A role for Rhesus factor Rhcg in renal ammonium excretion and male fertility.

The kidney has an important role in the regulation of acid-base homeostasis. Renal ammonium production and excretion are essential for net acid excretion under basal conditions and during metabolic acidosis. Ammonium is secreted into the urine by the collecting duct, a distal nephron segment where ammonium transport is believed to occur by non-ionic NH(3) diffusion coupled to H(+) secretion. Here we show that this process is largely dependent on the Rhesus factor Rhcg. Mice lacking Rhcg have abnormal urinary acidification due to impaired ammonium excretion on acid loading-a feature of distal renal tubular acidosis. In vitro microperfused collecting ducts of Rhcg(-/-) acid-loaded mice show reduced apical permeability to NH(3) and impaired transepithelial NH(3) transport. Furthermore, Rhcg is localized in epididymal epithelial cells and is required for normal fertility and epididymal fluid pH. We anticipate a critical role for Rhcg in ammonium handling and pH homeostasis both in the kidney and the male reproductive tract.

Mitochondrial aquaporin-8 in renal proximal tubule cells: evidence for a role in the response to metabolic acidosis.

Mitochondrial ammonia synthesis in proximal tubules and its urinary excretion are key components of the renal response to maintain acid-base balance during metabolic acidosis. Since aquaporin-8 (AQP8) facilitates transport of ammonia and is localized in inner mitochondrial membrane (IMM) of renal proximal cells, we hypothesized that AQP8-facilitated mitochondrial ammonia transport in these cells plays a role in the response to acidosis. We evaluated whether mitochondrial AQP8 (mtAQP8) knockdown by RNA interference is able to impair ammonia excretion in the human renal proximal tubule cell line, HK-2. By RT-PCR and immunoblotting, we found that AQP8 is expressed in these cells and is localized in IMM. HK-2 cells were transfected with short-interfering RNA targeting human AQP8. After 48 h, the levels of mtAQP8 protein decreased by 53% (P < 0.05). mtAQP8 knockdown decreased the rate of ammonia released into culture medium in cells grown at pH 7.4 (-31%, P < 0.05) as well as in cells exposed to acid (-90%, P < 0.05). We also evaluated mtAQP8 protein expression in HK-2 cells exposed to acidic medium. After 48 h, upregulation of mtAQP8 (+74%, P < 0.05) was observed, together with higher ammonia excretion rate (+73%, P < 0.05). In vivo studies in NH(4)Cl-loaded rats showed that mtAQP8 protein expression was also upregulated after 7 days of acidosis in renal cortex (+51%, P < 0.05). These data suggest that mtAQP8 plays an important role in the adaptive response of proximal tubule to acidosis possibly facilitating mitochondrial ammonia transport.

Weight, age and coefficients of variation in renal solute excretion.

BACKGROUND: Homoscedasticity (constant variance over axes or among statistical factors) is an integral assumption of most statistical analyses. However, a number of empirical studies in model organisms and humans demonstrate significant differences in residual variance (that component of phenotype unexplained by known factors) or intra-individual variation among genotypes. Our work suggests that renal traits may be particularly susceptible to randomization by genetic and non-genetic factors, including endogenous variables like age and weight. METHODS: We tested associations between age, weight and intra-individual variation in urinary calcium, citrate, chloride, creatinine, potassium, magnesium, sodium, ammonium, oxalate, phosphorus, sulfate, uric acid and urea nitrogen in 9,024 male and 6,758 female kidney stone patients. Coefficients of variation (CVs) were calculated for each individual for each solute from paired 24-hour urines. Analysis of CVs was corrected for inter-measurement collection variance in creatinine and urine volume. CVs for sodium and urea nitrogen were included to correct for dietary salt and protein. RESULTS: Age was positively associated with individual CVs for calcium and negatively associated with CVs for potassium, ammonium and phosphorus (p(FDR) < 0.01). Weight was associated with CVs for creatinine, magnesium and uric acid, and negatively associated with CVs for calcium, potassium and oxalate (p(FDR) < 0.05). CONCLUSION: Intra-individual variation changes over age and weight axes for numerous urinary solutes. Changing residual variance over age and weight could cause bias in the detection or estimation of genetic or environmental effects. New methodologies may need to account for such residual unpredictability, especially in diverse collections.

Preservation of nutrients during long-term storage of source-separated yellowwater.

Source separation of human urine (yellowwater) enhances the sustainability of wastewater management and efficiency of nutrient recovery and recycling. Storage of source-separated yellowwater is recommended prior to agronomic reuse. At this point, it is of immense interest to determine the effect of storage time on quality of yellowwater. Therefore, this study focused on examining changes in some chemical properties of raw, undiluted, freshly collected, source-separated yellowwater stored for a period of 1 year under different temperature regimes: cold (4 °C), mild (10 °C) and warm (22 °C). Chemical parameters (biochemical oxygen demand (BOD(5)), N-tot, N-NO(2), N-NO(3), N-NH(4), P-tot, K, S, and pH), with the main focus on fertiliser nutrient compounds intended for agricultural utilisation, were tested. The outcomes revealed that both nitrification and denitrification processes took place in the stored yellowwater, and an increase in the pH level of up to pH greater than 9 was observed. The study found that the main macronutrients can be well preserved in yellowwater, as there were no substantial changes in the contents of these elements over a 1 year storage period at the three temperatures tested.

Nitrogen recovery from source-separated human urine using clinoptilolite and preliminary results of its use as fertilizer.

The use of source separated human urine as fertilizer is one of the major suggestions of the new sanitation concept ECOSAN. Urine is rich in nitrogen, phosphorus and potassium which act as plant nutrients, however its salinity is high for agricultural and landscape purposes. Moreover, characteristics change significantly throughout storage where salinity increases to higher values as the predominant form of nitrogen shifts from urea to ammonium. Transferring nitrogen in human urine onto the natural zeolite clinoptilolite and using the subsequently recovered ammonium from the exhausted clinoptilolite for agricultural/landscape purposes is suggested as an indirect route of using urine in this work. Results reporting the outcome of the proposed process together with characterization of fresh and stored urine, and preliminary work on the application of the product on the landscape plant Ficus elastica are presented. Up to 97% of the ammonium in stored urine could be transferred onto clinoptilolite through ion exchange and about 88% could be recovered subsequently from exhausted clinoptilolite, giving an overall recovery of 86%. Another important merit of the suggested process was the successful elimination of salinity. Preliminary experiments with Ficus elastica had shown that the product, i.e. clinoptilolite exhausted with ammonium, was compatible with the synthetic fertilizer tested.

Laboratory diagnostics in the urine of young and pregnant ewes.

OBJECTIVE: The purpose of this study was to identify values for net acid base excretion (NABE) which are significant indicators of the acid-base equilibrium in pregnant and young ewes and to show its relationship with other parameters (base, acid, ammonium [NH4], base-acid quotient, sodium [Na], potassium [K], calcium [Ca]) in ovine urine. In contrast to dairy cows, data are rare on these parameters in ewes. MATERIAL AND METHODS: A total of 99 animals were used in the study, consisting of 56 young (average of 5.6±1.1 months) and 43 pregnant ewes (average of 35.2±18.8 months). Measurement of fractional NABE in urine samples was carried out according to the method reported by Kutas. The pH value of the urine was measured with a laboratory pH meter. Na, K and total Ca were measured with a flame photometer. RESULTS: For all values except Na significant differences occurred between urine samples of pregnant ewes and young ewes (p<0.001). Base, acid, NH4, NABE, K and Ca values were significantly higher in the urine of the youngs than in pregnant ewes. In young ewes, a strong correlation was found between NABE and base values while a weak correlation could be observed between pH and base values. In pregnant ewes, strong NABE-base, NABE-K, K-acid and K-base correlations were found as well as weak NH4-base, NH4-NABE and NH4-K correlations. There was a strongly positive correlation between NABE and NH4 in pregnant ewes, while a weak negative correlation between those values was observed in young ewes. CONCLUSION: For the first time, we established values for NABE and certain other parameters in urine of pregnant ewes and young ewes. It was shown that the acid-base balance in pregnant ewes and young ewes can be evaluated by measuring NABE and certain trace elements in urine like in cattle.

Deletion of the anion exchanger Slc26a4 (pendrin) decreases apical Cl(-)/HCO3(-) exchanger activity and impairs bicarbonate secretion in kidney collecting duct.

The anion exchanger Pendrin, which is encoded by SLC26A4 (human)/Slc26a4 (mouse) gene, is localized on the apical membrane of non-acid-secreting intercalated (IC) cells in the kidney cortical collecting duct (CCD). To examine its role in the mediation of bicarbonate secretion in vivo and the apical Cl(-)/HCO(3)(-) exchanger in the kidney CCD, mice with genetic deletion of pendrin were generated. The mutant mice show the complete absence of pendrin expression in their kidneys as assessed by Northern blot hybridization, Western blot, and immunofluorescence labeling. Pendrin knockout (KO) mice display significantly acidic urine at baseline [pH 5.20 in KO vs. 6.01 in wild type (WT); P < 0.0001] along with elevated serum HCO(3)(-) concentration (27.4 vs. 24 meq/l in KO vs. WT, respectively; P < 0.02), consistent with decreased bicarbonate secretion in vivo. The urine chloride excretion was comparable in WT and KO mice. For functional studies, CCDs were microperfused and IC cells were identified by their ability to trap the pH fluorescent dye BCECF. The apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells, as assessed by intracellular pH monitoring, was significantly reduced in pendrin-null mice. The basolateral Cl(-)/HCO(3)(-) exchanger activity in A-IC cells and in non-A, non-B-IC cells, was not different in pendrin KO mice relative to WT animals. Urine NH(4)(+) (ammonium) excretion increased significantly, consistent with increased trapping of NH(3) in the collecting duct in pendrin KO mice. We conclude that Slc26a4 (pendrin) deletion impairs the secretion of bicarbonate in vivo and reduces apical Cl(-)/HCO(3)(-) exchanger activity in B-IC and non-A, non-B-IC cells in CCD. Additional apical Cl(-)/HCO(3)(-) exchanger(s) is (are) present in the CCD.

Potassium restriction, high protein intake, and metabolic acidosis increase expression of the glutamine transporter SNAT3 (Slc38a3) in mouse kidney.

Kidneys produce ammonium to buffer and excrete acids through metabolism of glutamine. Expression of the glutamine transporter Slc38a3 (SNAT3) increases in kidney during metabolic acidosis (MA), suggesting a role during ammoniagenesis. Potassium depletion and high dietary protein intake are known to elevate renal ammonium excretion. In this study, we examined SNAT3, phosphate-dependent glutaminase (PDG), and phosphoenolpyruvate carboxykinase (PEPCK) regulation during a control (0.36%) or low-K(+) (0.02%) diet for 7 or 14 days or a control (20%) or high-protein (50%) diet for 7 days. MA was induced in control and low-K(+) groups by addition of NH(4)Cl. Urinary ammonium excretion increased during MA, after 14-day K(+) restriction alone, and during high protein intake. SNAT3, PDG, and PEPCK mRNA abundance were elevated during MA and after 14-day K(+) restriction but not during high protein intake. SNAT3 protein abundance was enhanced during MA (both control and low K(+)), after 14-day low-K(+) treatment alone, and during high protein intake. Seven-day dietary K(+) depletion alone had no effect. Immunohistochemistry showed SNAT3 staining in earlier parts of the proximal tubule during 14-day K(+) restriction with and without NH(4)Cl treatment and during high protein intake. In summary, SNAT3, PDG, and PEPCK mRNA expression were congruent with urinary ammonium excretion during MA. Chronic dietary K(+) restriction, high protein intake, and MA enhance ammoniagenesis, an effect that may involve enhanced SNAT3 mRNA and protein expression. Our data suggest that SNAT3 plays an important role as the glutamine uptake mechanism in ammoniagenesis under these conditions.

Bioelectronic tongue for the simultaneous determination of urea, creatinine and alkaline ions in clinical samples.

Urea and creatinine biosensors based on urease and creatinine deiminase, respectively, covalently immobilized onto ammonium selective electrodes, were included in an array together with sensors sensitive to ammonium, potassium and sodium. Generic sensors to alkaline ions were also included. All the sensors used were of all-solid-state type, employing polymeric membranes and having rather nonspecific response characteristics. A response model based on artificial neural networks was built and tested for the simultaneous determination of urea, creatinine, ammonium, potassium and sodium. The results show that it is possible to obtain a good multivariate calibration model. In this way, the developed bioelectronic tongue was successfully applied to multidetermination of the five species in raw and spiked urine samples. Predicted concentrations showed a good agreement with reference methods of analysis, allowing a simple direct method for determining urea and creatinine in real samples. At the same time, this method permitted to obtain the concentrations of the alkaline interferences (endogenous ammonium, potassium and sodium) without the need of eliminating them.

[Urinary ammonium: validation of an enzymatic method and reliability with an indirect urine ammonium estimation]. / Ammoniuries: validation d'une technique de dosage enzymatique et évaluation par rapport à une estimation par le calcul.

Urinary ammonium excretion is the best parameter to quantify net acid excretion by the kidney. As measurement of ammonium excretion is not routinely available, clinicians use formulas to estimate NH(4)+ excretion. However, the measurement of urinary NH(4)+ concentration can be performed by an automatically method, which is suitable for clinical practice. The aim of this study is to evaluate the validity of the enzymatic method of ammonium determination with Ammonia SL Elitech Diagnostics reagent on an Olympus AU 2700 analyzer, which use 1/100 diluted urine samples. A clinico-biological study allowed us to compare measurements obtained during a 30 months' period with the above enzymatic method with results obtained by a formula of calculation. Variations coefficients (CV%) of repeatability were less than 2.4% and, those of reproducibility tests less than 2,6%. Linearity was verified from 0.62 mmol/L to 158 mmol/L. Analytical sensitivity was 0.52 mmol/L and the correlation obtained with the assay used to date in the laboratory was excellent (y = 1.11 x - 1.72 ; r = 0.98). There is a significant positive correlation between measured concentrations obtained with this enzymatic method and urinary ammonium concentration estimates using the modified urine osmolal gap in two groups of patients, with and without mild chronic renal failure. As urine ammonium estimation is not reliable for detecting small changes in ammonium excretion, it must be absolutely measured when renal functional tests are performed. The assay described in this paper is simple, automatic and offers for the clinician accurate matter for the measurement of NH(4)+ excretion.

The effect of uncomplicated potassium depletion on urine acidification.

Studies were performed on normal human subjects to determine the effects of potassium depletion on urine acidification. Depletion was induced by ingestion of a low potassium diet either alone or in combination with a potassium-binding resin, and the response of each subject to an acute ammonium chloride load in the potassium-depleted state was compared to his normal response. Urine pH was significantly higher during potassium deficiency if sufficient potassium depletion had been induced. No differences in blood acid-base parameters, urinary flow rate, or urinary fixed buffer excretion rate were found to account for this change; however, the increase in urine pH was accompanied by a concomitant increase in net acid and ammonium excretion. It is proposed that these changes during potassium depletion reflect an increase in ammonia diffusion into the urine, presumably as a result of increased renal ammonia production. In addition, it is speculated that changes in ammonia metabolism may be a physiologic control mechanism for potassium conservation.

Antibacterial activity of human urine.

The fate of bacteria in human urine was studied after inoculation of small numbers of Escherichia coli and other bacterial strains commonly implicated in urinary tract infection. Urine from normal individuals was often inhibitory and sometimes bactericidal for growth of these organisms. Antibacterial activity of urine was not related to lack of nutrient material as addition of broth did not decrease inhibitory activity. Antibacterial activity was correlated with osmolality, urea concentration and ammonium concentration, but not with organic acid, sodium, or potassium concentration. Between a pH range of 5.0-6.5 antibacterial activity of urine was greater at lower pH. Ultrafiltration and column chromatography to remove protein did not decrease antibacterial activity. Urea concentration was a more important determinant of antibacterial activity than osmolality or ammonium concentration. Increasing the urea of a noninhibitory urine to equal that of an inhibitory urine made the urine inhibitory. However, increasing osmolality (with sodium chloride) or increasing ammonium to equal the osmolality or ammonium of an inhibitory urine did not increase antibacterial activity. Similarly, dialysis to decrease osmolality or ammonium but preserve urea did not decrease inhibitory activity. Decreasing urea with preservation of ammonium and osmolality decreased antibacterial activity. Removal of ammonium with an ion exchanger did not decrease antibacterial activity, whereas conversion of urea to ammonium with urease and subsequent removal of the ammonium decreased antibacterial activity. Urine collected from volunteers after ingestion of urea demonstrated a marked increase in antibacterial activity, as compared with urine collected before ingestion of urea.

Short-term exposure to a high-protein diet differentially affects glomerular filtration rate but not Acid-base balance in older compared to younger adults.

There is conflicting evidence regarding the effects of high protein intake on kidney health, especially as it relates to age. We investigated the short-term effects of a high-protein diet on kidney function and systemic acid-base homeostasis in older compared to younger adults. The subjects were healthy men and women either between the ages of 25 and 40 years (n=12) or 55 and 70 years (n=10). They underwent a two-period crossover trial with each period consisting of 2 weeks of usual diet followed by a 1-week experimental diet. During the experimental diet period subjects consumed metabolic meals that provided either low protein content (0.5 g protein/kg/day) or high protein content (2.0 g protein/kg/day). Outcome measures included blood and urine markers of renal function and acid-base balance. An analysis of variance was used to assess differences between age groups with respect to experimental diet. The older group, mainly women, showed an increase in glomerular filtration rate after the high-protein compared to low-protein diet; the younger group did not. Urinary pH was significantly lower, and ammonium excretion was significantly higher after the high-protein diet in both age groups, but neither group developed a clinically detectable acidosis after the week of receiving a high-protein diet.

Delayed hypokalemic paralysis following a convulsion due to alcohol abstinence.

We encountered three patients with hypokalemic paralysis following a convulsion in the early stages of alcohol abstinence. The transtubular potassium gradient was less than 2.0, suggesting intracellular potassium shift. Hypokalaemic paralysis may result from retention of intracellular cationic potassium bound by anionic phosphorylated compounds, precipitated by an acceleration of the (Na+)-(K+) pump in alcohol withdrawal and convulsions. These findings warn of the lethal hypokalemia that may occur after convulsions, particularly soon after alcohol abstinence associated with moderate withdrawal symptoms.

Simultaneous determination of phosphatidylcholine-derived quaternary ammonium compounds by a LC-MS/MS method in human blood plasma, serum and urine samples.

The determination of circulating trimethylamine-N-oxide (TMAO), choline, betaine, l-carnitine and O-acetyl-l-carnitine concentration in different human matrices is of great clinical interest. Recent results highlighted the prognostic value of TMAO and quaternary ammonium containing metabolites in the field of cardiovascular and kidney diseases. Herein, we report a method for the rapid and simultaneous measurement of closely related phosphatidylcholine-derived metabolites in three different biological matrices by stable isotope dilution assay. Plasma, serum and urine samples were simply deproteinized and separated by HILIC-chromatography. Detection and quantification were performed using LC-MS/MS with electrospray ionization in positive mode. For accuracy and precision, full calibration was performed covering more than the full reference range. Assay performance metrics include intra- and interday imprecision were below 10% for all analytes. To exclude matrix effects standard addition methods were applied for all matrices. It was shown that calibration standards and quality control prepared in water can be used instead of matrix-matched calibration and controls. The LC/MS/MS-based assay described in this article may improve future clinical studies evaluating TMAO and related substances as prognostic markers for cardiovascular risk and all-cause mortality in different patient populations.

Stimulation of ammonium ion excretion in the toad urinary bladder by an extract of human urine.

It is well known that ammonium ion excretion is increased during metabolic acidosis in mammals. The purpose of this study was to determine whether we could isolate from human urine during metabolic acidosis a factor that would stimulate NH+4 and/or H+ excretion in toad urinary bladder. Extracts of urine from six human subjects collected during NH4Cl-induced acidosis were prepared. These extracts were tested for their effect on NH+4 excretion in hemibladders mounted between plastic chambers. The extracts significantly increased NH+4 excretion in the toad urinary bladder. We found no effect on H+ excretion by these extracts. This ammoniuretic activity was not present in the urine when the same individuals were in metabolic alkalosis. We conclude that during metabolic acidosis a humoral factor is present which stimulates the excretion of NH+4. The factor could act as a permease in the bladder cell or as a stimulator of an NH+4 transport system.

The effect of oral protein loading on renal acidification in patients with heart failure.

BACKGROUND: The purpose of this study was to explore the renal acid-base response to acute protein load in patients with heart failure (HF). It was prompted by the fact that there are no data available regarding the role of renal tubules in maintaining acid-base balance following protein loading in HF patients. METHODS AND RESULTS: Nine male patients with HF and 12 healthy subjects (controls) were enrolled in this study. In the HF patients, average blood pH was 7.42 (0.03), average pCO2 was 36.6 mmHg (6.3) and average bicarbonate was 24.2 mmol/L (4.3). The acid-base status of patients was unaffected by meat ingestion. The values at peak glomerular filtration rate (GFR) did not differ significantly from baseline levels. An oral protein load did not influence the urinary pH, titratable acidity (TA) and ammonium excretion in the patients with HF, contrary to the findings in the controls. On the other hand, ammonium excretion in patients with HF reduced significantly compared with values from controls at baseline and following oral protein loading. Filtered and reabsorbed bicarbonate increased significantly in HF patients following meat ingestion, whereas there was no change in absolute and fractional bicarbonate excretion and fractional bicarbonate reabsorbed. CONCLUSIONS: This study demonstrated that in patients with HF, bicarbonate reabsorption increases following an oral protein load without a significant enhancement in bicarbonate excretion. The difference can be explained by the presence of respiratory alkalosis leading to bicarbonate conservation.

Renal acidification in sickle cell trait.

Nine sickle cell trait and nine control subjects underwent six-hour ammonium chloride acid loading. Maximal urine osmolality and renal hemodynamics were studied separately. Base line arterial pH, carbon dioxide pressure (Pco2), and [HCO3] were normal and comparable in the two groups. After ammonium chloride loading, urine pH decreased to 5.3 or less in all, and maximal excretion of ammonium and titratable and net acid was comparable as was urine minus blood Pco2 after bicarbonate loading. The ammonium chloride acidosis caused a small decrease in red blood cell 2,3-diphosphoglycerate levels but no alteration in oxygen pressure at 50% saturation at pH 7.4, sickling, or adverse effects. Control and sickle cell trait subjects had comparable renal hemodynamics but maximal urine osmolality was lower in sickle-cell trait subjects. Adults with sickle cell trait have diminished renal concentrating ability and normal renal acidification and hemodynamics.