Desoxycorticosterone pivalate-salt treatment leads to non-dipping hypertension in Per1 knockout mice.

AIM: Increasing evidence demonstrates that circadian clock proteins are important regulators of physiological functions including blood pressure. An established risk factor for developing cardiovascular disease is the absence of a blood pressure dip during the inactive period. The goal of the present study was to determine the effects of a high salt diet plus mineralocorticoid on PER1-mediated blood pressure regulation in a salt-resistant, normotensive mouse model, C57BL/6J. METHODS: Blood pressure was measured using radiotelemetry. After control diet, wild-type (WT) and Per1 (KO) knockout mice were given a high salt diet (4% NaCl) and the long-acting mineralocorticoid deoxycorticosterone pivalate. Blood pressure and activity rhythms were analysed to evaluate changes over time. RESULTS: Blood pressure in WT mice was not affected by a high salt diet plus mineralocorticoid. In contrast, Per1 KO mice exhibited significantly increased mean arterial pressure (MAP) in response to a high salt diet plus mineralocorticoid. The inactive/active phase ratio of MAP in WT mice was unchanged by high salt plus mineralocorticoid treatment. Importantly, this treatment caused Per1 KO mice to lose the expected decrease or 'dip' in blood pressure during the inactive compared to the active phase. CONCLUSION: Loss of PER1 increased sensitivity to the high salt plus mineralocorticoid treatment. It also resulted in a non-dipper phenotype in this model of salt-sensitive hypertension and provides a unique model of non-dipping. Together, these data support an important role for the circadian clock protein PER1 in the modulation of blood pressure in a high salt/mineralocorticoid model of hypertension.

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. LINKED ARTICLES This article is part of a themed section on Endothelin. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2013.168.issue-1.

Quinine induced HUS-TTP: an unusual presentation.

A 67-year-old white woman developed severe nausea, vomiting, diffuse abdominal cramping pain, and blurred vision followed by a syncopal episode after taking 1 tablet of quinine for leg cramps. Examination was significant for fever, elevated blood pressure, and confusion without any focal neurological deficits. Laboratory studies showed markedly elevated liver enzymes, elevated lactate dehydrogenase, anemia, thrombocytopenia, and acute renal failure. Peripheral smear showed many schistocytes and burr cells. She later recalled taking quinine more than 40 years before while on a trip to the Philippines. The patient was treated with 7 sessions of plasmapheresis with a rapid normalization of her hematological parameters. Three weeks of dialysis support were required before return of renal function to baseline. Re-exposure to quinine can cause a rapid onset of hemolytic uremic syndrome-like syndrome. We are not aware of any cases of hemolytic uremic syndrome-thrombotic thrombocytopenic purpura in response to re-exposure to a single tablet of the drug 40 years after first use.

Immunohistochemical localization of H-K-ATPase alpha(2c)-subunit in rabbit kidney.

The rabbit kidney possesses mRNA for the H-K-ATPase alpha(1)-subunit (HKalpha(1)) and two splice variants of the H-K-ATPase alpha(2)-subunit (HKalpha(2)). The purpose of this study was to determine the specific distribution of one of these, the H-K-ATPase alpha(2c)-subunit isoform (HKalpha(2c)), in rabbit kidney by immunohistochemistry. Chicken polyclonal antibodies against a peptide based on the NH(2) terminus of HKalpha(2c) were used to detect HKalpha(2c) immunoreactivity in tissue sections. Immunohistochemical localization of HKalpha(2c) revealed intense apical immunoreactivity in a subpopulation of cells in the connecting segment, cortical collecting duct, and outer medullary collecting duct in both the outer and inner stripe. An additional population of cells exhibited a thin apical band of immunolabel. Immunohistochemical colocalization of HKalpha(2c) with carbonic anhydrase II, the Cl(-)/HCO exchanger AE1, and HKalpha(1) indicated that both type A and type B intercalated cells possessed intense apical HKalpha(2c) immunoreactivity, whereas principal cells and connecting segment cells had only a thin apical band of HKalpha(2c). Labeled cells were evident through the middle third of the inner medullary collecting duct in the majority of animals. Immunolabel was also present in papillary surface epithelial cells, cells in the cortical thick ascending limb of Henle's loop (cTAL), and the macula densa. Thus in the rabbit kidney, apical HKalpha(2c) is present and may contribute to acid secretion or potassium uptake throughout the connecting segment and collecting duct in both type A and type B intercalated cells, principal cells, and connecting segment cells, as well as in cells in papillary surface epithelium, cTAL, and macula densa.

Increased CO(2) stimulates K/Rb reabsorption mediated by H-K-ATPase in CCD of potassium-restricted rabbit.

Apical H-K-ATPase in the cortical collecting duct (CCD) plays an important role in urinary acidification and K reabsorption. Our previous studies demonstrated that an H-K-ATPase mediates, in part, Rb reabsorption in rabbit CCD (Zhou X and Wingo CS. Am J Physiol Renal Fluid Electrolyte Physiol 263: F1134-F1141, 1992). The purpose of these experiments was to examine using in vitro microperfused CCD from K-restricted rabbits 1) whether an acute increase in PCO(2) and, presumably, intracellular acidosis stimulate K absorptive flux; and 2) whether this stimulation was dependent on the presence of a functional H-K-ATPase. Rb reabsorption was significantly increased after exposure to 10% CO(2) in CCD, and this effect was persistent for the entire 10% CO(2) period, whereas 10 microM SCH-28080 in the perfusate totally abolished the stimulation of Rb reabsorption by 10% CO(2). After stimulation of Rb reabsorption by 10% CO(2), subsequent addition of 0.1 mM methazolamide, an inhibitor of carbonic anhydrase, failed to affect Rb reabsorption. However, simultaneous exposure to 10% CO(2) and methazolamide prevented the stimulation of Rb reabsorption. Treatment with the intracellular calcium chelator MAPTAM (0.5 microM) inhibited the stimulation of Rb reabsorption by 10% CO(2). Similar inhibition was also observed in the presence of either a calmodulin inhibitor, W-7 (0.5 microM), or colchicine (0.5 mM), an inhibitor of tubulin polymerization. In time control studies, the perfusion time did not significantly affect Rb reabsorption. We conclude the following: 1) stimulation of Rb reabsorption on exposure to 10% CO(2) is dependent on the presence of a functional H-K-ATPase and appears to be regulated in part by the insertion of this enzyme into the apical plasma membrane by exocytosis; 2) insertion of H-K-ATPase requires changes in intracellular pH and needs a basal level of intracellular calcium concentration; and 3) H-K-ATPase insertion occurs by a microtubule-dependent process.

Apical membrane of native OMCD(i) cells has nonselective cation channels.

The purpose of this study was to examine cation channel activity in the apical membrane of the outer medullary collecting duct of the inner stripe (OMCD(i)) using the patch-clamp technique. In freshly isolated and lumen-opened rabbit OMCD(i), we have observed a single channel conductance of 23.3 +/- 0.6 pS (n = 17) in cell-attached (c/a) patches with high KCl in the bath and in the pipette at room temperature. Channel open probability varied among patches from 0.06 +/- 0.01 at -60 mV (n = 5) to 0.31 +/- 0.04 at 60 mV (n = 6) and consistently increased upon membrane depolarization. In inside-out (i/o) patches with symmetrical KCl solutions, the channel conductance (22.8 +/- 0.8 pS; n = 10) was similar as in the c/a configuration. Substitution of the majority of Cl- with gluconate from KCl solution in the pipette and bath did not significantly alter reversal potential (E(rev)) or the channel conductance (19.7 +/- 1.1 pS in asymmetrical potassium gluconate, n = 4; 21.4 +/- 0.5 pS in symmetrical potassium gluconate, n = 3). Experiments with 10-fold lower KCl concentration in bath solution in i/o patches shifted E(rev) to near the E(rev) of K+. The estimated permeability of K+ vs. Cl- was over 10, and the conductance was 13.4 +/- 0.1 pS (n = 3). The channel did not discriminate between K+ and Na+, as evidenced by a lack of a shift in the E(rev) with different K+ and Na+ concentration solutions in i/o patches (n = 3). The current studies demonstrate the presence of cation channels in the apical membrane of native OMCD(i) cells that could participate in K+ secretion or Na+ absorption.

Proteinuria: potential causes and approach to evaluation.

Proteinuria may be associated with a renal or systemic disease, or it may be isolated. The latter occurs in asymptomatic patients without evidence of any disease or abnormality of the urine sediment. Isolated proteinuria may be subdivided into two broad groups: (1) benign forms, with a favorable-to-excellent prognosis and (2) persistent forms, some of which have a worrisome prognosis. Functional proteinuria may occur in disorders with altered renal hemodynamics, usually resolves, and is not associated with progressive renal disease. Idiopathic transient proteinuria is typically discovered on routine screening and usually disappears on subsequent testing. In idiopathic intermittent proteinuria, a significant number (50%) of urine samples exhibit abnormal rates of protein excretion. Although structural abnormalities may be observed on renal biopsy, progressive renal insufficiency is unusual. In orthostatic proteinuria, the rate of protein excretion completely normalizes in the recumbent position. Long-term studies show this to be a benign condition. In persistent isolated proteinuria, at least 80% of random urine samples exhibit abnormal protein excretion. This represents a heterogeneous group, but a significant proportion of these patients have prominent renal pathologic findings and progress to serious renal disease. Proteinuria with significant renal disease may be non-nephrotic or nephrotic range. The former does not exclude glomerular disease, but tubulointerstitial or vascular disorders are also likely when proteinuria is less than 2 g/24 hours. Patients with nephrotic-range proteinuria generally have a glomerular disorder. Distinction between benign and more ominous forms of proteinuria requires careful evaluation.

Activation of H(+)-K(+)-ATPase by CO(2) requires a basolateral Ba(2+)-sensitive pathway during K restriction.

We studied the activation of H(+)-K(+)-ATPase by CO(2) in the renal cortical collecting duct (CCD) of K-restricted animals. Exposure of microperfused CCD to 10% CO(2) increased net total CO(2) flux (J(t CO(2))) from 4.9 +/- 2.1 to 14.7 +/- 4 pmol. mm(-1). min(-1) (P < 0. 05), and this effect was blocked by luminal application of the H(+)-K(+)-ATPase inhibitor Sch-28080. In the presence of luminal Ba, a K channel blocker, exposure to CO(2) still stimulated J(t CO(2)) from 6.0 +/- 1.0 to 16.8 +/- 2.8 pmol. mm(-1). min(-1) (P < 0.01), but peritubular application of Ba inhibited the stimulation. CO(2) substantially increased (86)Rb efflux (a K tracer marker) from 93.1 +/- 23.8 to 249 +/- 60.2 nm/s (P < 0.05). These observations suggest that during K restriction 1) the enhanced H(+)-K(+)-ATPase-mediated acidification after exposure to CO(2) is dependent on a basolateral Ba-sensitive mechanism, which is different from the response of rabbits fed a normal-K diet, where activation of the H(+)-K(+)-ATPase by exposure to CO(2) is dependent on an apical Ba-sensitive pathway; and 2) K/Rb absorption via the apical H(+)-K(+)-ATPase exits through a basolateral Ba-sensitive pathway. Together, these data are consistent with the hypothesis of cooperation between H(+)-K(+)-ATPase-mediated acidification and K exit pathways in the CCD that regulate K homeostasis.

Effects of ammonia on bicarbonate transport in the cortical collecting duct.

Both acidosis and hypokalemia stimulate renal ammoniagenesis, and both regulate urinary proton and potassium excretion. We hypothesized that ammonia might play an important role in this processing by stimulating H(+)-K(+)-ATPase-mediated ion transport. Rabbit cortical collecting ducts (CCD) were studied using in vitro microperfusion, bicarbonate reabsorption was measured using microcalorimetry, and intracellular pH (pH(i)) was measured using the fluorescent, pH-sensitive dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Ammonia caused a concentration-dependent increase in net bicarbonate reabsorption that was inhibited by luminal addition of either of the H(+)-K(+)-ATPase inhibitors, Sch-28080 or ouabain. The stimulation of net bicarbonate reabsorption was not mediated through apical H(+)-ATPase, basolateral Na(+)-K(+)-ATPase, or luminal electronegativity. Although ammonia caused intracellular acidification, similar changes in pH(i) induced by inhibiting basolateral Na(+)/H(+) exchange did not alter net bicarbonate reabsorption. We conclude that ammonia regulates CCD proton and potassium transport, at least in part, by stimulating apical H(+)-K(+)-ATPase.

Molecular identification of the renal H+,K+-ATPases.

The pharmacological properties of H+,K+-ATPase activity described in the kidney were not necessarily consistent with the properties of the well-characterized gastric H+,K+-ATPase. Recent molecular biology experiments suggest that renal H+,K+-ATPase activity may be the product of several closely related P-type ATPases. At least 3 different pumps containing the HKalpha1, HKalpha2a, and HKalpha2c subunits have been detected in rabbit kidney. The current view is that these HKalpha subunits arose through gene duplication early in evolution and the proteins evolved their differing activities over time. The HKbeta protein associates with HKalpha1 in gastric tissues and is the likely mate for the HKalpha1 subunit in renal tissues. Three distinct beta subunits have been implicated as possible partners for the HKalpha2 subunits, but it remains to be determined which beta subunit predominantly associates with the HKalpha2 subunits in vivo. Sequence analysis suggests the beta subunit was constrained by size and shape of the protein rather than specific amino acid content during the course of evolution. Multiple H+,K+-ATPases in the kidney may be an important adaptation providing redundancy for the essential physiological function of maintaining ionic balance.

Cation channels of the rabbit outer medullary collecting duct.

The outer medullary collecting duct (OMCD) produces high rates of proton secretion, and previous data show a significant role for an H+,K+-ATPase in luminal acidification. This mechanism of acidification requires proton secretion to occur in exchange for potassium absorption. Because net transport of potassium is small in the OMCD, pathways for potassium secretion would appear essential to explain the contribution of an H+,K+-ATPase under potassium-replete conditions. A significant issue is whether a potassium exit pathway (i.e., cation channel) is present at the apical and/or the basolateral membrane. In this report, using patch-clamp techniques to examine single channel conductances from the native rabbit OMCD, we show that the apical membrane has potassium permeable cation channels.

Apical proton secretion by the inner stripe of the outer medullary collecting duct.

The inner stripe of outer medullary collecting duct (OMCDis) is unique among collecting duct segments because both intercalated cells and principal cells secrete protons and reabsorb luminal bicarbonate. The current study characterized the mechanisms of OMCDis proton secretion. We used in vitro microperfusion, and we separately studied the principal cell and intercalated cell using differential uptake of the fluorescent, pH-sensitive dye, 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). Both the principal cell and intercalated cell secreted protons, as identified as Na+/H+ exchange-independent intracellular pH (pHi) recovery from an intracellular acid load. Two proton transport activities were identified in the principal cell; one was luminal potassium dependent and Sch-28080 sensitive and the other was luminal potassium independent and luminal bafilomycin A1 sensitive. Thus the OMCDis principal cell expresses both apical H+-K+-ATPase and H+-ATPase activity. Intercalated cell Na+/H+ exchange-independent pHi recovery was approximately twice that of the principal cell and was mediated by pharmacologically similar mechanisms. We conclude 1) the OMCDis principal cell may contribute to both luminal potassium reabsorption and urinary acidification, roles fundamentally different from those of the principal cell in the cortical collecting duct; and 2) the OMCDis intercalated cell proton transporters are functionally similar to those in the principal cell, raising the possibility that an H+-K+-ATPase similar to the one present in the principal cell may contribute to intercalated cell proton secretion.

H-K-ATPase in the RCCT-28A rabbit cortical collecting duct cell line.

In the present study, we demonstrate that the rabbit cortical collecting duct cell line RCCT-28A possesses three distinct H-K-ATPase catalytic subunits (HKalpha). Intracellular measurements of RCCT-28A cells using the pH-sensitive dye 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) indicated that the mechanism accounting for recovery from an acid load exhibited both K+ dependence and sensitivity to Sch-28080 characteristic of H-K-ATPases. Recovery rates were 0.022 +/- 0.005 pH units/min in the presence of K+, 0.004 +/- 0.002 in the absence of K+, and 0.002 +/- 0.002 in the presence of Sch-28080. The mRNAs encoding the HKalpha1 subunit and the H-K-ATPase beta-subunit (HKbeta) were detected by RT-PCR. In addition, two HKalpha2 species were found by RT-PCR and 5' rapid amplification of cDNA ends (5'-RACE) in the rabbit renal cortex. One was homologous to HKalpha2 cDNAs generated from other species, and the second was novel. The latter, referred to as HKalpha2c, encoded an apparent 61-residue amino-terminal extension that bore no homology to reported sequences. Antipeptide antibodies were designed on the basis of this extension, and these antibodies recognized a protein of the appropriate mass in both rabbit renal tissue samples and RCCT-28A cells. Such findings constitute very strong evidence for expression of the HKalpha2c subunit in vivo. The results suggest that the rabbit kidney and RCCT-28A cells express at least three distinct H-K-ATPases.

Chloride transport by the rabbit cortical collecting duct: dependence on H,K-ATPase.

The rabbit cortical collecting duct (CCD) exhibits the capacity for active chloride absorption when basolateral Na-K-ATPase is inhibited by ouabain. The present studies examine the contribution of H,K-ATPase to this ouabain-insensitive Cl absorption and to related ion fluxes. Rabbits were fed a KCl-rich diet with no measurable Na for 4 to 13 d before isolation of the CCD for microperfusion. Application of peritubular ouabain (0.1 mM) significantly increased (P < 0.001) net luminal absorptive chloride flux (J(N)Cl) without an effect on lumen-to-bath isotopic 36Cl flux (J(lb)Cl). The H,K-ATPase inhibitor Sch 28080 (1 to 10 microM) abolished ouabain-insensitive J(N)Cl, but transepithelial voltage (V(T)) was not significantly affected. The contribution of H,K-ATPase activity on active Cl flux (J(A)Cl) and passive Cl flux (J(P)Cl) was also assessed. Ouabain significantly increased J(A)Cl and Sch 28080 inhibited J(A)Cl, but J(P)Cl was not affected by Sch 28080. To assess the contribution of changes in net bicarbonate flux (JtCO2) to changes in J(N)Cl, JtCO2 was measured under identical conditions as for J(N)Cl. Ouabain significantly increased JtCO2, and this ouabain-insensitive bicarbonate flux was inhibited by Sch 28080 without significantly affecting V(T). To assess the possibility that the CCD may possess mechanisms for neutral salt absorption, lumen-to-bath 86Rb efflux (K(Rb)), and 22Na efflux (K(Na)) were also measured. Ouabain significantly increased K(Rb), and Sch 28080 inhibited this ouabain-insensitive K(Rb). Furthermore, Sch 28080 and A80915a (a structurally distinct H,K-ATPase inhibitor) significantly inhibited K(Na) in the presence of 1 mM luminal amiloride. These observations suggest that, in addition to potassium, sodium can be transported via the H,K-ATPase. Although the CCD contains more than one cell population, the data could be fitted very well to the function of the B-type intercalated cell. A cell model is proposed for the hypothesis that ouabain-insensitive chloride absorption is mediated by the parallel operation of an apical H,K-ATPase with an apical Cl-HCO3 exchanger and that the H,K-ATPase can function, under certain conditions, as a mechanism of Na absorption.

Increased superoxide anion generation and altered vasoreactivity in rabbits on low-potassium diet.

Potassium reduces blood pressure in populations at high risk of developing hypertension, which suggests that potassium depletion may increase vascular resistance. This study was designed to examine the effect of potassium depletion on the L-arginine-nitric oxide pathway in arterial tissues. New Zealand White rabbits were fed either a control diet, containing a normal amount of potassium, or a low-potassium diet for 1-3 wk. As expected, the low-potassium diet resulted in reduced serum and urinary potassium levels. Carotid arteries were excised, and their contractile and relaxant responses were determined in vitro. Carotid arterial ring contractile response to norepinephrine was enhanced, and relaxation in response to the endothelium-dependent vasodilators acetylcholine and calcium ionophore A-23187 was attenuated, in rabbits fed low-potassium diet (all P < 0.01 compared with responses in rabbits fed control diet). The vasomotor responses were similarly altered in rabbits fed low-potassium diet for 1 or 3 wk. Both the enhanced contraction and attenuated relaxation were abolished by treatment of arterial rings with superoxide dismutase but not by treatment with L-arginine or indomethacin. Carotid artery rings from rabbits fed the low-potassium diet showed approximately 100% greater superoxide anion formation than those from rabbits fed control diet (P < 0.01), whereas plasma and urinary nitrite levels were similar in both groups of rabbits. These observations indicate that low-potassium diet enhances the sensitivity of the carotid artery to vasoconstrictor stimuli and reduces the sensitivity to endothelium-dependent stimuli. Attenuation of endothelium-dependent relaxation appears to be secondary to increased free radical generation, which may degrade nitric oxide. Altered vasoreactivity may underlie the genesis of hypertension in populations consuming diets low in potassium.

Are academic medical societies needed in a changing healthcare arena?

Medical research has been demonstrated to increase the quality of life for all Americans and to be a sound investment with clear financial savings. However, academic health centers, which are the major source of medical research, have experienced continued erosion of their financial base of support. The author reviews some of the elements which have precipitated the current crisis for many academic health centers and proposes short- and long-term goals that should be endorsed by academic medical societies as a means of reducing these institutions' financial dependence on clinical income and restoring to them their primary social mission: to train the highest caliber of practicing physicians and to be centers of fundamental, not technological, research. Such research has been demonstrated to reduce, not increase, health care costs. Academic medical societies must work together to educate the public and Congress about the needs of academic health care centers, and to provide a cohesive and constructive set of practical recommendations that can strengthen their future financial stability.

The effects of potassium depletion and supplementation on blood pressure: a clinical review.

Nonpharmacologic treatment currently is recognized as an important part in the treatment of hypertension, and the role of dietary potassium intake in blood pressure (BP) control is becoming quite evident. Clinical studies have examined the mechanism by which hypokalemia can increase BP and the benefit of a large potassium intake on BP control. Epidemiologic data suggest that potassium intake and BP are correlated inversely. In normotensive subjects, those who are salt sensitive or who have a family history of hypertension appear to benefit most from the hypotensive effects of potassium supplementation. The greatest hypotensive effect of potassium supplementation occurs in patients with severe hypertension. This effect is pronounced with prolonged potassium supplementation. The antihypertensive effect of increased potassium intake appears to be mediated by several factors, which include enhancing natriuresis, modulating baroreflex sensitivity, direct vasodilation, or lowering cardiovascular reactivity to norepinephrine or angiotensin II. Potassium repletion in patients with diuretic-induced hypokalemia improves BP control. An increase in potassium intake should be included in the nonpharmacologic management of patients with uncomplicated hypertension.