S. J. White

Regulation of distal nephron K+ channels (ROMK) mRNA expression by aldosterone in rat kidney

1 The expression of ROMK mRNA isoforms in rat kidney was measured using competitive polymerase chain reaction. Under basal conditions the expression of ROMK2 and 3 mRNA was significantly higher than that of ROMK1 or 6. 2 Administration of aldosterone for a period of 1 week significantly increased the mRNA expression not only of the α1‐subunit of the Na+‐K+‐ATPase, but also of ROMK2, 3 and 6. 3 These data not only provide evidence that ROMK K+ channels may be involved with mineralocorticoid‐sensitive K+ secretion in the distal nephron, but also demonstrate for the first time that ROMK6 may be involved in this process.

Expression of sulphonylurea receptor protein in mouse kidney

Abstract The sulphonylurea receptor (SUR) is the site of action for sulphonylurea derivatives such as glibenclamide, which are widely used as oral hypoglycaemic agents. Sulphonylureas have also been shown to affect urine flow and salt excretion by the kidney; therefore, the use of these drugs may have important implications for the pharmacological manipulation of renal salt handling. The purpose of the present investigation was to increase our understanding of the possible role of SUR in the regulation of renal function by determining the distribution of SUR isoforms within mouse kidney. Immunostaining with anti-SUR antisera revealed specific staining of SUR2B in distal nephron segments of mouse kidney. A diffuse, low level staining was observed in proximal tubules in the inner cortical region. No evidence was found for the presence of SUR2B in intra-renal blood vessels. Reverse-transcription polymerase chain reaction and Western blotting experiments indicated that SUR2B is the only known isoform expressed. These data demonstrate that SUR2B in mouse kidney is expressed in tubule regions that are critical in determining renal salt excretion.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1

Non‐technical summary  Due to the conservation of developmental pathways and genetic material over the course of evolution, non‐mammalian ‘model organisms’ such as the zebrafish embryo are emerging as valuable tools to explore causes and potential treatments for human diseases. Ion channels are proteins that form pores and help to establish and control electrical gradients by allowing the flow of ions across biological membranes. A diverse range of key physiological mechanisms in every organ in the body depends on the activity of ion channels. In this paper, we show that a potassium‐selective channel that underlies salt reabsorption and potassium excretion in the human kidney is also expressed in zebrafish in cells that are important regulators of salt balance. Disruption of the channels expression in zebrafish leads to effects on the activity of the heart, consistent with a role for this channel in the control of potassium balance in the embryo.

Renal proximal tubule function is preserved in Cftrtm2camΔF508 cystic fibrosis mice

1 Changes in proximal tubule function have been reported in cystic fibrosis patients. The aim of this study was to investigate proximal tubule function in the Cftrtm2camΔF508 cystic fibrosis (CF) mouse model. A range of techniques were used including renal clearance studies, in situ microperfusion, RT‐PCR and whole‐cell patch clamping. 2 Renal Na+ clearance was similar in wild‐type (1.4 ± 0.3 μl min−1, number of animals, N= 12) and CF mice (1.6 ± 0.4 μl min−1, N= 7) under control conditions. Acute extracellular volume expansion resulted in significant natriuresis in wild‐type (7.0 ± 0.8 μl min−1, N= 8) and CF mice (9.3 ± 1.4 μl min−1, N= 9); no difference between genotypes was observed. 3 In situ microperfusion revealed that fluid absorptive rate (Jv) was similar under control conditions between wild‐type (2.2 ± 0.4 nl mm−1 min−1, n= 10) and CF mice (1.9 ± 0.3 nl mm−1 min−1, n= 11). Addition of a forskolin‐dibutyryl cAMP (db‐cAMP) cocktail to the perfusate caused no significant change in Jv in either wild‐type (2.6 ± 0.7 nl mm−1 min−1, n= 10) or Cftrtm2camΔF508 mice (2.0 ± 0.5 nl mm−1 min−1, n= 10). 4 CFTR expression was confirmed in samples of outer cortex using RT‐PCR. However, no evidence for functional CFTR was obtained when outer cortical cells were stimulated with protein kinase A or forskolin‐db‐cAMP using whole‐cell patch clamping. 5 In conclusion, no functional deficit in proximal tubule function was found in Cftrtm2camΔF508 mice. This may be a consequence of a lack of whole‐cell cAMP‐dependent Cl− conductance in mouse proximal tubule cells.

Volume regulation is defective in renal proximal tubule cells isolated from KCNE1 knockout mice

The membrane protein KCNE1 has been implicated in cell volume regulation. Using a knockout mouse model, this study examined the role of KCNE1 in regulatory volume decrease (RVD) in freshly isolated renal proximal tubule cells. Cell diameter was measured using an optical technique in response to hypotonic shock and stimulation of Na+‐alanine cotransport in cells isolated from wild‐type and KCNE1 knockout mice. In HEPES buffered solutions 64% of wild‐type and 56% of knockout cells demonstrated RVD. In HCO−3 buffered solutions 100% of the wild‐type cells showed RVD, while in the knockout cells the proportion of cells displaying RVD remained unchanged. RVD in the knockout cells was rescued by valinomycin, a K+ ionophore. In wild‐type HCO−3 dependent cells the K+ channel inhibitors barium and clofilium inhibited RVD. These data suggest that mouse renal proximal tubule is comprised of two cell populations. One cell population is capable of RVD in the absence of HCO−3, whereas RVD in the other cell population has an absolute requirement for HCO−3. The HCO−3 dependent RVD requires the normal expression of KCNE1.

Stable, polarised, functional expression of Kir1.1b channel protein in Madin-Darby canine kidney cell line.

1 The family of Kir1.1 (ROMK) channel proteins constitute a secretory pathway for potassium in principal cells of cortical collecting duct and thick ascending limb of Henles loop. Mutations in Kir1.1 account for some types of Bartters syndrome. 2 Here we report that stable transfection of Kir1.1b (ROMK2) in Madin‐Darby canine kidney (MDCK) cell line results in expression of inwardly rectifying K+ currents and transmonolayer electrical and transport properties appropriate to Kir1.1 function. When grown on permeable supports, transfected monolayers secreted K+ into the apical solution. This secretion was inhibited by application of barium to the apical membrane, or by reduction in expression temperature from 37 to 26°C. However, whole‐cell voltage clamp electrophysiology showed that K+ conductance was higher in cells expressing Kir1.1b at 26°C. 3 To investigate this further, Kir1.1b was tagged with (EGFP), a modification that did not affect channel activity. Protein synthesis was inhibited with cycloheximide. Spectrofluorimetry was used to compare protein degradation at 37 and 26°C. The increased level of Kir1.1b at the plasma membrane at 26°C was due to an increase in protein stability. 4 Confocal microscopic investigation of EGFP‐Kir1.1b fluorescence in transfected cells showed that the channel protein was targeted to the apical domain of the cell. 5 These results demonstrate that Kir1.1b is capable of appropriate trafficking and function in MDCK cell lines at physiological temperatures. In addition, expression of Kir1.1b in MDCK cell lines provides a useful and convenient tool for the study of functional activity and targeting of secretory K+ channels.

The expression and function of cadherin-mediated cell-to-cell adhesion in human embryonal carcinoma cells.

Human embryonal carcinoma (EC) cells typically require high cell densities to maintain their characteristic phenotype; they are generally subject to differentiation when cultured at low cell densities, marked by changes in morphology and expression of the surface antigen, SSEA-1. To test whether cadherin mediated cell-to-cell adhesion may be responsible for maintaining an EC phenotype we ascertained that human EC cells generally express E- and P-cadherins, and are subject to cadherin mediated, Ca2+ dependent aggregation. However, in the NTERA2 human EC cell line, inhibition of cadherin mediated adhesion by culture in low levels of Ca2+ did not result in the changes typically seen under low cell density conditions. Low Ca2+ levels also did not affect the pattern of differentiation in these cells following induction with retinoic acid. Therefore, cadherin-mediated cell adhesion does not appear to play a role in maintaining an EC phenotype. On the other hand, culture at both low cell density and in the absence of Ca2+ did result in changes in the patterns of cadherin expression suggesting a feedback regulatory effect of cell-to-cell adhesion. Further, lithium which inhibits the cytoplasmic kinase GSK3beta and hence influences beta-catenin levels did cause differentiation of NTERA2 cells. However, consideration of the phenotype of the resultant cells suggested that this effect may be because of lithium mimicking activation of a Wnt signalling pathway, rather than an effect on signalling consequent upon cadherin mediated cell to cell adhesion.

Renal defects in KCNE1 knockout mice are mimicked by chromanol 293B in vivo: identification of a KCNE1‐regulated K+ conductance in the proximal tubule

Non‐technical summary  The kidney plays a critical role in regulating body fluid volume and blood pressure by conserving ions, solutes and water. Knowing the processes that underpin the handling of ions, solutes and water by the kidney is essential to our understanding of fluid and blood pressure regulation. Movement of ions is mediated by specific transport proteins found in the membranes of kidney cells. These proteins are regulated by additional proteins, called accessory proteins. In the current study, we have examined the role of the accessory protein KCNE1 in regulating a channel, KCNQ1, which is important in kidney function. We have observed that in the absence of KCNE1 the kidney has difficulty conserving sodium, chloride and water. However, by using specific inhibitors of these proteins we have also determined that although KCNE1 has a role in kidney function, the mechanism of its action is unlikely to be by regulating the protein KCNQ1.

Renal function defects in KCNE1 knockout mice are mimicked by chromanol 293B infusion in vivo: identification of a KCNE1-regulated and chromanol-sensitive K+ conductance in mouse renal proximal tubule cells

Non‐technical summary  The kidney plays a critical role in regulating body fluid volume and blood pressure by conserving ions, solutes and water. Knowing the processes that underpin the handling of ions, solutes and water by the kidney is essential to our understanding of fluid and blood pressure regulation. Movement of ions is mediated by specific transport proteins found in the membranes of kidney cells. These proteins are regulated by additional proteins, called accessory proteins. In the current study, we have examined the role of the accessory protein KCNE1 in regulating a channel, KCNQ1, which is important in kidney function. We have observed that in the absence of KCNE1 the kidney has difficulty conserving sodium, chloride and water. However, by using specific inhibitors of these proteins we have also determined that although KCNE1 has a role in kidney function, the mechanism of its action is unlikely to be by regulating the protein KCNQ1.