Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger.

NHE3 is one of five plasma membrane Na+/H+ exchangers and is encoded by the mouse gene Slc9a3. It is expressed on apical membranes of renal proximal tubule and intestinal epithelial cells and is thought to play a major role in NaCl and HCO3- absorption. As the distribution of NHE3 overlaps with that of the NHE2 isoform in kidney and intestine, the function and relative importance of NHE3 in vivo is unclear. To analyse its physiological functions, we generated mice lacking NHE3 function. Homozygous mutant (Slc9a3-/-) mice survive, but they have slight diarrhoea and blood analysis revealed that they are mildly acidotic. HCO3- and fluid absorption are sharply reduced in proximal convoluted tubules, blood pressure is reduced and there is a severe absorptive defect in the intestine. Thus, compensatory mechanisms must limit gross perturbations of electrolyte and acid-base balance. Plasma aldosterone is increased in NHE3-deficient mice, and expression of both renin and the AE1 (Slc4a1) Cl-/HCO3- exchanger mRNAs are induced in kidney. In the colon, epithelial Na+ channel activity is increased and colonic H+,K+-ATPase mRNA is massively induced. These data show that NHE3 is the major absorptive Na+/H+ exchanger in kidney and intestine, and that lack of the exchanger impairs acid-base balance and Na+-fluid volume homeostasis.

Association between benzodiazepine receptor agonist use and mortality in patients hospitalised for COVID-19: a multicentre observational study.

AIMS: To examine the association between benzodiazepine receptor agonist (BZRA) use and mortality in patients hospitalised for coronavirus disease 2019 (COVID-19). METHODS: A multicentre observational study was performed at Greater Paris University hospitals. The sample involved 14 381 patients hospitalised for COVID-19. A total of 686 (4.8%) inpatients received a BZRA at hospital admission at a mean daily diazepam-equivalent dose of 19.7 mg (standard deviation (s.d.) = 25.4). The study baseline was the date of admission, and the primary endpoint was death. We compared this endpoint between patients who received BZRAs and those who did not in time-to-event analyses adjusted for sociodemographic characteristics, medical comorbidities and other medications. The primary analysis was a Cox regression model with inverse probability weighting (IPW). RESULTS: Over a mean follow-up of 14.5 days (s.d. = 18.1), the primary endpoint occurred in 186 patients (27.1%) who received BZRAs and in 1134 patients (8.3%) who did not. There was a significant association between BZRA use and increased mortality both in the crude analysis (hazard ratio (HR) = 3.20; 95% confidence interval (CI) = 2.74-3.74; p < 0.01) and in the IPW analysis (HR = 1.61; 95% CI = 1.31-1.98, p < 0.01), with a significant dose-dependent relationship (HR = 1.55; 95% CI = 1.08-2.22; p = 0.02). This association remained significant in sensitivity analyses. Exploratory analyses indicate that most BZRAs may be associated with an increased mortality among patients hospitalised for COVID-19, except for diazepam, which may be associated with a reduced mortality compared with any other BZRA treatment. CONCLUSIONS: BZRA use may be associated with an increased mortality among patients hospitalised for COVID-19, suggesting the potential benefit of decreasing dose or tapering off gradually these medications when possible.

A comparative analysis of four clinical guidelines for hypertension management.

We compared the structure and content of guidelines for hypertension management across countries to gain an understanding of where differences between them originate from. Four guidelines published between 2003 and 2006 were selected. Two were issued by national agencies in the United Kingdom and France, and two were issued by working groups or national medical societies in the United States and in Europe. The structure of guidelines, the content of each section and their underlying bibliographic references were compared between authoring bodies. If differences were found between guidelines in terms of content, we analysed the rationales. The guidelines were sufficiently similar in structure, showing common sections such as lifestyle interventions, cardiovascular risk assessment and drug therapies. However, contentwise, major differences were observed across the four hypertension guidelines in virtually every section of the document. The definition of hypertension was consistent, whereas the grade stratification was not. Information concerning the blood pressure self-measurement, the estimation of cardiovascular risk and the antihypertensive drugs proposed for initial treatment also varied. Most of the differences were present in both guidelines and their rationales, but some were only found in the guidelines. The bibliographic references for the rationales differed significantly, with only 1.2, 2.2 and 8.8% of the total number of references were common to four, three and two authoring bodies, accounting for the variability. We conclude that improving the selection process of bibliographic references and the extraction process of guidelines from the rationales might be the first step to harmonize guidelines' development.

High plasma aldosterone and low renin predict blood pressure increase and hypertension in middle-aged Caucasian populations.

Plasma aldosterone and renin levels have been associated with blood pressure increase and 3-4 year incidence of hypertension in a middle-aged North American community in Framingham. To confirm these findings in a different population, a nested case-control study was performed in a national sample of 1984 French non-hypertensive volunteers aged 45-64 year and followed for 5 years. Cases and controls (individuals becoming hypertensive or remaining non-hypertensive on follow-up) were individually matched on sex, diastolic and systolic pressures at baseline. Multivariable regression models show that plasma aldosterone and renin are respectively positively and negatively associated with the increase in systolic pressure (P=0.01 and 0.001) and the risk of hypertension (22% increase and 16% decrease per s.d. increment in the log, P=0.04 and 0.07). These associations are mostly observed in the lowest tertiles of dietary sodium and potassium intakes where plasma aldosterone is positively associated with the increase in systolic pressure (P=0.01 and 0.08) and the risk of hypertension (59 and 69% increase per s.d. increment in the log, P=0.02 and 0.01), whereas plasma renin is negatively associated with the increase in systolic pressure (P=0.0004 and 0.004) and the risk of hypertension (31 and 28% decrease per s.d. increment in the log, P=0.03 and 0.05). These results reinforce the hypothesis that high plasma aldosterone and low plasma renin levels precede blood pressure increase and the occurrence of hypertension in middle-aged Caucasian populations.

Cardioprotection and kallikrein-kinin system in acute myocardial ischaemia in mice.

1. Acute myocardial ischaemia and reperfusion trigger cardioprotective mechanisms that tend to limit myocardial injury. These cardioprotective mechanisms remain for a large part unknown, but can be potentiated by performing ischaemic preconditioning or by administering drugs such as angiotensin-I-converting enzyme (kininase II) inhibitors (ACEI). 2. This brief review summarizes the findings concerning the role of tissue kallikrein (TK), a major kinin-forming enzyme, kinins and kinin receptors in the cardioprotection afforded by ischaemic preconditioning (IPC) or by pharmacological postconditioning by drugs originally targeted at the renin-angiotensin system, ACEI and type 1 angiotensin-II receptor blockers (ARB) in acute myocardial ischaemia. Myocardial ischaemia was induced by left coronary occlusion and was followed after 30 min by a 3 h reperfusion period (IR), performed in vivo in mice. The role of the kallikrein-kinin system (KKS) was studied by using genetically engineered mice deficient in TK gene and their wild-type littermates, or by blocking B1 or B2 bradykinin receptors in wild-type mice using selective pharmacological antagonists. 3. Ischaemic preconditioning (three cycles: 3 min occlusion/5 min reperfusion) enhances the ability of the heart of wild-type mice to tolerate IR. Tissue kallikrein plays a major role in the cardioprotective effect afforded by IPC, which is largely reduced in TK-deficient mice. The B2 receptor is the main kinin receptor involved in the cardioprotective effect of IPC. 4. Tissue kallikrein is also required for the cardioprotective effects of pharmacological postconditioning with ACEI (ramiprilat) or ARB (losartan), which are abolished for both classes of drugs in TK-deficient mice. The B2 receptor mediates the cardioprotective effects of these drugs. Activation of angiotensin-II type 2 (AT2) receptor is involved in the cardioprotective effects of losartan, suggesting a functional coupling between AT2 receptor and TK during angiotensin-II type 1 (AT1) receptor blockade. 5. The demonstration of a cardioprotective effect of the KKS in acute myocardial ischaemia involving TK and the B2 receptor and playing a major role in IPC or pharmacological postconditioning by ACEI or ARB, suggests a potential therapeutic approach based on pharmacological activation of the B2 receptor.

Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion.

Multiple isoforms of the Na+/H+ exchanger (NHE) are expressed at high levels in gastric epithelium, but the physiological role of individual isoforms is unclear. To study the function of NHE2, which is expressed in mucous, zymogenic, and parietal cells, we prepared mice with a null mutation in the NHE2 gene. Homozygous null mutants exhibit no overt disease phenotype, but the cellular composition of the oxyntic mucosa of the gastric corpus is altered, with parietal and zymogenic cells reduced markedly in number. Net acid secretion in null mutants is reduced slightly relative to wild-type levels just before weaning and is abolished in adult animals. Although mature parietal cells are observed, and appear morphologically to be engaged in active acid secretion, many of the parietal cells are in various stages of degeneration. These results indicate that NHE2 is not required for acid secretion by the parietal cell, but is essential for its long-term viability. This suggests that the unique sensitivity of NHE2 to inhibition by extracellular H+, which would allow upregulation of its activity by the increased interstitial alkalinity that accompanies acid secretion, might enable this isoform to play a specialized role in maintaining the long-term viability of the parietal cell.

Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice.

Previous studies using isolated tissues suggest that the colonic H, K-ATPase (cHKA), expressed in the colon and kidney, plays an important role in K+ conservation. To test the role of this pump in K+ homeostasis in vivo, we generated a cHKA-deficient mouse and analyzed its ability to retain K+ when fed a control or K+-free diet. When maintained on a control diet, homozygous mutant (cHKA-/-) mice exhibited no deficit in K+ homeostasis compared to wild-type (cHKA+/+ greater, similar mice. Although fecal K+ excretion in cHKA-/- mice was double that of cHKA+/+ mice, fecal K+ losses were low compared with urinary K+ excretion, which was similar in both groups. When maintained on a K+-free diet for 18 d, urinary K+ excretion dropped over 100-fold, and to similar levels, in both cHKA-/- and cHKA+/+ mice; fecal K+ excretion was reduced in both groups, but losses were fourfold greater in cHKA-/- than in cHKA+/+ mice. Because of the excess loss of K+ in the colon, cHKA-/- mice exhibited lower plasma and muscle K+ than cHKA+/+ mice. In addition, cHKA-/- mice lost twice as much body weight as cHKA+/+ mice. These results demonstrate that, during K+ deprivation, cHKA plays a critical role in the maintenance of K+ homeostasis in vivo.

Role of tissue kallikrein in the cardioprotective effects of ischemic and pharmacological preconditioning in myocardial ischemia.

Tissue kallikrein (TK), a major kinin-forming enzyme, is synthesized in the heart and arteries. We tested the hypothesis that TK plays a protective role in myocardial ischemia by performing ischemia-reperfusion (IR) injury, with and without ischemic preconditioning (IPC) or ACE inhibitor (ramiprilat) pretreatment, in vivo in littermate wild-type (WT) or TK-deficient (TK-/-) mice. IR induced similar infarcts in WT and TK-/-. IPC reduced infarct size by 65% in WT, and by 40% in TK-/- (P<0.05, TK-/- vs WT). Ramiprilat also reduced infarct size by 29% in WT, but in TK-/- its effect was completely suppressed. Pretreatment of WT with a B2, but not a B1, kinin receptor antagonist reproduced the effects of TK deficiency. However, B2 receptor-deficient mice (B2-/-) unexpectedly responded to IPC or ramiprilat like WT mice. But pretreatment of the B2-/- mice with a B1 antagonist suppressed the cardioprotective effects of IPC and ramiprilat. In B2-/-, B1 receptor gene expression was constitutively high. In WT and TK-/- mice, both B2 and B1 mRNA levels increased several fold during IR, and even more during IPC+IR. Thus TK and the B2 receptor play a critical role in the cardioprotection afforded by two experimental maneuvers of potential clinical relevance, IPC and ACE inhibition, during ischemia.

Decreased flow-dependent dilation in carotid arteries of tissue kallikrein-knockout mice.

- Flow-dependent dilation is a fundamental mechanism by which large arteries ensure appropriate blood supply to tissues. We investigated whether or not the vascular kallikrein-kinin system, especially tissue kallikrein (TK), contributes to flow-dependent dilation by comparing wild-type and TK-knockout mice in which the presence or absence of TK expression was verified. We examined in vitro changes in the outer diameter of perfused carotid arteries from TK(+/+) and TK(-/-) mice. In both groups, exogenous bradykinin caused a similar dilation that was abolished by the B(2) receptor antagonist HOE-140, as well as by the NO synthase inhibitor N:(omega)-nitro-L-arginine methyl ester. However, purified kininogen dilated only TK(+/+) arteries, demonstrating the essential role of TK in the vascular formation of kinins. In TK(+/+) arteries, increasing intraluminal flow caused a larger endothelium-dependent dilation than that seen in TK(-/-). In both strains the flow response was mediated by NO and by endothelium-derived hyperpolarizing factor, whereas in TK(-/-) vasoconstrictor prostanoids participated as well. HOE-140 impaired flow-dependent dilation in TK(+/+) arteries while showing no effect in TK(-/-). This compound reduced the flow response in TK(+/+) arteries to a level similar to that in TK(-/-). After NO synthase inhibition, HOE-140 no longer affected the response of TK(+/+). Impaired flow-dependent dilation was also observed in arteries from knockout mice lacking bradykinin B(2) receptors as compared with wild-type animals. This study demonstrates the active contribution of the vascular kallikrein-kinin system to one-third of the flow-dependent dilation response via activation of B(2) receptors coupled to endothelial NO release.

Adipose angiotensinogen is involved in adipose tissue growth and blood pressure regulation.

White adipose tissue and liver are important angiotensinogen (AGT) production sites. Until now, plasma AGT was considered to be a reflection of hepatic production. Because plasma AGT concentration has been reported to correlate with blood pressure, and to be associated with body mass index, we investigated whether adipose AGT is released locally and into the blood stream. For this purpose, we have generated transgenic mice either in which adipose AGT is overexpressed or in which AGT expression is restricted to adipose tissue. This was achieved by the use of the aP2 adipocyte-specific promoter driving the expression of rat agt cDNA in both wild-type and hypotensive AGT-deficient mice. Our results show that in both genotypes, targeted expression of AGT in adipose tissue increases fat mass. Mice whose AGT expression is restricted to adipose tissue have AGT circulating in the blood stream, are normotensive, and exhibit restored renal function compared with AGT-deficient mice. Moreover, mice that overexpress adipose AGT have increased levels of circulating AGT, compared with wild-type mice, and are hypertensive. These animal models demonstrate that AGT produced by adipose tissue plays a role in both local adipose tissue development and in the endocrine system, which supports a role of adipose AGT in hypertensive obese patients.

Involvement of renal apical Na transport systems in the control of blood pressure.

Human genetic studies and gene targeting techniques in mice suggest that the genes encoding renal apical Na transport proteins play an essential role in the control of extracellular fluid volume and blood pressure. Particularly, very significant advancements in understanding the role of these genes in Mendelian forms of hypertension or hypotension have been achieved in recent years. However, much progress still needs to be made in understanding the more common forms of human essential hypertension. In addition to the mouse models that should be very useful for investigating the mechanisms by which a mutation provokes the hypertensive phenotype, improved clinical phenotyping of patients is needed as well as the use of DNA chip techniques to unravel global gene interactions. Indeed, it is likely that most chronic blood pressure disturbances in a given environment result from a specific combination of polymorphisms or mutations rather than from unique genetic variants. Of equal importance will be definition of the various factors that regulate the expression and activity of the Na transport systems. These regulatory pathways and the responses to environmental factors such as dietary salt, stress, etc, may play a central role in determining the appearance, severity, and complications of essential hypertension.

Potassium ATPases, channels, and transporters: an overview.

The identification of multigene families encoding K-ATPases, K channels, and K transporters is a major step in understanding the molecular mechanisms engaged in K homeostasis. These membrane proteins, which also transport Na, H, or Cl ions, have been shown to play fundamental roles in cellular housekeeping functions (volume regulation, uptake of nutrients) and in specialized tissue functions (transepithelial transport of solutes and water, uptake of neurotransmitters, control of vascular tone). The association of mutations (especially in the K channels) with human diseases and disorders as well as the creation of animal models harboring specific gene inactivation should allow investigators to reach nonambiguous conclusions about the roles of these genes. These approaches should be complemented by techniques such as DNA array and chip hybridization and computer-based simulation in order to form an integrated view of the functional interactions between the genes underlying physiological and pathological processes.

Genetic renal tubular disorders of renal ion channels and transporters.

This perspective on genetic renal tubular transport disorders selectively reviews the pathophysiology of renal apical Na(+) transport systems. These transporters play an essential role in the control of extracellular fluid volume and blood pressure. Significant advancements in the understanding of the role of these genes in Mendelian forms of extracellular volume homeostatic disorders have been achieved in the recent years. Of even greater importance will be the ongoing definition of the various factors that regulate the expression and activity of the Na(+) transport systems. These regulatory pathways, and the responses to environmental factors such as dietary salt, stress, and so on, may determine the appearance, severity and complexity of the clinical phenotypes that result from genetic disorders of the renal apical Na(+) transporters.

Dietary sources and correlates of sodium and potassium intakes in the French general population.

BACKGROUND/OBJECTIVES: To investigate the dietary sources of sodium and potassium and to explore the biological, behavioural and socio-demographic factors associated with a high sodium and low potassium diet in a general population. SUBJECTS/METHODS: Cross-sectional dietary survey carried out in 1998 and 1999 in nationally representative samples of adults (n=1474) and children (n=1018). Daily sodium and potassium intakes were estimated using a 7-day food record after exclusion of underreporters. RESULTS: Mean sodium intake was well above, whereas mean potassium intake was largely below the current recommendations in adults and children. The consumption of a high sodium and low potassium diet appeared very early in life and increased up to adulthood, especially in men living in small communities. Despite the fact that sodium and potassium intakes were positively correlated to each other and to total food intake, several food categories showed a sodium/potassium intake ratio well above one (cheeses, cooked pork meats, breads, breakfast cereals, soups, fast foods, pastries and sugary products) whereas others presented a ratio well below one (fruits, vegetables, dairy products, meats and hot beverages). CONCLUSIONS: High sodium and low potassium intakes were widespread in the population. The fact that the main dietary sources of sodium and potassium were, for the most part, not the same demonstrates the feasibility of simultaneously decreasing sodium intake and increasing potassium intake at the individual level [corrected].

Comparative roles of the renal apical sodium transport systems in blood pressure control.

Human genetic studies suggest that the genes encoding renal apical Na(+) transport proteins play an essential role in the control of extracellular fluid volume and BP. Mice with mutations in each of these genes provide the unique opportunity to directly assess their respective involvement in fluid homeostasis and BP control in vivo. Inactivation of either the epithelial Na(+) channel (ENaC) or the Na(+)-Cl(-) cotransporter decreases BP to the same extent in mice fed a low-salt diet, despite a more pronounced perturbation of fluid homeostasis in ENaC-deficient mice. In contrast, inactivation of Na(+)/H(+) exchanger 3 (NHE3) or the Na(+)-K(+)-2Cl(-) contransporter reduces BP with a normal-salt diet and renders mice unable to survive with a low-salt diet. Therefore, the general conception that ENaC in the collecting duct is the main renal controller of Na(+) balance and extracellular fluid volume should be tempered. For example, NHE3 in the proximal convoluted tubule seems to play a more substantial role in the control of fluid homeostasis. The overall effect of NHE3 inactivation on BP may also involve absorptive defects in the intestine and colon, where the exchanger normally reabsorbs significant amounts of Na(+) and water.

[Experimental genetic modifications in the renin-angiotensin and kallikrein-kinin systems: significance for understanding cardiovascular regulation and study of genetic determinism of human diseases]. / Modifications génétiques expérimentales dans les systèmes rénine-angiotensine et kallicréine-kinine: intérêt pour la compréhension des régulations cardio-vasculaires et l'étude du déterminisme génétique des maladies humaines.

The precise role of the different proteins that constitute the renin-angiotensin and kallikrein-kinin systems in the development of hypertension and some cardiac and renal diseases remains unclear. Genetic manipulations in animals is a powerful approach that provide the opportunity to explore the role of each of these proteins in vivo. Indeed it is possible in the rat and in the mouse to manipulate a specific gene without modifying the other genetic and environmental factors. A causal link can thus be established between the gene and a physiologic or pathologic alteration. The possibilities are either the overexpression of the gene in all or in specific tissues (transgenesis), or the modification (often the inactivation) of the endogenous gene by homologous recombination. The second technique has the advantage to be more specific but it can be used only in the mouse; it is performed by transfecting totipotent embryonic stem cells with a vector harboring identical sequences to those of the gene to be targeted. The embryonic stem cells are then injected into embryos in which they will participate in the generation of the different organs including the gonads. The resulting chimeric animals can therefore transmit the mutation to their offspring creating a new genetically modified mouse strain. Many strains targeted in the different components of the renin-angiotensin and kallikrein-kinin systems have been generated in this way. These animal models should allow to test many physiopathological hypotheses that have been put forward from the results of human genetics and clinical studies, and also to raise new ones.

Renal physiology of the mouse.

As the transgenic and gene-targeting technology has become an invaluable experimental approach to study the function of gene products, the need has been expanded to assess the physiology in the mouse, which is virtually the only animal species to which that new genetic technology can apply. In this regard, renal physiologists have also received fruits of success from modern technology in several key areas, and areas are expanding in both depth and scope.

Cholinergic stimulation of phosphoinositide metabolism in isolated rat glomeruli.

Cholinergic effects on kidney function have been observed in some mammals but the intrarenal localization and the cellular mechanisms of these effects are poorly defined to date. The aim of this work was to study the effects of carbachol on phosphoinositide metabolism in freshly isolated rat glomeruli labeled with myo-[3H]inositol. Carbachol rapidly and markedly stimulates phosphoinositide metabolism with a 50% effective concentration of 3 microM. The enormous magnitude of the response is enlightened by the use of 10 mM lithium, which provokes in the presence of the agonist a large accumulation of inositol phosphates and a corresponding depletion of cellular free inositol. The response is inhibited by 85% by pirenzepine, is pertussis toxin insensitive, and shows no desensitization at maximum dose of carbachol up to 40 min of stimulation.

The parietal sheet of Bowman's capsule of rat renal glomerulus: a target of endothelin and PAF.

On the basis of intracellular calcium concentration ([Ca2+]i) measurements, we have previously reported that the parietal sheet of Bowman's capsule was sensitive to cholinergic agonists. The aim of the present work was to investigate whether this structure could be also a target of endothelin and platelet-activating factor (PAF), since we observed [Ca2+]i increases in response to both agonists in the glomerulus, but which were very different from that induced by carbachol. For this purpose, we measured [Ca2+]i on single microdissected parietal sheets, using a fura 2 microfluorescence technique and compared the effects of maximal concentrations of the three agonists (10(-7), 10(-8), and 10(-4) M for endothelin, PAF, and carbachol, respectively) under various experimental conditions. We observed that, like in the glomerulus, endothelin and PAF induced, in the parietal sheet, [Ca2+]i responses that differed in many respects from those found with carbachol. Thus, in the presence of 2 mM external calcium, 1) endothelin and PAF responses spontaneously declined to basal level, whereas a stationary plateau was observed after a sharp peak of [Ca2+]i with carbachol; 2) the magnitude of [Ca2+]i peak was smaller with endothelin and PAF than with carbachol; and 3) endothelin and PAF, but not carbachol, induced a homologous dose-dependent desensitization. Moreover, in the absence of external calcium, endothelin and PAF responses were smaller than carbachol response, although all three responses apparently resulted from release of calcium ions from the same internal pool. In additional experiments, we observed that, like carbachol, endothelin and PAF contracted the parietal sheet, which is only composed of myoepithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)