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.     

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.     

Absence of small conductance K+ channel (SK) activity in apical membranes of thick ascending limb and cortical collecting duct in ROMK (Bartter's) knockout mice

The ROMK (Kir1.1; Kcnj1) gene is believed to encode the apical small conductance K(+) channels (SK) of the thick ascending limb (TAL) and cortical collecting duct (CCD). Loss-of-function mutations in the human ROMK gene cause Bartter's syndrome with renal Na(+) wasting, consistent with the role of this channel in apical K(+) recycling in the TAL that is crucial for NaCl reabsorption. However, the mechanism of renal K(+) wasting and hypokalemia that develop in individuals with ROMK Bartter's syndrome is not apparent given the proposed loss of the collecting duct SK channel. Thus, we generated a colony of ROMK null mice with approximately 25% survival to adulthood that provides a good model for ROMK Bartter's syndrome. The remaining 75% of null mice die in less than 14 days after birth. The surviving ROMK null mice have normal gross renal morphology with no evidence of significant hydronephrosis, whereas non-surviving null mice exhibit marked hydronephrosis. ROMK protein expression was absent in TAL and CCD from null mice but exhibited normal abundance and localization in wild-type littermates. ROMK null mice were polyuric and natriuretic with an elevated hematocrit consistent with mild extracellular volume depletion. SK channel activity in TAL and CCD was assessed by patch clamp analysis in ROMK wild-type ROMK(+/+), heterozygous ROMK(+/-), and null ROMK(-/-) mice. In 313 patches with successful seals from the three ROMK genotypes, SK channel activity in ROMK (+/+ and +/-) exhibited normal single channel kinetics. The expression frequencies are as follows: 67 (TAL) and 58% (CCD) in ROMK(+/+); about half that of the wild-type in ROMK(+/-), being 38 (TAL) and 25% (CCD); absent in both TAL or CCD in ROMK(-/-) between 2 and 5 weeks in 15 mice (61 and 66 patches, respectively). The absence of SK channel activity in ROMK null mice demonstrates that ROMK is essential for functional expression of SK channels in both TAL and CCD. Despite loss of ROMK expression, the normokalemic null mice exhibited significantly increased kaliuresis, indicating alternative mechanisms for K(+) absorption/secretion in the nephron.     

ENaC and ROMK channels in the connecting tubule regulate renal K+ secretion

We measured the activities of epithelial Na channels (ENaC) and ROMK channels in the distal nephron of the mouse kidney and assessed their role in the process of K⁺ secretion under different physiological conditions. Under basal dietary conditions (0.5% K), ENaC activity, measured as amiloride-sensitive currents, was high in cells at the distal end of the distal convoluted tubule (DCT) and proximal end of the connecting tubule (CNT), a region we call the early CNT (CNTe). In more distal parts of the CNT (aldosterone-sensitive portion [CNTas]), these currents were minimal. This functional difference correlated with alterations in the intracellular location of ENaC, which was at or near the apical membrane in CNTe and more cytoplasmic in the CNTas. ROMK activity, measured as TPNQ-sensitive currents, was substantial in both segments. A mathematical model of the rat nephron suggested that K⁺ secretion by the CNTe predicted from these currents provides much of the urinary K⁺ required for K balance on this diet. In animals fed a K-deficient diet (0.1% K), both ENaC and ROMK currents in the CNTe decreased by ∼50%, predicting a 50% decline in K⁺ secretion. Enhanced reabsorption by a separate mechanism is required to avoid excessive urinary K⁺ losses. In animals fed a diet supplemented with 3% K, ENaC currents increased modestly in the CNTe but strongly in the CNTas, while ROMK currents tripled in both segments. The enhanced secretion of K⁺ by the CNTe and the recruitment of secretion by the CNTas account for the additional transport required for K balance. Therefore, adaptation to increased K⁺ intake involves the extension of robust K⁺ secretion to more distal parts of the nephron.     

Differential regulation of ROMK (Kir1.1) in distal nephron segments by dietary potassium

ROMK channels are well-known to play a central role in renal K secretion, but the absence of highly specific and avid-ROMK antibodies has presented significant roadblocks toward mapping the extent of expression along the entire distal nephron and determining whether surface density of these channels is regulated in response to physiological stimuli. Here, we prepared new ROMK antibodies verified to be highly specific, using ROMK knockout mice as a control. Characterization with segmental markers revealed a more extensive pattern of ROMK expression along the entire distal nephron than previously thought, localizing to distal convoluted tubule regions, DCT1 and DCT2; the connecting tubule (CNT); and cortical collecting duct (CD). ROMK was diffusely distributed in intracellular compartments and at the apical membrane of each tubular region. Apical labeling was significantly increased by high-K diet in DCT2, CNT1, CNT2, and CD (P < 0.05) but not in DCT1. Consistent with the large increase in apical ROMK, dramatically increased mature glycosylation was observed following dietary potassium augmentation. We conclude 1) our new antibody provides a unique tool to characterize ROMK channel localization and expression and 2) high-K diet causes a large increase in apical expression of ROMK in DCT2, CNT, and CD but not in DCT1, indicating that different regulatory mechanisms are involved in K diet-regulated ROMK channel functions in the distal nephron.     

Tamm-Horsfall glycoprotein interacts with renal outer medullary potassium channel ROMK2 and regulates its function

Tamm-Horsfall glycoprotein (THGP) or Uromodulin is a membrane protein exclusively expressed along the thick ascending limb (TAL) and early distal convoluted tubule (DCT) of the nephron. Mutations in the THGP encoding gene result in Familial Juvenile Hyperuricemic Nephropathy (FJHN), Medullary Cystic Kidney Disease type 2 (MCKD-2), and Glomerulocystic Kidney Disease (GCKD). The physicochemical and biological properties of THGP have been studied extensively, but its physiological function in the TAL remains obscure. We performed yeast two-hybrid screening employing a human kidney cDNA library and identified THGP as a potential interaction partner of the renal outer medullary potassium channel (ROMK2), a key player in the process of salt reabsorption along the TAL. Functional analysis by electrophysiological techniques in Xenopus oocytes showed a strong increase in ROMK current amplitudes when co-expressed with THGP. The effect of THGP was specific for ROMK2 and did not influence current amplitudes upon co-expression with Kir2.x, inward rectifier potassium channels related to ROMK. Single channel conductance and open probability of ROMK2 were not altered by co-expression of THGP, which instead increased surface expression of ROMK2 as determined by patch clamp analysis and luminometric surface quantification, respectively. Despite preserved interaction with ROMK2, disease-causing THGP mutants failed to increase its current amplitude and surface expression. THGP(-/-) mice exhibited increased ROMK accumulation in intracellular vesicular compartments when compared with WT animals. Therefore, THGP modulation of ROMK function confers a new role of THGP on renal ion transport and may contribute to salt wasting observed in FJHN/MCKD-2/GCKD patients.     

Renal Proteinase-activated Receptor 2, a New Actor in the Control of Blood Pressure and Plasma Potassium Level

Proteinase-activated receptor 2 (PAR2) is a G protein-coupled membrane receptor that is activated upon cleavage of its extracellular N-terminal domain by trypsin and related proteases. PAR2 is expressed in kidney collecting ducts, a main site of control of Na⁺ and K⁺ homeostasis, but its function remains unknown. We evaluated whether and how PAR2 might control electrolyte transport in collecting ducts, and thereby participate in the regulation of blood pressure and plasma K⁺ concentration. PAR2 is expressed at the basolateral border of principal and intercalated cells of the collecting duct where it inhibits K⁺ secretion and stimulates Na⁺ reabsorption, respectively. Invalidation of PAR2 gene impairs the ability of the kidney to control Na⁺ and K⁺ balance and promotes hypotension and hypokalemia in response to Na⁺ and K⁺ depletion, respectively. This study not only reveals a new role of proteases in the control of blood pressure and plasma potassium level, but it also identifies a second membrane receptor, after angiotensin 2 receptor, that differentially controls sodium reabsorption and potassium secretion in the late distal tubule. Conversely to angiotensin 2 receptor, PAR2 is involved in the regulation of sodium and potassium balance in the context of either stimulation or nonstimulation of the renin/angiotensin/aldosterone system. Therefore PAR2 appears not only as a new actor of the aldosterone paradox, but also as an aldosterone-independent modulator of blood pressure and plasma potassium.     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Summary Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [³⁵S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

Emerging Role of the Calcium-Activated,Small Conductance,SK3 K+ Channel in Distal Tubule Function: Regulation by TRPV4

The Ca²⁺-activated, maxi-K (BK) K⁺ channel, with low Ca²⁺-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K⁺ secretion. In the present study we demonstrate that the Ca²⁺-activated, SK3 (K_Ca2.3) K⁺ channel, with high Ca²⁺-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K⁺ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca²⁺-dependent K⁺ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca²⁺-permeable TRPV4 channel, thereby inducing Ca²⁺ influx and elevating intracellular Ca²⁺ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca²⁺ influx, leading to elevated intracellular Ca²⁺ levels, activates this high Ca²⁺-affinity K⁺ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca²⁺-dependent regulation of membrane potential and K⁺ secretion.     

Expression of cytochrome P-450 4 enzymes in the kidney and liver: Regulation by PPAR and species-difference between rat and human

Members of the cytochrome P-450 4 (CYP4) family catalyze the ω-hydroxylation of fatty acids, and some of them have the PPAR response element in the promoter area of the genes. The localization of CYP4A and PPAR isoforms and the effect of PPAR agonists on CYP4A protein level and activity were determined in rat kidney and liver. Immunoblot analysis showed that CYP4A was expressed in the liver and proximal tubule, with lower expression in the preglomerular microvessel, glomerulus and thick ascending limb (TAL), but the expression was not detected in the collecting duct. PPARα was expressed in the liver, proximal tubule and TAL. PPARγ was expressed in the collecting duct, with lower expression in the TAL, but no expression in the proximal tubule and liver. The PPARα agonist clofibrate induced CYP4A protein levels and activity in the renal cortex and liver. The PPARγ agonist pioglitazone did not modulate them in these tissues. The localization of CYP4A and CYP4F were further determined in human kidney and liver by immunohistochemical technique. Immunostainings for CYP4A and CYP4F were observed in the hepatocytes of the liver lobule and the proximal tubules, with lower stainings in the TALs and collecting ducts, but no staining in the glomeruli or renal vasculatures. These results indicate that the inducibility of CYP4A by PPAR agonists in the rat tissues correlates with the expression of the respective PPAR isoforms, and that the localization of CYP4 in the kidney has a species-difference between rat and human.     

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.     

Expression and immunolocalization of ERG1 potassium channels in the rat kidney

Potassium (K⁺) channels participate in K⁺ secretion, K⁺ recycling, and cell volume regulation and help to maintain the resting potential in mammalian kidneys. Previously, we identified a set of voltage-gated K⁺ channels (Kv1) in the inner medullary collecting duct of the rat kidney. In the present work, we identified the voltage-gated K⁺ channel ether-à-go-go-related gene (ERG) in the rat kidney. mRNAs of ERG1a and its N-terminal splice-variant ERG1b were detected. Immunoblots of the cortex and medulla revealed two molecular mass proteins of 135 and 80 kDa, consistent in size with the nonglycosylated ERG1a and ERG1b isoforms, respectively. However, bands of 155 and 95 kDa, corresponding to mature glycosylated ERG1a and ERG1b, respectively, were also observed. In our immunohistochemical experiments, we could not differentiate the ERG1 isoforms because we used an antibody against a carboxy-terminal epitope. ERG1 was differentially localized in specific nephron segments: its localization was intracellular in the proximal tubule and medullary collecting ducts and in the apical membranes in the distal convoluted and connecting tubules. ERG1 was also abundant in glomerular arterioles and renal vessels. In summary, ERG1 displays a heterogeneous distribution in the rat kidney.     

Some reflections on the mechanism of renal tubular potassium transport.

Analysis of the driving forces acting on the movement of potassium across individual membranes of tubule cells shows that both active and passive components play an important role in the regulation of potassium transport. Distal and cortical collecting tubule and papillary collecting duct elements are the key nephron sites participating in a complex fashion to translate a wide variety of metabolic challenges into the appropriate excretory response. The latter involves both secretory and reabsorptive activity. The analysis of the factors modulating tubular potassium transfer has shown that the potassium concentration in the cells of the distal nephron is a dey factactors involved in setting the cellular potassium concentration are active potassium uptake at the peritubular and luminal membrane of the cells as well as electrogenic solium extrusion across the peritubular boundary of the cells. Additional factors regulating potassium transport involve the electrical potential difference, sensitive to changes in the sodium concentration in the lumen, the flow rate past the late distal tubular site of potassium secretion, and the activity of a reabsorptive potassium pump in the luminal membranes of the cells. In the cortical collecting tubule, active potassium secretion is also present at the luminal membrane of the cell, but the role of such an additional secretory mechanism in the late distal tubule is presently unknown. Most of these individual transport mechanisms exist along the whole distal nephron, but their relative prominence varies among the late distal tubule, the cortical collecting tubule, and the papilary collecting duct.     

Tamm-Horsfall protein-mRNA synthesis is localized to the thick ascending limb of Henle's loop in rat kidney

Tamm-Horsfall protein (THP) has been previously detected in cells of the thick ascending limb of Henle's loop (TAL) of different mammalian species using immunocytochemical methods. A nearly complete identity between THP and uromodulin, an immunosuppressive glycoprotein present in the urine of pregnant females, has been established recently. This paper describes the cellular location of THP mRNA by high-resolution in situ hybridization using a [35S]-labeled human uromodulin cRNA (antisense-) probe of a length of 665 base pairs. Control experiments were performed using an mRNA (sense-) probe of the same length. The probe was hybridized to frozen sections of the rat kidney. THP mRNA distribution in the kidney was found to be homologous to the immunocytochemical labeling pattern: Autoradiographic signal was present along the entire length of the TAL including the post-macula segment which leads to the distal convoluted tubule. Tubular cells of the macula densa were negative. Labeling intensity of the TAL epithelium was found to increase from the origin of the TAL at the transition between inner and outer medulla to its end beyond the macula densa. Labeling of the medullary segment in the inner stripe was weak, whereas outer medullary and cortical segments very strongly expressed THP mRNA. The glomerulus, the portions of the nephron proximal to the TAL, the distal convoluted tubule as well as the collecting duct system were negative.     

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.     

POSH Stimulates the Ubiquitination and the Clathrin-independent Endocytosis of ROMK1 Channels

POSH (plenty of SH3) is a scaffold protein that has been shown to act as an E3 ubiquitin ligase. Here we report that POSH stimulates the ubiquitination of Kir1.1 (ROMK) and enhances the internalization of this potassium channel. Immunostaining reveals the expression of POSH in the renal cortical collecting duct. Immunoprecipitation of renal tissue lysate with ROMK antibody and glutathione S-transferase pulldown experiments demonstrated the association between ROMK and POSH. Moreover, immunoprecipitation of lysates of HEK293T cells transfected with ROMK1 or with constructs encoding the ROMK-N terminus or ROMK1-C-Terminus demonstrated that POSH binds to ROMK1 on its N terminus. To study the effect of POSH on ROMK1 channels, we measured potassium currents with electrophysiological methods in HEK293T cells and in oocytes transfected or injected with ROMK1 and POSH. POSH decreased potassium currents, and the inhibitory effect of POSH on ROMK channels was dose-dependent. Biotinylation assay further showed that POSH decreased surface expression of ROMK channels in HEK293T cells transfected with ROMK1 and POSH. The effect of POSH on ROMK1 channels was specific because POSH did not inhibit sodium current in oocytes injected with ENaC-α, β, and γ subunits. Moreover, POSH still decreased the potassium current in oocytes injected with a ROMK1 mutant (R1Δ373–378), in which a clathrin-dependent tyrosine-based internalization signal residing between amino acid residues 373 and 378 is deleted. However, the inhibitory effect of POSH on ROMK channels was absent in cells expressing with dominant negative dynamin and POSHΔRING, in which the RING domain was deleted. Expression of POSH also increased the ubiquitination of ROMK1, whereas expression of POSHΔRING diminished its ubiquitination in HEK293T cells. The notion that POSH may serve as an E3 ubiquitin ligase is also supported by in vitro ubiquitination assays in which adding POSH increased the ROMK ubiquitination. We conclude that POSH inhibits ROMK channels by enhancing dynamin-dependent and clathrin-independent endocytosis and by stimulating ubiquitination of ROMK channels.ROMK channels (Kir1.1) are located in the apical membrane of the epithelial cells of the renal thick ascending limb (TAL)² and the CCD, where they are responsible for potassium recycling across the apical membrane in the TAL and potassium secretion in the CCD (1, 2). The expression of ROMK channels in the plasma membrane in the CCD is regulated by a variety of factors including protein kinases and dietary potassium intake (3–9). For instance, with-no-lysine kinase 4 (WNK4) and Src family protein-tyrosine kinase (PTK) reduce the expression of ROMK channels in the plasma membrane by stimulating dynamin-dependent endocytosis (10, 11). Several studies have demonstrated that potassium restriction decreased, and high potassium intake increased, the ROMK channel expression in the apical membrane of CCD epithelial cells (12, 13). Although the mechanism by which dietary potassium intake regulates surface expression is not completely understood, one possible mechanism is through modulating the ubiquitination of ROMK channels. The role for ubiquitination in regulating channel surface expression and endocytosis is best demonstrated by the observation that NEDD-4, an E3 ligase that contains the HECT domain (homologous to E6-AP C-terminal), regulates the ubiquitination of epithelial sodium channels (ENaC) (14–16). It has been shown that Nedd4 binds to ENaC on a PY motif (XPPXY) and causes channel internalization (17). Nedd-4 has also been reported to be responsible for ubiquitination of channels other than ENaC (18–21). We have previously demonstrated that ROMK1 channels can be monoubiquitinated and ubiquitinated ROMK channels were subjected to endocytosis (22). However, because ROMK channels lack a PY motif, it is unlikely that Nedd4 regulates ROMK channels in this fashion. POSH is a RING (really interesting new gene)-containing scaffold protein and has been suggested to be an E3 ligase for Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and Herp (homocystein-induced ER protein), and it has been shown to play an obligate role in cellular production of the human immunodeficiency virus, type 1 virus (23–25). Thus, the aim of the present study is to test whether POSH may act as an E3 ubiquitin ligase for the ubiquitination of ROMK channels.     

CFTR structure and function: is there a role in the kidney?

Cystic fibrosis (CF) is a lethal autosomal recessive genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR). Mutations in the CFTR gene may result in a defective protein processing that leads to changes in function and regulation of this chloride channel. Despite of the expression of CFTR in the kidney, patients with CF do not present major renal dysfunction, but it is known that both the urinary excretion of proteins and renal capacity to concentrate and dilute urine are altered in these patients. CFTR mRNA is expressed in all nephron segments of rat and human, and this abundance is more prominent in renal cortex and outer medulla renal areas. CFTR protein was detected in apical surface of both proximal and distal tubules of rat kidney but not in the outer medullary collecting ducts. Studies have demonstrated that CFTR does not only transport Cl⁻ but also ATP. ATP transport by CFTR could be involved in the control of other ion transporters such as Na⁺ (ENaC) and K⁺ (renal outer medullary potassium) channels, especially in TAL and CCD. In the kidney, CFTR also might be involved in the endocytosis of low-molecular-weight proteins by proximal tubules. This review is focused on the CFTR function and structure, its role in the renal physiology, and its modulation by hormones involved in the control of extracellular fluid volume.     

中华鳖肾脏的组织化学特性研究

利用光镜组织化学反应对中华鳖肾单位的结构和组织化学特性进行了详细的观察和分析。结果表明,中华鳖肾脏为分叶形的实质器官,肾小叶由被膜和实质组成,实质无髓质和皮质之分,但可以区分为外侧区和内侧区。外侧区嗜酸性,主要分布有近端小管和集合管。内侧区呈弱嗜酸性,肾小体、颈段、中间段和远端小管主要分布在内侧区。肾小球PAS反应呈阳性,但其琥珀酸脱氢酶(SDH)弱阳性,碱性磷酸酶(ALPase)、Na+/K+-ATPase和阿利新兰(AB)反应为阴性。足细胞酸性磷酸酶(ACPase)反应呈阳性。近端小管刷状缘嗜伊红,PAS反应以及ALPase、ACPase和Na+/K+-ATPase酶反应呈阳性,而SDH弱阳性。中间段、远端小管、集合管弱嗜酸性,SDH阳性。中间段Na+/K+-ATPase弱阳性。远端小管细胞侧面呈PAS阳性,腔面显示AB阳性。集合管胞质含有许多ACPase阳性颗粒,腔面呈PAS强阳性,AB阳性。甲苯胺兰(TB)染色可见集合管腔面有阳性颗粒,肾小管上皮含有亮、暗两种细胞。上述组化反应和分布结果表明,鳖的肾小管细胞类型较多,近端小管在原尿的重吸收中起主要作用,远端小管和集合管具有分泌黏液作用。中华鳖肾单位的结构与组化特性不仅与哺乳类和鸟类有一定差异,也与其他爬行动物不完全相同。