Stimulatory effect of insulin on H+-ATPase in the proximal tubule via the Akt/mTORC2 pathway

PurposeAcid-base transport in renal proximal tubules (PTs) is mainly sodium-dependent and conducted in coordination by the apical Na+/H+ exchanger (NHE3), vacuolar H+-adenosine triphosphatase (V-ATPase), and the basolateral Na+/HCO3- cotransporter. V-ATPase on PTs is well-known to play an important role in proton excretion. Recently we reported a stimulatory effect of insulin on these transporters. However, it is unclear whether insulin is involved in acid-base balance in PTs. Thus, we assessed the role of insulin in acid-base balance in PTs.MethodsV-ATPase activity was evaluated using freshly isolated PTs obtained from mice, and specific inhibitors were then used to assess the signaling pathways involved in the observed effects.ResultsV-ATPase activity in PTs was markedly enhanced by insulin, and its activation was completely inhibited by bafilomycin (a V-ATPase-specific inhibitor), Akt inhibitor VIII, and PP242 (an mTORC1/2 inhibitor), but not by rapamycin (an mTORC1 inhibitor). V-ATPase activity was stimulated by 1 nm insulin by approximately 20% above baseline, which was completely suppressed by Akt1/2 inhibitor VIII. PP242 completely suppressed the insulin-mediated V-ATPase stimulation in mouse PTs, whereas rapamycin failed to influence the effect of insulin. Insulin-induced Akt phosphorylation in the mouse renal cortex was completely suppressed by Akt1/2 inhibitor VIII and PP242, but not by rapamycin.ConclusionOur results indicate that stimulation of V-ATPase activity by insulin in PTs is mediated via the Akt2/mTORC2 pathway. These results reveal the mechanism underlying the complex signaling in PT acid-base balance, providing treatment targets for renal disease.

Prompt Rise in Urinary Ammonium Excretion Suffices To Mitigate Metabolic Acidosis in an Experimental Animal Model of Severe Normovolemic Hemodilution

Recently, we have established a model of severe stepwise normovolemic hemodilution to a hematocrit of 10 % in rats employing three different colloidal volume replacement solutions (Voluven, Volulyte and Gelafundin) that are routinely used in clinical practice at present. We did not see severe dilutional acidosis as to be expected, but a decline in urinary pH. We here looked on further mechanisms of renal acid excretion during normovolemic hemodilution. Bicarbonate, which had been removed during normovolemic hemodilution, was calculated with the help of the Henderson-Hasselbalch equation. The urinary amount of ammonium as well as phosphate was determined in residual probes. The absolute amount of free protons in urine was obtained from the pH of the respective samples. The amount of protons generated during normovolemic hemodilution was approximately 0.6 mmol. During experimental time (5.5 h), distinct urinary ammonium excretion occurred (Voluven 0.52 mmol, Volulyte 0.39 mmol and Gelafundin 0.77 mmol). Proton excretion via the phosphate buffer constituted 0.04 mmol in every experimental group. Excretion of free protons was in the range of 10-6 mmol. The present data prove that the prompt rise in urinary ammonium excretion is also valid for acute metabolic acidosis originating from severe normovolemic hemodilution.

Effect of NSAIDs on Na+/H+ exchanger activity in rat colonic crypts

Nonsteroidal anti-inflammatory drugs (NSAIDs; 1) are widely recommended for several acute and chronic conditions. For example, both indomethacin and aspirin are taken for pain relief. Aspirin is also used for prevention of myocardial infarction, and indomethacin can be administered orally or as a suppository for patients with rheumatoid disease and other chronic inflammatory states. However, use of NSAIDs can cause damage to the mucosal barrier surrounding the gastrointestinal (GI) tract, increasing the risk of ulcer formation. While microencapsulation of NSAIDs has been shown to reduce upper GI injury, sustained release in the lower GI tract and colon may cause epithelial erosion due to increased acidification. The use of suppositories has also been linked to rectal and lower GI bleeding. In this study, we investigated the role of NSAIDs aspirin and indomethacin on Na+/H+ exchanger (NHE) activity in rat colonic crypts. By comparing average rates of pH recovery between control and NSAID perfusion runs, we were able to determine that both aspirin and indomethacin increase hydrogen extrusion into the colonic lumen. Through treatment with 5-ethylisopropyl amiloride (EIPA), amiloride, and zoniporide dihydrochloride, we further demonstrated that indomethacin specifically enhances proton excretion through regulation of apical NHE-3 and NHE-2 and to a lesser extent on basolateral NHE-1 and NHE-4. Our results suggest that clinical exposure to NSAIDs may affect colonic tissue at the site of selected NHE isoforms, resulting in modulation of transport and barrier function.

New insights into ion regulation of cephalopod molluscs: a role of epidermal ionocytes in acid-base regulation during embryogenesis

The constraints of an active life in a pelagic habitat led to numerous convergent morphological and physiological adaptations that enable cephalopod molluscs and teleost fishes to compete for similar resources. Here, we show for the first time that such convergent developments are also found in the ontogenetic progression of ion regulatory tissues; as in teleost fish, epidermal ionocytes scattered on skin and yolk sac of cephalopod embryos appear to be responsible for ionic and acid-base regulation before gill epithelia become functional. Ion and acid-base regulation is crucial in cephalopod embryos, as they are surrounded by a hypercapnic egg fluid with a Pco2 between 0.2 and 0.4 kPa. Epidermal ionocytes were characterized via immunohistochemistry, in situ hybridization, and vital dye-staining techniques. We found one group of cells that is recognized by concavalin A and MitoTracker, which also expresses Na+/H+ exchangers (NHE3) and Na+-K+-ATPase. Similar to findings obtained in teleosts, these NHE3-rich cells take up sodium in exchange for protons, illustrating the energetic superiority of NHE-based proton excretion in marine systems. In vivo electrophysiological techniques demonstrated that acid equivalents are secreted by the yolk and skin integument. Intriguingly, epidermal ionocytes of cephalopod embryos are ciliated as demonstrated by scanning electron microscopy, suggesting a dual function of epithelial cells in water convection and ion regulation. These findings add significant knowledge to our mechanistic understanding of hypercapnia tolerance in marine organisms, as it demonstrates that marine taxa, which were identified as powerful acid-base regulators during hypercapnic challenges, already exhibit strong acid-base regulatory abilities during embryogenesis.

A comparison of proton excretion of twelve pasture legumes grown in nutrient solution

Summary.The use of legumes to improve the productivity of pastures has usually been associated with an increase in soil acidification. The present study compared the acidification potential of 12 N2-fixing pasture legume species/genotypes using a solution culture assay. The legumes were chosen because of their use or potential use in farming systems in the mediterranean zones of southern Australia. The species grown were Trifolium subterraneum L. (vars. Dalkeith, Yarloop and Clare), Trifolium glomeratum L., Trifolium tomentosum L., Medicago murex Willd., Medicago polymorpha L., Medicago truncatulaGaertn., Ornithopus compressus L., Ornithopus sativusBrot., Trifolium vesiculosum and Trifolium balansae. Proton excretion was measured over a period of 21 days from days 40 to 61 after germination. The amount of protons excreted into solution varied between 143 and 265 cmol H+ /kg dry matter for the different species and genotypes. In general, T. tomentosum and T. glomeratum excreted greater amounts of protons per unit biomass than Medicago species and T. subterraneum, which, in turn, excreted more protons than Ornithopus species. The amount of proton excreted per kilogram biomass was well correlated with the concentrations of excess cations over anions and ash alkalinity in plants across all the species. Total acid production was highly correlated with accumulation of excess cations and ash alkalinity, total dry matter production and total nitrogen fixation during the study period. It is suggested that the potential proton excretion by new pasture legume species should be considered when they are introduced into farming systems.