Amanda K. Welch

Endothelin-1 inhibits sodium reabsorption by ETA and ETB receptors in the mouse cortical collecting duct

The collecting duct (CD) is a major renal site for the hormonal regulation of Na homeostasis and is critical for systemic arterial blood pressure control. Our previous studies demonstrated that the endothelin-1 gene (edn1) is an early response gene to the action of aldosterone. Because aldosterone and endothelin-1 (ET-1) have opposing actions on Na reabsorption (JNa) in the kidney, we postulated that stimulation of ET-1 by aldosterone acts as a negative feedback mechanism, acting locally within the CD. Aldosterone is known to increase JNa in the CD, in part, by stimulating the epithelial Na channel (ENaC). In contrast, ET-1 increases Na and water excretion through its binding to receptors in the CD. To date, direct measurement of the quantitative effect of ET-1 on transepithelial JNa in the isolated in vitro microperfused mouse CD has not been determined. We observed that the CD exhibits substantial JNa in male and female mice that is regulated, in part, by a benzamil-sensitive pathway, presumably ENaC. ENaC-mediated JNa is greater in the cortical CD (CCD) than in the outer medullary CD (OMCD); however, benzamil-insensitive JNa is present in the CCD and not in the OMCD. In the presence of ET-1, ENaC-mediated JNa is significantly inhibited. Blockade of either ETA or ETB receptor restored JNa to control rates; however, only ETA receptor blockade restored a benzamil-sensitive component of JNa. We conclude 1) Na reabsorption is mediated by ENaC in the CCD and OMCD and also by an ENaC-independent mechanism in the CCD; and 2) ET-1 inhibits JNa in the CCD through both ETA and ETB receptor-mediated pathways.

Early progress in epigenetic regulation of endothelin pathway genes.

Control of gene transcription is a major regulatory determinant for function of the endothelin pathway. Epigenetic mechanisms act on tissue‐specific gene expression during development and in response to physiological stimuli. Most of the limited evidence available on epigenetic regulation of the endothelin pathway focuses on the EDN1 and EDNRB genes. Examination of whole genome databases suggests that both genes are influenced by histone modifications and DNA methylation. This interpretation is supported by studies directed at detecting epigenetic action on the two genes. The clearest illustration of epigenetic factors altering endothelin signalling is DNA methylation‐associated EDNRB silencing during tumourigenesis. This review summarizes our current understanding of epigenetic regulation of the endothelin pathway genes.

Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1

Aims The present study is designed to consider a role for the circadian clock protein Per1 in the regulation of the endothelin axis in mouse kidney, lung, liver and heart. Renal endothelin-1 (ET-1) is a regulator of the epithelial sodium channel (ENaC) and blood pressure (BP), via activation of both endothelin receptors, ETA and ETB. However, ET-1 mediates many complex events in other tissues. Main methods Tissues were collected in the middle of murine rest and active phases, at noon and midnight, respectively. ET-1, ETA and ETB mRNA expressions were measured in the lung, heart, liver, renal inner medulla and renal cortex of wild type and Per1 heterozygous mice using real-time quantitative RT-PCR. Key findings The effect of reduced Per1 expression on levels of mRNAs and the time-dependent regulation of expression of the endothelin axis genes appeared to be tissue-specific. In the renal inner medulla and the liver, ETA and ETB exhibited peaks of expression in opposite circadian phases. In contrast, expressions of ET-1, ETA and ETB in the lung did not appear to vary with time, but ET-1 expression was dramatically decreased in this tissue in Per1 heterozygous mice. Interestingly, ET-1 and ETA, but not ETB, were expressed in a time-dependent manner in the heart. Significance Per1 appears to regulate expression of the endothelin axis genes in a tissue-specific and time-dependent manner. These observations have important implications for our understanding of the best time of day to deliver endothelin receptor antagonists.

Hyperkalemia: getting to the heart of the matter

Hyperkalemia is a common and life-threatening complication frequently seen in patients with end-stage renal disease (ESRD), advanced chronic kidney disease (CKD) and acute kidney injury. Indeed, acute hyperkalemia is one of the most common reasons for patients requiring emergency dialysis [1]. However, hyperkalemia does not affect all patients in the same manner. Some of them persistently exhibit chronic hyperkalemia without ostensible signs or symptoms, whereas others are clearly symptomatic with the same plasma potassium concentration ([K]p). The former group appears to develop an undefined compensatory mechanism to mitigate the effects of long-term hyperkalemia. Thus, a significant variation appears to exist in the tolerance to hyperkalemia. A [K]p that causes no signs, symptoms or changes on the electrocardiogram (ECG) for one patient with chronic hyperkalemia may subject another patient to significant risk, presumably due to greater cell membrane depolarization. To date, there is no predictor of the degree of hyperkalemia a patient can tolerate without an adverse event, except for the ECG and even this direct assessment of electrical cardiac conduction has its limitations (see below). Thus, there is no diagnostic test that can determine what [K]p is acceptable for that particular patient. This knowledge would be particularly useful for the treatment of various diseases. For example, in CKD, certain drugs (angiotensin-converting enzyme inhibitors, angiotensin receptor blockers and mineralocorticoid receptors blocker) often cause hyperkalemia. Despite this, each of these drug classes has been shown to be beneficial for cardiac and renal protection. Thus, their potential to cause hyperkalemia can be significant and may outweigh the potential benefit for an individual patient. Indeed, hyperkalemia is a frequent end-point that requires discontinuation of these beneficial medicines. If there was a way to determine what degree of hyperkalemia was acceptable in an individual patient, then a more aggressive treatment plan could be enacted for that patient. As of now, most physicians feel obliged to treat hyperkalemia, particularly significant hyperkalemia ([K]p > 6.0 mEq/L) as potentially dangerous. This is based on epidemiological data that demonstrate an association of hyperkalemia with morbidity and mortality in different patient populations. Until a test is developed which would allow a physician to deem an elevated [K]p as tolerable for a particular patient, this practice should continue. All this is not to say that hyperkalemia never affects diagnostic tests. Hyperkalemia is manifested on an ECG, but the correlation between ECG changes and [K]p is imprecise: only in 50% of patients with [K]p >6.5 mEq/L changes will be seen on an ECG. A common manifestation of mild hyperkalemia (5.5–7.0 mmol/L) in ECG is the ‘tented T-waves,’ which are characterized as tall, peaked and narrow-based [2]. Unfortunately, not all patients with mild hyperkalemia exhibit tented T-waves. If there was another accurate ECG measurement that could predict whether the hyperkalemia was life-threatening, this would be a major advance to direct therapy. An ECG is a relatively noninvasive procedure that is widely available, which makes it an excellent candidate for this use. The article by Green et al. [3] from Manchester in this issue of Nephrology, Dialysis, and Transplantation aims to do just this. The authors propose a possible predictive tool based on ECG reading for hyperkalemia in ESRD and CKD stage 5. In this study, the authors examine the utility of the ratio of the T-wave to the R-wave (T:R) and whether it is more useful than the presence of tented T-waves as a predictor of hyperkalemia in patients with ESRD. The authors report that tenting was no more common in cases of hyperkalemia compared with normal serum potassium and was less common than left ventricular hypertrophy. T:R was less sensitive, but more specifically identified hyperkalemia with a serum potassium >6.0 mmol/L. They also noted that no clinical feature exhibited a correlation with the likelihood of developing abnormal T-waves in hyperkalemia. Finally, they report that abnormal T-waves in patients with hyperkalemia had greater all-cause mortality in their patient population with a mean follow-up of 3.5 years compared with those with normal T-waves. Unfortunately, this study did not provide us with a tool for guiding management of hyperkalemia within an individual patient. Neither T-wave shape or T:R ratio offered a good, predictive method for the management of hyperkalemia in ESRD. The authors remarked that younger individuals have a higher rate of ‘tented’ T-waves and that older patients naturally have lower T-waves. Because the majority of patients in the ESRD population are older

Effect of mineralocorticoid treatment in mice with collecting duct-specific knockout of endothelin-1

Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD-specific ET-1 knockout (CD ET-1 KO) mice. Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice, and by the end of the study the CD ET-1 KO mice were substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and that lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to and escape from the chronic effects of mineralocorticoids.

Manipulations in the Peripheral Stalk of the Saccharomyces cerevisiae F1F0-ATP Synthase

The Saccharomyces cerevisiae F1F0-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F1. In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F1F0-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F1F0-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.

The b arg36 contributes to efficient coupling in F 1 F O ATP synthase in Escherichia coli

In Escherichia coli, the F1FO ATP synthase b subunits house a conserved arginine in the tether domain at position 36 where the subunit emerges from the membrane. Previous experiments showed that substitution of isoleucine or glutamate result in a loss of enzyme activity. Double mutants have been constructed in an attempt to achieve an intragenic suppressor of the barg36→ile and the barg36→glu mutations. The barg36→ile mutation could not be suppressed. In contrast, the phenotypic defect resulting from the barg36→glu mutation was largely suppressed in the barg36→glu,glu39→arg double mutant. E. coli expressing the barg36→glu,glu39→arg subunit grew well on succinate-based medium. F1FO ATP synthase complexes were more efficiently assembled and ATP driven proton pumping activity was improved. The evidence suggests that efficient coupling in F1FO ATP synthase is dependent upon a basic amino acid located at the base of the peripheral stalk.

The H+- and H+, K+-ATPases of the Collecting Duct

The kidney functions to maintain ion balance via the activity of various channels, exchangers and adenosine triphosphate (ATP)-driven pumps. In the collecting duct, a H+-ATPase and two distinct H+,K+-ATPases have been localized on the apical membrane in the acid-secreting type A intercalating cells. All three of these pumps contribute to acid–base balance, and the H+,K+-ATPases may also participate in potassium conservation. The expression levels and activities of the ATP-driven H+ transporters may vary depending on physiological conditions in the organism. The goal of this chapter is to consider the molecular properties of the plasma membrane H+-ATPase and the H+,K+-ATPases of the collecting duct. The H+-ATPase is a member of the V-ATPase family. These enzymes are very large multi-subunit enzyme complexes that appear to have a common overall structure and a rotary mechanism similar to the mitochondrial F1F0-ATP synthases. In contrast, the H+,K+-ATPases are P-Type ATPases and specifically members of the P-type IIC family of cation translocating enzymes. The P-type IIC ATPases consist of an α and β subunit pair with the α subunit housing both the catalytic site and the ion channels through the membrane.