Françoise Roch-Ramel

An enzymic and fluorophotometric method for estimating urea concentrations in nanoliter specimens

Abstract An ultramicro method for estimating 2 × 10−11 to 10−10 mole urea or 4 × 10−11 to 2 × 10−10 equiv NH4+ is described. The method employs urease. Ammonia reacts with α-ketoglutarate and NADH in the presence of highly purified glutamate dehydrogenase (GLDH), yielding glutamate and oxidized NAD+. NAD+ is transformed into a fluorescent compound (340–460 mμ) either by treatment with strong alkali or by condensation with a ketone. The specificity and the sensitivity of the method are adequate for the analysis of micro puncture samples of normal tubular fluid, and may be assumed to allow the estimation of very small amounts of urea or NH4+ in small samples of other body fluids.

Transport of organic cations in brush border membrane vesicles from rabbit kidney cortex.

The transport of three organic cations, tetraethylammonium (TEA), morphine and N1-methylnicotinamide (NMN) was studied in brush border membrane vesicles from rabbit kidney cortex under voltage clamp conditions. A proton gradient (pHi=6.0, pHo=7.4) produced a large stimulation of TEA and morphine uptake, yielding a transient overshoot of 190 and 220% respectively, as compared to equilibrium uptake values. No overshoot was observed under pH equilibrium conditions (pHi=pHo=7.4, control). These data suggest the presence of a proton-organic cation exchange mechanism in the rabbit renal cortical brush border membrane. Identical experimental conditions (proton gradient) failed however to stimulate significantly NMN transport above control values measured under pH equilibrium conditions. Proton gradient driven TEA transport showed an inhibition of 21% in the presence of NMN (1 mM) and of 63% in the presence of TEA (1 mM), and TEA transport was stimulated by preloading the vesicles with 1 mM TEA (305%) but not with 1 mM NMN (128%). NMN transport showed an inhibition of 39% in the presence of 1 mM TEA and of 27% in the presence of 1 mM NMN and its transport was stimulated by preloading the vesicles with 1 mM TEA (228%) and 1 mM NMN (178%). Our data suggest that TEA, NMN and morphine are transported by a common transport mechanism for which NMN has only a low affinity.

Intrarenal urea and electrolyte concentrations as influenced by water diuresis and by hydrochlorothiazide

Abstract Concentrations of urea, ammonium, sodium and potassium as well as water content were measured in the renal papilla, medulla and cortex, the heart muscle, plasma and urine of rats, in the dehydrated state, the non-diuretic state and after 3 or 8 hr of sustained brisk water diuresis. The same measurements were made on rats which had received a natriuretic dose of hydrochlorothiazide during the 8th hr of water diuresis. Water content of renal tissue increased from cortex to inner medulla in all conditions. In hydrated rats the water content of all layers of renal tissue increased during the first 3 hr and remained stable thereafter. “Exaltation” of urea excretion continued during the first 2 hr of water diuresis, but subsided subsequently. Urinary urea concentrations were equal to, or higher than, urea concentrations in papillary water in the dehydrated and in the non-diuretic states. After 3 hr of water diuresis, urea concentrations decreased in all layers of renal tissue. The papillary urea concentration remained consistently higher than the urinary concentration. The papillo-urinary urea gradient of approximately 80 mmol/1 decreased to 19 mmol/1 after 8 hr of water diuresis, and disappeared in continuous water diuresis sustained over 30 days. These data are interpreted as a consequence of sequestration or tissue binding of urea in compartments of different accessibility to water flowing through papillary and medullary tissues. Cortical tissue water contained more urea than plasma, under all conditions investigated. A part of cortical urea may be sequestered in particular compartments. The cortico-papillary sodium concentration profile corresponded to a 2.8–5.1 fold concentration ratio for sodium in the non-diuretic and in the dehydrated states and decreased during 8 hr of water diuresis. The urinary sodium concentration consistently remained much lower than renal or plasma concentrations. The low sodium concentration in cortical tissue water allows an upper limit of approximately 1 2 to be set for the contribution of intratubular fluid to total tissue water. Potassium concentration in papillary tissue water was equal to or lower than cortical potassium concentration and did not change significantly in water diuresis. Calculated urinary osmolalities were higher than measured values in concentrated urine and in plasma, but were equal in dilute urine. The osmolar concentration profiles in renal tissue depended more on urea than on salt concentrations. Urinary ammonium concentration was higher than the papillary concentration in the dehydrated and the non-diuretic state, but lower in water diuresis. Hydrochlorothiazide, in water diuresis, caused a considerable increase in the urinary sodium concentration and excretion, and a slight increase in the urinary potassium excretion, but did not increase urine flow. Hydrochlorothiazide did not cause a significant decrease in the renal clearance of urea under these circumstances, but induced a slight depression of papillary urea concentration.

The effects of pressure on the water permeability of the descending limb of Henle's loops of rabbits.

Descending limbs of Henles loops from rabbits were perfused in vitro. Using techniques where the collecting pipets permitted cannulation of the tubule, we were able to maintain reasonable flow rates at lower perfusion reservoir heights than are required with a conventional “Sylgard seal” pipet. The bath was either isosmotic to the perfusate, or was made 300 mOsm hyperosmotic using urea. Net water reabsorption did not occur in tubules perfused at low pressure (average reservoir height = 26 cm H2O) even when the bath was hyperosmotic: ΔJv=−0.06 ±0.18 nl/min (n=7). Observed increases in sodium concentration and osmolality of collected fluid, when the bath was made hyperosmotic, were 16±8 mM (n=7) and 254±38 mOsm (n=7), respectively. Presumably the large increase in osmolality of the collected fluid was due to entrance of urea.When the “Sylgard seal” collecting end was utilized higher perfusion reservoir heights had to be used to maintain flow (mean height 66 cm H2O). These tubules were highly permeable to water as reported by others for this tubule segment. In the presence of a hyperosmotic bath water extrusion resulted in a dramatic increase in the osmolality of the collected fluid (312 ±5 mOsm; 7 tubules) which was almost completely accounted for by an increase in sodium concentration (153±8 mmole/l; 6 tubules).The14C urea permeability (measured lumen to bath) of descending limbs in a 300 mOsm bath was 0.64 ×10−7 cm2·s−1±0.23×10−7 (11 tubules). When the bath was made hyperosmotic using urea or raffinose the14C urea permeability increased significantly.

Net Sodium and Water Movements in the Newborn Rabbit Collecting Tubule: Lack of Modifications by Indomethacin

In contrast to adult tubules, a bath-to-lumen sodium gradient was followed by net sodium entry in the lumen of in vitro isolated perfused newborn (3-5 days) rabbit collecting tubules. In addition, transepithelial voltage and electrical resistance were lower in newborn tubules. The sodium flux into the lumen was unaffected by indomethacin. These observations suggest that the newborn collecting tubule may be unable to maintain an adequate sodium gradient for the optimal function of the urinary concentrating mechanism, and that endogenous prostaglandins are not involved in this defect.

Transport of Urate and Other Organic Anions by Anion Exchange in Human Renal Brush-Border Membrane Vesicles

Earlier studies demonstrated that an apical anion exchanger which accepts urate, chloride, aliphatic monocarboxylates, and the aromatic anions, pyrazinoate (PZA) and nicotinate, plays a major role in urate reabsorption in human proximal tubules. In substrate competition studies for transport, we investigated the effect of PZA on 50 µM [14C]-urate uptake in exchange of lactate, human brush-border membrane vesicles (BBMV) being loaded with 5 mM lactate. 1 mM PZA cis-inhibited, whereas 0.1 mM PZA cis-stimulated [14C]-urate uptake in exchange for lactate. Such paradoxical effects suggest the cooperation of two anion exchangers, PZA having more affinity for a ‘lactate/anion exchanger’ than for a ‘urate/ anion exchanger’. Apparent Km of [14C]-PZA uptake were measured in lactate- or urate-loaded BBMV, and were 0.33 ± 0.06 and 0.98 ± 0.44 mM, respectively. Unlike [14C]-PZA, [14C]-orotate and [14C]-nicotinate uptakes were stimulated only to a minor extent in BBMV loaded with lactate. In contrast, cis -inhibition studies in urate-loaded BBMV demonstrated that nicotinate and orotate had similar affinity as PZA for the ‘urate/anion exchanger’. Oxalate was shown to have affinity for the ‘lactate/anion exchanger’, but not for the ‘urate/anion exchanger’.

A micropuncture study of urate excretion byCebus monkeys employing high performance liquid chromatography with amperometric detection of urate

Net renal reabsorption of endogenous urate was studied by the micropuncture technique inCebus monkeys in the absence of osmotic diuresis. Most of filtered urate (more than 70%) was reabsorbed in the proximal convoluted tubules. Samples from early distal tubules contained 9% of filtered urate; approximately 18% being reabsorbed between the late proximal and early distal segments. There was no detectable reabsorption along the distal tubule. Fractional delivery of urate to late distal tubules was greater than fractional excretion, implying reabsorption of some 4% of filtered urate in the collecting system. However, we cannot exclude nephron heterogeneity as the cause of the difference.The foregoing results were obtained using the method of Pachla and Kissinger for the determination of urate. Urate is separated by high performance liquid chromatography and detected by an amperometric technique. We found the method to be sufficiently sensitive, precise and specific for renal micropuncture samples.

General and renal toxicity of phenacetin, paracetamol and some anti-mitotic agents in the rat.

The general and the renal toxicity of large doses of phenacetin, paracetamol, some antimitotic drugs and other constituents of analgesic mixtures was investigated in medium term toxicity tests in a large number of rats. Phenacetin and paracetamol depressed food intake and retarded growth. 800–1200 mg/kg · day paracetamol induced a larger mortality than 1500–3000 mg/kg · day phenacetin. Both analgesics and isophthalanilide, an antimitotic agent, induced hyperchromic anemia. Phenacetin induced methemoglobinemia more readily than paracetamol. Neither the analgesics, nor caffeine, sodium nitrite, isophthalanilide or mercaptopurine depressed GFR, maximal urinary concentration after dehydration plus vasopressin, urinary dilution after hypotonic expansion, or urinary acidification. Phenacetin, paracetamol and isophthalanilide depressed the fractional excretion of urea by the kidney. Very large doses of paracetamol slightly increased the proteinuria and the urinary excretion of tubular cells. Phenacetin and paracetamol induced degenerative histological alterations in cortical proximal and distal tubules, detected and quantitated under blind conditions. There were no inflammatory changes, nor any medullary or papillary lesions. The degenerative lesions could not be explained by the presence of methemoglobinemia or hemolysis. Isophthalanilide actually “improved” the histological appearance of the kidneys. The urinary excretion of tubular cells was not significantly correlated with the severity of the histological changes. It was concluded that neither phenacetin nor paracetamol exert major nephrotoxic effects in rats.ZusammenfassungDie allgemeine und renale Toxicität von großen Dosen von Phenacetin, Paracetamol und potentiell nephrotoxisehen Antimitotica, sowie anderen üblichen Bestandteilen von analgetischen Mischpräparaten wurde an Ratten in 30–60 Tage-Versuchen untersucht. Phenacetin und Paracetamol hemmten Futteraufnahme und Wachstum. 800–1200 mg/kg · Tag Paracetamol tötete mehr Tiere als 1500–3000 mg/kg · Tag Phenacetin. Die beiden Analgetica und das Antimitoticum Isophthalanilid riefen hyperchrome Anämie hervor. Phenacetin verursachte mehr Methämoglobinämie als Paracetamol. Weder dieser Analgetica, noch Coffein, noch Natriumnitrit, noch Isophthalanilid, noch Mercaptopurin verringerten das Glomerulusfiltrat, die maximale Harnkonzentrierungsfähigkeit nach Durst unter Vasopressin, die Harnverdünnuungsfähigkeit nach hypotonischen Infusionen oder die renale H+-Ionen-Sekretion. Phenacetin, Paracetamol und Isophthalanilid verringerten die fraktionelle renale Harnstoffausscheidung. Sehr große Dosen von Paracetamol steigerten leicht die Proteinurie und die Ausscheidung von Tubuluszellen im Harn. Phenacetin und Paracetamol verursachten degenerative Veränderungen im histologischen Bild der corticalen, proximalen und distalen Tubuli, die unter Blindbedingungen untersucht wurden. Es traten weder entzündliche Veränderungen, noch Schädigungen des Nierenmarks oder der Papille auf. Die corticalen degenerativen Veränderungen ließen sich nicht als Folge von Methämoglobinämie oder Hämolyse erklären. Isophthalanilid verursachte eine „Normalisierung” des histologischen Bilds der Nierenrinde über die Norm hinaus. Die Ausscheidung von Tubuluszellen im Harn war nicht signifikativ mit der Schwere der histologischen Veränderungen korreliert. Die Befunde führen zum Schluß, daß weder Phenacetin, noch Paracetamol bei der Ratte ausgeprägte nephrotoxische Wirkungen besitzt.

Pyrazinoate transport in the isolated perfused rabbit proximal tubule

The bidirectional tubular transport of pyrazinoate (PZA) was studied in the isolated perfused proximal S2 segment of rabbit kidney. PZA reabsorption was a mechanism of large capacity, temperature-dependent and requiring a normal Na+/K+-ATPase activity. PZA reabsorption was reversibly decreased when lactate was added to the perfusate, indicating that it might occur through the sodium-lactate cotransport. The addition of PAH to the bath had a slight stimulatory effect on PZA reabsorption, suggesting a component of anion exchange in the overall PZA reabsorption. However, SITS added to either the perfusate or the bathing medium induced a non-significant decrease in PZA reabsorption, confirming the minor part of an anion exchange mechanism in this reabsorptive process. PZA reabsorption was not affected by the establishment of a bath-to-lumen H+ gradient, and was only moderately decreased after carbonic anhydrase inhibition by ethoxyzolamide, in opposition to what is known for the reabsorbed anion salicylate. The secretory transport of PZA was saturable and also dependent on a normal Na+/K+-ATPase activity. It is concluded that PZA is bidirectionally transported by facilitated mechanisms in the rabbit proximal S2 segment, one major reabsorptive mechanism appearing to be a sodium-anion cotransport, which might be the sodium-lactate reabsorbing mechanism.

Establishment of renal cell lines derived from S2 segments of the proximal tubule

The aim of this study was to establish epithelial cell lines derived from defined nephron segments. Primary cultures were prepared from dissected proximal S2 segments of the rabbit kidney, and grown in monolayers. Immortalization was observed after nuclear microinjection of the cells with simian virus 40 DNA and resulted in the development of cell lines of epithelial morphology. These cell lines were maintained in culture for at least 24 passages, then cells were frozen. One of the cell lines, the RKPC-2, was selected and further characterized. RKPC-2 cells formed domes on impermeable supports, indicating fluid and solute transport. RKPC-2 cells formed continuous monolayers of low transepithelial resistance on collagen-coated filters. They were able to accumulate tetraethylammonium, an organic cation; however, no significant transcellular transport could be measured. We conclude that this cell line which shows characteristics of epithelial cells has maintained certain properties of intact proximal tubules, in particular the capacity to accumulate organic cations.