Annette Hus-Citharel

Production of Urea from Arginine in Pars recta and Collecting Duct of the Rat Kidney

Urea production from arginine was studied in vitro in the kidney of normal rats in tubule suspensions of the four different renal zones (cortex, outer and inner stripe of outer medulla, and inner medulla), and in individual microdissected nephron segments. Tissue was incubated with L-[guanido-14C]-arginine to measure cellular arginase activity. Addition of urease to the incubate freed 14CO2 from the 14C-urea formed by arginase and released from the cells. CO2 was trapped in KOH and counted. These experiments revealed that significant amounts of urea are produced in the outer stripe and in the inner medulla. This intrarenal urea generation takes place mainly in the proximal straight tubule and in the collecting duct, with increasing activity in these two structures from superficial to deep regions of the kidney. Urea is known to play a critical role in the urinary concentrating process. The fact that some urea can be produced in the mammalian kidney, and that the two structures showing this capacity are straight portions of the renal tubular system descending along the corticopapillary axis suggest that this urea production might play a role in the formation and/or maintenance of the medullary urea concentration gradient.

Coupling of metabolic CO2 production to ion transport in isolated rat thick ascending limbs and collecting tubules

Metabolic CO2 production from appropriate [U-14C]-labelled substrates (eitherl-lactate ord-glucose) was measured in single pieces of tubule as previously described (Le Bouffant et al. 1984). Changing the incubate osmotic pressure by mannitol addition resulted in an increase in oxidative metabolism which was more marked in outermedullary segments (MAL and MCT) than in cortical segments (CAL and CCT). Availability of metabolic substrate was not rate limiting under these conditions because FCCP addition (1 μmol·l−1) produced a marked rise in CO2 production in these structures.Ouabain (1 mmol·l−1) decreased by more than 50% the CO2 production by CAL, MAL, CCT and MCT samples, indicating that the larger part of oxidative metabolism was coupled to active Na transport. Furosemide addition (10−5 mol·l−1) to CAL and MAL samples, or amiloride addition (10−4 mol·l−1) to CCT and MCT samples reduced the rate of CO2 production to an extent almost similar to that obtained with ouabain, an observation suggesting that apical entry of Na+ was present in these non-perfused tubules.Finally, the effects of changing the concentration of either K+ or Cl− was tested in CAL samples. K+ suppression greatly depressed the rate of CO2 production. Replacement of chloride with sulfate also decreased this rate to an extent similar to that observed with furosemide. The CO2 production increased in a sigmoid way (apparentKa=41 mmol·l−1, Hill coefficient=2.12) as a function of [Cl−] in the incubate, suggesting that oxidative metabolism was coupled to bath chloride via the Cl−-requiring Na entry along the 1 Na+−1K+−2Cl− luminal contrasport system.

Metabolic CO2 production by isolated single pieces of rat distal nephron segments

A method is described which allowed in-vitro measurements of metabolic CO2 production from [U-14C]-substrates by single pieces of kidney tubules. The tubules were isolated by microdissection from collagenase treated rat kidneys. Single pieces of various distal nephrons portions were incubated in 1 μl of bicarbonate free minimum essential medium containing the required [U-14C]-substrate (about 0.2 μCi per sample), and the14CO2 produced was continuously trapped into a 2-μl KOH droplet. The KOH droplets were replaced every 30 min. Metabolic CO2 production from the labelled substrate used was calculated as picomoles CO2 per mm of tubular length per minute, by dividing the KOH radioactivity by the specific radioactivity per carbon of the substrate present in the incubate ([U-14C] plus cold substrate concentrations). Under these conditions, it was established that single pieces of tubule could sustain almost constant CO2 production for at least 2 h at 31° C. Experiments testing four different conditions with five to six replicate samples per condition were performed in order to compare oxidative metabolism in medullary (MAL) and cortical (CAL) thick ascending limbs, medullary (MCT) and cortical (CCT) collecting tubules and, in a few instances, proximal convoluted tubules (PCT) and early distal convoluted tubules (DCT). The results show that: a) PCT poorly oxidized glucose; b) in contrast, CAL, MAL, CCT, MCT and DCT oxidized efficiently glucose and lactate; c) however, when both substrates were offered simultaneously, CAL and MAL oxidized lactate preferentially to glucose, whereas CCT oxidized glucose preferentially to lactate; d) when each segment was offered its preferential substrate, the rate of oxidative metabolism was observed to vary from about 4 to 0.5 pmol·mm−1·min−1 according to the following sequence: DCT>MAL>CAL≈PCT>CCT ≈MCT.

Ornithine decarboxylase along the mouse and rat nephron

Renal arginase activity is a potent source of ornithine (Orn) for polyamine synthesis. Ornithine decarboxylase (ODC) was localized along the mouse and rat nephron by incubating viable nephron segments isolated by microdissection from collagenase-treated kidneys with or without D,L-2-(difluoromethyl)ornithine (DFMO), a selective inactivator of ODC. Tubules from either control or DFMO-treated animals were incubated with 100 ¿M L-[1-14C]Orn. In control mice, Orn decarboxylation occurred mainly in the proximal convoluted tubule (PCT). In DFMO-treated mice, Orn decarboxylation was dramatically reduced in PCT and in proximal straight tubules (PST). In rats, Orn decarboxylation also occurred predominantly in the proximal tubule. Addition of 10 mM DFMO to isolated tubules dramatically decreased Orn decarboxylation in PCT and in PST. Thereafter, ODC activity was demonstrated in permeabilized tubules. In Triton X-100-treated tubules from control mice, ODC was exclusively found in proximal tubules (PCT > PST). This ODC activity was strongly inhibited in DFMO-treated mice. In conclusion, the highest ODC activity was found in rat and mouse PCT, a segment devoid of arginase. We hypothesize that the filtered Orn, which is reabsorbed along the PCT,is the main source of Orn for ODC.Renal arginase activity is a potent source of ornithine (Orn) for polyamine synthesis. Ornithine decarboxylase (ODC) was localized along the mouse and rat nephron by incubating viable nephron segments isolated by microdissection from collagenase-treated kidneys with or withoutd,l-2-(difluoromethyl)ornithine (DFMO), a selective inactivator of ODC. Tubules from either control or DFMO-treated animals were incubated with 100 μMl-[1-14C]Orn. In control mice, Orn decarboxylation occurred mainly in the proximal convoluted tubule (PCT). In DFMO-treated mice, Orn decarboxylation was dramatically reduced in PCT and in proximal straight tubules (PST). In rats, Orn decarboxylation also occurred predominantly in the proximal tubule. Addition of 10 mM DFMO to isolated tubules dramatically decreased Orn decarboxylation in PCT and in PST. Thereafter, ODC activity was demonstrated in permeabilized tubules. In Triton X-100-treated tubules from control mice, ODC was exclusively found in proximal tubules (PCT > PST). This ODC activity was strongly inhibited in DFMO-treated mice. In conclusion, the highest ODC activity was found in rat and mouse PCT, a segment devoid of arginase. We hypothesize that the filtered Orn, which is reabsorbed along the PCT, is the main source of Orn for ODC.

Urea production by kidney collecting ducts in vitro: effect of amino acid addition

Urea production by cortical (CCD) and medullary (OMCD) collecting ducts of the rat kidney was measured in vitro by incubating single microdissected pieces of tubule in the presence of L-[guanido-14C]arginine (0.2 mM). The [14C]urea released from the cells was hydrolysed in presence of urease added to the incubation medium and the 14CO2 formed was trapped in KOH and counted. The effect of various amino acids (AA) on urea production was investigated by adding unlabelled AA (either in combination or singly) at concentrations close to those present in blood plasma. A mixture of 17 AA decreased urea production from [14C]arginine by 46% in CCD and by 58% in OMCD. When lysine and proline were omitted from the mixture, the inhibition was less marked (19% in CCD and 43% in OMCD, respectively). When AA were tested singly, lysine induced the larger inhibition (40% in CCD and 45% in OMCD), than ornithine and glutamine (about 15% each, in CCD and OMCD), whereas proline inhibition (7% in CCD, 10% in OMCD) was not statistically significant. Branched-chain amino acids (BCAA) in combination (leucine, isoleucine and valine) also markedly reduced urea production by CCD and OMCD. Their effect was dose dependent. Solubilization of CCD and OMCD cell membranes with Triton X-100 resulted in a twofold increase in urea production by control samples; the relative inhibition (per cent) induced by BCAA was enhanced, whereas that induced by lysine was decreased. The data suggest that, in living tubules, the inhibition obtained with lysine resulted, for a large part, from competition between lysine and arginine for cell uptake via a common membrane carrier, whereas the inhibition induced by BCAA corresponded to an effect on arginase activity itself.

AT1 receptor expression in glomeruli from NO-deficient rats.

Chronic inhibition of nitric oxide synthase promotes renin-dependent hypertension and renal injury. The present study examines how renal angiotensin II receptors are expressed in this model. NG-nitro-L-arginine methyl ester (L-NAME) was given orally to rats for 1 month and was associated or not with captopril during the 4 last days of the administration. 125I-[Sar1, Ile8]-Ang II binding, AT1 mRNA and cytosolic calcium were studied in isolated glomeruli from L-NAME and control rats and in cultured mesangial cells from normal rats. Renal injury was marked in rats receiving L-NAME. Type I angiotensin II (AT1) receptor number and mRNA expression were decreased (p < 0.05) in glomeruli isolated from L-NAME-treated rats compared with controls, unless they received captopril in combination. The low level of AT1 receptor expression was associated with an attenuated rise of cytosolic calcium in response to angiotensin II. Angiotensin-converting enzyme activity in glomeruli and angiotensin II concentration in renal cortex were increased (p < 0.05) in rats receiving L-NAME alone, whereas aminopeptidase A activity was not modified. To better discriminate between the direct and indirect effects of nitric oxide deficiency, rat mesangial cells were exposed or not for 24 h to S-nitroso-N-acetyl penicillamine, a nitric oxide donor. Angiotensin II binding, AT1 mRNA expression and calcium response to angiotensin II were decreased in presence of the nitric oxide donor (p < 0.01). These results suggest that the decrease of AT1 receptor expression after 1 month of L-NAME treatment does not depend on a direct effect of nitric oxide deficiency but results from the high local angiotensin II concentration due to the stimulated angiotensin-converting enzyme activity. They also show that the renin-angiotensin dependence of this model of hypertension does not result from the overexpression of AT1 receptors.

Sites of arginine synthesis and urea production along the nephron of a desert rodent species,Meriones shawi

The distribution of arginine synthase and arginase activities along the successive nephron segments ofMeriones kidney was measured in vitro with single tubule enzymatic microtechniques making use of eitherl-[ureido-14C] Citrulline (0.108 mM) orl-[guanidion-14C]arginine (0.2 mM) as the respective substrates. Arginase activity (fmol urea formed per min per mm of tubule) was very low (5–25 fmol.min−1.mm−1) in most nephron segments including the early portions of proximal convoluted tubules (early PCT). It increased progressively after 3 mm of the PCT to reach a value of 200 fmol.min−1.mm−1 in the cortical portion of the straight proximal tubule (CPST), with a further increase, along the pars recta, of up to 250 fmol.min−1.mm−1 in the outer medullary portion (OSPST). In addition, arginase activity in OSPST and the adjacent descending thin limb (DTL) was higher in juxtamedullary nephrons (JN) than in the corresponding portions of superficial nephrons (SN). Arginine synthase activity (fmol arginine formed per mm of tubule per min) was present in proximal tubules exclusively, with a gradient decreasing along the PCT (about 600 fmol.min−1.mm−1 in the 1st mm, 65 fmol.min−1.mm−1 in CPST and 30 fmol. min−1. mm−1 in OSPST). It has been checked that CPST and OSPST (where the two enzyme systems are present) are able to convert citrulline directly into urea with a yield of 65%. It is suggested that: (1) in early PCT cells, arginine synthase activity permits the conversion of the reabsorbed citrulline into arginine (which then diffuses towards blood vessels); and (2) in pars recta cells, arginase activity results in a net entry of arginine across the basolateral membranes and in a net exit of the formed urea into the tubular fluid, if the permeability to urea of luminal membranes is greater than that of basolateral membranes. Such a mechanism of urea secretion might contribute to the maintenance of urea recycling in the medulla and, thereby, participate in the process of concentrating the urine.

Genetically increased angiotensin I-converting enzyme alters peripheral and renal vascular reactivity to angiotensin II and bradykinin in mice

Angiotensin I-converting enzyme (ACE) levels in humans are under strong genetic influence. Genetic variation in ACE has been linked to risk for and progression of cardiovascular and renal diseases. Causality has been documented in genetically modified mice, but the mechanisms underlying causality are not completely elucidated. To further document the vascular and renal consequences of a moderate genetic increase in ACE synthesis, we studied genetically modified mice carrying three copies of the ACE gene (three-copy mice) and littermate wild-type animals (two-copy mice). We investigated peripheral and renal vascular reactivity to angiotensin II and bradykinin in vivo by measuring blood pressure and renal blood flow after intravenous administration and also reactivity of isolated glomerular arterioles by following intracellular Ca2+ mobilization. Carrying three copies of the ACE gene potentiated the systemic and renal vascular responses to angiotensin II over the whole range of peptide concentration tested. Consistently, the response of isolated glomerular afferent arterioles to angiotensin II was enhanced in three-copy mice. In these mice, signaling pathways triggered by endothelial activation by bradykinin or carbachol in glomerular arterioles were also altered. Although the nitric oxide (NO) synthase (NOS)/NO pathway was not functional in arterioles of two-copy mice, in muscular efferent arterioles of three-copy mice NOS3 gene expression was induced and NO mediated the effect of bradykinin or carbachol. These data document new and unexpected vascular consequences of a genetic increase in ACE synthesis. Enhanced vasoconstrictor effect of angiotensin II may contribute to the risk for cardiovascular and renal diseases linked to genetically high ACE levels. NEW & NOTEWORTHY A moderate genetic increase in angiotensin I-converting enzyme (ACE) in mice similar to the effect of the ACE gene D allele in humans unexpectedly potentiates the systemic and renal vasoconstrictor responses to angiotensin II. It also alters the endothelial signaling pathways triggered by bradykinin or carbachol in glomerular efferent arterioles.