Recent concepts concerning the renal handling of NH3/NH4+.

To be appropriately excreted in urine, NH4+ , the major component of urinary acid excretion, must be synthesized by proximal tubular cells, secreted into the proximal tubular fluid, reabsorbed by the medullary thick ascending limb (MTAL) to accumulate in the medullary interstitium, and finally be secreted in the medullary collecting ducts. Each of the various steps of this particular renal pathway is highly regulated, and the control of gene expression explains how the renal handling of NH 4 + becomes fully adapted to chronic acid-base changes. Several targets have been identified to account for the adaptation of renal NH 4 + synthesis and transport in response to an acid load. These are the key enzymes of ammoniagenesis and the apical Na+/H+ (NH4+) exchanger NHE3 in the proximal tubule, the apical Na + -K + (NH 4 + )-2Cl - cotransporter of the MTAL, and the basolateral Na+-K+ (NH4+)-2Cl- cotransporter and the epithelial Rh B and C glycoproteins in the collecting ducts. An acid pH appears to be a major factor in the control of gene expression during metabolic acidosis probably through the activation of pH sensors. Glucocorticoids can contribute to coordinate the adaptation of various tubular cell types. This review focuses on some new aspects of NH3/NH4+ transport and of gene expression regulation that have recently emerged.

Mechanisms of tandem repeat instability in bacteria.

Hypermutable tandem repeat sequences (TRSs) are present in the genomes of both prokaryotic and eukaryotic organisms. Numerous studies have been conducted in several laboratories over the past decade to investigate the mechanisms responsible for expansions and contractions of microsatellites (a subset of TRSs with a repeat length of 1-6 nucleotides) in the model prokaryotic organism Escherichia coli. Both the frequency of tandem repeat instability (TRI), and the types of mutational events that arise, are markedly influenced by the DNA sequence of the repeat, the number of unit repeats, and the types of cellular pathways that process the TRS. DNA strand slippage is a general mechanism invoked to explain instability in TRSs. Misaligned DNA sequences are stabilized both by favorable base pairing of complementary sequences and by the propensity of TRSs to form relatively stable secondary structures. Several cellular processes, including replication, recombination and a variety of DNA repair pathways, have been shown to interact with such structures and influence TRI in bacteria. This paper provides an overview of our current understanding of mechanisms responsible for TRI in bacteria, with an emphasis on studies that have been carried out in E. coli. In addition, new experimental data are presented, suggesting that TLS polymerases (PolII, PolIV and PolV) do not contribute significantly to TRI in E. coli.

[Role of the Na(+)-K+(NH4+)-2Cl cotransporter of the medullary ascending limb in the regulation of renal acid-base equilibrium]. / Rôle du cotransporteur Na(+)-K+(NH4+)-2Cl- de la branche large ascendante dans la régulation rénale de l'équilibre acido-basique.

NH4+ absorption by the medullary thick ascending limb (MTAL) of Henle's loop, which causes the accumulation of NH4+/NH3 in the medullary interstitium, is a key step in the renal handling of ammonia. Accumulation of NH4+/NH3 in the medullary interstitium is necessary to the secretion of ammonia in the medullary collecting ducts and then to NH4+ excretion in the urine. The MTAL apical Na(+)-K+(NH4+)-2Cl- cotransporter BSC1/NKCC2 is responsible for the majority of the MTAL luminal NH4+ uptake. Stimulation of BSC1 expression by metabolic acidosis accounts for the increase of the MTAL ability to absorb NH4+ during this condition. Metabolic acidosis increases the mRNA and protein abudance and the transport activity of BSC1. Two factors have been demonstrated to mediate the effects of acidosis, an acid pH and glucocorticoids whose production augments during metabolic acidosis. These two factors thus control in a coordinated manner ammoniagenesis in the proximal tubule and MTAL NH4+ transport to ensure urinary acid excretion rates appropriate to the acid-base status.

Alteration of cardiac and renal functions in transgenic mice overexpressing human mineralocorticoid receptor.

The mineralocorticoid receptor (MR), a ligand-dependent transcription factor, mediates aldosterone actions in a large variety of tissues. To explore the functional implication of MR in pathophysiology, transgenic mouse models were generated using the proximal human MR (hMR) promoter to drive expression of hMR in aldosterone target tissues. Tissue-specific analysis of transgene expression in two independent transgenic animal (TG) lines by ribonuclease protection assays revealed that hMR is expressed in all mineralocorticoid-sensitive tissues, most notably in the kidney and the heart. TG exhibit both renal and cardiac abnormalities. Enlarged kidneys were histologically associated with renal tubular dilation and cellular vacuolization whose prevalence increased with aging. Renal clearance studies also disclosed a significant decrease in urinary potassium excretion rate in TG. hMR-expressing animals had normal blood pressure but developed mild dilated cardiomyopathy (increased left ventricle diameters and decreased shortening fraction), which was accompanied by a significant increase in heart rate. Differential gene expression analysis revealed a 2- to 5-fold increase in cardiac expression of atrial natriuretic peptide, serum- and glucocorticoid-induced kinase, and early growth response gene 1 as detected by microarrays; renal serum- and glucocorticoid-induced kinase was also induced significantly. Altogether, TG exhibited specific alteration of renal and cardiac functions, thus providing useful pathophysiological models to gain new insights into the tissue-specific mineralocorticoid signaling pathways.

Modulation of transcription reveals a new mechanism of triplet repeat instability in Escherichia coli.

Many human hereditary disease genes are associated with the expansion of triplet repeat sequences. In Escherichia coli (CTG/CAG) triplet repeat sequences are unstable and we have developed a plasmid-based assay enabling us to observe and quantify both expansions and deletions. In this work, we have investigated the role of transcription on the instability of a (CTG/CAG) insert containing 64 repeats. Using this assay, we show that induction of transcription results in a significant increase in the frequency of long deletions and a reduction in the frequency of long expansions. On the other hand, overproduction of transcription repressor molecules leads to an increase in both expansions and deletions. In this latter case, we propose that the increased instability is due to the arrest of replication progression by the interaction of the repressor molecule with its cognate operator and subsequent generations of DNA strand breaks.

Ammonium carriers in medullary thick ascending limb.

Absorption of NH(4)(+) by the medullary thick ascending limb (MTAL) is a key event in the renal handling of NH(4)(+), leading to accumulation of NH(4)(+)/NH(3) in the renal medulla, which favors NH(4)(+) secretion in medullary collecting ducts and excretion in urine. The Na(+)-K(+)(NH(4)(+))-2Cl(-) cotransporter (BSC1/NKCC2) ensures approximately 50-65% of MTAL active luminal NH(4)(+) uptake under basal conditions. Apical barium- and verapamil-sensitive K(+)/NH(4)(+) antiport and amiloride-sensitive NH(4)(+) conductance account for the rest of active luminal NH(4)(+) transport. The presence of a K(+)/NH(4)(+) antiport besides BSC1 allows NH(4)(+) and NaCl absorption by MTAL to be independently regulated by vasopressin. At the basolateral step, the roles of NH(3) diffusion coupled to Na(+)/H(+) exchange or Na(+)/NH(4)(+) exchange, which favors NH(4)(+) absorption, and of Na(+)/K(+)(NH(4)(+))-ATPase, NH(4)(+)-Cl(-) cotransport, and NH(4)(+) conductance, which oppose NH(4)(+) absorption, have not been quantitatively defined. The increased ability of the MTAL to absorb NH(4)(+) during chronic metabolic acidosis involves an increase in BSC1 expression, but fine regulation of MTAL NH(4)(+) transport probably requires coordinated effects on various apical and basolateral MTAL carriers.

[Knowledge and attitudes of health care professionals with respect to AIDS and the risk of occupational transmission of HIV in 2 Moroccan hospitals]. / Connaissances et attitudes du personnel soignant face au sida et au risque de transmission professionnelle du VIH dans deux hôpitaux marocains.

We carried out a survey of 266 health care workers at two hospitals, in Rabat and Casablanca, to evaluate the level of knowledge, attitudes and behavior of these individuals with respect to AIDS. We also analyzed working conditions presenting a risk of occupational transmission of HIV, with the aim of developing appropriate preventive measures. We carried out a cross-sectional study, using a standardized questionnaire. The study population consisted of 91 doctors (34.2%), 106 nurses (39.8%), 12 laboratory technicians (8. 8%) and 47 support staff (17.6%) working in various departments. The mean age was 32.7 years. This study population was young, with 83% less than 40 years old and more than half having worked in the hospital for less than ten years. We found that the personnel knew a great deal about the usual means of transmission of HIV, but much less about possible occupational contamination. One person in two was unaware of the ways in which HIV in the hospital environment can be inactivated (bleach - 70% alcohol) and only 18.4% knew that HIV is sensitive to heat. Half the study population thought that the systematic exclusion of patients with HIV was essential and two thirds suggested that every patient admitted to the hospital should undergo systematic HIV testing. Anxiety when caring for seropositive patients was expressed by 56% of doctors and 62% of paramedical workers and 85% thought that health workers were at high risk of contamination during their work. The frequency of occupational injuries was found to be high and such accidents were rarely declared (declaration rate 7%). Protection measures were not in place in more than 50% of cases and too little information and resources were available to increase the awareness of the health care workers. These data show that greater efforts should be made to educate and inform health workers by means of the occupational medicine units recently set up for the benefit of the staff.

[Prevalence of respiratory systems and evaluation of sensitization levels in traditional grain market workers in Casablanca]. / Prévalence des symptômes respiratoires et évaluation du degré de sensibilisation chez les ouvriers du souk de céréales de Casablanca.

Our study proposes to evaluate the prevalence of clinical respiratory symptoms, spirometric abnormalities and allergy skin test sensitivities in two groups: on exposed to grain dust in a big traditional grain market in Casablanca and the other unexposed. The inquiry which concerned 277 exposed workers and 230 non exposed consisted of a questionnaire, spirometric examinations and skin prick testings. Exposed and no exposed groups are statically similar as far as physical data (sex, age, weight, heignt) and smoking habits. The atopy was found among 18% of the exposed. The prevalence of clinical respiratory symptomatology among exposed is 64.3% against 24.8% among non exposed. Respiratory symptoms (cough, expectoration), rhinitis, asthma, conjonctivitis, dermatitis, chronic bronchitis were significantly more frequent in those exposed than in the non exposed. Smoking is at the origin of additional morbidity. Atopy seems to be a potentiating factor as all the atopic people exposed are symptomatic. Respiratory function was altered in 37.1% of those exposed versus 12.8% of those no exposed. Among exposed workers with decline of lung function parameters 68.9% have only light anomalies. Tabacco interferes significantly in the alteration of respiratory function parameters. Work exposure to grain associated with smoking resulted in a reduction in respiratory function values. In grain workers, the prevalence of allergy skin test sensitivities of occupational allergens is 30.3% versus 6.9% among those no exposed. The enquiry in the workplace shows complete absence of means of protection for the work force and elevated levels of dust. It is imperative to implement an occupational health service and to develop means for collective and individual prevention to maximally reduce the risk.

Effect of locally applied drugs on the pH of luminal fluid in the endolymphatic sac of guinea pig.

The aim of the present work was to assess the effect of various drugs applied locally on the pH of the luminal fluid (pH(lum)) in guinea pig endolymphatic sac. pH(lum) and transepithelial potential, when measured in vivo by means of double-barrelled pH-sensitive microelectrodes, were 7.06 +/- 0.08 and +6.1 +/- 0.34 mV (mean +/- SE; n = 84), respectively, which is consistent with a net acid secretion in the luminal fluid of the endolymphatic sac. Bafilomycin and acetazolamide increased and decreased, respectively, pH(lum). Amiloride, ethylisopropylamiloride, ouabain, and Schering 28080 had no effect on pH(lum). Results obtained with inhibitors of anionic transport systems were inconclusive; e.g., DIDS reduced pH(lum), whereas neither SITS nor triflocin had any effect. We conclude that bafilomycin-sensitive H(+)-ATPase activity accounts for the transepithelial acid gradient measured in the endolymphatic sac and that intracellular and membrane-bound carbonic anhydrase probably participates in regulating endolymphatic sac pH(lum). The relationship between acid pH, endolymph volume, and Ménière's disease remains to be further investigated.

Regulation by glucocorticoids of expression and activity of rBSC1, the Na+-K+(NH4+)-2Cl- cotransporter of medullary thick ascending limb.

To assess whether glucocorticoids regulate rBSC1, the apical Na(+)-K(+)(NH(4)(+))-2Cl(-) cotransporter of kidney medullary thick ascending limb (MTAL), studies were performed in normal rats, adrenalectomized (ADX) rats, and ADX rats infused with dexamethasone for 6 days. The effects of dexamethasone on rBSC1 were also studied in vitro using isolated rat MTAL segments. Cotransport activity was estimated by intracellular pH measurements; rBSC1 protein was quantified in MTAL crude membranes by immunoblotting analysis, and mRNA was quantified by quantitative reverse transcription-polymerase chain reaction. The abundance of rBSC1 protein and mRNA increased in ADX rats infused with dexamethasone compared with ADX rats (p < 0. 04). In addition, application of dexamethasone for 1-3 h to MTALs caused rBSC1 protein and mRNA abundance and cotransport activity to significantly increase in a hyperosmotic medium (450 mosmol/kg of H(2)O) containing 0.7 nm arginine vasopressin, which is an in vitro experimental condition that resembles the in vivo MTAL environment. Results obtained in various media and with 8-bromo-cAMP indicated that stimulation of rBSC1 expression by glucocorticoids required interactions between glucocorticoid receptor- and cAMP-dependent factors. Up to 100 nm d-aldosterone had no effect on cotransport activity in vitro. Thus glucocorticoids directly stimulate MTAL rBSC1 expression and activity, which contributes to glucocorticoid-dependent effects on the renal regulation of acid-base balance and urinary concentrating ability.

Mechanisms of dinucleotide repeat instability in Escherichia coli.

The high level of polymorphism of microsatellites has been used for a variety of purposes such as positional cloning of genes associated with diseases, forensic medicine, and phylogenetic studies. The discovery that microsatellites are associated with human diseases, not only as markers of risk but also directly in disease pathogenesis, has triggered a renewed interest in understanding the mechanism of their instability. In this work we have investigated the role of DNA replication, long patch mismatch repair, and transcription on the genetic instability of all possible combinations of dinucleotide repeats in Escherichia coli. We show that the (GpC) and (ApT) self-complementary sequence repeats are the most unstable and that the mode of replication plays an important role in their instability. We also found that long patch mismatch repair is involved in avoiding both short deletion and expansion events and also in instabilities resulting from the processing of bulges of 6 to 8 bp for the (GpT/ApC)- and (ApG/CpT)- containing repeats. For each dinucleotide sequence repeat, we propose models for instability that involve the possible participation of unusual secondary structures.

Adaptation of NHE-3 in the rat thick ascending limb: effects of high sodium intake and metabolic alkalosis.

The present studies examined the effects of chronic NaCl administration and metabolic alkalosis on NHE-3, an apical Na+/H+ exchanger of the rat medullary thick ascending limb of Henle (MTAL). NaCl administration had no effect on NHE-3 mRNA abundance as assessed by competitive RT-PCR, as well as on NHE-3 transport activity estimated from the Na+-dependent cell pH recovery of Na+-depleted acidified MTAL cells, in the presence of 50 microM Hoe-694, which specifically blocks NHE-1 and NHE-2. Two models of metabolic alkalosis were studied, one associated with high sodium intake, i.e., NaHCO3 administration, and one not associated with high sodium intake, i.e., chloride depletion alkalosis (CDA). In both cases, the treatment induced a significant metabolic alkalosis that was associated with a decrease in NHE-3 transport activity (-27% and -25%, respectively). Negative linear relationships were observed between NHE-3 activity and plasma pH or bicarbonate concentration. NHE-3 mRNA abundance and NHE-3 protein abundance, assessed by Western blot analysis, also decreased by 35 and 25%, respectively, during NaHCO3-induced alkalosis, and by 47 and 33%, respectively, during CDA. These studies demonstrate that high sodium intake has per se no effect on MTAL NHE-3. In contrast, chronic metabolic alkalosis, regardless of whether it is associated with high sodium intake or not, leads to an appropriate adaptation of NHE-3 activity, which involves a decrease in NHE-3 protein and mRNA abundance.

Stimulation by in vivo and in vitro metabolic acidosis of expression of rBSC-1, the Na+-K+(NH4+)-2Cl- cotransporter of the rat medullary thick ascending limb.

To assess whether metabolic acidosis per se regulates rBSC-1, the rat medullary thick ascending limb (MTAL) apical Na+-K+(NH4+)-2Cl- cotransporter, rat MTALs were incubated for 16 h in an acid 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's medium. Cotransport activity was estimated in intact cells and membrane vesicles by intracellular pH and 22Na+ uptake measurements, respectively; rBSC-1 protein was quantified by immunoblotting analysis and mRNA by quantitative reverse transcription-polymerase chain reaction. As compared with incubation at pH approximately 7.35, acid incubation (pH approximately 7.10) up-regulated by 35-100% rBSC-1 transport activity in cells and membrane vesicles, and rBSC-1 protein and mRNA abundance. In contrast, acid incubation did not alter alkaline phosphatase and Na+/K+-ATPase enzyme activities or beta-actin protein abundance. After 3 h of in vivo chronic metabolic acidosis (CMA) rBSC-1 mRNA abundance increased in freshly harvested MTALs, which was accompanied after 1-6 days of CMA with enhanced rBSC-1 protein abundance. These results demonstrate that both in vivo and in vitro CMA stimulate rBSC-1 expression, which would contribute to the adaptive increase in MTAL absorption and urinary excretion of NH4+ in response to CMA.

Expansion of CTG repeats from human disease genes is dependent upon replication mechanisms in Escherichia coli: the effect of long patch mismatch repair revisited.

Many human hereditary disease genes have been recently associated with the expansion of CTG/GAC repeats. We have used a plasmid-based assay in Escherichia coli to investigate the instability of a (CTG/GAC) insert containing 64 repeats. Using this assay, expansions were biochemically detected and subsequently quantified. We show that the occurence of expansions within these trinucleotide repeats is dependent upon replicative mechanisms. Expansions of up to 30 repeats and deletions of almost all possible sizes occured regardless of the orientation of the insert relative to the replication origin. In contradiction to a previous report, the mismatch repair pathway was found to strongly stabilize these repeat stretches.

ANG II controls Na(+)-K+(NH4+)-2Cl- cotransport via 20-HETE and PKC in medullary thick ascending limb.

Cell pH was monitored in medullary thick ascending limbs to determine effects of ANG II on Na(+)-K+(NH4+)-2Cl- cotransport. ANG II at 10(-16) to 10(-12) M inhibited 30-50% (P < 0.005), but higher ANG II concentrations were stimulatory compared with the 10(-12) M ANG II level cotransport activity; eventually, 10(-6) M ANG II stimulated 34% cotransport activity (P < 0.003). Inhibition by 10(-12) M ANG II was abolished by phospholipase C (PLC), diacylglycerol lipase, or cytochrome P-450-dependent monooxygenase blockade; 10(-12) M ANG II had no effect additive to inhibition by 20-hydroxyeicosatetranoic acid (20-HETE). Stimulation by 10(-6) M ANG II was abolished by PLC and protein kinase C (PKC) blockade and was partially suppressed when the rise in cytosolic Ca2+ was prevented. All ANG II effects were abolished by DUP-753 (losartan) but not by PD-123319. Thus < or = 10(-12) M ANG II inhibits via 20-HETE, whereas > or = 5 x 10(-11) M ANG II stimulates via PKC Na(+)-K+(NH4+)-2Cl- cotransport; all ANG II effects involve AT1 receptors and PLC activation.

Long-term shake suspension and membrane vesicles of medullary thick ascending limb.

Cultured medullary thick ascending limb (MTAL) cells may lack some of the main carriers of fresh MTAL cells, such as apical Na+-K+(NH4+)-2Cl- cotransporter (BSC-1) and Na+/H+ exchanger (NHE-3). We have developed a technique to maintain rat MTALs several hours in suspension and in a good state of viability. Medullary thick ascending limbs were suspended in a 1:1 mixture of Ham's nutrient mixture F-12 and Dulbecco's modified Eagle's essential medium (HDMEM) supplemented with 25 mM HCO3- and gassed with 95% O2/5% CO2; the resulting mixture was placed in a rotary shaking water bath at 37 degrees C for 16 hours. As seen by electron microscopy, MTALs from the HDMEM-suspension retained a virtually normal tubular organization. Na+-K+(NH4+)-2Cl- cotransport activity and NHE consistent with both apical NHE-3 and basolateral NHE-1 activities were underscored both in intact cells by intracellular pH measurements and in a membrane fraction enriched in apical and basolateral membranes by 22Na+ uptake experiments. These results demonstrate that freshly harvested MTALs can be maintained in a well differentiated state for at least 16 hours; this preparation should make long-term in vitro studies of MTAL transport regulations possible.

Apical location and inhibition by arginine vasopressin of K+/H+ antiport of the medullary thick ascending limb of rat kidney.

To characterize and localize a K+/H+ antiport mechanism in the renal medullary thick ascending limb (MTAL), membrane vesicles were isolated from a rat MTAL homogenate. K+/H+ antiport (in > out H+ gradient-stimulated 86Rb+ uptake) was abolished by barium and verapamil (apparent Ki of 55 microM) but unaffected by other K+ channel blockers such as quinidine and high amiloride concentrations. SCH 28080, a H+/K+-ATPase blocker, did not affect K+/H+ antiport. K+/H+ antiport activity was correlated positively with the enrichment factor of the membranes in the apical marker enzyme alkaline phosphatase (r = 0.875, p < 0.01) and negatively correlated with the enrichment factor in basolateral Na+/K+-ATPase (r = -0.665, p < 0.05). Moreover, a functional interaction occurred with Na+/H+ exchange (NHE) consistent with colocation of K+/H+ antiport and apical NHE-3, not basolateral NHE-1. K+/H+ antiport was shown by intracellular pH measurements to be inhibited by arginine vasopressin and 8-bromo-cAMP through cAMP-dependent protein kinase (protein kinase A) activation. These results demonstrate the presence of a K+/H+ antiport mechanism, which is inhibited by arginine vasopressin via protein kinase A, in the apical membrane of the MTAL.

Two distinct models account for short and long deletions within sequence repeats in Escherichia coli.

In Escherichia coli, (GpC)n sequences cloned into plasmid DNA molecules are deletion-prone with the occurrence of both short (<2 bp) and long (>2 bp) deletion events. These repetitive tracts can be stabilized by interrupting the strict monotony of the repetition with a variant dinucleotide sequence. The stabilization of short deletion events that is mediated by the variant sequence is completely lost in E. coli mismatch repair-deficient strains. In contrast, this repair pathway has no influence on the frequency of occurrence of long deletion events, even in sequences containing the variant repeat. These results lead us to propose two distinct models to account for short and long deletions within repetitive sequences in E. coli. Furthermore, this study reveals that the deletions occur preferentially at the end of the repeat sequence that is distal with respect to the origin of replication.

Chronic metabolic acidosis enhances NHE-3 protein abundance and transport activity in the rat thick ascending limb by increasing NHE-3 mRNA.

Chronic metabolic acidosis (CMA) is associated with an adaptive increase in the bicarbonate absorptive capacity of the rat medullary thick ascending limb (MTAL). To specify whether NHE-3, the apical MTAL Na/H exchanger, is involved in this adaptation, NHE-3 mRNA was quantified by a competitive RT-PCR using an internal standard which differed from the wild-type NHE-3 mRNA by an 80-bp deletion. CMA increased NHE-3 mRNA from 0.025+/-0.003 to 0.042+/-0.009 amol/ng total RNA (P < 0.005). NHE-3 transport activity was measured as the initial proton flux rate calculated from the Na-dependent cell pH recovery of Na-depleted acidified MTAL cells in the presence of 50 microM HOE694 which specifically blocks NHE-1, the basolateral MTAL NHE isoform. CMA caused a 68% increase in NHE-3 transport activity (P < 0.001). In addition, CMA was associated with a 71% increase in NHE-3 protein abundance (P < 0.05) as determined by Western blot analysis on MTAL membranes using a polyclonal antiserum directed against a cytoplasmic epitope of rat NHE-3. Thus, NHE-3 adapts to CMA in the rat MTAL via an increase in the mRNA transcript that enhances NHE-3 protein abundance and transport activity.