Sebastian Frische

Regulation of the Cl−/HCO3− Exchanger AE2 in Rat Thick Ascending Limb of Henle’s Loop in Response to Changes in Acid-Base and Sodium Balance

The Cl(-)/HCO(3)(-) exchanger AE2 is believed to be involved in transcellular bicarbonate reabsorption that occurs in the thick ascending limb of Henles loop (TAL). The purpose of this study was to test whether chronic changes in acid-base status and sodium intake regulate AE2 polypeptide abundance in the TAL of the rat. Rats were subjected to 6 d of loading with NaCl, NH(4)Cl, NaHCO(3), KCl, or KHCO(3). AE2 protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex and the outer medulla of treated and control rats. In the renal cortex, AE2 abundance was markedly increased in response to oral loading with NH(4)Cl or with NaCl. In contrast, AE2 abundance was unchanged in response to loading with KCl or with NaHCO(3) and was decreased by loading with KHCO(3). The response of AE2 in the outer medulla differed from that in the cortex in that HCO(3)(-) loading increased AE2 abundance when administered with Na(+) but had no effect when administered with K(+). Immunohistochemistry revealed that NaCl loading increased AE2 abundance in the basolateral membrane of both the cortical and the medullary TAL. In contrast, NH(4)Cl loading increased AE2 abundance only in the cortical TAL but not in the medullary TAL. These results suggest that regulation of the basolateral Cl(-)/HCO(3)(-) exchanger AE2 plays an important role in the adaptation of bicarbonate absorption in the TAL during chronic acid-base disturbances and high sodium intake. The present study also emphasizes the contribution of cortical TAL adaptation in the renal regulation of acid-base status.

A Role of Myosin Vb and Rab11-FIP2 in the Aquaporin-2 Shuttle

Arginine‐vasopressin (AVP) regulates water reabsorption in renal collecting duct principal cells. Its binding to Gs‐coupled vasopressin V2 receptors increases cyclic AMP (cAMP) and subsequently elicits the redistribution of the water channel aquaporin‐2 (AQP2) from intracellular vesicles into the plasma membrane (AQP2 shuttle), thereby facilitating water reabsorption from primary urine. The AQP2 shuttle is a paradigm for cAMP‐dependent exocytic processes. Using sections of rat kidney, the AQP2‐expressing cell line CD8, and primary principal cells, we studied the role of the motor protein myosin Vb, its vesicular receptor Rab11, and the myosin Vb‐ and Rab11‐binding protein Rab11‐FIP2 in the AQP2 shuttle. Myosin Vb colocalized with AQP2 intracellularly in resting and at the plasma membrane in AVP‐treated cells. Rab11 was found on AQP2‐bearing vesicles. A dominant‐negative myosin Vb tail construct and Rab11‐FIP2 lacking the C2 domain (Rab11‐FIP2‐ΔC2), which disrupt recycling, caused condensation of AQP2 in a Rab11‐positive compartment and abolished the AQP2 shuttle. This effect was dependent on binding of myosin Vb tail and Rab11‐FIP2‐ΔC2 to Rab11. In summary, we identified myosin Vb as a motor protein involved in AQP2 recycling and show that myosin Vb‐ and Rab11‐FIP2‐dependent recycling of AQP2 is an integral part of the AQP2 shuttle.

Ion transporters in secretory and cyclically modulating ameloblasts: a new hypothesis for cellular control of preeruptive enamel maturation

Mature enamel consists of densely packed and highly organized large hydroxyapatite crystals. The molecular machinery responsible for the formation of fully matured enamel is poorly described but appears to involve oscillative pH changes at the enamel surface. We conducted an immunohistochemical investigation of selected transporters and related proteins in the multilayered rat incisor enamel organ. Connexin 43 (Cx-43) is found in papillary cells and ameloblasts, whereas Na(+)-K(+)-ATPase is heavily expressed during maturation in the papillary cell layer only. Given the distribution of Cx-43 channels and Na(+)-K(+)-ATPase, we suggest that ameloblasts and the papillary cell layer act as a functional syncytium. During enamel maturation ameloblasts undergo repetitive cycles of modulation between ruffle-ended (RA) and smooth-ended (SA) ameloblast morphologies. Carbonic anhydrase II and vacuolar H(+)-ATPase are expressed simultaneously at the beginning of the maturation stage in RA cells. The proton pumps are present in the ruffled border of RA and appear to be internalized during the SA stage. Both papillary cells and ameloblasts express plasma membrane acid/base transporters (AE2, NBC, and NHE1). AE2 and NHE1 change position relative to the enamel surface as localization of the tight junctions changes during ameloblast modulation cycles. We suggest that the concerted action of the papillary cell layer and the modulating ameloblasts regulates the enamel microenvironment, resulting in oscillating pH fluctuations. The pH fluctuations at the enamel surface may be required to keep intercrystalline spaces open in the surface layers of the enamel, enabling degraded enamel matrix proteins to be removed while hydroxyapatite crystals grow as a result of influx of calcium and phosphate ions.

P2Y6 receptor mediates colonic NaCl secretion via differential activation of cAMP-mediated transport

Extracellular nucleotides are important regulators of epithelial ion transport. Here we investigated nucleotide-mediated effects on colonic NaCl secretion and the signal transduction mechanisms involved. Basolateral UDP induced a sustained activation of Cl(-) secretion, which was completely inhibited by 293B, a specific inhibitor of cAMP-stimulated basolateral KCNQ1/KCNE3 K(+) channels. We therefore speculated that a basolateral P2Y(6) receptor could increase cAMP. Indeed UDP elevated cAMP in isolated crypts. We identified an epithelial P2Y(6) receptor using crypt [Ca(2+)](i) measurements, RT-PCR, and immunohistochemistry. To investigate whether the rat P2Y(6)elevates cAMP, we coexpressed the P2Y(1) or P2Y(6) receptor together with the cAMP-regulated cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel in Xenopus oocytes. A two-electrode voltage clamp was used to monitor nucleotide-induced Cl(-) currents. In oocytes expressing the P2Y(1) receptor, ATP transiently activated the endogenous Ca(2+)-activated Cl(-) current, but not CFTR. In contrast, in oocytes expressing the P2Y(6)receptor, UDP transiently activated the Ca(2+)-activated Cl(-) current and subsequently CFTR. CFTR Cl(-) currents were identified by their halide conductance sequence. In summary we find a basolateral P2Y(6) receptor in colonic epithelial cells stimulating sustained NaCl secretion by way of a synergistic increase of [Ca(2+)](i) and cAMP. In support of these data P2Y(6) receptor stimulation differentially activates CFTR in Xenopus oocytes.

Basolateral Na+-dependent HCO3− transporter NBCn1-mediated HCO3− influx in rat medullary thick ascending limb

The electroneutral Na+‐dependent HCO3− transporter NBCn1 is strongly expressed in the basolateral membrane of rat medullary thick ascending limb cells (mTAL) and is up‐regulated during NH4+‐induced metabolic acidosis. Here we used in vitro perfusion and BCECF video‐imaging of mTAL tubules to investigate functional localization and regulation of Na+‐dependent HCO3− influx during NH4+‐induced metabolic acidosis. Tubule acidification was induced by removing luminal Na+ (ΔpHi: 0.88 ± 0.11 pH units, n= 10). Subsequently the basolateral perfusion solution was changed to CO2/HCO3− buffer with and without Na+. Basolateral Na+–H+ exchange function was inhibited with amiloride. Na+‐dependent HCO3− influx was determined by calculating initial base flux of Na+‐mediated re‐alkalinization. In untreated animals base flux was 8.4 ± 0.9 pmol min−1 mm−1. A 2.4‐fold increase of base flux to 21.8 ± 3.2 pmol min−1 mm−1 was measured in NH4+‐treated animals (11 days, n= 11). Na+‐dependent re‐alkalinization was significantly larger when compared to control animals (0.38 ± 0.03 versus 0.22 ± 0.02 pH units, n= 10). In addition, Na+‐dependent HCO3− influx was of similar magnitude in chloride‐free medium and also up‐regulated after NH4+ loading. Na+‐dependent HCO3− influx was not inhibited by 400 μm DIDS. A strong up‐regulation of NBCn1 staining was confirmed in immunolabelling experiments. RT‐PCR analysis revealed no evidence for the Na+‐dependent HCO3− transporter NBC4 or the two Na+‐dependent CI−/HCO3− exchangers NCBE and NDCBE. These data strongly indicate that rat mTAL tubules functionally express basolateral DIDS‐insensitive NBCn1. Function and protein are strongly up‐regulated during NH4+‐induced metabolic acidosis. We suggest that NBCn1‐mediated basolateral HCO3− influx is important for basolateral NH3 exit and thus NH4+ excretion by means of setting pHi to a more alkaline value.

Tubulovascular Cross-Talk by Vascular Endothelial Growth Factor A Maintains Peritubular Microvasculature in Kidney

Vascular endothelial growth factor A (VEGFA) production by podocytes is critical for glomerular endothelial health. VEGFA is also expressed in tubular epithelial cells in kidney; however, its physiologic role in the tubule has not been established. Using targeted transgenic mouse models, we found that Vegfa is expressed by specific epithelial cells along the nephron, whereas expression of its receptor (Kdr/Vegfr2) is largely restricted to adjacent peritubular capillaries. Embryonic deletion of tubular Vegfa did not affect systemic Vegfa levels, whereas renal Vegfa abundance was markedly decreased. Excision of Vegfa from renal tubules resulted in the formation of a smaller kidney, with a striking reduction in the density of peritubular capillaries. Consequently, elimination of tubular Vegfa caused pronounced polycythemia because of increased renal erythropoietin (Epo) production. Reducing hematocrit to normal levels in tubular Vegfa-deficient mice resulted in a markedly augmented renal Epo production, comparable with that observed in anemic wild-type mice. Here, we show that tubulovascular cross-talk by Vegfa is essential for maintenance of peritubular capillary networks in kidney. Disruption of this communication leads to increased renal Epo production and resulting polycythemia, presumably to counterbalance microvascular losses.

NBCn1 is a basolateral \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{Na}^{+}\mathrm{-HCO}_{3}^{-}\) \end{document} cotransporter in rat kidney inner medullary collecting ducts

Primary cultures of rat inner medullary collecting duct (IMCD) cells Na(+) dependently import HCO(3)(-) across the basolateral membrane through an undefined transport protein. We used RT-PCR, immunoblotting, and immunohistochemistry to identify candidate proteins for this basolateral Na(+)-HCO(3)(-) cotransport. The mRNA encoding the electroneutral Na(+)-HCO(3)(-) cotransporter NBCn1 was detected as the only Na(+)-HCO(3)(-) cotransporter in the rat inner medulla (IM) among the five characterized Na(+)-dependent HCO(3)(-) transporters. The mRNA of a yet uncharacterized transporter-like protein, BTR1, was also present in the IM, but its expression in microdissected tubules seemed restricted to the thin limbs of Henles loop. Immunoblotting confirmed the presence of NBCn1 as an approximately 180-kDa protein of the rat IM. Anti-NBCn1 immunolabeling was confined to the basolateral plasma membrane domain of IMCD cells in the papillary two-thirds of the IM. Consistent with the presence of NBCn1, IMCD cells possessed stilbene-insensitive, Na(+)- and HCO(3)(-)-dependent pH recovery after acidification, as assessed by fluorescence microscopy using a pH-sensitive intracellular dye. In furosemide-induced alkalotic rats, NBCn1 protein abundance was decreased in both the IM and inner stripe of outer medulla (ISOM) as determined by immunoblotting and immunohistochemistry. In contrast, NBCn1 abundance in the IM and ISOM was unchanged in NaHCO(3)-loaded animals, and the NBCn1 abundance increased only in the ISOM after NH(4)Cl loading. In conclusion, NBCn1 is a basolateral Na(+)-HCO(3)(-) cotransporter of IMCD cells and is differentially regulated in IMCD and medullary thick ascending limb.

Tissue kallikrein permits early renal adaptation to potassium load

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK−/−) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK−/− mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK−/− mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK−/− mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.

Targeted disruption of the Cl-/HCO3-exchanger Ae2 results in osteopetrosis in mice

Osteoclasts are multinucleated bone-resorbing cells responsible for constant remodeling of bone tissue and for maintaining calcium homeostasis. The osteoclast creates an enclosed space, a lacuna, between their ruffled border membrane and the mineralized bone. They extrude H+ and Cl− into these lacunae by the combined action of vesicular H+-ATPases and ClC-7 exchangers to dissolve the hydroxyapatite of bone matrix. Along with intracellular production of H+ and HCO3− by carbonic anhydrase II, the H+-ATPases and ClC-7 exchangers seems prerequisite for bone resorption, because genetic disruption of either of these proteins leads to osteopetrosis. We aimed to complete the molecular model for lacunar acidification, hypothesizing that a HCO3− extruding and Cl− loading anion exchange protein (Ae) would be necessary to sustain bone resorption. The Ae proteins can provide both intracellular pH neutrality and serve as cellular entry mechanism for Cl− during bone resorption. Immunohistochemistry revealed that Ae2 is exclusively expressed at the contra-lacunar plasma membrane domain of mouse osteoclast. Severe osteopetrosis was encountered in Ae2 knockout (Ae2−/−) mice where the skeletal development was impaired with a higher diffuse radio-density on x-ray examination and the bone marrow cavity was occupied by irregular bone speculae. Furthermore, osteoclasts in Ae2−/− mice were dramatically enlarged and fail to form the normal ruffled border facing the lacunae. Thus, Ae2 is likely to be an essential component of the bone resorption mechanism in osteoclasts.

Clinical review: Renal tubular acidosis – a physicochemical approach

The Canadian physiologist PA Stewart advanced the theory that the proton concentration, and hence pH, in any compartment is dependent on the charges of fully ionized and partly ionized species, and on the prevailing CO2 tension, all of which he dubbed independent variables. Because the kidneys regulate the concentrations of the most important fully ionized species ([K+], [Na+], and [Cl-]) but neither CO2 nor weak acids, the implication is that it should be possible to ascertain the renal contribution to acid–base homeostasis based on the excretion of these ions. One further corollary of Stewarts theory is that, because pH is solely dependent on the named independent variables, transport of protons to and from a compartment by itself will not influence pH. This is apparently in great contrast to models of proton pumps and bicarbonate transporters currently being examined in great molecular detail. Failure of these pumps and cotransporters is at the root of disorders called renal tubular acidoses. The unquestionable relation between malfunction of proton transporters and renal tubular acidosis represents a problem for Stewart theory. This review shows that the dilemma for Stewart theory is only apparent because transport of acid–base equivalents is accompanied by electrolytes. We suggest that Stewart theory may lead to new questions that must be investigated experimentally. Also, recent evidence from physiology that pH may not regulate acid–base transport is in accordance with the concepts presented by Stewart.