Interaction between bradykinin and natriuretic peptides via RGS protein activation in HEK-293 cells.

In this study, the interaction of natriuretic peptides (NP) and bradykinin (BK) signaling pathways was identified by measuring membrane potential (V(m)) and intracellular Ca(2+) using the patch-clamp technique and flow cytometry in HEK-293 cells. BK and NP receptor mRNA was identified using RT-PCR. BK (100 nM) depolarized cells activating bradykinin receptor type 2 (B(2)R) and Ca(2+)-dependent Cl(-) channels inhibitable by 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 10 µM). The BK-induced Ca(2+) signal was blocked by the B(2)R inhibitor HOE 140. [Des-Arg(9)]-bradykinin, an activator of B(1)R, had no effect on intracellular Ca(2+). NP [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP), and urodilatin] depolarized HEK-293 cells inhibiting K(+) channels. ANP, urodilatin, BNP [binding to natriuretic peptide receptor (NPR)-A] and 8-bromo-(8-Br)-cGMP inhibited the BK-induced depolarization while CNP (binding to NPR-Bi) failed to do so. The inhibitory effect on BK-triggered depolarization could be reversed by blocking PKG using the specific inhibitor KT 5823. BK-stimulated depolarization as well as Ca(2+) signaling was completely blocked by the phospholipase C (PLC) inhibitor U-73122 (10 nM). The inositol 1,4,5-trisphosphate receptor blocker 2-aminoethoxydiphenyl borate (2-APB; 50 µM) completely inhibited the BK-induced Ca(2+) signaling. UTP, another activator of the PLC-mediated Ca(2+) signaling pathway, was blocked by U-73122 as well but not by 8-Br-cGMP, indicating an intermediate regulatory step for NP via PKG in BK signaling such as regulators of G-protein signaling (RGS) proteins. When RGS proteins were inhibited by CCG-63802 in the presence of BK and 8-Br-cGMP, cells started to depolarize again. In conclusion, as natural antagonists of the B(2)R signaling pathway, NP may also positively interact in pathological conditions caused by BK.

Cellular localization of THIK-1 (K(2P)13.1) and THIK-2 (K(2P)12.1) K channels in the mammalian kidney.

K(+)-channels fulfill several important functions in the mammalian kidney such as volume regulation, recirculation and secretion of K(+) ions, and maintaining the resting potential. In this study we used immunocytochemical methods, in situ hybridization, and nephron segment-specific RT-PCR to obtain a detailed picture of the cellular localization of two tandem pore domain potassium (K(2P)) channels, THIK-1 (K(2P)13.1, KCNK13) and THIK-2 (K(2P)12.1, KCNK12). Monospecific antibodies against C-terminal domains of rat THIK-1 and THIK-2 proteins (GST-fusion proteins) were raised in rabbits, freed from cross-reactivity, and affinity purified. All antibodies were validated by Western blot analysis, competitive ELISA, and preabsorption experiments. The expression of THIK channels in specific nephron segments was confirmed by double staining with marker proteins. Results indicate that in rat and mouse THIK-1 and THIK-2 were expressed in the proximal tubule (PT), thick ascending limb (TAL), connecting tubule (CNT), and cortical collecting duct (CCD). In human kidney THIK-1 and THIK-2 were localized in PT, TAL and CCD. Immunostaining of rat tissue revealed an intracellular expression of THIK-1 and THIK-2 throughout the identified nephron segments. However in mouse kidney THIK-2 was identified in basolateral membranes. Overall, the glomerulus, thin limbs and medullary collecting ducts were devoid of THIK-1 and THIK-2 signal. In summary, THIK-1 and THIK-2 are abundantly expressed in the proximal and distal nephron of the mammalian kidney.

Uroguanylin and guanylin regulate transport of mouse cortical collecting duct independent of guanylate cyclase C.

BACKGROUND: Electrolyte and water homeostasis mostly depend on differentially regulated intestinal and renal transport. Guanylin and uroguanylin were proposed as first hormones linking intestinal with renal electrolyte and water transport, which is disturbed in pathophysiology. Guanylate cyclase C is the intestinal receptor for these peptides, but in guanylate cyclase C-deficient mice renal effects are retained. Unlike for the intestine the sites of renal actions and cellular mechanisms of guanylin peptides are still unclear. METHODS: After first data on proximal tubular effects in this study their effects are examined in detail in mouse cortical collecting duct (CCD). Effects of guanylin peptides on principal cells of isolated mouse CCD were studied by slow whole-cell patch-clamp analysis, reverse transcription-polymerase chain reaction (RT-PCR), and microfluorimetric measurements of intracellular Ca2+. RESULTS: Guanylin peptides depolarized or hyperpolarized principal cells. Whereas 8-Br-cyclic guanosine monophosphate (8-Br-cGMP) hyperpolarized, 8-Br-cyclic adenosine monophosphate (8-Br-cAMP) depolarized principal cells. All effects of guanylin peptides were inhibited by Ba2+. Hyperpolarizations were blocked by clotrimazole or protein kinase G (PKG) inhibition, suggesting an involvement of basolateral Ca2+- and cGMP-dependent K+ channels. Effects remained in CCD isolated from guanylate cyclase C-deficient mice. Depolarizations were inhibited by arachidonic acid or inhibition of phospholipase A2 (PLA2), but not by protein kinase A (PKA) inhibition. Conclusion. These results suggest the existence of two signaling pathways for guanylin peptides in principal cells of mouse CCD. One pathway is cGMP- and PKG-dependent but not mediated by guanylate cyclase C, the second involves PLA2 and arachidonic acid. The first pathway most likely leads to an activation of the basolateral K+-conductance while the latter probably results in decreased activity of ROMK channels in the luminal membrane.

Guanylin and uroguanylin regulate electrolyte transport in isolated human cortical collecting ducts.

BACKGROUND: Guanylin and uroguanylin link intestinal and renal electrolyte and water transport. Their function in intestine is well studied, but renal actions are less understood. Uroguanylin concentrations are increased in patients with chronic renal failure, nephrotic syndrome, or those on dialysis. Guanylate cyclase C (GC-C) is the receptor first described for these peptides. In guanylate cyclase C-deficient mice guanylin- and uroguanylin-induced renal natriuresis, kaliuresis, and diuresis are retained. METHODS: Effects of guanylin and uroguanylin on principal cells of human cortical collecting ducts (CCD) isolated from kidneys after tumor nephrectomy were investigated. Reverse transcription-polymerase chain reaction (RT-PCR), slow whole-cell patch-clamp, and microfluorimetric analysis of intracellular Ca(2+) were used. Here we present first functional measurements of isolated human CCD. RESULTS: Principal cells of CCD were identified by the amiloride-induced hyperpolarization of principal cells (-3.8 +/- 0.3 mV) (N= 52). Cells depolarized upon guanylin or uroguanylin (each 10 nmol/L) by 3.3 +/- 0.8 mV (N= 12) and 3.4 +/- 0.5 mV (N= 18), respectively, but were hyperpolarized by 8Br-cyclic guanosine monophosphate (cGMP) (100 micromol/L) (-3.0 +/- 0.2 mV) (N= 4). mRNA for GC-C was not detected in CCD. Effects of both peptides were inhibited by Ba(2+) (1 mmol/L) or phospholipase A(2) (PLA(2)) inhibition (AACOCF(3)) (5 micromol/L). CONCLUSION: These findings suggest a new cGMP- and GC-C-independent but PLA(2)-dependent signaling pathway for these peptides in the kidney. Most likely guanylin and uroguanylin inhibit luminal K(+) channels of principal cells of human CCD via this pathway. This depolarization of principal cells consequently reduces the driving force of Na(+) and water reabsorption, explaining natriuresis and diuresis caused by these peptides.

Acute rejection after rat renal transplantation leads to downregulation of NA+ and water channels in the collecting duct.

Renal transplantation is associated with alterations of tubular functions and of the renin-angiotensin-aldosterone system. The underlying cellular and molecular mechanisms are unclear. We used an allogeneic rat renal transplantation model of acute rejection with and without immunosuppression by cyclosporine A (CsA) and a syngeneic model as control. Uninephrectomized Lewis or Lewis-Brown-Norway (LBN) rats received a kidney from LBN-rats. Renal transporters and receptors were analyzed by immunohistochemistry, semiquantitative RT-PCR and Western-blot analysis. Intracellular Na(+) was analyzed microfluorimetrically in isolated cortical collecting ducts. mRNA expression and function of the epithelial Na(+)-channel (ENaC) and mRNA and protein expression of the water-channel AQP2 were downregulated in transplanted kidneys undergoing rejection. Expression of the serum- and glucocorticoid-kinase (Sgk1) was decreased and that of the ubiquitin-protein ligase Nedd4-2 was increased. These changes were absent under CsA-therapy and in syngeneic model. Expression and function of the Na(+)-K(+)-ATPase, expression of the secretory K(+)-channel and of the mineralocorticoid receptor remained unchanged. Reduced ENaC function is likely due to decreased Sgk1- and increased Nedd4-2 mRNA expression leading to reduced ENaC expression in the membrane. These acute downregulations of ENaC and AQP2 may be triggered to reduce energy consumption in the distal nephron to protect the kidney immediately after transplantation.

Signaling and distribution of NPR-Bi, the human splice form of the natriuretic peptide receptor type B.

Recently, we described a splice variant of the human natriuretic peptide receptor type B (NPR-Bi) in human proximal tubule cells [immortalized human kidney epithelial cells (IHKE-1) that lacks a functional guanylate cyclase domain (Hirsch JR, Meyer M, Mägert HJ, Forssmann WG, Mollerup S, Herter P, Weber G, Cermak R, Ankorina-Stark I, Schlatter E, and Kruhøffer M. J Am Soc Nephrol 10: 472-480, 1999). Its signaling pathway does not include cGMP, cAMP, or Ca2+ but leads to inhibition of K+ channels. In patch-clamp experiments, effects of tyrosine kinase receptor blockers on C-type natriuretic peptide (CNP)-mediated depolarizations of membrane voltages (Vm) of IHKE-1 cells were tested. The epidermal growth factor (EGF) receptor blocker genistein (10 microM) abolished the effect of CNP (0.2 +/- 0.4 mV, n = 7), and comparable results were obtained with 10 microM daidzein (n = 8). Aminogenistein (10 microM, n = 5) and tyrphostin AG1295 (10 microM, n = 5) had no significant effects. EGF (1 nM) hyperpolarized cells by -5.3 +/- 0.8 mV (n = 5). This effect was completely blocked by genistein or daidzein. The Cl- channel blocker NPPB (10 microM, n = 5) inhibited the EGF-mediated hyperpolarization. mRNA expression of NPR-B and NPR-Bi shows reversed patterns along the human nephron. NPR-B is highly expressed in glomeruli and proximal tubules, whereas NPR-Bi shows strong signals in the distal nephron. Expression of NPR-Bi in the cortical collecting duct was also confirmed with immunohistochemistry. In other human tissues, NPR-Bi shows strongest expression in pancreas and lung, whereas in the heart and liver NPR-B is the dominating receptor. In conclusion, CNP inhibits an apical K+ channel in IHKE-1 cells independently of cGMP and so far this effect can only be blocked by genistein and daidzein. Tyrosine phosphorylation might be the missing link in the signaling pathway of CNP/NPR-Bi.

Inhibition of Na(+)-dependent transporters in cystine-loaded human renal cells: electrophysiological studies on the Fanconi syndrome of cystinosis.

Cystinosis is the most common cause of the renal Fanconi syndrome in children, leading to severe electrolyte disturbances and growth failure. A defective lysosomal transporter, cystinosin, results in intralysosomal accumulation of cystine. Loading cells with cystine dimethyl ester (CDME) is the only available model for this disease. This model was used to present electrophysiologic studies on immortalized human kidney epithelial (IHKE-1) cells that had been derived from the proximal tubule with the slow whole-cell patch clamp technique. Basal membrane voltages (V(m)) of IHKE-1 cells were -30.7 +/- 0.4 mV (n = 151). CDME concentration-dependently altered V(m) with an initial depolarization (2.7 +/- 0.2 mV;n = 76; 1 mM CDME) followed by a more pronounced hyperpolarization (-9.9 +/- 1.0 mV;n = 49). Three Na(+)-dependent transporters were examined. Alanine (1 mM) depolarized IHKE-1 cells by 17.6 +/- 0.7 mV (n = 59), and phosphate (1.8 mM) depolarized by 9.7 +/- 1.1 mV (n = 18). Acidification of IHKE-1 cells with propionate (20 mM) resulted in a depolarization of V(m) by 7.1 +/- 0.3 mV (n = 21) followed by a repolarization by 2.9 +/- 0.3 mV/min (n = 17), reflecting Na(+)/H(+)-exchanger activity. Acute addition of 1 mM CDME did not alter the alanine- and propionate-induced changes in V(m), but it reduced the phosphate-induced depolarization by 37 +/- 9% (n = 10). Incubation with 1 mM CDME reduced the activity of all three transporters. Depolarizations by alanine and phosphate and the repolarization after propionate were inhibited by 57 +/- 4% (n =30), 45 +/- 9% (n = 9), and 78 +/- 15% (n = 8), respectively. In conclusion, this study demonstrates that CDME acutely alters V(m) of IHKE-1 cells and that at least three Na(+)-dependent transporters are inhibited, the Na(+)-phosphate cotransporter most sensitively. This might suggest that phosphate depletion and dissipation of the Na(+)-gradient are involved in the development of the Fanconi syndrome of cystinosis.

Regulation of the human organic cation transporter hOCT1.

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO-hOCT1) and in human embryonic kidney cells (HEK293-hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)) as substrate. hOCT1 expressed in CHO-cells was inhibited by protein kinase A (PKA) activation (1 microM forskolin, -58 +/- 6%, n = 12), calmodulin inhibition (0.1 microM calmidazolium, -68 +/- 3%, n = 6; 10 microM ophiobolin A, -48 +/- 10%, n = 7), calmodulin-dependent kinase II inhibition (1 microM KN62, -78 +/- 4%, n = 12), and inhibition of p56(lck) tyrosine kinase (10 microM aminogenistein, -35 +/- 7%, n = 12). The apparent affinities for TEA(+) were lower in CHO-hOCT1 than in HEK293-hOCT1, while those for TPA(+) and quinine were almost identical; the rank order of EC(50) values (TPA(+) > quinine > TEA(+)) was independent of the expression system. EC(50) values for TEA(+) in CHO-hOCT1 or HEK293-hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin-dependent kinase II, and p56(lck) tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.

Up-regulation of early growth response gene-1 via the CXCR3 receptor induces reactive oxygen species and inhibits Na+/K+-ATPase activity in an immortalized human proximal tubule cell line.

The CXCR3 chemokine receptor, a member of the CXCR family, has been linked to a pathological role in autoimmune disease, inflammatory disease, allograft rejection, and ischemia. In the kidney, expression of the CXCR3 receptor and its ligands is up-regulated in states of glomerulonephritis and in allograft rejection, but little is known about the expression and functional role the CXCR3 receptor might play. Here, we study the function of the CXCR3 chemokine receptor in an immortalized human proximal tubular cell line (IHKE-1). Stimulation of the CXCR3 receptor by its selective agonist monokine induced by IFN-gamma leads via a Ca(2+)-dependent mechanism to an up-regulation of early growth response gene (EGR)-1. Overexpression of EGR-1 induces down-regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase and stimulates the generation of reactive oxygen species (ROS) via the NADH/NADPH-oxidase system. EGR-1 overexpression or treatment with monokine induced by IFN-gamma resulted in a ROS-dependent inhibition of basolateral Na(+)/K(+)-ATPase activity, compromising sodium transport in these cells. Thus, activation of the CXCR3 receptor in proximal tubular cells might disturb natriuresis during inflammatory and ischemic kidney disease via EGR-1-mediated imbalance of ROS.

Guanylin, uroguanylin, and heat-stable euterotoxin activate guanylate cyclase C and/or a pertussis toxin-sensitive G protein in human proximal tubule cells.

Membrane guanylate cyclase C (GC-C) is the receptor for guanylin, uroguanylin, and heat-stable enterotoxin (STa) in the intestine. GC-C-deficient mice show resistance to STa in intestine but saluretic and diuretic effects of uroguanylin and STa are not disturbed. Here we describe the cellular effects of these peptides using immortalized human kidney epithelial (IHKE-1) cells with properties of the proximal tubule, analyzed with the slow-whole-cell patch clamp technique. Uroguanylin (10 or 100 nm) either hyperpolarized or depolarized membrane voltages (V(m)). Guanylin and STa (both 10 or 100 nm), as well as 8-Br-cGMP (100 microm), depolarized V(m). All peptide effects were absent in the presence of 1 mm Ba(2+). Uroguanylin and guanylin changed V(m) pH dependently. Pertussis toxin (1 microg/ml, 24 h) inhibited hyperpolarizations caused by uroguanylin. Depolarizations caused by guanylin and uroguanylin were blocked by the tyrosine kinase inhibitor, genistein (10 microm). All three peptides increased cellular cGMP. mRNA for GC-C was detected in IHKE-1 cells and in isolated human proximal tubules. In IHKE-1 cells GC-C was also detected by immunostaining. These findings suggest that GC-C is probably the receptor for guanylin and STa. For uroguanylin two distinct signaling pathways exist in IHKE-1 cells, one involves GC-C and cGMP as second messenger, the other is cGMP-independent and connected to a pertussis toxin-sensitive G protein.

Expression pattern and functional characteristics of two novel splice variants of the two-pore-domain potassium channel TREK-2.

Two novel alternatively spliced isoforms of the human two-pore-domain potassium channel TREK-2 were isolated from cDNA libraries of human kidney and fetal brain. The cDNAs of 2438 base pairs (bp) (TREK-2b) and 2559 bp (TREK-2c) encode proteins of 508 amino acids each. RT-PCR showed that TREK-2b is strongly expressed in kidney (primarily in the proximal tubule) and pancreas, whereas TREK-2c is abundantly expressed in brain. In situ hybridization revealed a very distinct expression pattern of TREK-2c in rat brain which partially overlapped with that of TREK-1. Expression of TREK-2b and TREK-2c in human embryonic kidney (HEK) 293 cells showed that their single-channel characteristics were similar. The slope conductance at negative potentials was 163 +/- 5 pS for TREK-2b and 179 +/- 17 pS for TREK-2c. The mean open and closed times of TREK-2b at -84 mV were 133 +/- 16 and 109 +/- 11 micros, respectively. Application of forskolin decreased the whole-cell current carried by TREK-2b and TREK-2c. The sensitivity to forskolin was abolished by mutating a protein kinase A phosphorylation site at position 364 of TREK-2c (construct S364A). Activation of protein kinase C (PKC) by application of phorbol-12-myristate-13-acetate (PMA) also reduced whole-cell current. However, removal of the putative TREK-2b-specific PKC phosphorylation site (construct T7A) did not affect inhibition by PMA. Our results suggest that alternative splicing of TREK-2 contributes to the diversity of two-pore-domain K+ channels.

Renal transplantation modulates expression and function of receptors and transporters of rat proximal tubules.

Kidney transplantation often leads to disturbances of solute and volume maintenance in humans. To investigate underlying mechanisms, expression and function of renal transporters and receptors of the proximal tubule (PT) were analyzed in an acute rejection model of rat kidney transplantation. Semiquantitative RT-PCR and Western blot, histology, immunohistochemistry, and microfluorometry were performed on whole kidneys and isolated PT. With acute rejection, Na+/H+-exchanger type-3 (NHE-3) was markedly downregulated. Na+-HCO(3)(-)-cotransporter (NBC-1) and Na+-glucose transporter type-2 (SGLT2) were upregulated after transplantation. Expressions of Na+/H+-exchanger type-1 (NHE-1), Na+/K+-ATPase (NKA), angiotensin II (AngII) receptor (AT-1), or natriuretic peptide receptor (GC-A) were unaltered. Microfluorometric analyses of intracellular pH, Na+, and Ca2+ demonstrated a decrease in NHE-3 function and AngII-mediated stimulation of NHE-3. AngII-mediated inhibition of NHE-1 and function of all other transporters tested remained unaltered. Function of AT-1 and GC-A were unaffected. Reduced expression of NHE-3 was also confirmed by semiquantitative immunohistochemistry. These findings suggest that expression and function of transmembrane proteins involved in Na+-transport after transplantation and rejection is specifically modulated. The local renin-angiotensin-system is apparently not altered. Downregulation of NHE-3 may be a protective mechanism occurring in the graft.

Regulation of human organic cation transporter hOCT2 by PKA, PI3K, and calmodulin-dependent kinases.

Properties and regulation of the human organic cation (OC) transporter type 2 (hOCT2) expressed in HEK-293 cells were extensively characterized using the fluorescent OC 4-[4-(dimethylamino)styryl]-N-methylpyridinium (ASP(+)). ASP(+) uptake was electrogenic and inhibited by TPA(+) (EC(50) = 2.7 microM), tetraethylammonium (EC(50) = 35 microM), cimetidine (EC(50) = 36 microM), or quinine (EC(50) = 6.7 microM). Stimulation with carbachol or ATP decreased initial uptake by 44 +/- 3 (n = 14) and 34 +/- 4% (n = 21), respectively, independently of PKC but dependent on phosphatidylinositol 3-kinase (PI3K). PKA stimulation decreased uptake by 18 +/- 4% (n = 40). Inhibition of calmodulin (CaM), Ca(2+)/CaM-dependent kinase II, or myosin light chain kinase decreased uptake by 63 +/- 2 (n = 15), 40 +/- 4 (n = 30), and 31 +/- 4% (n = 16), respectively. Inhibition of CaM resulted in a significant change in the EC(50) value for the inhibition of ASP(+) uptake by tetraethylammonium. In conclusion, we demonstrate that the hOCT2 is inhibited by PI3K and PKA and activated by a CaM-dependent signaling pathway, probably via a change in substrate affinity.