved exercise tolerance, diastolic and systolic function, 4HNE detoxification and cardiac liver kinase B1 (LKB1)-AMP-activated protein kinase (AMPK) pathways in ALDH2*2 mice with diabetes-associated HFpEF. This combination was even more effective than EMP alone. Our data indicate that ALDH2 activation along with the treatment of hypoglycemic agents may be a salient strategy to alleviate diabetes-associated HFpEF.

The deleterious role of the prostaglandin E2 EP3 receptor in angiotensin II hypertension.

Angiotensin II (ANG II) plays a key role in regulating blood pressure and inflammation. Prostaglandin E2 (PGE2) signals through four different G protein-coupled receptors, eliciting a variety of effects. We reported that activation of the EP3 receptor reduces cardiac contractility. More recently, we have shown that overexpression of the EP4 receptor is protective in a mouse myocardial infarction model. We hypothesize in this study that the relative abundance of EP3 and EP4 receptors is a major determinant of end-organ damage in the diseased heart. Thus EP3 is detrimental to cardiac function and promotes inflammation, whereas antagonism of the EP3 receptor is protective in an ANG II hypertension (HTN) model. To test our hypothesis, male 10- to 12-wk-old C57BL/6 mice were anesthetized with isoflurane and osmotic minipumps containing ANG II were implanted subcutaneously for 2 wk. We found that antagonism of the EP3 receptor using L798,106 significantly attenuated the increase in blood pressure with ANG II infusion. Moreover, antagonism of the EP3 receptor prevented a decline in cardiac function after ANG II treatment. We also found that 10- to 12-wk-old EP3-transgenic mice, which overexpress EP3 in the cardiomyocytes, have worsened cardiac function. In conclusion, activation or overexpression of EP3 exacerbates end-organ damage in ANG II HTN. In contrast, antagonism of the EP3 receptor is beneficial and reduces cardiac dysfunction, inflammation, and HTN.NEW & NOTEWORTHY This study is the first to show that systemic treatment with an EP3 receptor antagonist (L798,106) attenuates the angiotensin II-induced increase in blood pressure in mice. The results from this project could complement existing hypertension therapies by combining blockade of the EP3 receptor with antihypertensive drugs.

Urinary concentrating defect in mice lacking Epac1 or Epac2.

cAMP is a universal second messenger regulating a plethora of processes in the kidney. Two downstream effectors of cAMP are PKA and exchange protein directly activated by cAMP (Epac), which, unlike PKA, is often linked to elevation of [Ca2+]i. While both Epac isoforms (Epac1 and Epac2) are expressed along the nephron, their relevance in the kidney remains obscure. We combined ratiometric calcium imaging with quantitative immunoblotting, immunofluorescent confocal microscopy, and balance studies in mice lacking Epac1 or Epac2 to determine the role of Epac in renal water-solute handling. Epac1-/- and Epac2-/- mice developed polyuria despite elevated arginine vasopressin levels. We did not detect major deficiencies in arginine vasopressin [Ca2+]i signaling in split-opened collecting ducts or decreases in aquaporin water channel type 2 levels. Instead, sodium-hydrogen exchanger type 3 levels in the proximal tubule were dramatically reduced in Epac1-/- and Epac2-/- mice. Water deprivation revealed persisting polyuria, impaired urinary concentration ability, and augmented urinary excretion of Na+ and urea in both mutant mice. In summary, we report a nonredundant contribution of Epac isoforms to renal function. Deletion of Epac1 and Epac2 decreases sodium-hydrogen exchanger type 3 expression in the proximal tubule, leading to polyuria and osmotic diuresis.-Cherezova, A., Tomilin, V., Buncha, V., Zaika, O., Ortiz, P. A., Mei, F., Cheng, X., Mamenko, M., Pochynyuk, O. Urinary concentrating defect in mice lacking Epac1 or Epac2.

Superoxide increases surface NKCC2 in the rat thick ascending limbs via PKC.

The apical Na+-K+-2Cl- cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The free radical superoxide ( O2- ) stimulates TAL NaCl absorption by enhancing NKCC2 activity. In contrast, nitric oxide (NO) scavenges O2- and inhibits NKCC2. NKCC2 activity depends on the number of NKCC2 transporters in the TAL apical membrane and its phosphorylation. We hypothesized that O2- stimulates NKCC2 activity by enhancing apical surface NKCC2 expression. We measured surface NKCC2 expression in rat TALs by surface biotinylation and Western blot analysis. Treatment of TALs with O2- produced by exogenous xanthine oxidase (1 mU/ml) and hypoxanthine (500 µM) stimulated surface NKCC2 expression by ~18 ± 5% (P < 0.05). O2- -stimulated surface NKCC2 expression was blocked by the O2- scavenger tempol (50 µM). Scavenging H2O2 with 100 U/ml catalase did not block the stimulatory effect of xanthine oxidase-hypoxanthine (22 ± 8% increase from control, P < 0.05). Inhibition of endogenous NO production with Nω-nitro-l-arginine methyl ester enhanced surface NKCC2 expression by 21 ± 6% and, when added together with xanthine oxidase-hypoxanthine, increased surface NKCC2 by 41 ± 10% (P < 0.05). Scavenging O2- with superoxide dismutase (300 U/ml) decreased this stimulatory effect by 60% (39 ± 4% to 15 ± 10%, P < 0.05). Protein kinase C inhibition with Gö-6976 (100 nM) blocked O2- -stimulated surface NKCC2 expression (P < 0.05). O2- did not affect NKCC2 phosphorylation at Thr96/101 or its upstream kinases STE20/SPS1-related proline/alanine-rich kinase-oxidative stress-responsive kinase 1. We conclude that O2- increases surface NKCC2 expression by stimulating protein kinase C and that this effect is blunted by endogenous NO. O2- -stimulated apical trafficking of NKCC2 may be involved in the enhanced surface NKCC2 expression observed in Dahl salt-sensitive rats.

Fructose acutely stimulates NKCC2 activity in rat thick ascending limbs by increasing surface NKCC2 expression.

The thick ascending limb (TAL) reabsorbs 25% of the filtered NaCl through the Na+-K+-2Cl- cotransporter (NKCC2). NKCC2 activity is directly related to surface NKCC2 expression and phosphorylation. Higher NaCl reabsorption by TALs is linked to salt-sensitive hypertension, which is linked to consumption of fructose in the diet. However, little is known about the effects of fructose on renal NaCl reabsorption. We hypothesized that fructose, but not glucose, acutely enhances TAL-dependent NaCl reabsorption by increasing NKCC2 activity via stimulation of surface NKCC2 levels and phosphorylation at Thr96/101. We found that fructose (5 mM) increased transport-related O2 consumption in TALs by 11.1 ± 3.2% ( P < 0.05). The effect of fructose on O2 consumption was blocked by furosemide. To study the effect of fructose on NKCC2 activity, we measured the initial rate of NKCC2-dependent thallium influx. We found that 20 min of treatment with fructose (5 mM) increased NKCC2 activity by 58.5 ± 16.9% ( P < 0.05). We then used surface biotinylation to measure surface NKCC2 levels in rat TALs. Fructose increased surface NKCC2 expression in a concentration-dependent manner (22 ± 5, 49 ± 10, and 101 ± 59% of baseline with 1, 5, and 10 mM fructose, respectively, P < 0.05), whereas glucose or a glucose metabolite did not. Fructose did not change NKCC2 phosphorylation at Thre96/101 or total NKCC2 expression. We concluded that acute fructose treatment increases NKCC2 activity by enhancing surface NKCC2 expression, rather than NKCC2 phosphorylation. Our data suggest that fructose consumption could contribute to salt-sensitive hypertension by stimulating NKCC2-dependent NaCl reabsorption in TALs.

Molecular regulation of NKCC2 in blood pressure control and hypertension.

PURPOSE OF REVIEW: The apical Na/K/2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb, contributing to maintenance of blood pressure (BP). Despite effective NKCC2 inhibition by loop diuretics, these agents are not viable for long-term management of BP due to side effects. Novel molecular mechanisms that control NKCC2 activity reveal an increasingly complex picture with interacting layers of NKCC2 regulation. Here, we review the latest developments that shine new light on NKCC2-mediated control of BP and potential new long-term therapies to treat hypertension. RECENT FINDINGS: Emerging molecular NKCC2 regulators, often binding partners, reveal a complex overlay of interacting mechanisms aimed at fine tuning NKCC2 activity. Different factors achieve this by shifting the balance between trafficking steps like exocytosis, endocytosis, recycling and protein turnover, or by balancing phosphorylation vs. dephosphorylation. Further molecular details are also emerging on previously known pathways of NKCC2 regulation, and recent in-vivo data continues to place NKCC2 regulation at the center of BP control. SUMMARY: Several layers of emerging molecular mechanisms that control NKCC2 activity may operate simultaneously, but they can also be controlled independently. This provides an opportunity to identify new pharmacological targets to fine-tune NKCC2 activity for BP management.

Single-molecule labeling for studying trafficking of renal transporters.

The ability to detect and track single molecules presents the advantage of visualizing the complex behavior of transmembrane proteins with a time and space resolution that would otherwise be lost with traditional labeling and biochemical techniques. Development of new imaging probes has provided a robust method to study their trafficking and surface dynamics. This mini-review focuses on the current technology available for single-molecule labeling of transmembrane proteins, their advantages, and limitations. We also discuss the application of these techniques to the study of renal transporter trafficking in light of recent research.

Vesicle-associated Membrane Protein 3 (VAMP3) Mediates Constitutive Trafficking of the Renal Co-transporter NKCC2 in Thick Ascending Limbs: ROLE IN RENAL FUNCTION AND BLOOD PRESSURE.

Renal cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na+/K+/2Cl- co-transporter NKCC2. Trafficking of NKCC2 to the apical surface regulates NKCC2-mediated NaCl absorption and blood pressure. The molecular mechanisms by which NKCC2 reaches the apical surface and their role in renal function and maintenance of blood pressure are poorly characterized. Here we report that NKCC2 interacts with the vesicle fusion protein VAMP3, and they co-localize at the TAL apical surface. We observed that silencing VAMP3 in vivo blocks constitutive NKCC2 exocytic delivery, decreasing the amount of NKCC2 at the TAL apical surface. VAMP3 is not required for cAMP-stimulated NKCC2 exocytic delivery. Additionally, genetic deletion of VAMP3 in mice decreased total expression of NKCC2 in the TAL and lowered blood pressure. Consistent with these results, urinary excretion of water and electrolytes was higher in VAMP3 knock-out mice, which produced more diluted urine. We conclude that VAMP3 interacts with NKCC2 and mediates its constitutive exocytic delivery to the apical surface. Additionally, VAMP3 is required for normal NKCC2 expression, renal function, and blood pressure.

Real-time monitoring of NKCC2 endocytosis by total internal reflection fluorescence (TIRF) microscopy.

The apical Na-K-2Cl cotransporter (NKCC2) mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TAL cells is determined by exocytic delivery, recycling, and endocytosis. Surface biotinylation allows measurement of NKCC2 endocytosis, but it has low time resolution and does not allow imaging of the dynamic process of endocytosis. We hypothesized that total internal reflection fluorescence (TIRF) microscopy imaging of labeled NKCC2 would allow monitoring of NKCC2 endocytosis in polarized Madin-Darby canine kidney (MDCK) and TAL cells. Thus we generated a NKCC2 construct containing a biotin acceptor domain (BAD) sequence between the transmembrane domains 5 and 6. Once expressed in polarized MDCK or TAL cells, surface NKCC2 was specifically biotinylated by exogenous biotin ligase (BirA). We also demonstrate that expression of a secretory form of BirA in TAL cells induces metabolic biotinylation of NKCC2. Labeling biotinylated surface NKCC2 with fluorescent streptavidin showed that most apical NKCC2 was located within small discrete domains or clusters referred to as "puncta" on the TIRF field. NKCC2 puncta were observed to disappear from the TIRF field, indicating an endocytic event which led to a decrease in the number of surface puncta at a rate of 1.18 ± 0.16%/min in MDCK cells, and a rate 1.09 ± 0.08%/min in TAL cells (n = 5). Treating cells with a cholesterol-chelating agent (methyl-ß-cyclodextrin) completely blocked NKCC2 endocytosis. We conclude that TIRF microscopy of labeled NKCC2 allows the dynamic imaging of individual endocytic events at the apical membrane of TAL cells.

Angiotensin II-mediated hypertension impairs nitric oxide-induced NKCC2 inhibition in thick ascending limbs.

In thick ascending limbs (THALs), nitric oxide (NO) decreases NaCl reabsorption via cGMP-mediated inhibition of Na-K-2Cl cotransporter (NKCC2). In angiotensin (ANG II)-induced hypertension, endothelin-1 (ET-1)-induced NO production by THALs is impaired. However, whether this alters NO's natriuretic effects and the mechanisms involved are unknown. In other cell types, ANG II augments phosphodiesterase 5 (PDE5)-mediated cGMP degradation. We hypothesized that NO-mediated inhibition of NKCC2 activity and stimulation of cGMP synthesis are blunted via PDE5 in ANG II-induced hypertension. Sprague-Dawley rats were infused with vehicle or ANG II (200 ng·kg-1·min-1) for 5 days. ET-1 reduced NKCC2 activity by 38 ± 13% (P < 0.05) in THALs from vehicle-treated rats but not from ANG II-hypertensive rats (Δ: -9 ± 13%). A NO donor yielded similar results as ET-1. In contrast, dibutyryl-cGMP significantly decreased NKCC2 activity in both vehicle-treated and ANG II-hypertensive rats (control: Δ-44 ± 15% vs. ANG II: Δ-41 ± 10%). NO increased cGMP by 2.08 ± 0.36 fmol/µg protein in THALs from vehicle-treated rats but only 1.06 ± 0.25 fmol/µg protein in ANG II-hypertensive rats (P < 0.04). Vardenafil (25 nM), a PDE5 inhibitor, restored NO's ability to inhibit NKCC2 activity in THALs from ANG II-hypertensive rats (Δ: -60 ± 9%, P < 0.003). Similarly, NO's stimulation of cGMP was also restored by vardenafil (vehicle-treated: 1.89 ± 0.71 vs. ANG II-hypertensive: 2.02 ± 0.32 fmol/µg protein). PDE5 expression did not differ between vehicle-treated and ANG II-hypertensive rats. We conclude that NO-induced inhibition of NKCC2 and increases in cGMP are blunted in ANG II-hypertensive rats due to PDE5 activation. Defects in the response of THALs to NO may enhance NaCl retention in ANG II-induced hypertension.

Vesicle-associated membrane protein 2 (VAMP2) but Not VAMP3 mediates cAMP-stimulated trafficking of the renal Na+-K+-2Cl- co-transporter NKCC2 in thick ascending limbs.

In the kidney, epithelial cells of the thick ascending limb (TAL) reabsorb NaCl via the apical Na(+)/K(+)/2Cl(-) co-transporter NKCC2. Steady-state surface NKCC2 levels in the apical membrane are maintained by a balance between exocytic delivery, endocytosis, and recycling. cAMP is the second messenger of hormones that enhance NaCl absorption. cAMP stimulates NKCC2 exocytic delivery via protein kinase A (PKA), increasing steady-state surface NKCC2. However, the molecular mechanism involved has not been studied. We found that several members of the SNARE family of membrane fusion proteins are expressed in TALs. Here we report that NKCC2 co-immunoprecipitates with VAMP2 in rat TALs, and they co-localize in discrete domains at the apical surface. cAMP stimulation enhanced VAMP2 exocytic delivery to the plasma membrane of renal cells, and stimulation of PKA enhanced VAMP2-NKCC2 co-immunoprecipitation in TALs. In vivo silencing of VAMP2 but not VAMP3 in TALs blunted cAMP-stimulated steady-state surface NKCC2 expression and completely blocked cAMP-stimulated NKCC2 exocytic delivery. VAMP2 was not involved in constitutive NKCC2 delivery. We concluded that VAMP2 but not VAMP3 selectively mediates cAMP-stimulated NKCC2 exocytic delivery and surface expression in TALs. We also demonstrated that cAMP stimulation enhances VAMP2 exocytosis and promotes VAMP2 interaction with NKCC2.

Direct renal effects of a fructose-enriched diet: interaction with high salt intake.

Consumption of fructose has increased during the last 50 years. Excessive fructose consumption has a detrimental effect on mammalian health but the mechanisms remain unclear. In humans, a direct relationship exists between dietary intake of added sugars and increased risk for cardiovascular disease mortality (52). While the causes for this are unclear, we recently showed that fructose provided in the drinking water induces a salt-dependent increase in blood pressure in Sprague-Dawley rats in a matter of days (6). However, little is known about the effects of fructose in renal salt handling and whether combined intake of high fructose and salt can lead to salt-sensitive hypertension before the development of metabolic abnormalities. The long-term (more than 4 wk) adverse effects of fructose intake on renal function are not just due to fructose but are also secondary to alterations in metabolism which may have an impact on renal function. This minireview focuses on the acute effect of fructose intake and its effect on salt regulation, as they affect blood pressure.

Regulation of renal NaCl transport by nitric oxide, endothelin, and ATP: clinical implications.

NaCl absorption along the nephron is regulated not just by humoral factors but also by factors that do not circulate or act on the cells where they are produced. Generally, nitric oxide (NO) inhibits NaCl absorption along the nephron. However, the effects of NO in the proximal tubule are controversial and may be biphasic. Similarly, the effects of endothelin on proximal tubule transport are biphasic. In more distal segments, endothelin inhibits NaCl absorption and may be mediated by NO. Adenosine triphosphate (ATP) inhibits sodium bicarbonate absorption in the proximal tubule, NaCl absorption in thick ascending limbs via NO, and water reabsorption in collecting ducts. Defects in the effects of NO, endothelin, and ATP increase blood pressure, especially in a NaCl-sensitive manner. In diabetes, disruption of NO-induced inhibition of transport may contribute to increased blood pressure and renal damage. However, our understanding of how NO, endothelin, and ATP work, and of their role in pathology, is rudimentary at best.

Dissociation of Hypertension and Renal Damage After Cessation of High-Salt Diet in Dahl Rats.

BACKGROUND: Every year, thousands of patients with hypertension reduce salt consumption in an effort to control their blood pressure. However, hypertension has a self-sustaining character in a significant part of the population. We hypothesized that chronic hypertension leads to irreversible renal damage that remains after removing the trigger, causing an elevation of the initial blood pressure. METHODS: Dahl salt-sensitive rat model was used for chronic, continuous observation of blood pressure. Rats were fed a high salt diet to induce hypertension, and then the diet was switched back to normal sodium content. RESULTS: We found that developed hypertension was irreversible by salt cessation: after a short period of reduction, blood pressure grew even higher than in the high-salt phase. Notably, the self-sustaining phase of hypertension was sensitive to benzamil treatment due to sustaining epithelial sodium channel hyperactivity, as shown with patch-clamp analysis. Glomerular damage and proteinuria were also irreversible. In contrast, some mechanisms, contributing to the development of salt-sensitive hypertension, normalized after salt restriction. Thus, flow cytometry demonstrated that dietary salt reduction in hypertensive animals decreased the number of total CD45+, CD3+CD4+, and CD3+CD8+ cells in renal tissues. Also, we found tubular recovery and improvement of glomerular filtration rate in the postsalt period versus a high-salt diet. CONCLUSIONS: Based on earlier publications and current data, poor response to salt restriction is due to the differential contribution of the factors recognized in the developmental phase of hypertension. We suggest that proteinuria or electrolyte transport can be prioritized over therapeutic targets of inflammatory response.

Dynamin2, clathrin, and lipid rafts mediate endocytosis of the apical Na/K/2Cl cotransporter NKCC2 in thick ascending limbs.

Steady-state surface levels of the apical Na/K/2Cl cotransporter NKCC2 regulate NaCl reabsorption by epithelial cells of the renal thick ascending limb (THAL). We reported that constitutive endocytosis of NKCC2 controls NaCl absorption in native THALs; however, the pathways involved in NKCC2 endocytosis are unknown. We hypothesized that NKCC2 endocytosis at the apical surface depends on dynamin-2 and clathrin. Measurements of steady-state surface NKCC2 and the rate of NKCC2 endocytosis in freshly isolated rat THALs showed that inhibition of endogenous dynamin-2 with dynasore blunted NKCC2 endocytosis by 56 ± 11% and increased steady-state surface NKCC2 by 67 ± 27% (p < 0.05). Expression of the dominant negative Dyn2K44A in THALs slowed the rate of NKCC2 endocytosis by 38 ± 8% and increased steady-state surface NKCC2 by 37 ± 8%, without changing total NKCC2 expression. Inhibition of clathrin-mediated endocytosis with chlorpromazine blunted NKCC2 endocytosis by 54 ± 6%, while preventing clathrin from interacting with synaptojanin also blunted NKCC2 endocytosis by 52 ± 5%. Disruption of lipid rafts blunted NKCC2 endocytosis by 39 ± 4% and silencing caveolin-1 by 29 ± 4%. Simultaneous inhibition of clathrin- and lipid raft-mediated endocytosis completely blocked NKCC2 internalization. We concluded that dynamin-2, clathrin, and lipid rafts mediate NKCC2 endocytosis and maintain steady-state apical surface NKCC2 in native THALs. These are the first data identifying the endocytic pathway for apical NKCC2 endocytosis.

Knockout of ACE-N facilitates improved cardiac function after myocardial infarction.

Angiotensin-converting enzyme (ACE) hydrolyzes N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) into inactive fragments through its N-terminal site (ACE-N). We previously showed that Ac-SDKP mediates ACE inhibitors' cardiac effects. Whether increased bioavailability of endogenous Ac-SDKP caused by knocking out ACE-N also improves cardiac function in myocardial infarction (MI)-induced heart failure (HF) is unknown. Wild-type (WT) and ACE-N knockout (ACE-NKO) mice were subjected to MI by ligating the left anterior descending artery and treated with vehicle or Ac-SDKP (1.6 mg/kg/day, s.c.) for 5 weeks, after which echocardiography was performed and left ventricles (LV) were harvested for histology and molecular biology studies. ACE-NKO mice showed increased plasma Ac-SDKP concentrations in both sham and MI group compared to WT. Exogenous Ac-SDKP further increased its circulating concentrations in WT and ACE-NKO. Shortening (SF) and ejection (EF) fractions were significantly decreased in both WT and ACE-NKO mice post-MI, but ACE-NKO mice exhibited significantly lesser decrease. Exogenous Ac-SDKP ameliorated cardiac function post-MI only in WT but failed to show any additive improvement in ACE-NKO mice. Sarcoendoplasmic reticulum calcium transport ATPase (SERCA2), a marker of cardiac function and calcium homeostasis, was significantly decreased in WT post-MI but rescued with Ac-SDKP, whereas ACE-NKO mice displayed less loss of SERCA2 expression. Our study demonstrates that gene deletion of ACE-N resulted in improved LV cardiac function in mice post-MI, which is likely mediated by increased circulating Ac-SDKP and minimally reduced expression of SERCA2. Thus, future development of specific and selective inhibitors for ACE-N could represent a novel approach to increase endogenous Ac-SDKP toward protecting the heart from post-MI remodeling.

Development of Novel Phase-Change Materials Derived from Methoxy Polyethylene Glycol and Aromatic Acyl Chlorides.

In this research, novel, organic, solid-liquid phase-change materials (PCMs) derived from methoxy polyethylene glycol (MPEG) and aromatic acyl chlorides (ACs) were prepared through a condensation reaction. The MPEGs were used as phase-change functional chains with different molecular weights (350, 550, 750, 2000, and 5000 g/mol). The aromatic ACs, terephthaloyl chloride (TPC) and isophthaloyl chloride (IPC), were employed as bulky linker cores. Solubility tests demonstrated that this family of PCMs is soluble in protic polar solvents such as H2O and MeOH, and insoluble in nonpolar solvents such as n-hexane. Fourier-ransform infrared spectroscopy (FT-IR UATR) and nuclear magnetic resonance (1H, 13C, DEPT 135°, COSY, HMQC, and HMBC NMR) were used to confirm the bonding of MPEG chains to ACs. The crystalline morphology of the synthesized materials was examined using polarized optical microscopy (POM), revealing the formation of spherulites with Maltese-cross-extinction patterns. Furthermore, it was confirmed that PCMs with higher molecular weights were crystalline at room temperature and exhibited an increased average spherulite size compared to their precursors. Thermal stability tests conducted through thermogravimetric analysis (TGA) indicated decomposition temperatures close to 400 °C for all PCMs. The phase-change properties were characterized by differential scanning calorimetry (DSC), revealing that the novel PCMs melted and crystallized between -23.7 and 60.2 °C and -39.9 and 45.9 °C, respectively. Moreover, the heat absorbed and released by the PCMs ranged from 57.9 to 198.8 J/g and 48.6 to 195.6 J/g, respectively. Additionally, the PCMs exhibited thermal stability after undergoing thermal cycles of melting-crystallization, indicating that energy absorption and release occurred at nearly constant temperatures. This study presents a new family of high-performance organic PCMs and demonstrates that the orientation of substituent groups in the phenylene ring influences supercooling, transition temperatures, and thermal energy storage capacity depending on the MPEG molecular weight.