Mathematical Model of Ammonia Handling in the Rat Renal Medulla

The kidney is one of the main organs that produces ammonia and release it into the circulation. Under normal conditions, between 30 and 50% of the ammonia produced in the kidney is excreted in the urine, the rest being absorbed into the systemic circulation via the renal vein. In acidosis and in some pathological conditions, the proportion of urinary excretion can increase to 70% of the ammonia produced in the kidney. Mechanisms regulating the balance between urinary excretion and renal vein release are not fully understood. We developed a mathematical model that reflects current thinking about renal ammonia handling in order to investigate the role of each tubular segment and identify some of the components which might control this balance. The model treats the movements of water, sodium chloride, urea, NH₃ and NH4+, and non-reabsorbable solute in an idealized renal medulla of the rat at steady state. A parameter study was performed to identify the transport parameters and microenvironmental conditions that most affect the rate of urinary ammonia excretion. Our results suggest that urinary ammonia excretion is mainly determined by those parameters that affect ammonia recycling in the loops of Henle. In particular, our results suggest a critical role for interstitial pH in the outer medulla and for luminal pH along the inner medullary collecting ducts.     

Diurnal fluctuations of protein contents and the pH dependence of β<Subscript>2</Subscript>-microglobulin stability in urine

Diurnal fluctuations of protein excretion into urine and the effect of urinary pH on the urinary protein concentrations were studied in patients with various kidney diseases. The diurnal kinetics of γ-immunoglobulin, transferrin, albumin, α₁-microglobulin, γ-immunoglobulin light chains, and the retinol-binding protein proved to positively correlate with the diurnal fluctuations of proteinuria and to negatively correlate with urinary pH. Diurnal changes in urinary β₂-microglobulin content did not correlate with those of any other protein. Oral bicarbonate intake alkalinized the urine, increased the urinary β₂-microglobulin content, and led to a direct correlation between β₂-microglobulin excretion and excretion of other low-molecular proteins. Thus, proteinuria, single protein excretion, and urinary pH displayed diurnal rhythmicity in the patients; β₂-microglobulin was unstable in acid urine and its urinary level depended on the urinary pH.     

Quantitative studies on prostaglandin synthesis in man. 3. Excretion of the major urinary metabolite of prostaglandins F 1 alpha and F 2 alpha during pregnancy

The mean urinary excretion of 5α, 7α-dihydroxy-11-ketotetranor-prostane-1,16-dioic acid, the major urinary metabolite of prostaglandins F_1α and F_2α, in eight women in pregnancy weeks 37–40 was 32.5 ± 12.2 μg/24 hours, representing a 2–5 fold increase compared to non-pregnant women. Determination of the metabolite throughout pregnancy in three subjects showed that there was a gradual increase in the urinary excretion as pregnancy progressed with maximum excretion (4–5 times the individual pre-pregnant value) at the end of pregnancy. After pregnancy there was a rapid normalization back to the pre-pregnant excretion value.     

Isolation, identification and determination of sulfamethoxazole and its known metabolites in human plasma and urine by high-performance liquid chromatography

From human urine the following metabolites of sulfamethoxazole (S) were isolated by preparative HPLC: 5-methylhydroxysulfamethoxazole (SOH), N₄-acetyl-5-methylhydroxysulfamethoxazole (N₄SOH) and sulfamethoxazole-N₁-glucuronide (Sgluc). The compounds were identified by NMR, mass spectrometry, infrared spectrometry, hydrolysis by β-glucuronidase and ratio of capacity factors. The analysis of S and the metabolites N₄-acetylsulfamethoxazole (N₄), SOH, N₄-hydroxysulfamethoxazole (N₄OH), N₄SOH, and Sgluc in human plasma and urine samples was performed with reversed-phase gradient HPLC with UV detection. In plasma, S and N₄ could be detected in high concentrations, while the other metabolites were present in only minute concentrations. In urine, S and the metabolites and conjugates were present. The quantitation limit of the compounds in plasma are respectively: S and N₄ 0.10 μg/ml; N₄SOH 0.13 μg/ml; N₄OH 0.18 μg/ml; SOH 0.20 μg/ml; and Sgluc 0.39 μg/ml. In urine the quantitation limits are: N₄ and N₄OH 1.4 μg/ml; S 1.5 μg/ml; N₄SOH 1.9 μg/ml; SOH 3.5 μg/ml; and Sgluc 4.1 μg/ml. The method was applied to studies with healthy subjects and HIV positive patients.     

Urinary prostaglandin E2 excretion in renal allotransplantation in man

The urinary prostaglandin E₂ excretion was measured daily for 28 days in 15 patients (10 men and 5 women) after renal allotransplantation. Patients with acute oliguric renal failure immediately after the transplantation showed high urinary PGE₂ concentrations, but no or minimal increase in the total excretion rates. The median PGE₂ excretion was 211 μg/24 h after establishment of stable renal function, but with great individual variations. Rejection crises were characterized by a two-fold increase in PGE₂ excretion, with a subsequent fall induced by the steroid treatment. The PGE₂ excretion correlated better with urinary sodium excretion than diuresis.The pathophysiological role of the renal prostaglandin ssynthesis remains incompletely defined. The prostaglandin E₂ (PGE₂) appears to act as a modulator of the renal salt and water excretion (1,2) and prostaglandins are important mediators of the immunresponses (3,4). The eraly renal allograft rejection is an event characterized by salt and water retention together with decreasing renal function (5). Antibodies against renal tissue as well as cytotoxic leukocytes (“killer cells”) are active in the process (6,7) and many hormonal systems are involved, among them renin and vasopressin (8). Both hormones are known to stimulate the synthesis of prostaglandin in the kidneys and interact with its effect (9,10,11). The present material was therefore designed to study the urinary excretion of PGE₂ in the kidney allografts before and during rejection crises.     

The diuretic effects of CL 115,129 (d,1-15-deoxy-16-hydroxy-16(α/β)-vinyl-prostaglandin E2) and ℓ-prostaglandin E2 in dogs

CL 115,129, the corresponding carboxylic acid and major metabolite of CL 115,347 (d,1-15-deoxy-16-hydroxy-16(α/β)-vinyl-prostaglandin E₂ methyl ester), a potent orally and transdermally long acting antihypertensive agent, infused at 0.1 μg/kg/min into the left renal artery of sodium pentobarbital anesthetized beagle dogs increased urinary volume, sodium (Na⁺), potassium (K⁺) and chloride (Cl⁻) excretion of the left kidney 289, 201, 101 and 229%, respectively, over the 30 min vehicle-treated control periods. At 0.3 μg/kg/min CL 115,129 caused a 475 and 336% increase in urinary volume and Na , respectively. ℓ-Prostaglandin E₂ (ℓ-PGE₂) infused at 0.1 μg/kg/min into the left renal artery increased urinary volume, Na⁺, K⁺ and Cl⁻ excretion of the left kidney of anesthetized beagle dogs 416, 234, 112 and 255%, respectively, over the control. Both CL 115,129 and ℓ-PGE₂ did not affect the systemic arterial blood pressure or the electrolyte excretion of the contralateral kidney. It is concluded that in contrast to other conventional vasodilators, which may cause severe water and electrolyte retention, CL 115,347, via its metabolite CL 115,129, may cause diuresis and natriuresis in many clinical settings when used as an antihypertensive.     

Long-Term Responses to Loss of Renal Mass: Pathophysiological Aspects: The Effects of Functional Adaptation of Residual Nephrons on the Urinary Excretion of Drugs

In patients with chronic renal failure due to glomerulonephritis, pyelonephritis or polycystic kidneys the urinary clearance of free chloramphenicol (C_CHL) was depressed proportionally to GFR (C_In). The ordinate intercept of the regression line of C_CHL on C_In, however, consistently was positive (+3 to +5 ml/min). The fractional excretion of chloramphenicol in renal failure increased from its normal value of 50 percent as an exponential function of the decrease of GFR, and as a linear function of the fractional excretion of water or of sodium. Dietary sodium restriction had no influence on C_CHL in the patients, while water diuresis, in normal subjects, enhanced the urinary excretion of chloramphenicol. The data suggest that chloramphenicol is reabsorbed by back-diffusion and that increases of the rate of flow of urine and tubular fluid prevent back-diffusion.     

Dose response relation of the renal effects of PGA1, PGE2, and PGF2α in dogs

The effects of the three prostaglandins A₁, E₂, and F_2α on renal blood flow, glomerular filtration rate (GFR), fluid excretion, and urinary output of Na, K, Ca, Cl, and solutes were evaluated at a dose range of 0.01 – 10 μg/min. The prostaglandins were infused into the renal artery of dogs. GFR was not significantly altered by the PGs. PGA₁ increased renal blood flow by approximately of the control at 0.01 μg/min without dose dependence at higher infusion rates. It had only little effects which were not dose dependent on fluid and electrolyte output. The effects of PGE₂ on renal blood flow, fluid, sodium, and chloride excretion were dose dependent with a steep slope of the dose response curve between 0.1 and 1.0 μg/min. Blood flow was increased maximally by 80 %, urine volume by more than 400 %. PGF_2α had no effect on renal blood flow, whereas urinary output was increased to approximately the same maximal level as by E₂ although ten times higher doses were needed. Potassium excretion was less influenced than the excretion of Na and Cl and osmolar clearance was less increased than urine volume by all three prostaglandins.It is concluded that if a PG is involved in the regulation of the renal fluid or electrolyte excretion it is likely to be of the PGE-type. A PGA could only be involved in regulation of renal hemodynamics, whereas PGF although effective in the kidney exerts its effects at doses too high to have physiological significance.     

Gas chromatographic—mass spectrometric assay to measure urinary N-methylhistamine excretion in man

An assay has been developed for N^τ-methylhistamine, a major metabolite of the autocoid histamine, based on gas chromatography—electron-capture negative-ion chemical ionisation mass spectrometry. N^τ-Methylhistamine was extracted from urine by cation-exchange chromatography and converted to its di-(3,5-bistrifluoromethylbenzoyl) derivative. The latter has good chromatographic properties and gives a negative-ion mass spectrum with the molecular ion (M, m/z 605) as base peak. A commercially available trideuterated analogue of N^τ-methylhistamine was used as internal standard. Basal urinary excretion of N^τ-methylhistamine in five normal subjects was found to be 0.21 ± 0.05 μmol/h (289 ± 74 μmol/mol of creatinine). This value was not significantly altered in these subjects following the infusion of a sub-pharmacological dose of histamine. In eight atopic volunteers, basal urinary excretion of N^τ-methyl-histamine was also not significantly changed following challenge with inhaled allergen.     

The effect of suppression of prostaglandin synthesis on renal function in rats with intact and reduced renal mass

The present study was undertaken to assess the role of prostaglandin system in the compensatory response to reduced nephron population, respective to renal function and electrolyte excretion. Intact and nephrectomized rats were divided in 4 groups: 1) rats pretreated with indomethacin, 2) rats pretreated with the vehicle of indomethacin, 3) rats pretreated with sulindac, and 4) rats pretreated with the vehicle of sulindac.In normal rats, indomethacin administration resulted in a mild decrease in creatinine clearance and a significant reduction of the urinary Na excretion. In the rats with reduced renal mass treated with indomethacin, the creatinine clearance did not differ from that in the control group. The 24 h urinary sodium excretion and the fractional excretion of sodium, however, were significantly lower in the indomethacin treated animals than in the control rats. No change in the creatinine clearance or in the sodium excretion was observed in all groups pretreated with sulindac.The urinary PGE₂ and thromboxane excretion was significantly lower in the indomethacin treated intact rats and the rats with reduced renal mass. Sulindac induced a slight decrease in urinary excretion of PGE₂ in intact rats. No significant change in urinary excretion of PGE₂ or thromboxane was seen after sulindac in the rats with reduced renal mass.The antinatriuretic effect of indomethacin was dissociated from changes in urine flow in all groups of animals, suggesting that the increase in Na reabsorption tool place in a water impermeable segment of nephron.These results suggest that the compensatory increase in urinary Na excretion per nephron in rats with reduced nephron population at least partly depends on an intact prostaglandin synthesis.     

Excretion of primary prostanoids and their metabolites during acute volume expansion

Simultaneous determination of urinary excretion rates of primary unmetabolized prostanoids and their enzymatic metabolites were performed by gas chromatography-mass spectrometry (GC/MS) or tandem mass spectrometry (GC/MS/MS). Changes in kidney function were induced by acute (4 h) volume expansion. Despite marked changes in urine flow, GFR, urinary pH, osmolality, sodium and potassium excretion, only a insignificant or transient rise in the enzymatic prostanoid metabolites (2,3-dinor-6-keto-PGF_1α, PGE-M, 2,3-dinor-TxB₂ and 11-dehydro-TxB₂) was observed. The excretion rates of the primary prostanoids were elevated in parallel with the rise in urine flow: PGE₂ rose (p < 0.05) from 14.2 ± 4.0 to 86.2 ± 20.7, PGF_2α from 60.0 ± 4.9 to 119.8 ± 24.0, 6-keto-PGF_2α from 7.2 ± 1.3 to 51.5 ± 17.0, and txB₂ from 11.2 ± 3.3 to 13.6 ± 3.6 ng/h/1.73 m² ( ) at the maximal urine flow. Except for 6-keto-PGF_1α and TxB₂, this rise in urinary prostanoid levels was only transient despite a sustained fourfold elevated urine flow. We conclude that urine flow rate acutely affect urine prostanoid excretion rates, however, over a prolonged peroid of time these effects are not maintained. The present data support the concept that urinary levels of primary prostanoids mainly reflect renal concentrations whereas those of enzymatic metabolites reflect systemic prostanoid activity. From the excretion pattern of TxB₂ one can assume that this prostanoid represents renal as well as systemic TxA₂ activity.     

Effect of dietary sodium bicarbonate supplementation on the toxicokinetics of ochratoxin A in pigs

The mycotoxin ochratoxin A (OA) is regarded as a causative agent for endemic nephropathy in farm animals and humans. Reabsorption of OA along the nephron results from nonionic diffusion and by carrier-mediated mechanisms indicating that urine alkalinization may help to accelerate OA excretion and thus reduce its toxicity. The aim of the present study was to investigate the effect of a dietary sodium bicarbonate (NaHCO₃) supplementation as a means to increase urinary pH on the systemic availability and excretion of OA in pigs. Dietary supplementation of 2% NaHCO₃ increased urinary pH (5.7±0.2 to 8.3±0.1) and daily urine volume (1108±276 to 2479±912ml) significantly. The systemic availability of OA and its dechloro-analog Ochratoxin B (OB) in the NaHCO₃ group calculated as the area under the serum concentration-time curve (AUC) was reduced to 75 and 68%, respectively, of the control (P<0.05). This effect was mainly due to an accelerated elimination of OA and OB in the urine. The faster renal elimination might be explained by a reduced reabsorption of the ochratoxins by nonionic diffusion, and other H⁺-dependent mechanisms. Thus, urinary alkalinization might be an efficient means to partially reduce the toxic effects and carry-over of OA in pigs. Presented at the 26^th Mykotoxin Workshop in Herrsching, Germany, May 17–19, 2004     

Effect of polyaspartic acid on CdCl2-induced nephrotoxicity in the rat

We produced an animal model of CdCl₂ nephrotoxicity in rats, and treated them with polyaspartic acid (PAA) to prevent renal damage. Male Sprague-Dawley (SD) rats (190–200 g) were used to induce proximal renal tubular damage by daily injection of CdCl₂ 3.0 mg/1,000 g body wt for 2 wk. CdCl₂-exposed SD rats exhibited significant increases in urine volume, urinary excretion ofN-acetyl-β-D-glucosaminidase (NAG), alanine aminopeptidase (AAP), and fractional excretion of sodium (FENa) and a decrease in the percentage of tubular reabsorption of phosphate (%TRP). Of these indicators of proximal tubular function, AAP and %TRP are more sensitive than NAG or FENa. No glycosuria or aminoaciduria, however, were observed. PAA markedly improved these indicators of proximal tubular function. Daily urinary protein excretion and creatinine clearance, on the other hand, did not change after administration of PAA. Cd concentrations in the cortex were 3 times higher than in the medulla, however, there were no differences between Cd-treated rats and PAA-treated rats. Our animal model is an excellent one for determining the effect of cadmium on renal proximal tubule damage. PAA appears to be useful in the treatment of CdCl₂ nephrotoxicity.     

Effects of Vitamin B6 Deficiency on the Conversion Ratio of Tryptophan to Niacin

To investigate how vitamin B₆ (B₆) deficiency affects the whole metabolism of tryptophan-niacin, rats were fed for 19 days with each of the following four kinds of diets; a complete 20% casein diet (control diet), the control diet without B₆, the control diet without nicotinic acid, and the control diet without nicotinic acid and B₆, and the urinary excretion of such tryptophan metabolites as kynurenic acid, xanthurenic acid, nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5-carboxamide, and N¹-methyl-4-pyridone3- carboxamide each and the enzyme activities involved in tryptophan-niacin pathway were measured. The urinary excretion of kynurenic acid decreased while that of xanthurenic acid increased drastically in the two B₆-deficient groups, when compared with the B₆-containing groups. These results indicate that the rats fed with the B₆-free diets were in the vitamin-deficient state. The conversion ratio was calculated from the ratio of the urinary excretion of sum of nicotinamide, N¹-methylnicotinamide, N¹-methyl-2-pyridone-5carboxamide, and N¹-methyl-4-pyridone-3-carboxamide, to the Trp intake. The ratio was statistically lower in the B₆-free diet than in the B₆-containing diet under the niacin-free conditions.     

Regulation of urinary thromboxane B2 in man: Influence of urinary flow rate and tubular transport

Thromboxane B₂ (TxB) is excreted in human urine, but the mechanism of renal excretion and the quantitative relationship of urinary TxB to the active parent compound, thromboxane A₂, of renal or extrarenal origin is not established. To determine the effects of vasoactive hormones, uricosuric agents and urinary flow rate on TxB excretion, urinary TxB was measured by radioimmunoassay and mass spectrometry, and renal metabolism of blood TxB was determined by radiochromatography of urine after i.v. [³H]-TxB infusions. Basal TxB was 6.7 ± 1.1 ng/h during an oral water load, and TxB fell with s.q. antidiuretic hormone (to 3.4 ± 0.4 ng/h, P<0.01) and with fluid restriction (to 2.6 ± 0.5 ng/hr, P=0.001) in parallel with urinary volume. Urinary excretion of unmetabolized [³H]-TxB also fell (by 56%) with fluid restriction, implicating altered metabolism rather than synthesis as the mechanism of the urinary flow effect. Angiotensin II infusions slightly reduced both TxB and urine volume, consistent with a flow effect. In contrast, probenecid did not alter urine volume, but increased urinary uric acid (by 244%), TxB (from 5.6 ± 0.9 to 11.1 ± 2.9 ng/h) and urinary excretion of blood [³H]-TxB (by 243%) by similar amounts (all P<0.05), suggesting that TxB is actively reabsorbed in the proximal tubule, similarly to uric acid. Thus, urinary excretion of TxB of renal and extrarenal origin is regulated by proximal and distal tubule factors.     

Metabolism of PG in man: Effect of furosemide on the excretion of the main metabolite of PG F2α

The excretion rates of main urinary metabolite of PG F₂α were measured radioimmunologically in 4 healthy persons and in 13 essential hypertensives. The resting values were 9.3±0.73 in the former and 10.4±2.17 ng/min in the latter. There was no significant differences between them. The excretion of the metabolite decresed prominently after the administration of furosemide. The percent decrease was 57% in healthy persons and 70% in essential hypertension. The percent result supports that furosemide inhibit the catabolism of PG F₂α.     

A bout of resistance exercise increases urinary calcium independently of osteoclastic activation in men

Ashizawa, Noriko, Rei Fujimura, Kumpei Tokuyama, andMasashige Suzuki. A bout of resistance exercise increases urinary calcium independently of osteoclastic activation in men.J. Appl. Physiol. 83(4):1159-1163, 1997.Metabolic acidosis increases urinary calciumexcretion in humans as a result of administration of ammonium chloride,an increase in dietary protein intake, and fasting-inducedketoacidosis. An intense bout of exercise, exceeding aerobic capacity, also causes significant decrease in blood pH as aresult of increase in blood lactate concentration. In this study weinvestigated changes in renal calcium handling, plasma parathyroidhormone concentration, and osteoclastic bone resorption after a singlebout of resistance exercise. Ten male subjects completed about of resistance exercise with an intensity of 60% of one repetitionmaximum for the first set and 80% of one repetition maximum for thesecond and third sets. After exercise, blood and urine pH shiftedtoward acidity and urinary calcium excretion increased.Hypercalciuria was observed in the presence of an increased fractionalcalcium excretion and an unchanged filtered load of calcium. Therefore,the observed increase in urinary calcium excretion was due primarily todecrease in renal tubular reabsorption of calcium. Likely causes of theincrease in renal excretion of calcium are metabolic acidosis itselfand decreased parathyroid hormone. When urinary calcium excretionincreased, urinary deoxypyridinoline, a marker of osteoclastic boneresorption, decreased. These results suggest that1) strenuous resistance exerciseincreased urinary calcium excretion by decreasing renal tubular calciumreabsorption, 2) urinary calciumexcretion increased independently of osteoclast activation, and3) the mechanism resulting inpostexercise hypercalciuria might involve non-cell-mediatedphysicochemical bone dissolution.

     

High urine flow rate increases prostaglandin E excretion in the conscious dog

Although previous studies from this and other laboratories have shown that urinary prostaglandin E excretion (U_PGEV) can vary independent of urine flow rate, recent studies during water diuresis in the conscious dog have suggested that high urine flow rate per se may increase U_PGEV. To examine the effect of urine flow rate on U_PGEV we administered either mannitol, chlorothiazide or Ringer's solution to mongrel dogs and measured U_PGEV. During anesthesia neither mannitol or chlorothiazide increased U_PGEV. There was, however, a consistent increase with all three agents in awake animals. This increase in U_PGEV was independent of alterations in glomerular filtration rate. There was a consistent increase in urinary sodium excretion and decrease in urinary osmolality with all three agents. The changes in PGE, however, were similar to those found during water diuresis when no increase in sodium excretion was found. It is not presently clear whether these findings reflect a true increase in renal PGE synthesis due to some change in flow or pressure within the renal medulla or rather represent unchanged PGE synthesis by renal tubular cells, the high tubule fluid flow rate causing increased entry into the tubular lumen in contrast to the renal interstitium.     

The effect of dehydroepiandrosterone (DHEA) on renal function and metabolism in diabetic rats

Dehydroepiandrosterone (DHEA) is an endogenous steroid hormone involved in a number of biological actions in humans and rodents, but its effects on renal tissue have not yet been fully understood. The aim of this study is to assess the effect of DHEA treatment on diabetic rats, mainly in relation to renal function and metabolism. Diabetic rats were treated with subcutaneous injections of a 10 mg/kg dose of DHEA diluted in oil. Plasma glucose and creatinine, in addition to urine creatinine, were quantified espectophotometrically. Glucose uptake and oxidation were quantified using radioactive glucose, the urinary Transforming Growth Factor β₁ (TGF-β₁) was assessed by enzyme immunoassay, and the total glutathione in the renal tissue was also measured. The diabetic rats displayed higher levels of glycemia, and DHEA treatment reduced hyperglycemia. Plasmatic creatinine levels were higher in the diabetic rats treated with DHEA, while creatinine clearance was lower. Glucose uptake and oxidation were lower in the renal medulla of the diabetic rats treated with DHEA, and urinary TGF-β₁, as well as total gluthatione levels, were higher in the diabetic rats treated with DHEA. DHEA treatment was not beneficial to renal tissue, since it reduced the glomerular filtration rate and renal medulla metabolism, while increasing the urinary excretion of TGF-β₁ and the compensatory response by the glutathione system, probably due to a mechanism involving a pro-oxidant action or a pro-fibrotic effect of this androgen or its derivatives. In conclusion, this study reports that DHEA treatment may be harmful to renal tissue, but the mechanisms of this action have not yet been fully understood.     

Effects of acid challenge on in vivo and in vitro rat renal ammoniagenesis.

This study compares the ability of different strengths of NH₄Cl, CaCl₂, and HCl to affect the termporal excretion of ammonium in rats. Oral NH₄Cl given in a single dose of 0.5 mmole, 1.0 mmole and 1.5 mmole/100 g BW steadily increases ammonium excretion in rats. The majority of the augmented ammonium excretion is secondary to increased renal production — not to changes in urine pH or urine volume. Acute challenges greater than 1.5 mmole/100 g BW do not increase ammonium excretion further. Results were similar when chronic acid challenge was investigated — greater NH₄Cl challenges cause greater ammonium excretion. Challenges beyond 1.5 mmole/100 g BW bid frequently cause death unless the rats are preconditioned (made mildly acidotic) prior to initiation of this dose. At the 1.5 mmole/100 g BW dose, maximal ammonium excretion is reached by day 2 or 3. Thus, maximal renal ammoniagenesis during acid stress occurs rapidly, and at different times depending on the strength of the acid challenge. CaCl₂ or HCl offer no advantages over NH₄Cl as acidifying agents. In addition to the above, there is a significant correlation between ammonium excretion $\text{in vivo}$ and the ability of rat renal slices to produce ammonia from glutamine or glutamate $\text{in vitro}$.