Search

Primary focal segmental glomerulosclerosis (FSGS) recurs in up to 55% of patients after kidney transplantation. Herein we report the successful management of recurrent FSGS. A 5‐year‐old boy with primary FSGS received a deceased donor renal transplant. Immediate and fulminant recurrence of FSGS caused anuric graft failure that was resistant to plasmapheresis and rituximab. After exclusion of structural or immunologic damage to the kidney by repeated biopsies, the allograft was retrieved from the first recipient on day 27 and transplanted into a 52‐year‐old second recipient who had vascular nephropathy. Immediately after retransplantation, the allograft regained function with excellent graft function persistent now at 3 years after transplant. After 2 years on hemodialysis, the boy was listed for kidney retransplantation. To prevent FSGS recurrence, pretreatment with ofatumumab was performed. Nephrotic range proteinuria still occurred after the second transplantation, which responded, however, to daily plasma exchange in combination with ofatumumab. At 8 months after kidney retransplantation graft function is good. The clinical course supports the hypothesis of a circulating permeability factor in the pathogenesis of FSGS. Successful ofatumumab pretreatment implicates a key role of B cells. Herein we provide a description of successful management of kidney failure by FSGS, carefully avoiding waste of organs., Successful management of recurrent primary focal segmental glomerulosclerosis after kidney transplantation includes retransplantation of an allograft that failed in the first recipient due to disease recurrence into a second recipient and ofatumumab pretreatment before kidney retransplantation in the patient with fulminant recurrence of focal segmental glomerulosclerosis in the first graft.

Children presenting with proliferative lupus nephritis (LN) are treated with intensified immunosuppressive protocols. Data on renal outcome and treatment toxicity is scare. Twelve-month renal outcome and comorbidity were assessed in 79 predominantly Caucasian children with proliferative LN reported to the Lupus Nephritis Registry of the German Society of Paediatric Nephrology diagnosed between 1997 and 2015. At the time of diagnosis, median age was 13.7 (interquartile range 11.8–15.8) years; 86% showed WHO histology class IV, nephrotic range proteinuria was noted in 55%, and median estimated glomerular filtration rate amounted to 75 ml/min/1.73 m2. At 12 months, the percentage of patients with complete and partial remission was 38% and 41%, respectively. Six percent of patients were non-responders and 15% presented with renal flare. Nephrotic range proteinuria at the time of diagnosis was associated with inferior renal outcome (odds ratio 5.34, 95% confidence interval 1.26–22.62, p = 0.02), whereas all other variables including mode of immune-suppressive treatment (e.g., induction treatment with cyclophosphamide (IVCYC) versus mycophenolate mofetil (MMF)) were not significant correlates. Complications were reported in 80% of patients including glucocorticoid toxicity in 42% (Cushingoid appearance, striae distensae, cataract, or osteonecrosis), leukopenia in 37%, infection in 23%, and menstrual disorder in 20%. Growth impairment, more pronounced in boys than girls, was noted in 78% of patients. In this cohort of juvenile proliferative LN, renal outcome at 12 months was good irrespectively if patients received induction treatment with MMF or IVCYC, but glucocorticoid toxicity was very high underscoring the need for corticoid sparing protocols.

Elevated blood pressure remains a major cause of cardiovascular disease, disability, and premature death in Austria, with suboptimal rates of detection, treatment and control also in recent years. Management of hypertension is a common challenge for physicians with different spezializations. In an attempt to standardize diagnostic and therapeutic strategies and, ultimately, to increase the rate of patients with controlled blood pressure and to decrease the burden of cardiovascular disease, 13 Austrian medical societies reviewed the evidence regarding prevention, detection, workup, treatment and consequences of high blood pressure in general and in various clinical scenarios. The result is presented as the first national consensus on blood pressure. The authors and societies involved are convinced that a joint national effort is needed to decrease hypertension-related morbidity and mortality in our country.

Over the last decades, a growing spectrum of monogenic disorders of human magnesium homeostasis has been clinically characterized, and genetic studies in affected individuals have identified important molecular components of cellular and epithelial magnesium transport. Here, we describe three infants who are from non-consanguineous families and who presented with a disease phenotype consisting of generalized seizures in infancy, severe hypomagnesemia, and renal magnesium wasting. Seizures persisted despite magnesium supplementation and were associated with significant intellectual disability. Whole-exome sequencing and conventional Sanger sequencing identified heterozygous de novo mutations in the catalytic Na(+), K(+)-ATPase α1 subunit (ATP1A1). Functional characterization of mutant Na(+), K(+)-ATPase α1 subunits in heterologous expression systems revealed not only a loss of Na(+), K(+)-ATPase function but also abnormal cation permeabilities, which led to membrane depolarization and possibly aggravated the effect of the loss of physiological pump activity. These findings underline the indispensable role of the α1 isoform of the Na(+), K(+)-ATPase for renal-tubular magnesium handling and cellular ion homeostasis, as well as maintenance of physiologic neuronal activity.

Calcineurin inhibitor (CNI)-induced thrombotic microangiopathy (TMA) is a rare complication after renal transplantation. It may be difficult to distinguish from CNI toxicity and acute antibody-mediated rejection (AMR). Its clinical presentation may vary from isolated localised forms up to catastrophic systemic presentations.We report a case of tacrolimus-induced TMA soon after renal transplantation in an 11-year-old boy who received his second renal transplantation. His first graft was lost because of AMR. On day 12 after his second renal transplantation, his renal function started worsening and a kidney biopsy was performed, which showed histopathological signs of TMA. The diagnosis of tacrolimus-induced TMA was established after excluding AMR and other causes of de novo TMA. Genetic complement investigation disclosed two complement factor H risk polymorphisms as possible modifiers of TMA emergence. Treatment was based on replacing tacrolimus with everolimus, with a subsequent normalisation of renal function.A prompt diagnosis of de novo TMA by early allograft biopsy is essential for the allograft outcome and genetic investigations for possible complement abnormalities are reasonable, not only for patients with a systemic aspect of their post-transplant TMA. Replacing tacrolimus with everolimus effectively controlled the TMA and stabilised renal function in our patient.

Diagnostische und therapeutische Algorithmen zu tubularen Erkrankungen werden in diesem Kapitel dargestellt.

Unter dem Sammelbegriff Tubulopathien werden Erkrankungen der Niere zusammengefasst, die aus einem gestorten Substrattransport uber das tubulare Epithel des Nephrons resultieren. Die wesentlichen Transporteigenschaften der von der Erkrankung betroffenen Tubulussegmente sowie die moglichen kompensatorischen Mechanismen in den gesunden Nephronsegmenten bestimmen das Bild der Tubulopathie. Fur die Diagnosestellung essenziell ist die Evaluation der Urinzusammensetzung, die – insbesondere im Vergleich mit den entsprechenden Blutwerten – haufig bereits mit einfachen Laborparametern eine eindeutige Zuordnung der Erkrankung ermoglicht. Ziel dieses Kapitels ist es, Kenntnisse zur Diagnosestellung von Tubulopathien sowie deren Therapie zu vermitteln.

Background/aims ROMK channels mediate potassium secretion and regulate NaCl reabsorption in the kidney. The aim was to study the functional implications of the interaction between ROMK2 (Kir1.1b) and two glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and enolase-α, which were identified as potential regulatory subunits of the channel complex. Methods We performed a membrane yeast-two-hybrid screen of a human kidney cDNA library with ROMK2 as a bait. Interaction of ROMK2 with GAPDH and enolase was verified using GST pull-down, co-immunoprecipitation, immunohistochemistry and co-expression in Xenopus oocytes. Results Confocal imaging showed co-localisation of enolase and GAPDH with ROMK2 in the apical membrane of the renal epithelial cells of the thick ascending limb. Over-expression of GAPDH or enolase-α in Xenopus oocytes markedly reduced the amplitude of ROMK2 currents but did not affect the surface expression of the channels. Co-expression of the glycolytically inactive GAPDH mutant C149G did not have any effect on ROMK2 current amplitude. Conclusion Our results suggest that the glycolytic enzymes GAPDH and enolase are part of the ROMK2 channel supramolecular complex and may serve to couple salt reabsorption in the thick ascending limb of the loop of Henle to the metabolic status of the renal epithelial cells.

Claudins are tight junction integral membrane proteins that are key regulators of the paracellular pathway. Defects in claudin-16 (CLDN16) and CLDN19 function result in the inherited human renal disorder familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC). Previous studies showed that siRNA knockdown of CLDN16 in mice results in a mouse model for FHHNC. Here, we show that CLDN19-siRNA mice also developed the FHHNC symptoms of chronic renal wasting of magnesium and calcium together with defective renal salt handling. siRNA knockdown of CLDN19 caused a loss of CLDN16 from tight junctions in the thick ascending limb (TAL) without a decrease in CLDN16 expression level, whereas siRNA knockdown of CLDN16 produced a similar effect on CLDN19. In both mouse lines, CLDN10, CLDN18, occludin, and ZO-1, normal constituents of TAL tight junctions, remained correctly localized. CLDN16- and CLDN19-depleted tight junctions had normal barrier function but defective ion selectivity. These data, together with yeast two-hybrid binding studies, indicate that a heteromeric CLDN16 and CLDN19 interaction was required for assembling them into the tight junction structure and generating cation-selective paracellular channels.

Knowledge regarding this important system of renal chloride transporters has rapidly accumulated. A severe salt-losing tubulopathy—Bartter syndrome type III—develops when ClCKB is non-functional, whereas a common genetic variant of theCLCNKBgene results in salt-dependent hypertension. Disruption of the Barttin gene manifests as Bartter syndrome type IV with sensorineural deafness and an especially severe salt-losing phenotype. It is timely, therefore, to review the properties of these transporters. Rodent ClC-K1 and ClC-K2, and their respective human orthologs ClCKA and ClCKB, are chloride channels specific to the kidney (and inner ear); Barttin is their functionally important subunit. ClC-K1 is predominantly localized to the thin ascending limb of the loop of Henle. ClC-K2 is expressed more broadly in the distal nephron; expression levels are highest along the thick ascending limb of the loop of Henle and distal convoluted tubule. Expression of ClC-K1 is upregulated by dehydration and downregulated by the diuretic furosemide, whereas expression of ClC-K2 is upregulated by furosemide and downregulated by high salt levels. ClCKA is important for maintenance of the corticomedullary osmotic gradient and the kidney's capacity to concentrate urine. If its ortholog, ClC-K1, is nonfunctional in mice, renal diabetes insipidus develops. ClCKB is a key determinant of tubular reabsorption of chloride and electrolytes along the distal tubule. A severe salt-losing tubulopathy (Bartter syndrome type III) develops if ClCKB is nonfunctional, whereas a common genetic variant of the CLCNKB gene that leads to increased activity of ClCKB results in salt-dependent hypertension. Disruption of the gene encoding Barttin, BSND, results in a 'double knockout' of the functions of both ClCKA and ClCKB, manifesting as Bartter syndrome type IV with sensorineural deafness and an especially severe salt-losing phenotype.

In the past decade, research in genetic disorders of hypophosphatemic disorders has significantly expanded our understanding of phosphate metabolism. X-linked hypophosphatemic rickets is the most common inherited form of rickets due to renal phosphate wasting. The common denominator for all types of rickets is hypophosphatemia, leading to inadequate supply of the mineral to the growing bone. Recent understanding of the mechanisms of disease and role of fibroblast growth factor 23 hypophosphatemic disorders disorders have opened new potential therapeutic avenues. We will discuss the genetic and clinical features of hypophosphatemic disorders and provide understanding and treatment options.

The focus of this chapter are different disorders of primary renal salt-wasting caused by inherited dysfunction of ion-transporting proteins expressed along the thick ascending limb (TAL) of Henle’s loop and along the early distal convoluted tubule (DCT). The clinical presentation, the underlying pathophysiology resulting from the different gene defects and the therapeutic approaches for the Bartter-like syndromes, Gitelman syndrome and EAST syndrome will be discussed in detail. Renal salt-wasting due to impaired sodium reabsorption along the aldosterone-sensitive distal nephron (ASDN) composed of the late DCT, the connecting tubule (CNT), and the collecting duct (CD) is accompanied by hyperkalemia and is discussed elsewhere.

Human magnesium homeostasis primarily depends on the balance between intestinal absorption and renal excretion. Magnesium transport processes in both organ systems – next to passive paracellular magnesium flux – involve active transcellular magnesium transport consisting of an apical uptake into the epithelial cell and a basolateral extrusion into the interstitium. Whereas the mechanism of basolateral magnesium extrusion remains unknown, recent molecular genetic studies in patients with hereditary hypomagnesemia helped gain insight into the molecular nature of apical magnesium entry into intestinal brush border and renal tubular epithelial cells. Patients with Hypomagnesemia with Secondary Hypocalcemia (HSH), a primary defect in intestinal magnesium absorption, were found to carry mutations in TRPM6, a member of the melastatin-related subfamily of transient receptor potential (TRP) ion channels. Before, a close homologue of TRPM6, TRPM7, had been characterized as a magnesium and calcium permeable ion channel vital for cellular magnesium homeostasis. Both proteins share the unique feature of an ion channel fused to a kinase domain with homology to the family of atypical alpha kinases. The aim of this review is to summarize the data emerging from clinical and molecular genetic studies as well as from electrophysiologic and biochemical studies on these fascinating two new proteins and their role in human magnesium metabolism.

Hearing depends on functional ClC-K-type chloride channels composed of barttin with ClC-Ka or ClC-Kb. Loss-of-function mutations of the barttin gene BSND or of both, the ClC-Ka gene CLNKA and the ClC-Kb gene CLNKB lead to congenital deafness and renal salt wasting. Recently, we identified the gain-of-function mutation ClC-Kb(T481S) which is associated with increased blood pressure. To explore the impact of ClC-Kb(T481S) on hearing, healthy volunteers (n=329) and individuals suffering from tinnitus (n=246) volunteered for hearing tests (n=348) and genetic analysis (n=575). 19.1% of the individuals were heterozygote (ClC-Kb(T481S)/ClC-Kb) and 1.7% homozygote carriers. Pure tone average hearing threshold (PTAt) for air conduction was significantly (p0.033) lower in ClC-Kb(T481S) carriers (13.2+/-1.2dB) than in wild-type individuals (17.1+/-0.9dB). The prevalence of ClC-Kb(T481S) carriers was significantly increased (29.7%) in individuals with PTAt15dB (p0.05) and significantly decreased (13.2%) in individuals with PTAt30 dB (p0.017). The difference was largely due to the female population. Bone conduction was less affected pointing to an effect of the mutation on middle ear function. Tinnitus tended to be more frequent in ClC-Kb(T481S) carriers, a difference, however, not statistically significant. In conclusion, hearing thresholds are slightly lower in carriers of ClC-Kb(T481S), i.e., the gain-of-function polymorphism ClC-Kb(T481S) exerts a subtle but significant protective effect against hearing loss.

Several monogenic hypertensive disorders are caused by genetic mutations leading to the deranged function and/or regulation of renal tubular NaCl transport, such as mutations of the renal epithelial Na+ channel (ENaC) in Liddle syndrome, of the kinase WNK1 (with no K) in Gordon syndrome, and of the mineralocorticoid receptor, or of 11β-hydroxysteroid dehydrogenase. Moreover, excessive formation of aldosterone in glucocorticoid-remediable hypertension leads to severe hypertension. Conversely, impaired function of the Na+,K+,2Cl- cotransporter (NKCC2), the renal outer medullary K+ channel (ROMK1), and the renal epithelial Cl- channel ClCKb/Barttin causes Bartter syndrome and defective Na+,Cl+ cotransporter (NCCT) Gitelman syndrome, salt-wasting disorders with hypotension. These monogenic disorders are rare, but illustrate the significance of renal tubular transport in blood pressure regulation. There is little doubt, however, that deranged renal salt reabsorption significantly contributes to essential hypertension polymorphisms of several genes participating in the regulation of renal Na + transport have been shown to be associated with blood pressure and prevalence of hypertension. Two common genes will be discussed in more detail. The first encodes the renal Cl- channel ClCKb. A gain-of-function mutation of ClCKb, increasing channel activity by 7- to 20-fold is found in approximately 20% of unselected Caucasians and 40% of an unselected African population. The second common gene variant (prevalence, 3%-5% in unselected Caucasians), to be discussed in more detail, affects the serum and glucocorticoid inducible kinase SGK1, a kinase upregulated by mineralocorticoids and enhancing the activity of ENaC, ROMK, and Na+/K +ATPase. Both gene variants are associated with slightly increased blood pressure. SGK1 further stimulates the glucose transporter SGLT1, and the SGK1 gene variant correlates, in addition, with increased body mass index. © Japanese Society of Nephrology 2005.

Impaired magnesium reabsorption in patients with TRPM6 gene mutations stresses an important role of TRPM6 (melastatin-related TRP cation channel) in epithelial magnesium transport. While attempting to isolate full-length TRPM6, we found that the human TRPM6 gene encodes multiple mRNA isoforms. Full-length TRPM6 variants failed to form functional channel complexes because they were retained intracellularly on heterologous expression in HEK 293 cells and Xenopus oocytes. However, TRPM6 specifically interacted with its closest homolog, the Mg 2+ -permeable cation channel TRPM7, resulting in the assembly of functional TRPM6/TRPM7 complexes at the cell surface. The naturally occurring S141L TRPM6 missense mutation abrogated the oligomeric assembly of TRPM6, thus providing a cell biological explanation for the human disease. Together, our data suggest an important contribution of TRPM6/TRPM7 heterooligomerization for the biological role of TRPM6 in epithelial magnesium absorption.

Classification and rescue of ROMK mutations underlying hyperprostaglandin E syndrome/antenatal Bartter syndrome. Background Mutations in the renal K + channel ROMK (Kir 1.1) cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), a severe tubular disorder leading to renal salt and water wasting. Several studies confirmed the predominance of alterations of current properties in ROMK mutants. However, in most of these studies, analysis was restricted to nonmammalian cells and electrophysiologic methods. Therefore, for the majority of ROMK mutations, disturbances in protein trafficking remained unclear. The aim of the present study was the evaluation of different pathogenic mechanisms of 20 naturally occurring ROMK mutations with consecutive classification into mutational classes and identification of distinct rescue mechanisms according to the underlying defect. Methods Mutated ROMK potassium channels were expressed in Xenopus oocytes and a human kidney cell line and analyzed by two electrode voltage clamp analysis, immunofluorescence, and Western blot analysis. Results We identified 14 out of 20 ROMK mutations that did not reach the cell surface, indicating defective membrane trafficking. High expression levels rescued six out of 14 ROMK mutants, leading to significant K + currents. In addition, two early inframe stop mutations could be rescued by aminoglycosides, resulting in full-length ROMK and correct trafficking to the plasma membrane in a subset of transfected cells. Conclusion In contrast to previous reports, most of the investigated ROMK mutations displayed a trafficking defect that might be rescued by pharmacologic agents acting as molecular chaperones. The evaluation of different disease-causing mechanisms will be essential for establishing new and more specific therapeutic strategies for HPS/aBS patients.

The serum and glucocorticoid-inducible kinase SGK1 stimulates the Na+ channels ENaC and SCN5A, the K+ channels ROMK1, Kv1.3, and KCNE1/KCNQ1, the cation conductance induced by 4F2/LAT1 and the chloride conductance induced by CFTR. The isoforms SGK2 and SGK3 have similarly been shown to regulate ENaC, SCN5A, Kv1.3 and KCNE1/KCNQ1. The kinases regulate channel abundance in the plasma membrane in part by inhibition of the ubiquitin ligase Nedd4-2 and in part by interaction with trafficking molecules such as the Na+/H+ exchanger regulating factor NHERF2. An in vivo role of SGK1 mediated ENaC channel regulation in renal salt excretion and blood pressure control is documented by the impaired ability of SGK1 knockout mice to adequately reduce renal Na+ output and maintain blood pressure during dietary salt restriction and by enhanced blood pressure in individuals carrying certain polymorphisms in the SGK1 gene. The in vivo physiological significance of SGK dependent regulation of the other channels remains to be shown even though circumstantial evidence points to involvement in the regulation of epithelial transport, cell volume, cell proliferation, cardiac action potential and neuroexcitability. There is little doubt that further channels will be identified which are modulated by the SGKs and that further in vivo physiological functions will be defined where channel regulation by the SGKs plays a critical role.

The human serine/threonine kinase hSGK1 is expressed ubiquitously with highest transcript levels in pancreas and liver. This study has been performed to determine the hSGK1 distribution in normal liver and its putative role in fibrosing liver disease. HSGK1-localization was determined by in situ hybridization, regulation of hSGK1-transcription by Northern blotting, fibronectin synthesis and hSGK1 phosphorylation by Western blotting. In normal liver hSGK1 was mainly transcribed by Kupffer cells. In liver tissue from patients with chronic viral hepatitis, hSGK1 transcript levels were excessively high in numerous activated Kupffer cells and inflammatory cells localized within fibrous septum formations. HSGK1 transcripts were also detected in activated hepatic stellate cells. Accordingly, Western blotting revealed that tissue from fibrotic liver expresses excessive hSGK1 protein as compared to normal liver. TGF-beta1 (2 ng/ml) increases hSGK1 transcription in both human U937 macro-phages and HepG2 hepatoma cells. H(2)O(2) (0.3 mM) activated hSGK1 and increased fibronectin formation in HepG2 cells overexpressing hSGK1 but not in HepG2 cells expressing the inactive mutant hSGK1(K127R). In conclusion hSGK1 is upregulated by TGF-beta1 during hepatitis and may contribute to enhanced matrix formation during fibrosing liver disease.

Ample pharmacological evidence points to a role of kinases in the regulation of cell volume. Given the limited selectivity of most inhibitors, however, the specific molecules involved have remained largely elusive. The search for cell volume regulated genes in liver HepG2 cells led to the discovery of the human serum- and glucocorticoid-dependent serine/threonine kinase hsgk1. Transcription and expression of hsgk1 is markedly and rapidly upregulated by osmotic and isotonic cell shrinkage. The effect of osmotic cell shrinkage on hsgk1 is mediated by p38 kinase. Further stimuli of hsgk1 transcription include glucocorticoids, aldosterone, TGF-β1, serum, increase of intracellular Ca 2+ and phorbolesters, whereas cAMP downregulates hsgk1 transcription. The hsgk1 protein is expressed in several epithelial tissues including human pancreas, intestine, kidney, and shark rectal gland. Co-expression of hsgk1 with the renal epithelial Na + -channel ENaC or the Na + /K + /2Cl − -cotransporter NKCC2 (BSC1) in Xenopus oocytes , accelerates insertion of the transport proteins into the cell membrane and thus, stimulates channel or transport activity. Thus, hsgk1 participates in the regulation of transport by steroids and secretagogues increasing intracellular Ca 2+ -activity. The stimulation of hsgk1 transcription by TGF-β1 may further bear pathophysiological relevance.

Outer hair cells (OHCs) of the mammalian cochlea actively change their cell length in response to changes in membrane potential. This electromotility, thought to be the basis of cochlear amplification, is mediated by a voltage-sensitive motor molecule recently identified as the membrane protein prestin. Here, we show that voltage sensitivity is conferred to prestin by the intracellular anions chloride and bicarbonate. Removal of these anions abolished fast voltage-dependent motility, as well as the characteristic nonlinear charge movement (“gating currents”) driving the underlying structural rearrangements of the protein. The results support a model in which anions act as extrinsic voltage sensors, which bind to the prestin molecule and thus trigger the conformational changes required for motility of OHCs.

Cystic fibrosis (CF) is characterized by impaired Cl(-) secretion and increased Na(+) reabsorption in several tissues including respiratory epithelium. Many CFTR mutations have been identified over the past years. However, only a poor correlation between the genotype and lung phenotype was found suggesting additional factors influencing the phenotype and course of the disease. The serine/threonine kinase SGK1 has recently been shown to stimulate the activity of the epithelial Na(+) channel ENaC. A variety of stimuli such as aldosterone, cell shrinkage, insulin or TGF-beta1 stimulate transcription and activate the SGK1 kinase. Here we further examined the effects of SGK1 on ENaC and CFTR which have mutual interactions and we analyzed sgk1 mRNA abundance in lung tissue from CF patients. Coexpression of CFTR and h-SGK1 in Xenopus oocytes increased ENaC currents as previously described. In addition CFTR mediated currents were also stimulated. h-SGK1 accelerated the expression of the amiloride sensitive Na(+)- current in Xenopus oocytes paralleled by increased ENaC-protein abundance in the oocyte membrane, an effect which was reversed by a h-SGK1(K127R) mutation lacking the ATP-binding site. The cation selectivity or Na(+) affinity were not affected. However, coexpression of h-SGK1 with ENaC altered the sensitivity of the Na(+)-channel to the inhibitors amiloride and triamterene. The inhibitory effect of CFTR expression on ENaC current was not affected by coexpression of h-SGK1 in Xenopus oocytes. Lung tissue from CF patients strongly expressed the serine/threonine kinase h-sgk1 which was not the case for non-CF lung tissue. Loss of CFTR function itself in a CF lung epithelial cell line did not increase SGK1 expression. In summary, enhanced expression of h-SGK1 in epithelial cells of CF-lung tissue may be a novel pathophysiological factor contributing to increased Na(+) channel activity and thus to increased Na(+) transport in CF.

The effects of the fluoresceine derivative, phloxine B, on the Cl− current through the cystic fibrosis transmembrane conductance regulator (CFTR) were examined in Xenopus oocytes expressing human CFTR. In whole oocytes, the CFTR Cl− current (ICFTR) was activated by superfusion with isobutylmethylxanthine and forskolin. ICFTR was stable during activation and deactivated rapidly upon washout of the activation solution. Phloxine B slowed deactivation and, at high concentrations, inhibited ICFTR weakly. In excised inside-out macropatches, ICFTR was activated by the catalytic subunit of protein kinase A (cPKA) and MgATP. Phloxine B (0.01–3 μM), applied after activation, increased ICFTR within 30 s followed by a slow decrease which became dominant at high concentrations. Slowing of deactivation of the CFTR was observed at all concentrations. The effect of phloxine B after 30 s had a bell-shaped concentration-dependence with midpoints at 45 and 1600 nM for the stimulatory and the inhibitory limb, respectively; maximum stimulation was about 1.8 times. The slow inhibitory component, measured after 6 min, occurred with an IC50 value of ∼1 μM. In the absence of cPKA, phloxine B did not stimulate ICFTR. In the presence of cPKA and MgATP, the effects of phloxine B were more prominent at low (0.02 mM) than at high ATP (2 mM). The data show that phloxine B modulates ICFTR by increasing channel activity and slowing channel deactivation; at high concentrations inhibition dominates. The effects may be mediated by direct interactions with CFTR from the inside of the cell. British Journal of Pharmacology (2000) 131, 433–440; doi:10.1038/sj.bjp.0703600

Transforming growth factor β (TGF-β) has been shown to participate in the pathophysiology of diabetic complications. As shown most recently, TGF-β stimulates the expression of a distinct serine/threonine kinase (hSGK) which had previously been cloned as an early gene transcriptionally regulated by cell volume alterations. The present study was performed to elucidate transcription and function of hSGK in diabetic nephropathy. As shown by Northern blotting, an increase of extracellular glucose concentration increased hSGK mRNA levels in cultured cells, an effect qualitatively mimicked by osmotic cell shrinkage or treatment with TGF-β (2 μg/liter), phorbol 12,13-didecanoate (1 μM), or the Ca 2+ ionophore ionomycin (1 μM) and blunted by high concentrations of nifedipine (10 and 100 μM). In situ hybridization revealed that hSGK transcription was markedly enhanced in diabetic nephropathy, with particularly high expression in mesangial cells, interstitial cells, and cells in thick ascending limbs of Henle's loop and distal tubules. According to voltage clamp and tracer flux studies in Xenopus oocytes expressing the renal epithelial Na + channel ENaC or the mouse thick ascending limb Na + ,K + ,2Cl − cotransporter BSC-1, coexpression with hSGK stimulated ENaC and BSC-1 11-fold and 6-fold, respectively, effects reversed by kinase inhibitors staurosporine (1 μM) and chelerythrine (1 μM) and not elicited by inactive hSGK. In conclusion, excessive extracellular glucose concentrations enhance hSGK transcription, which in turn stimulates renal tubular Na + transport. These observations disclose an additional element in the pathophysiology of diabetic nephropathy.

The present study was performed to elucidate the effects of urea on vascular smooth muscle cells (SMC). Addition of urea (20, 50, 100 mM) to physiological salt solution blunted the vasoconstrictory effect of phenylephrine (by 17, 25 and 30%, respectively) and of an increased extracellular K+ concentration (by 7, 14 and 19%, respectively) without affecting the basal tone of rabbit arterial rings. According to Fura-2 fluorescence in cultured SMC (A7r5), urea had no effect on basal intracellular calcium activity ([Ca2+]i), but significantly blunted the increase of [Ca2+]i following an increase of extracellular K+. Whole-cell patch-clamp studies revealed that the Ca2+ current through voltage-sensitive Ca2+ channels is significantly inhibited in the presence of urea. As evident from calcein fluorescence, addition of urea leads to sustained cell shrinkage. The effects of urea on vascular tone, [Ca2+]i activity, voltage-gated Ca2+ channels and cell volume are mimicked by addition of raffinose or NaCl. However, the cell shrinkage induced by urea is sustained, whereas the addition of equiosmolar NaCl is only transient and followed by a regulatory cell volume increase. Moreover, hypertonic NaCl increases, whereas urea decreases, the transcription of cell-volume-regulated kinase hsgk. In conclusion, urea leads to sustained shrinkage of vascular smooth muscle cells, which is followed by inhibition of voltage-gated Ca2+ channels, a decrease of [Ca2+]i and thus blunts the vasoconstrictory action of phenylephrine and increased extracellular K+ concentration.

Mutations in all four known KCNQ potassium channel alpha-subunit genes lead to human diseases. KCNQ1 (KvLQT1) interacts with the beta-subunit KCNE1 (IsK, minK) to form the slow, depolarization-activated potassium current I(Ks) that is affected in some forms of cardiac arrhythmia. Here we show that the novel beta-subunit KCNE3 markedly changes KCNQ1 properties to yield currents that are nearly instantaneous and depend linearly on voltage. It also suppresses the currents of KCNQ4 and HERG potassium channels. In the intestine, KCNQ1 and KCNE3 messenger RNAs colocalized in crypt cells. This localization and the pharmacology, voltage-dependence and stimulation by cyclic AMP of KCNQ1/KCNE3 currents indicate that these proteins may assemble to form the potassium channel that is important for cyclic AMP-stimulated intestinal chloride secretion and that is involved in secretory diarrhoea and cystic fibrosis.

The effect of verapamil and its enantiomers and metabolites on cardiac action potential repolarizing potassium channels was tested. For this purpose, the potassium channels Kv1.1, Kv1.5, Kir2.1, and HERG, and the IsK subunit of the IKs-channel complex were expressed in Xenopus oocytes and two-electrode voltage-clamp experiments were performed. Verapamil induced a concentration-dependent block of Kv1. 1-, Kv1.5-, IKs-, and HERG-induced currents with IC50 values of 14.0 +/- 2.7 microM (n = 4), 5.1 +/- 0.5 microM (n = 6), 161.0 +/- 26.3 microM (n = 4), and 3.8 +/- 0.2 microM (n = 5), respectively. The same potency of HERG channel inhibition was observed for the optical enantiomers (+)-verapamil (IC50 = 3.5 +/- 0.4 microM, n = 5) and (-)-verapamil (IC50 = 4.0 +/- 0.7 microM, n = 4), as well as the derivatives norverapamil (D591; IC50 = 3.8 +/- 0.3 microM, n = 4) and D703 (IC50 = 2.2 +/- 0.4 microM, n = 4). The verapamil metabolites D620 and D617 did not block HERG-induced currents at concentrations of up to 30 microM (n = 3). These results demonstrate that cardiac delayed rectifier potassium currents are sensitive targets to calcium channel blockers.

Functional and pharmacological data point to the involvement of KCNQ1/IsK potassium channels in the basolateral potassium conductance of secretory epithelia. In this study, we report the cloning and electrophysiological characterization of the KCNQ1 protein from the salt secretory rectal gland of the spiny dogfish (Squalus acanthias). The S. acanthias KCNQ1 (s-KCNQ1) cDNA was cloned by polymerase chain reaction (PCR) intensive techniques and showed overall sequence similarities with the KCNQ1 potassium channel subunits of Man, mouse and Xenopus laevis of 64, 70 and 77%, respectively, at the translated amino acid level. Analysis of s-KCNQ1 expression on a Northern blot containing RNA from heart, rectal gland, kidney, brain, intestine, testis, liver and gills revealed distinct expression of 7.4-kb s-KCNQ1 transcripts only in rectal gland and heart. Voltage-clamp analysis of s-KCNQ1 expressed in Xenopus oocytes showed pronounced electrophysiological similarities to human and murine KCNQ1 isoforms, with a comparable sensitivity to inhibition by the chromanol 293B. Coexpression of s-KCNQ1 with human-IsK (h-IsK) induced currents with faster activation kinetics and stronger rectification than observed after coexpression of human KCNQ1 with h-IsK, with the voltage threshold of activation shifted to more negative potentials. The low activation threshold at approximately -60 mV in combination with the high expression in rectal gland cells make s-KCNQ1 a potential candidate responsible for the basolateral potassium conductance.

Recently, we cloned the human cation transporter hOCT2, a member of a new family of polyspecific transporters from kidney, and demonstrated electrogenic uptake of tetraethylammonium, choline, N1-methylnicotinamide, and 1-methyl-4-phenylpyridinium. Using polymerase chain reaction amplification, cDNA sequencing, in situ hybridization, and immunohistochemistry, we now show that hOCT2 message and protein are expressed in neurons of the cerebral cortex and in various subcortical nuclei. In Xenopus laevis oocytes expressing hOCT2, electrogenic transport of norepinephrine, histamine, dopamine, serotonin, and the antiparkinsonian drugs memantine and amantadine was demonstrated by tracer influx, tracer efflux, electrical measurements, or a combination. Apparent Km values of 1.9 +/- 0.6 mM (norepinephrine), 1.3 +/- 0.3 mM (histamine), 0.39 +/- 0.16 mM (dopamine), 80 +/- 20 microM (serotonin), 34 +/- 5 microM (memantine), and 27 +/- 3 microM (amantadine) were estimated. Measurement of trans-effects in depolarized oocytes and human embryonic kidney cells expressing hOCT2 suggests that there were different rates and specificities for cation influx and efflux. The hypothesis is raised that hOCT2 plays a physiological role in the central nervous system by regulating interstitial concentrations of monoamine neurotransmitters that have evaded high affinity uptake mechanisms. We show that amantadine does not interact with the expressed human Na+/Cl- dopamine cotransporter. However, concentrations of amantadine that are effective for the treatment of Parkinson's disease may increase the interstitial concentrations of dopamine and other aminergic neurotransmitters by competitive inhibition of hOCT2.

Recently, the cell-volume-regulated serine-threonine protein kinase h-sgk was cloned from a human hepatoma cell line. The sgk gene was shown to be induced by cell shrinkage in many different mammalian cell lines. In this study, two highly conserved serine-threonine protein kinases, sgk-1 and sgk-2, were cloned from rectal gland tissue of the spiny dogfish (Squalus acanthias). Both kinases showed a distinct pattern of tissue specificity, with high expression levels in kidney, intestine, liver and heart. In rectal gland slices sgk-1 transcription was induced by exposure to hypertonic solution, reduction of the extracellular urea concentration, and addition of the secretagogues vasoactive intestinal polypeptide (VIP) and carbachol. The shark sgk-1 serine-threonine protein kinase may therefore provide a link between cell volume, Cl–secretion and protein phosphorylation state in shark rectal gland cells.

The SGK protein kinase is a novel member of the serine/threonine protein kinase family. Its corresponding gene belongs to the group of immediate-early genes. SGK transcription is controlled by cell volume alterations in different cell lines. To analyze the genomic structure and chromosomal location of the SGK gene, a human P1 clone was isolated by screening a human genomic library with a SGK cDNA probe. This clone was confirmed to encode the authentic SGK gene by the detection of exon-intron structures and the correspondence between the nucleotide sequences of exons and human cDNA. Using this P1 clone as a probe for fluorescence in situ hybridization, a single chromosomal locus for SGK was assigned to band 6q23, a region frequently affected by deletion in various human neoplasms.

A wide variety of factors challenge constancy of cell volume. Alterations of cell volume activate diverse cell volume regulatory mechanisms including ion transport, osmolyte accumulation, metabolism and expression of appropriate genes. A wealth of cellular signalling pathways link cell volume to the respective regulatory mechanisms. Cell volume emerges as a pathophysiologically important parameter in several diseases including diabetes mellitus, uraemia, hepatic insufficiency and hypercatabolic states. The role of altered cell volume in disease is a challenge which requires more experimental research and clinical investigation.

In the proximal tubule Na+-dependent (SDAT) and Na+-independent (SIAT) amino acid (AA) transporters are present. The effects of neutral, basic, and acidic AA on membrane voltage (Vm) of immortalized human kidney epithelial (IHKE-1) cells derived from the proximal tubule were examined using the slow whole-cell patch-clamp technique. In the presence of Na+ AA depolarized Vm in a concentration-dependent manner (0.05–5 mM) with Asp = Arg = Glu =2Cys < Pro = Leu < Phe = AIB = Ala = Pro = Asn < Gly. In the absence of extracellular Na+ a decreased depolarization was seen with most neutral AA (Ala, Pro, Asn, Gly, Phe, and Leu), and the depolarization was increased with Asp, Glu, Arg, and 2Cys (1 mM each). In the absence of Na+ and a reduction in Cl– (5 mM) the depolarization by Arg was reduced. Unlike that predicted for transport by system b0,+ which exchanges neutral against dibasic amino acids, Leu does not hyperpolarize but depolarize Vm of IHKE-1 cells in the absence of extracellular Na+. After removal of Na+ (0 mM) and a reduction in Cl– (5 mM) in the extracellular solution, Leu or Glu hyperpolarized Vm, indicating that IHKE-1 cells possess two different SIAT systems, one Cl–-independent and similar to system b0,+ and one novel Cl–-dependent system, which might be a Cl–/AA exchanger and can be blocked by the Cl–-channel blockers 5-nitro-2-(3-phenylpropylamino)-benzoate (10 μM) and 4,4′-diisothiocyanostibene-2,2′-disulfonic acid (50 μM). B system-related AA transporters might be responsible for the C–-independent SIAT, since we were able to detect its signal by Northern blot analysis.

Lang, Florian, Gillian L. Busch, Markus Ritter, Harald Völkl, Siegfried Waldegger, Erich Gulbins, and Dieter Häussinger. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. 78: 247–306, 1998. — To survive, cells have to avoid excessive alterations of cell volume that jeopardize structural integrity and constancy of intracellular milieu. The function of cellular proteins seems specifically sensitive to dilution and concentration, determining the extent of macromolecular crowding. Even at constant extracellular osmolarity, volume constancy of any mammalian cell is permanently challenged by transport of osmotically active substances across the cell membrane and formation or disappearance of cellular osmolarity by metabolism. Thus cell volume constancy requires the continued operation of cell volume regulatory mechanisms, including ion transport across the cell membrane as well as accumulation or disposal of organic osmolytes and metabolites. The various cell volume regulatory mechanisms are triggered by a multitude of intracellular signaling events including alterations of cell membrane potential and of intracellular ion composition, various second messenger cascades, phosphorylation of diverse target proteins, and altered gene expression. Hormones and mediators have been shown to exploit the volume regulatory machinery to exert their effects. Thus cell volume may be considered a second message in the transmission of hormonal signals. Accordingly, alterations of cell volume and volume regulatory mechanisms participate in a wide variety of cellular functions including epithelial transport, metabolism, excitation, hormone release, migration, cell proliferation, and cell death.

Alloimmune antenatal membranous nephropathy (MN) during pregnancy results from antibodies produced by a neutral endopeptidase (NEP)–deficient mother. Here we report two recent cases that provide clues to the severity of renal disease. Mothers of the two children had circulating antibodies against NEP showing the characteristic species-dependent pattern by immunofluorescence on kidney slices. A German mother produced predominantly anti-NEP IgG4 accompanied by a low amount of IgG1. Her child recovered renal function within a few weeks. In sharp contrast, an Italian mother mainly produced complement-fixing anti-NEP IgG1, which also inhibits NEP enzymatic activity, whereas anti-NEP IgG4 has a weak inhibitory potency. Her child was dialyzed for several weeks. A kidney biopsy performed at 12 days of age showed MN, ischemic glomeruli, and arteriolar and tubular lesions. A second biopsy performed at 12 weeks of age showed aggravation with an increased number of collapsed capillary tufts. Both mothers were homozygous for the truncating deletion mutation 466delC and were thus NEP deficient. The 466delC mutation, identified in three previously described families, suggests a founder effect. Because of the potential severity of alloimmune antenatal MN, it is essential to identify families at risk by the detection of anti-NEP antibodies and NEP antigen in urine. On the basis of the five families identified to date, we propose an algorithm for the diagnosis of the disease and the prevention of complications.

GF-β is a pathogenic factor in patients with acute respiratory distress syndrome (ARDS), a condition characterized by alveolar edema. A unique TGF-β pathway is described, which rapidly promoted internalization of the αβγ epithelial sodium channel (ENaC) complex from the alveolar epithelial cell surface, leading to persistence of pulmonary edema. TGF-β applied to the alveolar airspaces of live rabbits or isolated rabbit lungs blocked sodium transport and caused fluid retention, which-together with patch-clamp and flow cytometry studies-identified ENaC as the target of TGF-β. TGF-β rapidly and sequentially activated phospholipase D1, phosphatidylinositol-4-phosphate 5-kinase 1α, and NADPH oxidase 4 (NOX4) to produce reactive oxygen species, driving internalization of βENaC, the subunit responsible for cell-surface stability of the αβγENaC complex. ENaC internalization was dependent on oxidation of βENaC Cys(43). Treatment of alveolar epithelial cells with bronchoalveolar lavage fluids from ARDS patients drove βENaC internalization, which was inhibited by a TGF-β neutralizing antibody and a Tgfbr1 inhibitor. Pharmacological inhibition of TGF-β signaling in vivo in mice, and genetic ablation of the nox4 gene in mice, protected against perturbed lung fluid balance in a bleomycin model of lung injury, highlighting a role for both proximal and distal components of this unique ENaC regulatory pathway in lung fluid balance. These data describe a unique TGF-β-dependent mechanism that regulates ion and fluid transport in the lung, which is not only relevant to the pathological mechanisms of ARDS, but might also represent a physiological means of acutely regulating ENaC activity in the lung and other organs.

Die Funktion der Nieren lasst sich in folgende wesentliche Aufgaben gliedern: Die Ausscheidung von Wasser und hydrophilen Substanzen, die Konstanterhaltung des Wasser-, Mineral- und Saure-Basen-Haushalts, die endokrine Regulation des Blutdrucks, der Kalzium-Phosphat-Homoostase und der Erythropoese sowie die Beteiligung am Intermediarstoffwechsel (z. B. durch Glukoneogenese aus Aminosauren und Laktat).

As shown previously, expression of Ha-ras oncogene in NIH 3T3 fibroblasts (+ras cells) increases cellular concentrations of Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and enhances bradykinin induced Ca2+ entry 1 , 2 , 3 . These cells respond to low concentrations of serum or bradykinin with sustained oscillations of the cell membrane potential due to pulsatile release of calcium from internal stores and subsequent activation of calcium sensitive K+ channels [1] . Furthermore Ha-ras oncogene expression leads to depolymerization of the actin filament network and delayed increase of cell volume 4 , 5 , 6 . Pretreatment of the same cells not expressing the oncogene (−ras cells) with Li+ similarly increases Ins(1,4,5)P3 and Ins(1,3,4,5)P4 [2] . As shown in the present study, −ras cells pretreated with Li+ similar to Ha-ras oncogene expressing cells respond to bradykinin with sustained oscillations of cell membrane potential, depolymerization of the actin filament network and increase of cell volume. The oscillations of the cell membrane potential and the depolymerization of the actin cytoskeleton can be inhibited by the calcium channel blocker lanthanum and the bradykinin induced increase of cell volume is inhibited by HOE 694, pointing to involvement of Na+/H+ exchange. The data indicate a close functional linkage of the calcium oscillations, cytoskeletal rearrangement and activation of the Na+/H+ exchanger. Thus, Li+ pretreatment mimicks crucial cellular events triggered by expression of the Ha-ras oncogene. However, unlike in cells expressing the Ha-ras oncogene, Li+ pretreatment alone does not allow for growth factor-independent proliferation of the cells.

The human rBAT protein elicits sodium-independent, high affinity obligatory exchange of cystine, dibasic amino acids, and some neutral amino acids in Xenopus oocytes (Chillarón, J., Estévez, R., Mora, C., Wagner, C. A., Suessbrich, H., Lang, F., Gelpí, J. L., Testar, X., Busch, A. E., Zorzano, A., and Palacín, M. (1996) J. Biol. Chem. 271, 17761-17770). Mutations in rBAT have been found to cause cystinuria (Calonge, M. J., Gasparini, P., Chillarón, J., Chillón, M., Galluci, M., Rousaud, F., Zelante, L., Testar, X., Dallapiccola, B., Di Silverio, F., Barceló, P., Estivill, X., Zorzano, A., Nunes, V., and Palacín, M. (1994) Nat. Genet. 6, 420-426). We have performed functional studies with the most common point mutation, M467T, and its relative, M467K, using the oocyte system. The Km and the voltage dependence for transport of the different substrates were the same in both M467T and wild type-injected oocytes. However, the time course of transport was delayed in the M467T mutant: maximal activity was accomplished 3-4 days later than in the wild type. This delay was cRNA dose-dependent: at cRNA levels below 0.5 ng the M467T failed to achieve the wild type transport level. The M467K mutant displayed a normal Km, but the Vmax was between 5 and 35% of the wild type. The amount of rBAT protein was similar in normal and mutant-injected oocytes. In contrast to the wild type, the mutant proteins remained endoglycosidase H-sensitive, suggesting a longer residence time in the endoplasmic reticulum. We quantified the amount of rBAT protein in the plasma membrane by surface labeling with biotin 2 and 6 days after injection. Most of the M467T and M467K protein was located in an intracellular compartment. The converse situation was found in the wild type. Despite the low amount of M467T protein reaching the plasma membrane, the transport activity at 6 days was the same as in the wild type-injected oocytes. The increase in plasma membrane rBAT protein between 2 and 6 days was completely dissociated from the rise in transport activity. These data indicate impaired maturation and transport to the plasma membrane of the M467T and M467K mutant, and suggest that rBAT alone is unable to support the transport function.

Heavy metal intoxication with Hg2+, Pb2+ and Cd2+ commonly leads to phosphaturia. In this study, we examined the effects of these heavy metals on Pi-induced currents (Ip) through NaPi-3, the human renal cotransporter for Na+ and Pi. Hg2+ inhibited Ip in a dose- and time-dependent fashion. Hg2+ decreased the extrapolated maximal current but did not alter the apparent affinity for Pi. This inhibition was also observed with the membrane-permeable oxidizing agent 2,2'-dithio-bis(5-nitropyridine) (DTNP) but not with the membrane-impermeable 5,5'-dithiobis(2-nitrobenzoic acid). Hg(2+)- and DTNP-mediated inhibition of Ip was reversible only in the presence of the reducing agent 2,3-dihydroxybutane-1,4-dithiol. Cd2+ and Pb2+ also inhibited Ip. However, while CD2+ did not significantly alter the apparent affinity for Pi, the apparent concentration needed for half-maximal current (Km) for Pi was increased by Pb2+. In contrast to Hg2+, the inhibition of Ip by Cd2+ and Pb2+ was rapidly reversible upon washout. In the presence of the Na(+)-K(+)-adenosinetriphosphatase inhibitor ouabain, Ip was not reduced, and the effects of the heavy metals were maintained. In summary, the three heavy metals Hg2+, Cd2+, and Pb2+ inhibit Ip through the Na+/Pi cotransporter NaPi-3 by distinct mechanisms. Heavy metal-mediated inhibition of NaPi-3 may be responsible for the phosphaturia observed after intoxication with these compounds.

The widely used histamine receptor antagonists terfenadine and astemizole were shown to prolong the QT interval in electrocardiographic recordings in cases of overdose or inappropriate co-medications, indicating a possible interaction with cardiac K+ channels. Here, terfenadine and astemizole both inhibited the human ether-a-go-go related gene (HERG) encoded channels expressed in Xenopus oocytes at nanomolar concentrations in a use- and voltage-dependent fashion. In contrast, inhibition of other delayed rectifier (Kvl.1 and IsK) or inward rectifier K+ channels (IRK1) was much weaker and occurred only at high micromolar concentrations. These results suggest that blockade of HERG channels by terfenadine and astemizole might contribute to the cardiac side effects of these compounds.

The availability of cloned transport molecules achieved by efforts in expression cloning has allowed their electrophysiological analysis in the Xenopus oocyte expression system. We describe the electrogenic uptake of various substrates by their corresponding transport molecules originally expressed in brush border membranes of proximal tubules. The functional properties of the following transport molecules are discussed: the sodium-coupled glucose transporter, the sodium-coupled phosphate transporter, the sodium-coupled sulfate transporter and the sodium-independent transporter of neutral and dibasic amino acids. Additionally, functional consequences of naturally occurring disease-causing mutations in some of these transport molecules are described.

Tubular phosphate reabsorption is strongly regulated by parathyroid hormone (PTH) via intracellular activation of protein kinase A (PKA) and C (PKC). In the present study, we expressed the human renal brush border Na+-dependent phosphate (Pi) transporter (NaPi-3) in Xenopus oocytes and analyzed its regulation by coexpressed PTH receptors, activation of PKA and PKC and increase of [Ca2+]i. Stimulation of coexpressed PTH receptors (with 10 nMPTH 1-34) or activation of PKA (with dibutyryl-cAMP) had no effect on Ip. Increasing [Ca2+]i with the calcium ionophore A23187 reduced Ip. Ca2+-mediated Ip inhibition was prevented in the presence of staurosporine, but not by the calmodulin-antagonist calmidazo-lium. Activation of PKC by the racemate of phorbol-12, 13-didecanoate (PDD) mimicked the effect of A23187 and decreased Pi-induced currents (Ip). The inactive enantiomere α-phorbol-12, 12-didecanoate (α-PDD) had no effects on IP. An increase in [Ca2+]i accelerated PDD-mediated IP-inhibition, but [Ca2+]i and PDD effects were not additive. It is concluded that [Ca2+]i and PDD inhibit IP via activation of PKC, while activation of PKA has no functional consequence for NaPi-3 expressed Xenopus oocytes.

Inhibition of human IsK channels expressed in Xenopus oocytes by the novel class III antiarrhythmic azimilide was studied under distinct treatments known to increase IsK (hypotonic solution, A23187 and isoproterenol) Azimilide inhibited IsK under all conditions with similar potency. Reduction of ionic strength or pH changes from pH 6.5 to 8.5 did not alter IsK amplitude. However, inhibition of IsK by azimilide was decreased by reduced pH, but not by reduced ionic strength. Further, the apparent affinity of azimilide was increased more than tenfold by increasing pH from 6.5 to 8.5. The data suggest that the neutral form of azimilide, a weak base, inhibits IsK via a lipophilic protein-drug interaction. pH-dependence of azimilide may significantly alter its effects on IsK under distinct pathophysiological conditions (acidosis vs. alkalosis) and in distinct locations (heart vs. kidney).