by
Maria Chavez-Canales;
Juan Pablo Arroyo;
Benjamin Ko;
Norma Vazquez;
Rocio Bautista;
Maria Castaneda-Bueno;
Norma A. Bobadilla;
Robert S. Hoover Jr;
Gerardo Gamba
Objectives: Insulin is recognized to increase renal salt reabsorption in the distal nephron and hyperinsulinemic states have been shown to be associated with increased expression of the renal NaCl cotransporter (NCC). However, the effect of insulin on NCC functional activity has not been reported.
Methods: Using a heterologous expression system of Xenopus laevis oocytes, a mouse distal convoluted cell line, mDCT15 cells, endogenously expressing NCC, and an ex-vivo kidney perfusion technique, we assessed the effect of insulin on the activity and phosphorylation of NCC. The signaling pathway involved was analyzed.
Results: In Xenopus oocytes insulin increases the activity of NCC together with its phosphorylation at threonine residue 58. Activation of NCC by insulin was also observed in mDCT15 cells. Additionally, insulin increased the NCC phosphorylation in kidney under the ex-vivo perfusion technique. In oocytes and mDCT15 cells, insulin effect on NCC was prevented with inhibitors of phosphatidylinositol 3-kinase (PI3K), mTORC2, and AKT1 kinases, but not by inhibitors of MAP or mTORC1 kinases, suggesting that PI3K-mTORC2-AKT1 is the intracellular pathway required. Additionally, activation of NCC by insulin was not affected by wild-type or mutant versions of with no lysine kinase 1, with no lysine kinase 4, or serum glucocorticoid kinase 1, but it was no longer observed in the presence of wild-type or the dominant negative, catalytically inactive with no lysine kinase 3, implicating this kinase in the process.
Conclusion: Insulin induces activation and phosphorylation of NCC. This effect could play an important role in arterial hypertension associated with hyperinsulinemic states, such as obesity, metabolic syndrome, or type 2 diabetes mellitus.
Volume depletion due to persistent glucosuria-induced osmotic diuresis is a significant problem in uncontrolled diabetes mellitus (DM). Angiotensin II receptor blockers (ARBs), such as candesartan, slow the progression of chronic kidney disease in patients with DM. However, mice with genetic knockout of components of the renin-angiotensin system have urine concentrating defects, suggesting that ARBs may exacerbate the volume depletion. Therefore, the effect of candesartan on UT-A1, UT-A3, NKCC2, and aquaporin-2 (AQP2) protein abundances was determined in control and 3-wk DM rats. Aldosterone levels in control rats (0.36 ± 0.06 nM) and candesartan-treated rats (0.34 ± 0.14 nM) were the same. DM rats had higher aldosterone levels (1.48 ± 0.37 nM) that were decreased by candesartan (0.97 ± 0.26 nM). Western analysis showed that UT-A1 expression was increased in DM rats compared with controls in inner medullary (IM) tip (158 ± 13%) and base (120 ± 25%). UT-A3 abundance was increased in IM tip (123 ± 11%) and base (146 ± 17%) of DM rats vs. controls. UT-A3 was unchanged in candesartan-treated control rats. In candesartan-treated DM rats, UT-A3 increased in IM tip (160 ± 14%) and base (210 ± 19%). Candesartan-treated DM rats had slightly higher AQP2 in IM (46%, P < 0.05) vs. control rats. NKCC2/BSC1 was increased 145 ± 10% in outer medulla of DM vs. control rats. We conclude that candesartan augments compensatory changes in medullary transport proteins, reducing the losses of solute and water during uncontrolled DM. These changes may represent a previously unrecognized beneficial effect of type 1 ARBs in DM.
Background: Epithelial sodium channels (ENaC) are activated by proteolytic cleavage. Several proteases including furin and prostasin cleave ENaC.
Results: Cathepsin B also cleaves and activates ENaC. Cathepsin B cleaves ENaC α but not β or γ subunits.
Conclusion: Cathepsin B is a secreted protease, so it may cleave ENaC at the cell surface.
Significance: Cathepsin B cleavage represents a novel ENaC regulatory mechanism.
The elderly population in the United States continues to grow and is expected to double by 2050. With aging there are degenerative changes in many organs and the kidney is no exception. After age forty there is an increase in cortical glomerulosclerosis and a decline in both glomerular filtration rate and renal plasma flow. These changes may be associated with an inability to excrete a concentrated or a dilute urine, ammonium, sodium, or potassium. Hypernatremia and hyponatremia are the most common electrolyte abnormalities found in the elderly and both are associated with a high mortality. Under normal conditions the elderly are able to maintain water and electrolyte balance but this may be jeopardized by an illness, a decline in cognitive ability, and with certain medications. Therefore, it is important to be aware of the potential electrolyte abnormalities in the elderly that can arise under these various conditions in order to prevent adverse outcomes.
Acute lung injury leading to acute respiratory distress (ARDS) is a global health concern. ARDS patients have significant pulmonary inflammation leading to flooding of the pulmonary alveoli. This prevents normal gas exchange with consequent hypoxemia and causes mortality. A thin fluid layer in the alveoli is normal. The maintenance of this thin layer results from fluid movement out of the pulmonary capillaries into the alveolar interstitium driven by vascular hydrostatic pressure and then through alveolar tight junctions. This is then balanced by fluid reabsorption from the alveolar space mediated by transepithelial salt and water transport through alveolar cells. Reabsorption is a two-step process: first, sodium enters via sodium-permeable channels in the apical membranes of alveolar type 1 and 2 cells followed by active extrusion of sodium into the interstitium by the basolateral Na + , K + -ATPase. Anions follow the cationic charge gradien t and water follows the salt-induced osmotic gradient. The proximate cause of alveolar flooding is the result of a failure to reabsorb sufficient salt and water or a failure of the tight junctions to prevent excessive movement of fluid from the interstitium to alveolar lumen. Cytokine- and chemokine-induced inflammation can have a particularly profound effect on lung sodium transport since they can alter both ion channel and barrier function. Cytokines and chemokines affect alveolar amiloride-sensitive epithelial sodium channels (ENaCs), which play a crucial role in sodium transport and fluid reabsorption in the lung. This review discusses the regulation of ENaC via local and systemic cytokines during inflammatory disease and the effect on lung fluid balance.
by
Lihe Chen;
Jae Wook Lee;
Chung-Lin Chou;
Anil V. Nair;
Maria A. Battistone;
Teodor G. Paunescu;
Maria Merkulova;
Sylvie Breton;
Jill W. Verlander;
Susan M Wall;
Dennis Brown;
Maurice B. Burg;
Mark A. Knepper
Prior RNA sequencing (RNA-seq) studies have identified complete transcriptomes for most renal epithelial cell types. The exceptions are the cell types that make up the renal collecting duct, namely intercalated cells (ICs) and principal cells (PCs), which account for only a small fraction of the kidney mass, but play critical physiological roles in the regulation of blood pressure, extracellular fluid volume, and extracellular fluid composition. To enrich these cell types, we used FACS that employed well-established lectin cell surface markers for PCs and type B ICs, as well as a newly identified cell surface marker for type A ICs, c-Kit. Single-cell RNA-seq using the IC- and PC-enriched populations as input enabled identification of complete transcriptomes of A-ICs, B-ICs, and PCs. The data were used to create a freely accessible online gene-expression database for collecting duct cells. This database allowed identification of genes that are selectively expressed in each cell type, including cell-surface receptors, transcription factors, transporters, and secreted proteins. The analysis also identified a small fraction of hybrid cells expressing aquaporin-2 and anion exchanger 1 or pendrin transcripts. In many cases, mRNAs for receptors and their ligands were identified in different cells (e.g., Notch2 chiefly in PCs vs. Jag1 chiefly in ICs), suggesting signaling cross-talk among the three cell types. The identified patterns of gene expression among the three types of collecting duct cells provide a foundation for understanding physiological regulation and pathophysiology in the renal collecting duct.
The thiazide-sensitive sodium chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) are two of the most important determinants of salt balance and thus systemic blood pressure. Abnormalities in either result in profound changes in blood pressure. There is one segment of the nephron where these two sodium transporters are coexpressed, the second part of the distal convoluted tubule. This is a key part of the aldosterone-sensitive distal nephron, the final regulator of salt handling in the kidney. Aldosterone is the key hormonal regulator for both of these proteins. Despite these shared regulators and coexpression in a key nephron segment, associations between these proteins have not been investigated. After confirming apical localization of these proteins, we demonstrated the presence of functional transport proteins and native association by blue native PAGE. Extensive coimmunoprecipitation experiments demonstrated a consistent interaction of NCC with α-And γ-ENaC. Mammalian two-hybrid studies demonstrated direct binding of NCC to ENaC subunits. Fluorescence resonance energy transfer and immunogold EM studies confirmed that these transport proteins are within appropriate proximity for direct binding. Additionally, we demonstrate that there are functional consequences of this interaction, with inhibition of NCC affecting the function of ENaC. This novel finding of an association between ENaC and NCC could alter our understanding of salt transport in the distal tubule.
AIM: This study evaluates the effect of dapagliflozin, a SGLT2 inhibitor, on fluid or electrolyte balance and its effect on urea transporter-A1 (UT-A1), aquaporin-2 (AQP2) and Na-K-2Cl cotransporter (NKCC2) protein abundance in diabetic rats. METHODS: Diabetes mellitus (DM) was induced by injection of streptozotocin into the tail vein. Serum Na+, K+, Cl- concentration, urine Na+, K+, Cl- excretion, blood glucose, urine glucose excretion, urine volume, urine osmolality and urine urea excretion were analyzed after the administration of dapagliflozin. UT-A1, AQP2 and NKCC2 proteins were detected by western blot. RESULTS: Dapagliflozin treatment decreased blood glucose concentration by 38% at day 7 and by 47% at day 14 and increased the urinary glucose excretion rate compared with the untreated diabetic animals. Increased 24-hour urine volume, decreased urine osmolality and hyponatremia, hypokalemia and hypochloremia observed in diabetic rats were attenuated by dapagliflozin treatment. Western blot analysis showed that UT-A1, AQP2 and NKCC2 proteins are upregulated in DM rats over control rats; dapagliflozin treatment results in a further increase in inner medulla tip UT-A1 protein abundance by 42% at day 7 and by 46% at day 14, but it did not affect the DM-induced upregulation of AQP2 and NKCC2 proteins. CONCLUSION: Dapagliflozin treatment augmented the compensatory changes in medullary transport proteins in DM. These changes would tend to conserve solute and water even with persistent glycosuria. Therefore, diabetic rats treated with dapagliflozin have a mild osmotic diuresis compared to nondiabetic animals, but this does not result in an electrolyte disorder or significant volume depletion.
Regulation of the epithelial sodium channel (ENaC), which regulates fluid homeostasis and blood pressure, is complex and remains incompletely understood. The TIP peptide, a mimic of the lectin-like domain of TNF, activates ENaC by binding to glycosylated residues in the extracellular loop of ENaC-α as well as to a hitherto uncharacterized internal site. Molecular docking studies suggested three residues, Val 567 , Glu 568 , and Glu 571 , located at the interface between the second transmembrane and C-terminal domains of ENaC-α, as a critical site for binding of the TIP peptide. We generated Ala replacement mutants in this region of ENaC-α and examined its interaction with TIP peptide (3M, V567A/E568A/E571A; 2M, V567A/E568A; and 1M, E571A). 3M and 2M ENaC-α but not 1M ENaC-α, displayed significantly reduced binding capacity to TIP peptide and to TNF. When overexpressed in H441 cells,3M mutant ENaC-α formed functional channels with similar gating and density characteristics as the WT subunit and efficiently associated with the β and γ subunits in the plasma membrane. We subsequently assayed for increased open probability time and membrane expression, both of which define ENaC activity, following addition of TIP peptide. TIP peptide increased open probability time in H441 cells overexpressing wild type and 1M ENaC-α channels, but not 3M or 2M ENaC-α channels. On the other hand, TIP peptide-mediated reduction in ENaC ubiquitination was similar in cells overexpressing either WT or 3M ENaC-α subunits. In summary, this study has identified a novel site in ENaC-α that is crucial for activation of the open probability of the channel, but not membrane expression, by the lectin-like domain of TNF.
The urea transporter UT-B is widely expressed and has been studied in erythrocyte, kidney, brain and intestines. Interestingly, UT-B gene has been found more abundant in bladder than any other tissue. Recently, gene analyses demonstrate that SLC14A1 (UT-B) gene mutations are associated with bladder cancer, suggesting that urea transporter UT-B may play an important role in bladder carcinogenesis. In this study, we examined UT-B expression in bladder cancer with human primary bladder cancer tissues and cancer derived cell lines. Human UT-B has two isoforms. We found that normal bladder expresses long form of UT-B2 but was lost in 8 of 24 (33%) or significantly downregulated in 16 of 24 (67%) of primary bladder cancer patients. In contrast, the short form of UT-B1 lacking exon 3 was detected in 20 bladder cancer samples. Surprisingly, a 24-nt in-frame deletion in exon 4 in UT-B1 (UT-B1Δ24) was identified in 11 of 20 (55%) bladder tumors. This deletion caused a functional defect of UT-B1. Immunohistochemistry revealed that UT-B protein levels were significantly decreased in bladder cancers. Western blot analysis showed a weak UT-B band of 40 kDa in some tumors, consistent with UT-B1 gene expression detected by RT-PCR. Interestingly, bladder cancer associate UT-B1Δ24 was barely sialylated, reflecting impaired glycosylation of UT-B1 in bladder tumors. In conclusion, SLC14A1 gene and UT-B protein expression are significantly changed in bladder cancers. The aberrant UT-B expression may promote bladder cancer development or facilitate carcinogenesis induced by other carcinogens.