Physiological Mechanisms Regulating Lens Transport

The transparency and refractive properties of the lens are maintained by the cellular physiology provided by an internal microcirculation system that utilizes spatial differences in ion channels, transporters and gap junctions to establish standing electrochemical and hydrostatic pressure gradients that drive the transport of ions, water and nutrients through this avascular tissue. Aging has negative effects on lens transport, degrading ion and water homeostasis, and producing changes in lens water content. This alters the properties of the lens, causing changes in optical quality and accommodative amplitude that initially result in presbyopia in middle age and ultimately manifest as cataract in the elderly. Recent advances have highlighted that the lens hydrostatic pressure gradient responds to tension transmitted to the lens through the Zonules of Zinn through a mechanism utilizing mechanosensitive channels, multiple sodium transporters respond to changes in hydrostatic pressure to restore equilibrium, and that connexin hemichannels and diverse intracellular signaling cascades play a critical role in these responses. The mechanistic insight gained from these studies has advanced our understanding of lens transport and how it responds and adapts to different inputs both from within the lens, and from surrounding ocular structures.

Particition of Sodium-potassium Adenosine Triphosphatases in Homeostasis Regulation

A literature review presented an analysis of data regarding the mechanisms of the Na pump in nephron and hormonal regulators of enzyme activity, including enzymatic catalysts. Investigating the regulatory mechanisms of metabolic processes can facilitate the development of new strategies to repair various pathological conditions. Among these functional proteins, Na+/K+ATPase is responsible for the regulation of hydroionic homeostasis and signaling. Ion transport in different parts of the nephron is mediated via sodium transporters, which are characterized by a clear topographical expression. In the oligomeric Na+/K+ATPase molecule, the α-subunit comprises 10 transmembrane domains and performs a catalytic function. The signal function of Na+/K+ATPase and its interaction with the molecular environment in lipid microdomains involve rafts and caveolae. Analysis of the literature data demonstrated an important function of Na+/K+ATPase, along with its interaction with caveolin-1, in the regulation of intracellular cholesterol traffic. Moreover, reciprocal interactions of enzymes and cholesterol have been indicated. The status of Na+/K+ATPase activity is affected by hypoxia, reactive oxygen species, lipid peroxidation (LPO), increased cholesterol concentrations, and the viscosity of the cytoplasmic membrane. Ecological pollutants, including heavy metals, have significant effects on enzyme activity in nephron, hepatocytes and cardiomyocytes. Thus, available literature data indicate an important role of Na+/K+ATPase in the regulation of metabolic processes.

γENaC/CD9 in urinary extracellular vesicles as a potential biomarker of MR activity

Primary aldosteronism (PA) is caused by autonomous overproduction of aldosterone, which induces organ damage directly via activation of the mineralocorticoid receptor (MR); however, no specific or sensitive biomarkers are able to reflect MR activity. Recently, it is found that urinary extracellular vesicles (uEVs) are secreted by multiple cell types in the kidney and are an enriched source of kidney-specific proteins. Here, we evaluate sodium transporters in uEVs as candidates of biomarkers of MR activity in the clinical setting. Sixteen patients were examined to determine their plasma aldosterone concentration (PAC) and renin activity, and their morning urine was collected. The protein levels of two sodium transporters in uEVs, γ-epithelial sodium channel (γENaC) and thiazide-sensitive sodium chloride cotransporter (NCC), were quantified by Western blot analysis, and their clinical correlation with PAC was determined. Consequently, we found PAC was significantly correlated with the γENaC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.71). PAC was also correlated with the NCC protein level adjusted by the CD9 protein level in uEVs (correlation coefficient = 0.61). In two PA patients, treatment with an MR antagonist or adrenalectomy reduced γENaC/CD9 in uEVs. In conclusion, γENaC/CD9 in uEVs is a valuable biomarker of MR activity in PA patients and may be a useful biomarker for other MR-associated diseases.

Renocardiac protective effects of SGLT2 inhibitor combined with angiotensin receptor blocker in salt sensitive Dahl rats

Background Kidney plays a central role in regulating salt-sensitivity of blood pressure (BP) to governs sodium excretion via several mechanisms including pressure natriuresis and the actions of renal sodium transporters. Purpose We clarified the effects of combination treatment of sodium-glucose cotransporter 2 (SGLT2) inhibitor and angiotensin receptor blocker (ARB) on BP and the pathogenesis of renocardiac injuries, and elucidated underlying molecular mechanisms involved in the regulation of renal sodium handling in the development of salt-sensitivity by comparing with each monotreatment in Dahl salt-sensitive (DSS) hypertensive rats. Methods DSS rats were treated orally for 8-weeks with normal salt diet (0.3% NaCl) (NS/Cont group), high salt diet (8% NaCl) (HS/Cont group), high salt diet with ipragliflozin (0.04%) (HS/Ipra group), high salt diet with losartan (0.05%) (HS/Los group), or high salt diet with combination of ipragliflozin and losartan (HS/Ipra+Los group). Results The combination group significantly reduced systolic BP compared with either high salt diet control group, losartan or ipragliflozin monotreatment groups (HS/Ipra+Los: 182.5±18.4mmHg vs HS/Cont: 227.7±26.1; HS/Ipra: 216.6±26.9; HS/Los: 208.6±21.6, at 8-weeks of treatment, P<0.05, respectively) (Figure 1A). The slope of pressure-natriuresis curve was significantly increased in the HS/Ipra+Los group compared to that in the HS/Cont group (interaction P=0.024), HS/Ipra group (P=0.009), and HS/Los group (P=0.084) using the linear regression model (Figure 1B), which indicated that only the combination treatment of ipragliflozin and losartan improved salt-sensitivity. The combined treatment significantly improved creatinine clearance (HS/Ipra+Los: 3.3±0.9mL/min vs HS/Cont: 1.1±0.5; HS/Ipra: 1.7±0.6; HS/Los: 1.9±0.8, P<0.05, respectively). The combination treatment also significantly ameliorated glomerulosclerosis, and improved cardiomyocyte hypertrophy and perivascular fibrosis (Figure 1C). Angiotensin II type 1 receptor (AT1R) protein expression level in the kidney was remarkably suppressed in the combination treatment group compared to the other high salt diet groups. The protein expression level of Na+/H+ exchanger isoform 3 (NHE3) and Na+-K+-Cl– cotransporter 2 (NKCC2), two of major sodium transports in the renal tubules, were significantly decreased with losartan monotreatment and combination treatment, but not with ipragliflozin monotreatment (Figure 2). Conclusions The dual inhibition of SGLT2 and AT1R effectively improved salt-sensitivity via reducing renal expression levels of the sodium transporters, which eventually lead to renocardiac protection. Thus, the combination treatment could be a novel and useful therapeutic strategy for treating salt-sensitive hypertension and renal injury in non-diabetic patients. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Grant-in-Aid for Scientific Research

Butter clam genome assembly and analysis reveals the historical adaptation of shellfish genome to changes in the marine environment

Purple butter clam (Saxidomus purpuratus) is an economically important bivalve shellfish. This species belongs to the subclass Heterodonta that diverged in calcite seas with low magnesium concentrations. We sequenced and assembled its genome and performed an evolutionary comparative analysis. A total of 911 Mb assembly of S. purpuratus was anchored into 19 chromosomes and a total of 48,090 protein-coding genes were predicted. We identified its repeat-based expanded genes that are associated with the sodium/potassium-exchange ATPase complex. In addition, different types of ion transporters were enriched in the common ancestor of Heterodonta (calcium, sulfate, and lipid transporters) and the specific evolution of S. purpuratus (calcium and sodium transporters). These differences seem to be related to the divergence times of Heterodonta (calcitic sea) and Veneraidea (aragonitic sea). Furthermore, we analyzed the evolution of scavenger receptor (SR) proteins in S. purpuratus, which are involved in a wide range of immune responses, and compared them to the closely related Cyclina sinensis. We showed that a small number of SR proteins, exhibited collinearity between the two genomes, which is indicative of independent gene evolution. Our genomic study provides an evolutionary perspective on the genetic diversity of bivalves and their adaptation to historical changes in the marine environment.

Local and Downstream Actions of Proximal Tubule Angiotensin II Signaling on Sodium Transporters in the Mouse Nephron

The renal nephron consists of a series of distinct cell types which function in concert to maintain fluid and electrolyte balance and blood pressure. The renin angiotensin system (RAS) is central to sodium and volume balance. We aimed to determine how loss of angiotensin II signaling in the proximal tubule (PT), which reabsorbs the bulk of filtered sodium and volume, impacts solute transport throughout the nephron. We hypothesized that proximal tubule (PT) RAS disruption would not only depress PT sodium transporters, but also impact downstream Na+ transporters. Utilizing a mouse model in which the type 1a angiotensin receptor (AT1aR) is deleted specifically within the PT (AT1aR PTKO), we profiled the abundance of sodium transporters, channels, and claudins along the nephron. Absence of PT AT1aR signaling was associated with lower abundance of PT transporters (NHE3, NBCe2 and claudin 2) as well as lower abundance of downstream transporters (total and phosphorylated NKCC2, medullary Na,K-ATPase, phosphorylated NCC and claudin 7) versus controls. However, transport activities of NKCC2 and NCC (assessed with diuretics) were similar between groups in order to maintain electrolyte balance. Together, these results demonstrate the primary impact of angiotensin II regulation on sodium reabsorption in PT at baseline and the associated influence on downstream Na+ transporters, highlighting the ability of the nephron to integrate sodium transport along the nephron to maintain homeostasis.

Vascular Control of Kidney Epithelial Transporters

A major pathway in hypertension pathogenesis involves direct activation of Ang II (AT1) receptors in the kidney, stimulating sodium reabsorption. AT1 receptors in tubular epithelia control expression and stimulation of sodium transporters and channels. Recently, we found reduced blood pressure and enhanced natriuresis in mice with cell-specific deletion of AT1 receptors in smooth muscle (SMKOs). While impaired vasoconstriction and preserved renal blood flow might contribute to exaggerated urinary sodium excretion in SMKOs, we considered whether alterations in sodium transporter expression might also play a role and therefore carried out a proteomic analysis of key sodium transporters and associated proteins. Here we show that levels of Na+-K+-2Cl- cotransporter isoform 2 (NKCC2) and Na+/H+ exchanger isoform 3 (NHE3) are reduced at baseline in SMKOs, accompanied by attenuated natriuretic and diuretic responses to furosemide. During Ang II hypertension, we find widespread remodeling of transporter expression in wild-type mice with significant increases in levels of total NCC, phosphorylated-NCCps71, and NKCC2-P, along with the cleaved, activated forms of the a- and g-ENaC. However, the increases in a- and g-ENaC with Ang II were substantially attenuated in SMKOs. This was accompanied by reduced natriuretic response to amiloride. Thus, enhanced urinary sodium excretion observed after cell-specific deletion of AT1 receptors from smooth muscle cells is associated with altered sodium transporter abundance across epithelia in multiple nephron segments. These findings suggest a system of vascular-epithelial cross-talk in the kidney, modulating expression of sodium transporters and contributing to regulation of pressure-natriuresis.

Impact of casein- versus grain-based diets on rat renal sodium transporters' abundance and regulation

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Abstract P008: Sex Differences In The Development Of Diabetic Kidney Disease And Salt Sensitivity In Db/db Mice

Introduction: Clinical studies show that men have higher risk than premenopausal women for the development of diabetic kidney disease. However, the mechanisms behind the sexual dimorphism associated with renal injury and salt sensitivity during diabetes remain unknown. Methods: 7-month-old male and female diabetic (db/db) and non-diabetic (db/+) mice received either a high-salt (HS, 4% NaCl w/w) or a control (0.7% NaCl w/w) diet for 4 weeks (n=6). Mean arterial pressure (MAP) was measured by telemetry. A subgroup of male mice was exposed to HS and the anti-inflammatory drug mycophenolate mofetil (MMF, 30 mg/Kg/day, IP). At the end, mice were euthanized, and kidneys were preserved to assess renal sodium transporters and inflammation. Results: Male db/db mice display hypertension when exposed to HS whereas female db/db mice remain normotensive during the whole treatment (MAP: 125±7 vs. 103±3 mmHg, P<0.05). Hypertension in male db/db mice was associated with an improper suppression of key sodium transporters: NKCC2 and ENaC. Renal angiotensin II and plasma aldosterone were suppressed after HS in all experimental groups negating a role of these hormones in sodium transporters dysregulation and salt sensitivity. Compared to females, male db/db mice show higher levels of proinflammatory cytokines (IL-1β: 2.3-fold, IL-6: 1.7-fold and TNFα: 2.3-fold, P<0.05 by ELISA) and more leucocyte infiltration (CD45+ cells: 1.6-fold, P<0.05 by flow cytometry) in the kidney. Blocking inflammation with MMF reduced NKCC2 and ENaC expression in response to HS and prevented salt sensitivity in male db/db mice (MAP: 105±10 mmHg). Conclusion: Renal inflammation in male, but not in female, db/db mice induces salt-sensitive hypertension by preventing the suppression of key sodium transporters in response to HS. Thus, we provide a mechanistic explanation of the sexual dimorphism associated with the development of salt sensitivity during diabetes.