Masashi Imai

Transcriptional activation of RACTK1 K + channel gene by apical alkalization in renal cortical collecting duct cells

We have previously demonstrated that RACTK1 cDNA encodes a pH sensitive K+ channel expressed in the apical side of renal collecting tubule cells. To determine whether extracellular pH induces the RACTK1 gene expression in the renal cortical collecting duct (CCD) cells, we measured mRNA of the RACTK1 using cultured rabbit CCD cells. Alkalization of incubation medium activated the transcription of the RACKTK1 gene in a time- and dose-dependent manner after 1 h, and reached a maximal level after 12 h. To examine whether the stimulation of mRNA by alkalization of body fluid occurs also in vivo, mRNA levels were measured in mice loaded with acid or alkali. The RACTK1 mRNA was increased in association with the rise in urinary pH. To examine side face of the effect of pH on stimulation of mRNA, we observed the effect of pH in the apical or the basolateral side in the preparation where CCD cells were cultured on filter membrane supports. Alkalization of the apical side but not of the basolateral side, was shown to be a determinant in inducting the RACTK1 mRNA. These findings suggest that, in addition to rapid direct regulation of RACTK1 K+ channel conductance by intracellular pH, this channel is also regulated by the changes in luminal pH through synthesis of channel protein by transcriptional activation.

Mechanism of iodide transport in the rabbit cortical collecting duct

BackgroundPendrin, an anion exchanger known to participate in iodide transport in the apical membrane of follicular cells of the thyroid gland, has recently been shown to exist in the apical membrane of the β- and γ-intercalated (β/γ-IC) cells of the cortical collecting duct (CCD). We examined mechanisms of iodide transport in the CCD.MethodsRabbit CCD was perfused in vitro, and lumen-to-bath flux coefficients for both 125I− (KI (lb)) and 36Cl− (KCl (lb)) were measured simultaneously. The intracellular pH (pHi) of β/γ-IC cells in the perfused CCD was measured by microscopic fluorometory, by loading 2′,7′-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein tetraacetoxy methylester (BCECF-AM), a fluorescent marker for pHi. The effects on pHi of the replacement of NaCl with Na cyclamate, NaI, or NaBr in the lumen or bath were observed.ResultsKI (lb) was comparable to or slightly higher than KCl (lb). Both iodide and chloride in the lumen caused self- and cross-inhibitions to both fluxes. The addition of 5-nitro-2-(-3-phenylpropylamino)-benzoate (NPPB), a Cl− channel inhibitor, to the bath significantly reduced KCl (lb), but not KI (lb). Replacement of luminal fluid NaCl with Na cyclamate, NaI, or NaBr caused alkalization of pHi, no change in pHi, and slight acidification of pHi, respectively. Replacement of bath NaCl with Na cyclamate, NaI, or NaBr caused alkalization, alkalization, and acidification of pHi, respectively. Luminal NaI prevented the acidification of pHi caused by bath Na cyclamate.ConclusionsThe data are consistent with the model that iodide is transported via the Cl−/HCO3− exchanger in the apical membrane of β/γ-IC cells and exits the basolateral membrane via an electroneutral transporter that is distinct from the Cl− channel. We could not, however, identify which type of β/γ-IC cell was mainly responsible.

Computer analysis of the significance of the effective osmolality for urea across the inner medullary collecting duct in the operation of a single effect for the counter-current multiplication system

BackgroundAlthough urea and water are transported across separate pathways in the apical membrane of the inner medullary collecting duct (IMCD), the existence of a cellular diffusion barrier as an unstirred layer makes it possible to use coefficients of effective osmotic force (σ*) as equivalent to reflection coefficients. The difference in effective osmolality between urea and NaCl across the IMCD becomes a driving force for water if the compositions of solutes are different between tubular lumen and interstitium. Since reported values for σ*urea are discrepant, we compared the efficiency of a single effect in the counter-current system between an ascending thin limb (ATL) and the IMCD, with the interposition of capillary networks (CNW), between two models with σurea* = 0.7 (model 1) and σurea* = 1.0 (model 2).MethodsThe time courses (within 3 s) of solute and the water transport profiles among ATL, CNW, and IMCD were simulated with a computer in the absence of flow in each compartment.ResultsIn spite of small differences in the profiles of urea and NaCl concentrations between the two models, model 1 displayed a larger volume flux in the IMCD than model 2, resulting in an increase of osmolality in the IMCD and a decrease of osmolality in the ATL. These findings are vital for the operation of the counter-current multiplication system.ConclusionsThe concept of coefficients for effective osmotic force can be applied to the counter-current model between the IMCD and the ATL with the interposition of CNW. The model of σurea* = 0.7 is more efficient than that of σurea* = 1.0.

Mechanism of Cd-induced inhibition of Na-glucose cotransporter in rabbit proximal tubule cells: roles of luminal pH and membrane-bound carbonic anhydrase.

Background/Aims: We have previously reported that a complex of cadmium-metallothionein (Cd-MT) directly affects the apical Na-glucose cotransporter on the luminal side in proximal tubules, suggesting that Cd-MT is more toxic than CdCl2 in causing tubulopathy. To find the potential mechanisms, we evaluated the effect of luminal pH alteration and carbonic anhydrase (CA) inhibition on Cd-MT-induced reduction of glucose-dependent transmural voltage in rabbit S2 segments perfused in vitro. Methods: Before and after the addition of Cd-MT (1 µg Cd/ml) to the lumen, the deflections of transmural voltage upon the elimination of glucose from the perfusate (ΔVtglu) were measured as a parameter of activity of the Na-glucose cotransporter. Results: During perfusion with a control solution of pH 7.4, the ΔVtglu significantly decreased after addition of Cd-MT for 10 min. A reduction in pH to 6.8 significantly shortened the time needed to reduce the ΔVtglu to <5 min, whereas an increase of pH to 7.7 significantly prolonged the time to >20 min. Furthermore, simultaneous addition of acetazolamide with control perfusate prevented the reduction. P-Fluorobenzyl-aminobenzolamide (pFB-ABZ), a membrane-impermeable CA inhibitor, added to the lumen also completely prevented the reduction in ΔVtglu. In rabbits with chronic Cd exposure, acetazolamide prevented the glucosuria. Conclusion: Cd-MT-induced inhibition of Na-glucose cotransporter activity in the S2 segment strongly depends on luminal pH, and that an increase in pH by inhibition of luminal membrane-bound CA is useful to prevent renal Cd toxicity.

Electrophysiological Localization of Transporters in the Distal Nephron Segments

The distal convoluted tubule (DCT) and the connecting tubule (CNT) from the rabbit kidney were investigated by electrophysiological techniques, isotopic flux measurement, and microfluorometry, in order to characterize the ion transport properties of cell membranes. When the DCT and the CNT were per-fused in vitro, the transepithelial voltage (VT) displayed lumen negative, and the transepithelial resistances (RT) were relatively low, as in the category of leaky epithelia. Random cellular impalement revealed that the basolateral membrane voltage (VB) of the DCT showed Gaussian distribution, whereas the CNT consisted of two cell populations, having different VB and different fractional resistance of the apical membrane (fRA). The CNT cell had a high VB. and lower fRA and the intercalated (IC) cell in the CNT had a low VB and higher fRA Using ion substitution and channel inhibitors, the conductive properties of DCT cells and CNT cells revealed that the luminal membrane had both Na+ and K+ conductances and that the basolateral membrane had both K+ and Cl-conductances. Intercalated cells of CNT had only a Cl-conductance in the basolateral membrane. Besides Na+ conductance in the luminal membrane of CNT cells, two other modes of Na+ entry process were observed. One pathway was the Na/Cl cotransporter, revealed by isotopic ion flux studies, which was inhibitable with thiazide diuretics, and the other was a nonselective cation conductance, which was not sensitive to amiloride and was opened by parathyroid hormone (PTH). From intracellular calcium measurement and calcium flux studies, it was found that the latter mode serves as a route for calcium entry pathway in CNT cells.

Function of Thin Segments of Henle’s Loop

It is well known that the renal medulla plays an important role in the generation of concentrated urine, which is critical in the maintenance of body fluid osmolality. Since the proposal of the operation of countercurrent systems in the renal medulla by Kuhn and his associates [1–3], it has been widely accepted that the loop structures of the nephron in the renal medulla are essential for the generation and maintenance of a steep osmotic gradient along the axis of the renal medulla. However, the detailed mechanisms by which a steep osmotic gradient is generated by the countercurrent multiplication system remain to be established.