EGF and its related growth factors mediate sodium transport in mpkCCDc14 cells via ErbB2 (neu/HER‐2) receptor

Amiloride‐sensitive sodium entry, via the epithelial sodium channel (ENaC), is the rate‐limiting step for Na⁺ absorption. Epidermal growth factor (EGF) is involved in the regulation of Na⁺ transport and ENaC activity. However it is still controversial exactly how EGF regulates ENaC and Na⁺ absorption. The aim of the present study was to characterize the EGF regulation of Na⁺ transport in cultured mouse renal collecting duct principal mpkCCD_c14 cells, a highly differentiated cell line which retains many characteristics of the cortical collecting duct (CCD). EGF dose dependently regulates basal transepithelial Na⁺ transport in two phases: an acute phase (<4 h) and a chronic phase (>8 h). Similar effects were observed with TGF‐α, HB‐EGF, and amphiregulin which also belong to the EGF‐related peptide growth factor family. Inhibition of MEK1/2 by PD98059 or U0126 increased acute effects and disrupted chronic effects of EGF on Na⁺ reabsorption. Inhibition of PI3‐kinase with LY294002 abolished acute effect of EGF. As assessed by Western blotting, ErbB2 is the most predominant member of the ErbB family detected in mpkCCD_c14 cells. Immunohistochemistry analysis revealed localization of ErbB2 in the CCD in Sprague–Dawley rat kidneys. Both acute and long‐term effects of EGF were abolished when cells were treated with tyrphostin AG‐825 and ErbB2 inhibitor II, chemically dissimilar selective inhibitors of the ErbB2 receptor. Thus, we conclude that EGF and its related growth factors are important for maintaining transepithelial Na⁺ transport and that EGF biphasically modulates sodium transport in mpkCCD_c14 cells via the ErbB2 receptor. J. Cell. Physiol. 223: 252–259, 2010. © 2009 Wiley‐Liss, Inc.     

Expression of sodium/hydrogen exchanger 3 and cation-chloride cotransporters in the kidney of Japanese eel acclimated to a wide range of salinities

Reabsorption of monovalent ions in the kidney is essential for adaptation to freshwater and seawater in teleosts. To assess a possible role of Na⁺/H⁺ exchanger 3 (NHE3) in renal osmoregulation, we first identified a partial sequence of cDNA encoding NHE3 from the Japanese eel kidney. For comparison, we also identified cDNAs encoding kidney specific Na⁺–K⁺–2Cl^? cotransporter 2 (NKCC2α) and Na⁺–Cl^? cotransporter (NCCα). In eels acclimated to a wide range of salinities from deionized freshwater to full-strength seawater, the expression of NHE3 in the kidney was the highest in eel acclimated to full-strength seawater. Meanwhile, the NCCα expression exhibited a tendency to increase as the environmental salinity decreased, whereas the NKCC2α expression was not significantly different among the experimental groups. Immunohistochemical studies showed that NHE3 was localized to the apical membrane of epithelial cells composing the second segments of the proximal renal tubule in seawater-acclimated eel. Meanwhile, the apical membranes of epithelial cells in the distal renal tubule and collecting duct showed more intense immunoreactions of NKCC2α and NCCα, respectively, in freshwater eel than in seawater eel. These findings suggest that renal monovalent-ion reabsorption is mainly mediated by NKCC2α and NCCα in freshwater eel and by NHE3 in seawater eel.     

Differential expression of KCNQ1 K+ channel in tubular cells of frog kidney

The aim of this study was to evaluate KCNQ1 K⁺ channel expression in the frog kidney of Rana esculenta. KCNQ1 K⁺ channel, also known as KvLQT1, is the pore forming α-subunit of the I_Ks K⁺ channel, a delayed rectifier voltage-gated K⁺ channel, which has an important role in water and salt transport in the kidney and gastrointestinal tract. The expression of KCNQ1 K⁺ channel along tubular epithelium differs from species to species. In the present study the expression of KCNQ1 K⁺ channel in the frog kidney has been demonstrated by immunohistochemistry. The presence of KCNQ1 K⁺ channel was demonstrated in the epithelial cells of distal convoluted tubule and collecting duct. However, the pattern of expression of KCNQ1 K⁺ channel differs between distal convoluted tubules and collecting duct. All epithelial cells of distal convoluted tubules revealed basolateral expression of KCNQ1 K⁺ channel. On the contrary, only the single cells of collecting duct, probably intercalated cells, showed diffuse cell surface staining with antibodies against KCNQ1 K⁺ channel. These findings suggest that KCNQ1 K⁺ channel has cell-specific roles in renal potassium ion transport.Key words: KCNQ1 K⁺ channel, rana esculenta, frog kidney, immunohistochemistry.     

Mineralocorticoids modulate the expression of the β-3 subunit of the Na+, K+-ATPase in the renal collecting duct

Renal sodium reabsorption depends on the activity of the Na⁺,K⁺-ATPase α/β heterodimer. Four α (α_1–4) and 3 β (β_1–3) subunit isoforms have been described. It is accepted that renal tubule cells express α₁/β₁ dimers. Aldosterone stimulates Na⁺,K⁺-ATPase activity and may modulate α₁/β₁ expression. However, some studies suggest the presence of β₃ in the kidney. We hypothesized that the β₃ isoform of the Na⁺,K⁺-ATPase is expressed in tubular cells of the distal nephron, and modulated by mineralocorticoids. We found that β₃ is highly expressed in collecting duct of rodents, and that mineralocorticoids decreased the expression of β₃. Thus, we describe a novel molecular mechanism of sodium pump modulation that may contribute to the effects of mineralocorticoids on sodium reabsorption.     

Localization of Na+, K+-ATPase to the inside of the basolateral cell membranes of epithelial cells of proximal and distal tubules in rabbit kidney

Summary Cysteine-sensitive alkaline phosphatase and/or ouabain-sensitive Na⁺, K⁺-ATPase were studied by ultrastructure cytochemistry in epithelial cells of proximal and distal kidney tubules. Alkaline phosphatase reactivity was confined to the surface of the microvillous luminal cell membrane of proximal tubule cells, whereas distal tubules and collecting ducts were unreactive. The Na⁺, K⁺-ATPase reactivity was localized evenly along the cytoplasmic side of the basolateral cell membrane of cells of proximal and distal tubules and in collecting ducts. In the proximal tubules, where the activity was strongest, the Na⁺, K⁺-ATPase deposits were also found in the 10–50 nm gap between the cell membrane and the cisternae of tubulo-cisternal endoplasmic reticulum (TER) underlying a major part of the basolateral cell membrane. The restriction of Na⁺, K⁺-ATPase sites, which are involved in extrusion of Na⁺ from the cell, to a narrow cytoplasmic compartment located between the cell membrane and the cisternae of TER, is consistent with a transport role for the TER.     

Purinergic Modulation of Na+,K+,Cl− Cotransport and MAP Kinases is Limited to C11-MDCK Cells Resembling Intercalated Cells from Collecting Ducts

We demonstrated recently that in renal epithelial cells from collecting ducts of Madin-Darby canine kidneys (MDCK), Na⁺,K⁺,Cl⁻ cotransport is inhibited up to 50% by ATP via its interaction with P_2Y purinoceptors (Biochim. Biophys. Acta 1998. 1369:233–239). In the present study we examined which type of renal epithelial cells possesses the highest sensitivity of Na⁺,K⁺,Cl⁻ cotransport to purinergic regulation. We did not observe any effect of ATP on Na⁺,K⁺,Cl⁻ cotransport in renal epithelial cells from proximal and distal tubules, whereas in renal epithelial cells from rabbit and rat collecting ducts ATP decreased the carrier's activity by ∼30%. ATP did not affect Na⁺,K⁺,Cl⁻ cotransport in C7 subtype MDCK cells possessing the properties of principal cells but led to ∼85% inhibition of this carrier in C11-MDCK cells in which intercalated cells are highly abundant. Both C7- and C11-MDCK exhibited ATP-induced IP₃ and cAMP production and transient elevation of [Ca²⁺] _i . In contrast to the above-listed signaling systems, ATP-induced phosphorylation of ERK and JNK MAP kinases was observed in C11-MDCK only. Thus, our results reveal that regulation of renal Na⁺,K⁺,Cl⁻ cotransport by P_2Y receptors is limited to intercalated cells from collecting ducts and indicate the involvement of the MAP kinase cascade in purinergic control of this ion carrier's activity. Received: 10 June 1999/Revised: 23 August 1999     

Regulation of Epithelial Na+ Transport by Soluble Adenylyl Cyclase in Kidney Collecting Duct Cells

Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na⁺ transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD_c14) cells. These cells exhibit aldosterone-stimulated transepithelial Na⁺ currents that depend on both the apical epithelial Na⁺ channel (ENaC) and basolateral Na⁺,K⁺-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I_sc) in mpkCCD_c14 cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I_sc in these cells by 50-60% within 30 min. 8-Bromoadenosine-3′,5′-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na⁺ current. Aldosterone doubled ENaC-dependent I_sc over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I_sc was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na⁺ current was fully inhibited by basolateral ouabain treatment, suggesting that the Na⁺,K⁺-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD_c14 cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na⁺,K⁺-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na⁺ reabsorption in the kidney collecting duct, acting to enhance Na⁺,K⁺-ATPase activity.Maintenance of intracellular pH depends in part on the extracellular to intracellular Na⁺ gradient, and elevation of intracellular [Na⁺] can lead to acidification of the cytoplasm. It has been shown that acidification of the cytoplasm of cells from frog skin and toad bladder by increased partial pressure of CO₂ reduces Na⁺ transport and permeability (1, 2). Conversely, the rise in plasma bicarbonate caused by metabolic alkalosis with chronic diuretic use has been shown to increase net renal Na⁺ reabsorption independently of volume status, electrolyte depletion, and/or increased aldosterone secretion (3, 4). However, the underlying mechanisms involved in these phenomena remain unclear.The soluble adenylyl cyclase (sAC)² is a chemosensor that mediates the elevation of cAMP in intracellular microdomains (5-7). Unlike transmembrane adenylyl cyclases (tmACs), sAC is insensitive to regulation by forskolin or heterotrimeric G proteins (8) and is directly activated by elevations of intracellular calcium (9, 10) and/or bicarbonate ions (11). Thus, sAC mediates localized intracellular increases in cAMP in response to variations in bicarbonate levels or its closely related parameters, partial pressure of CO₂ and pH. Mammalian sAC is more similar to bicarbonate-regulated cyanobacterial adenylyl cyclases than to other mammalian nucleotidyl cyclases, which may indicate that there is a unifying mechanism for the regulation of cAMP signaling by bicarbonate across biological systems. Although sAC appears to be encoded by a single gene, there is significant isoform diversity for this ubiquitously expressed enzyme (11, 12) generated by alternative splicing (reviewed in Ref. 13). sAC has been shown to regulate the subcellular localization and/or activity of membrane transport proteins such as the vacuolar H⁺-ATPase (V-ATPase) and cystic fibrosis transmembrane conductance regulator in epithelial cells (14, 15). Functional activity of sAC has been reported in the kidney (16), and sAC has been localized to epithelial cells in the distal nephron (14, 17).Given that natriuresis is decreased during metabolic alkalosis, when bicarbonate is elevated, and Na⁺ reabsorption is impaired by high partial pressure of CO₂, we hypothesized that bicarbonate-regulated sAC may play a key role in the regulation of transepithelial Na⁺ transport in the distal nephron. Reabsorption of Na⁺ in the kidney and other epithelial tissues is mediated by the parallel operation of apical ENaC and basolateral Na⁺,K⁺-ATPase, and both transport proteins can be stimulated by cAMP via the cAMP-dependent protein kinase (PKA) (18, 53). The aims of this study were to investigate the role of sAC in the regulation of transepithelial Na⁺ transport in the kidney through the use of specific sAC inhibitors and electrophysiological measurements. We found that sAC inhibition blocks transepithelial Na⁺ reabsorption in polarized mpkCCD_c14 cells under both basal and hormone-stimulated conditions. Selective membrane permeabilization studies revealed that although ENaC activity appears to be unaffected by sAC inhibition, flux through the Na⁺,K⁺-ATPase is sensitive to sAC modulation. Inhibiting sAC decreases ATPase activity without affecting plasma membrane expression of the pump; thus, tonic sAC activity appears to be required for Na⁺ reabsorption in kidney collecting duct.     

Differential expression of the Kv1 voltage-gated potassium channel family in the rat nephron

Several potassium (K⁺) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K⁺ channels allow sodium reabsorption in the proximal tubule (PT), K⁺ recycling and K⁺ reabsorption in the thick ascending limb (TAL) and K⁺ secretion and K⁺ reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K⁺ to function as a secretory K⁺ channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K⁺ channels. Our results expand the repertoire of K⁺ channels that contribute to K⁺ homeostasis to include the Kv1 family.     

Spatial,Cellular, and Intracellular Localization of Na+/K+-ATPase in the Sterically Disposed Renal Tubules of Japanese Eel

The kidney plays a crucial role in the regulation of water and ion balances in both freshwater and seawater fishes. However, the complicated structures of the kidney hamper comprehensive understanding of renal functions. In this study, to investigate the structure of sterically disposed renal tubules, we examined spatial, cellular, and intracellular localization of Na⁺/K⁺-ATPase in the kidney of the Japanese eel. The renal tubule was composed of the first (PT-I) and second (PT-II) segments of the proximal tubule and the distal tubule (DT), followed by the collecting ducts (CDs). Light microscopic immunocytochemistry detected Na⁺/K⁺-ATPase along the renal tubules and CD; however, the subcellular distribution of the Na⁺/K⁺-ATPase immunoreaction varied among different segments. Electron microscopic immunocytochemistry further revealed that Na⁺/K⁺-ATPase was distributed on the basal infoldings of PT-I, PT-II, and DT cells. Three-dimensional analyses showed that the renal tubules meandered in a random pattern through lymphoid tissues, and then merged into the CD, which was aligned linearly. Among the different segments, the DT and CD cells showed more-intense Na⁺/K⁺-ATPase immunoreaction in freshwater eel than in seawater-acclimated eel, confirming that the DT and CD segments are important in freshwater adaptation, or hyperosmoregulation. (J Histochem Cytochem 58:707–719, 2010)     

Effects of extracellular nucleotides on renal tubular solute transport

A range of P2 receptor subtypes has been identified along the renal tubule, in both apical and basolateral membranes. Furthermore, it has been shown that nucleotides are released from renal tubular cells, and that ectonucleotidases are present in several nephron segments. These findings suggest an autocrine/paracrine role for nucleotides in regulating tubular function. The present review catalogues the known actions of extracellular nucleotides on tubular solute transport. In the proximal tubule, there is firm evidence that stimulation of apical P2Y₁ receptors inhibits bicarbonate reabsorption, whilst basolaterally applied ATP has the opposite effect. Clearance studies suggest that systemic diadenosine polyphosphates profoundly reduce proximal tubular fluid transport, through as yet unidentified P2 receptors. To date, only circumstantial evidence is available for an action of nucleotides on transport in the loop of Henle; and no studies have been made on native distal tubules, though observations in cell lines suggest an inhibitory effect on sodium, calcium and magnesium transport. The nephron segment most studied is the collecting duct. Apically applied nucleotides inhibit the activity of small-conductance K⁺ channels in mouse collecting duct, apparently through stimulation of P2Y₂ receptors. There is also evidence, from cell lines and native tissue, that apically (and in some cases basolaterally) applied nucleotides inhibit sodium reabsorption. In mice pharmacological profiling implicates P2Y₂ receptors; but in rats, the receptor subtype(s) responsible is/are unclear. Recent patch-clamp studies in rat collecting ducts implicate apical P2Y and P2X subtypes, with evidence for both inhibitory and stimulatory effects. Despite considerable progress, clarification of the physiological role of the tubular P2 receptor system remains some way off.     

Expression of the potassium channel ROMK in adult and fetal human kidney

The renal potassium channel ROMK is a crucial element of K⁺ recycling and secretion in the distal tubule and the collecting duct system. Mutations in the ROMK gene (KCNJ1) lead to hyperprostaglandin E syndrome/antenatal Bartter syndrome, a life-threatening hypokalemic disorder of the newborn. The localization of ROMK channel protein, however, remains unknown in humans. We generated an affinity-purified specific polyclonal anti-ROMK antibody raised against a C-terminal peptide of human ROMK. Immunoblotting revealed a 45 kDa protein band in both rat and human kidney tissue. In human kidney sections, the antibody showed intense staining of epithelial cells in the cortical and medullary thick ascending limb (TAL), the connecting tubule, and the collecting duct. Moreover, a strong expression of ROMK protein was detected in cells of the macula densa. In epithelial cells of the TAL expression of ROMK protein was mainly restricted to the apical membrane. In human fetal kidney expression of ROMK protein was detected mainly in distal tubules of mature nephrons but not or only marginally in the collecting system. No expression was found in early developmental stages such as comma or S shapes, indicating a differentiation-dependent expression of ROMK protein. In summary, these findings support the proposed role of ROMK channels in potassium recycling and in the regulation of K⁺ secretion and present a rationale for the phenotype observed in patients with ROMK deficiency.     

The ubiquitin ligase NEDD4-2/NEDD4L regulates both sodium homeostasis and fibrotic signaling to prevent end-stage renal disease

Kidney disease progression can be affected by Na⁺ abundance. A key regulator of Na⁺ homeostasis is the ubiquitin ligase NEDD4-2 and its deficiency leads to increased Na⁺ transport activity and salt-sensitive progressive kidney damage. However, the mechanisms responsible for high Na⁺ induced damage remain poorly understood. Here we show that a high Na⁺ diet compromised kidney function in Nedd4-2-deficient mice, indicative of progression toward end-stage renal disease. Injury was characterized by enhanced tubule dilation and extracellular matrix accumulation, together with sustained activation of both Wnt/β-catenin and TGF-β signaling. Nedd4-2 knockout in cortical collecting duct cells also activated these pathways and led to epithelial–mesenchymal transition. Furthermore, low dietary Na⁺ rescued kidney disease in Nedd4-2-deficient mice and silenced Wnt/β-catenin and TGF-β signaling. Our study reveals the important role of NEDD4-2-dependent ubiquitination in Na⁺ homeostasis and protecting against aberrant Wnt/β-catenin/TGF-β signaling in progressive kidney disease.Subject terms: Stress signalling, End-stage renal disease     

The natriuretic mechanism of Gamma-Melanocyte-Stimulating Hormone

Gamma-Melanocyte Stimulating Hormone (Gamma-MSH) regulates sodium (Na⁺) balance and blood pressure through activation of the melanocortin receptor 3 (MC3-R). The mechanism of the natriuretic effect is proposed to involve binding of MC3-R either in the kidney to directly inhibit tubular Na⁺ transport or in the brain to inhibit central neural pathways that control renal tubular Na⁺ absorption. This study aimed to clarify the mechanism involved in the natriuretic effect of Gamma-MSH on MC3-R in kidney cells. In Ussing chamber studies, we observed no effects of Gamma-MSH on NaCl transport in the mouse inner medullary collecting duct cell line (mIMCD-K2). We also found that neither MC3-R protein nor mRNA was expressed in mouse kidney, suggesting that renal Gamma-MSH action may not be mediated through direct effects on tubular Na⁺ transport but rather through effects on central neural pathways that innervate the kidney.     

Expression of binding sites for Dolichos biflorus agglutinin at the apical aspect of collecting duct cells in rat kidney

Summary To identify precisely the structural and functional cell type in the collecting duct of the rat kidney expressing binding sites for Dolichos biflorus agglutinin (DBA), we stained serial paraffin sections of kidney with horseradish peroxidase-labeled DBA and with immunocytochemical methods for localizing (Na⁺+K⁺)-ATPase and carbonic anhydrase II (CA II), enzymes found preferentially in principal and intercalated cells, respectively. Most principal cells expressing a strong basolateral staining for (Na⁺ + K⁺)-ATPase showed binding sites for DBA at their luminal surfaces. However, a minority of cells rich in CA II and showing morphologic characteristics of intercalated cells also expressed DBA binding sites at their luminal surface and apical cytoplasm. These data suggest that DBA cytochemistrycan provide a useful tool for studying the functional polarity of the main cell types of the collecting duct of the rat kidney.     

Molecular mechanisms in proximal tubular and small intestinal phosphate reabsorption (Plenary Lecture)

Renal and small intestinal (re-)absorption contribute to overall phosphate(P_i)-homeostasis. In both epithelia, apical sodium (Na⁺)/P_i-cotransport across the luminal (brush border) memi brane is rate limiting and the target for physiological/pathophysiological alterations. Three different Na/P_i-cotransporters have been identified: (i) type I cotransporter(s) - present in the proximal tubule - also show anion channel function and may play a role in secretion of organic anions; in the brain, it may serve vesicular glutamate uptake functions; (ii) type II cotransporter(s) seem to serve rather specific epithelial functions; in the renal proximal tubule (type IIa)and in the small intestine (type IIb), isoform determines Na⁺-dependent transcellular P_i-movements; (iii) type III cotransporters are expressed in many different cells/tissues where they could serve housekeeping functions. In the small intestine, alterations in P_i-absorption and, thus, apical expression of IIb protein are mostly in response to longer term (days) situations (altered P_i-intake, levels of 1.25 (OH₂) vitamin D₃, growth, etc), whereas in renal proximal tubule, in addition, hormonal effects (e.g. Parathyroid Hormone, PTH) acutely control (minutes/hours) the expression of the IIa cotransporter. The type II Na/P_i-cotransporters operate (as functional monomers) in a 3 Na⁺:1 P_i stoichiometry, including transfer of negatively charged (-1) empty carriers and electroneutral transfers of partially loaded carriers (1 Na⁺, slippage)and of the fully loaded carriers (3 Na⁺, 1 P_i). By a chimera (IIa/IIb) approach, and by site-directed mutagenesis (including cysteine-scanning), specific sequences have been identified contributing to either apical expression, PTH-induced membrane retrieval, Na⁺-interaction or specific pH-dependence of the IIa and IIb cotransporters. For the COOH-terminal tail of the IIa Na/P_i -cotransporter, several interacting PDZ-domain proteins have been identified which may contribute to either its apical expression (NaPi-Cap1) or to its subapical/lysosomal traffic (NaPi-Cap2).     

Sodium-calcium exchange in renal epithelial cells: Dependence on cell sodium and competitive inhibition by magnesium

Summary Kinetic properties of Na⁺–Ca²⁺ exchange in a renal epithelial cell line (LLC-MK₂) were assessed by measuring cytosolic free Ca²⁺ with fura-2 and⁴⁵Ca²⁺ influx. Replacing external Na⁺ with K⁺ produced relatively small increases in free Ca²⁺ and⁴⁵Ca²⁺ uptake unless the cells were incubated with ouabain. Ouabain markedly increased cell Na⁺ and strongly potentiated the effect of replacing external Na⁺ with K⁺ on free Ca²⁺ and⁴⁵Ca²⁺ uptake.⁴⁵Ca²⁺ influx in 140mm K⁺ or N-methyl-d-glucamine minus influx in 140mm Na⁺ was used to quantify Na⁺–Ca²⁺ exchange activity of Na⁺-loaded cells. The dependence of exchange on cell Na⁺ was sigmoidal; theK _0.5 was 26±3 mmol/liter cell water space, and the Hill coefficient was 3.1±0.2. The kinetic features of the dependence of exchange on cell Na⁺ partly account for the small increase in Ca²⁺ influx when all external Na⁺ is replaced by K⁺. Besides raising cell Na⁺ ouabain appears to activate the exchanger. Magnesium competitively inhibited exchange activity. The potency of Mg²⁺ was 8.2-fold lower with potassium instead of N-methyl-d-glucamine or choline as the replacement for external Na⁺. Potassium also increased theV _max of exchange by 86% and had no effect on theK _m for Ca²⁺. The exchanger does not cause detectable²²Na⁺–Mg²⁺ exchange and does not appear to require K⁺ or transport⁸⁶Rb⁺. Although exchange activity was plentiful in the epithelial cells from monkey kidney, others from amphibian, canine, opossum, and porcine kidney had no detectable exchange activity. All of the measured kinetic properties of Na⁺–Ca²⁺ exchange in the renal epithelial cells are very similar to those of the exchanger in rat aortic myocytes.     

Immunohistochemical localization of a protein fraction derived from rabbit renal papilla

Summary Studies were carried out to define antigenic characteristics of the rabbit renal collecting duct. Renal papillae of adult rabbits were homogenized, centrifuged, and the 600×g pellet was extracted with 0.5% Triton X-100 in the presence of 1 M NaCl. The crude extract was fractionated on an anion exchange column (DEAE cellulose). A fraction enriched in acidic proteins that co-purified with a radioactive 150 kd glycoprotein from cultured collecting duct cells (Minuth 1982), was used for immunization of guinea pigs. The antiserum shows the following characteristics as revealed by indirect immunofluorescence on the rabbit kidney: 1) Among all tubular epithelial cells only principal cells of the collecting duct and the connecting tubule cell show immunoreactivity. 2) The antiserum decorates the epithelial-interstitial interface of the whole collecting duct as well as of connecting tubule and thick ascending limb of Henle's loop. 3) There is immunoreactivity of interstitial fibers throughout the kidney. 4) Epithelial cells in a variety of other organs in rabbit did not react with the antiserum.Our data demonstrate an antigenic distinction of both, the connecting tubule cell and the principal cell, discriminating these cells from other tubular epithelial cells including the intercalated cells of the collecting duct system. Furthermore, our findings point to a heterogeneity along the distal nephron with respect to the constituents of the epithelial-interstitial interface.     

Modeling Proximal Tubule Cell Homeostasis: Tracking Changes in Luminal Flow

During normal kidney function, there are routinely wide swings in proximal tubule fluid flow and proportional changes in Na⁺ reabsorption across tubule epithelial cells. This “glomerulotubular balance” occurs in the absence of any substantial change in cell volume, and is thus a challenge to coordinate luminal membrane solute entry with peritubular membrane solute exit. In this work, linear optimal control theory is applied to generate a configuration of regulated transporters that could achieve this result. A previously developed model of rat proximal tubule epithelium is linearized about a physiologic reference condition; the approximate linear system is recast as a dynamical system; and a Riccati equation is solved to yield the optimal linear feedback that stabilizes Na⁺ flux, cell volume, and cell pH. The first observation is that optimal feedback control is largely consigned to three physiologic variables, cell volume, cell electrical potential, and lateral intercellular hydrostatic pressure. Parameter modulation by cell volume stabilizes cell volume; parameter modulation by electrical potential or interspace pressure act to stabilize Na⁺ flux and cell pH. This feedback control is utilized in a tracking problem, in which reabsorptive Na⁺ flux varies over a factor of two, in order to represent a substantial excursion of glomerulotubular balance. The resulting control parameters consist of two terms, an autonomous term and a feedback term, and both terms include transporters on both luminal and peritubular cell membranes. Overall, the increase in Na⁺ flux is achieved with upregulation of luminal Na⁺/H⁺ exchange and Na⁺–glucose cotransport, with increased peritubular Na⁺–3HCO₃⁻ and K⁺–Cl⁻ cotransport, and with increased Na⁺, K⁺–ATPase activity. The configuration of activated transporters emerges as a testable hypothesis of the molecular basis for glomerulotubular balance. It is suggested that the autonomous control component at each cell membrane could represent the cytoskeletal effects of luminal flow.     

Ceramide-1-Phosphate as a Potential Regulator of the Second Sodium Pump from Kidney Proximal Tubules by Triggering Distinct Protein Kinase Pathways in a Hierarchic Way

Kidney proximal tubules are a key segment in the reabsorption of solutes and water from the glomerular ultrafiltrate, an essential process for maintaining homeostasis in body fluid compartments. The abundant content of Na⁺ in the extracellular fluid determines its importance in the regulation of extracellular fluid volume, which is particularly important for different physiological processes including blood pressure control. Basolateral membranes of proximal tubule cells have the classic Na⁺ + K⁺-ATPase and the ouabain-insensitive, K⁺-insensitive, and furosemide-sensitive Na⁺-ATPase, which participate in the active Na⁺ reabsorption. Here, we show that nanomolar concentrations of ceramide-1 phosphate (C1P), a bioactive sphingolipid derived in biological membranes from different metabolic pathways, promotes a strong inhibitory effect on the Na⁺-ATPase activity (C1P₅₀ ≈ 10 nM), leading to a 72% inhibition of the second sodium pump in the basolateral membranes. Ceramide-1-phosphate directly modulates protein kinase A and protein kinase C, which are known to be involved in the modulation of ion transporters including the renal Na⁺-ATPase. Conversely, we did not observe any effect on the Na⁺ + K⁺-ATPase even at a broad C1P concentration range. The significant effect of ceramide-1-phosphate revealed a new potent physiological and pathophysiological modulator for the Na⁺-ATPase, participating in the regulatory network involving glycero- and sphingolipids present in the basolateral membranes of kidney tubule cells.