Motoei Kunimi

Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities.

Mutations in SLC4A4 cause permanent isolated proximal renal tubular acidosis with ocular abnormalities

Molecular basis of ocular abnormalities associated with proximal renal tubular acidosis

Proximal renal tubular acidosis associated with ocular abnormalities such as band keratopathy, glaucoma, and cataracts is caused by mutations in the Na(+)-HCO(3)(-) cotransporter (NBC-1). However, the mechanism by which NBC-1 inactivation leads to such ocular abnormalities remains to be elucidated. By immunological analysis of human and rat eyes, we demonstrate that both kidney type (kNBC-1) and pancreatic type (pNBC-1) transporters are present in the corneal endothelium, trabecular meshwork, ciliary epithelium, and lens epithelium. In the human lens epithelial (HLE) cells, RT-PCR detected mRNAs of both kNBC-1 and pNBC-1. Although a Na(+)-HCO(3)-cotransport activity has not been detected in mammalian lens epithelia, cell pH (pH(i)) measurements revealed the presence of Cl(-)-independent, electrogenic Na(+)-HCO(3)-cotransport activity in HLE cells. In addition, up to 80% of amiloride-insensitive pH(i) recovery from acid load in the presence of HCO(3)(-)/CO(2) was inhibited by adenovirus-mediated transfer of a specific hammerhead ribozyme against NBC-1, consistent with a major role of NBC-1 in overall HCO(3)-transport by the lens epithelium. These results indicate that the normal transport activity of NBC-1 is indispensable not only for the maintenance of corneal and lenticular transparency but also for the regulation of aqueous humor outflow.

Roles of Insulin Receptor Substrates in Insulin-Induced Stimulation of Renal Proximal Bicarbonate Absorption

Insulin resistance is frequently associated with hypertension, but the mechanism underlying this association remains speculative. Although insulin is known to modify renal tubular functions, little is known about roles of insulin receptor substrates (IRS) in the renal insulin actions. For clarifying these issues, the effects of insulin on the rate of bicarbonate absorption (JHCO3-) were compared in isolated renal proximal tubules from wild-type, IRS1-deficient (IRS1-/-), and IRS2-deficient (IRS2-/-) mice. In wild-type mice, physiologic concentrations of insulin significantly increased JHCO3-. This stimulation was completely inhibited by wortmannin and LY-294002, indicating that the phosphatidylinositol 3-kinase pathway mediates the insulin action. The stimulatory effect of insulin on JHCO3- was completely preserved in IRS1-/- mice but was significantly attenuated in IRS2-/- mice. Similarly, insulin-induced Akt phosphorylation was preserved in IRS1-/- mice but was markedly attenuated in IRS2-/- mice. Furthermore, insulin-induced tyrosine phosphorylation of IRS2 was more prominent than that of IRS1. These results indicate that IRS2 plays a major role in the stimulation of renal proximal absorption by insulin. Because defects at the level of IRS1 may underlie at least some forms of insulin resistance, sodium retention, facilitated by hyperinsulinemia through the IRS1-independent pathway, could be an important factor in pathogenesis of hypertension in insulin resistance.

Functional and molecular evidence for Na+-HCO3– cotransporter in human corneal endothelial cells

Abstract. Although bicarbonate transport in corneal endothelium has been suggested to be coupled to Na+, the underlying molecular mechanism has not been clarified. In the present study we investigated whether a recently cloned Na+-HCO3– cotransporter (NBC-1) is responsible for this process, and, if so, whether the endothelium expresses a separate isoform or one of the other two isoforms that have recently been identified (kNBC-1 from kidney and pNBC-1 from pancreas). Using primers designed for specific and common regions we demonstrated by reverse transcriptase polymerase chain reaction (RT-PCR) that both kNBC-1 and pNBC-1 are expressed in cultured human corneal endothelial cells. In addition functional studies with a pH-sensitive fluorescence probe were performed. In the presence of HCO3–/CO2 a pH regulatory process was demonstrated which depends on the presence of Na+ and membrane potential, but is independent of Cl– and is inhibited by the disulfonic stilbene DIDS. These results support the presence of NBC-1 as the major bicarbonate transport system in corneal endothelium.

Thiazolidinediones Enhance Sodium-Coupled Bicarbonate Absorption from Renal Proximal Tubules via PPARγ-Dependent Nongenomic Signaling

Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion.

Biphasic Regulation of Na+-HCO3− Cotransporter by Angiotensin II Type 1A Receptor

Abstract—Although angiotensin (Ang) II is known to regulate renal proximal transport in a biphasic way, the receptor subtype(s) mediating these Ang II effects remained to be established. To clarify this issue, we compared the effects of Ang II in wild-type mice (WT) and Ang II type 1A receptor–deficient mice (AT1A KO). The Na+-HCO3− cotransporter (NBC) activity, analyzed in isolated nonperfused tubules with a fluorescent probe, was stimulated by 10−10 mol/L Ang II but was inhibited by 10−6 mol/L Ang II in WT. Although valsartan (AT1 antagonist) blocked both stimulation and inhibition by Ang II, PD 123,319 (AT2 antagonist) did not modify these effects of Ang II. In AT1A KO, in contrast, this biphasic regulation was lost, and only stimulation of NBC activity by 10−6 mol/L Ang II was observed. This stimulation was blocked by valsartan but not by PD 123,319. More than 10−8 mol/L Ang II induced a transient increase in cell Ca2+ concentrations in WT, which was again blocked by valsartan but not by PD 123,319. However, up to 10−5 mol/L Ang II did not increase cell Ca2+ concentrations in AT1A KO. Finally, the addition of arachidonic acid inhibited the NBC activity similarly in WT and AT1A KO, suggesting that the inhibitory pathway involving P-450 metabolites is preserved in AT1A KO. These results indicate that AT1A mediates the biphasic regulation of NBC. Although low-level expression of AT1B could be responsible for the stimulation by 10−6 mol/L Ang II in AT1A KO, no evidence was obtained for AT2 involvement.

Biphasic Regulation of Renal Proximal Bicarbonate Absorption by Luminal AT1A Receptor

Angiotensin II (AngII) regulates renal proximal transport in a biphasic way. It has been recently shown that the basolateral type 1A receptor (AT(1A)) mediates the biphasic regulation of Na(+)-HCO(3)(-) cotransporter (NBC) by AngII. However, the receptor subtype(s) responsible for the luminal AngII actions remained to be established. To clarify this issue, the luminal AngII effects in isolated proximal tubules from wild-type (WT) and AT(1A)-deficient mice (AT(1A) KO) were compared. In WT, the rate of bicarbonate absorption (JHCO(3)(-)), analyzed with a stop-flow microspectrofluorometric method, was stimulated by 10(-10) mol/L luminal AngII but was inhibited by 10(-6) mol/L luminal AngII. Both stimulatory and inhibitory effects of AngII were completely blocked by valsartan (AT(1) antagonist) but unaffected by PD 123,319 (AT(2) antagonist). In AT(1A) KO, in contrast, luminal AngII (10(-10) - 10(-6) mol/L) did not change JHCO(3)(-). In WT, 10(-6) mol/L luminal AngII increased cell Ca(2+) concentrations ([Ca(2+)](i)), which was again blocked by valsartan but not by PD 123,319. However, luminal AngII did not increase [Ca(2+)](i) in AT(1A) KO. On the other hand, the addition of arachidonic acid similarly inhibited JHCO(3)(-) in WT and AT(1A) KO. Furthermore, the acute activation of protein kinase C by phorbol 12-myristate 13-acetate similarly stimulated JHCO(3)(-) in WT and AT1A KO, indicating that the inhibitory and stimulatory pathways necessary for the AngII actions were preserved in AT(1A) KO. These results indicate that the luminal AT(1A) mediates the biphasic regulation of bicarbonate absorption by luminal AngII, while no evidence was obtained for a role of AT(2).

Roles of ERK and cPLA2 in the Angiotensin II-Mediated Biphasic Regulation of Na+-HCO3− Transport

Regulation of renal proximal transport by angiotensin II (Ang II) is biphasic: low concentrations (picomolar to nanomolar) stimulate reabsorption, but higher concentrations (nanomolar to micromolar) inhibit reabsorption. Traditionally, the stimulatory effect has been attributed to activation of protein kinase C and/or a decrease in intracellular cAMP, whereas the inhibitory action has been attributed to the activation of phospholipase A2 (PLA2) and the subsequent release of arachidonic acid. The Ang II receptor subtype responsible for these effects and the intracellular signaling pathways involved are not completely understood. We isolated proximal tubules from wild-type, Ang II type 1A receptor (AT1A)-deficient, and group IVA cytosolic phospholipase A2 (cPLA2alpha)-deficient mice, and compared their responses to Ang II. In wild-type mice, we found that the stimulatory and inhibitory effects of Ang II on Na+-HCO3(-) cotransporter activity are both AT1-mediated but that ERK activation only plays a role in the former. The stimulatory effect of Ang II was also observed in AT1A-deficient mice, suggesting that this occurs through AT1B. In contrast, the inhibitory effects of Ang II appeared to be mediated by cPLA2alpha activation because high-concentration Ang II stimulated Na+-HCO3(-) cotransporter activity when cPLA2alpha activity was abrogated by pharmacological means or genetic knockout. Consistent with this observation, we found that activation of the cPLA2alpha/P450 pathway suppressed ERK activation. We conclude that Ang II activates ERK and cPLA2alpha in a concentration-dependent manner via AT1, and that the balance between ERK and cPLA2alpha activities determines the ultimate response to Ang II in intact proximal tubules.

Intracellular pH regulatory mechanism in a human renal proximal cell line (HKC-8): evidence for Na+/H+ exchanger, Cl–/HCO3– exchanger and Na+-HCO3– cotransporter

In the present study we investigated whether an immortalized human renal proximal cell line, HKC-8, expresses a recently cloned Na+-HCO3– cotransporter (NBC-1) and, if so, which isoform (kNBC-1 from kidney or pNBC-1 from pancreas) is expressed in this cell line. Cell pH (pHi) measurements using a pH-sensitive fluorescence probe in the absence of HCO3–/CO2 revealed the presence of a Na+/H+ exchanger that required high concentrations of amiloride for full inhibition. In the presence of HCO3–/CO2 another pHi recovery process, dependent on Na+ but independent of Cl–, was identified. This process was electrogenic and was inhibited by 4,4′-diisothiocyanatodihydrostilbene-2,2′-disulphonic acid (DIDS), being consistent with the Na+-HCO3– cotransporter. In addition, the pHi responses to Cl– removal were compatible with the presence of a Na+-independent Cl–/HCO3– exchanger that was also inhibited by DIDS. Reverse transcriptase polymerase chain reaction (RT-PCR) using primers designed for specific and common regions detected mRNAs of both kNBC-1 and pNBC-1 and Western blot analysis confirmed the expression of NBC-1 protein. These results indicate that HKC-8 has transport activities similar to intact proximal tubules and also suggest that both kNBC-1 and pNBC-1 may contribute to the Na+-HCO3– cotransport activity in this cell line.