Medullary Collecting Tubules Research Articles

Poly-ADP ribose polymerase, xanthine oxidase and nitric oxide synthase expression in kidney tissue of experimental diabetes

Background The spatial distribution of inflammatory and DNA reparation markers in the kidney tissue in diabetes is poorly understood. Aim The present study investigated the role of endothelial and inducible nitric oxide synthase (eNOS and iNOS), Poly ADP ribose polymerase (PARP) xanthine oxidase (XO) in the pathogenesis of streptozotocin-induced diabetic changes in the kidney tissue. Methods Diabetes mellitus was induced in rats by a single injection of streptozotocin (STZ) at a dose of 50 mg/kg. The XO, PARP, eNOS and iNOS protein expression in the kidney was studied by immunohistochemistry. Results Obtained results showed that STZ administration incresed the numbers of PARP and XO-positive cells in kidney tissue compared to control group (respectively, 42 ± 8 vs 10 ± 2, p = 0.01; and 27 ± 7 vs 8 ± 2 cells/mm 2 , p = 0.002). At contrast, the eNOS expression was also observed in medullary collecting tubules, and it was decreased in STZ-treated rats. Conclusion The present study suggests that increased PARP, XO in proximal tubules and decreased eNOS expression in medullary collection tubules, with concomitant increased eNOS and iNOS expression in the microvasculature plays a pivotal role in the kidney damage in diabetes mellitus. Supported by LCS grant Number 278/2012.

VACM-1 receptor is specifically expressed in rabbit vascular endothelium and renal collecting tubule.

The vasopressin-activated calcium-mobilizing (VACM-1) protein is a novel arginine vasopressin (AVP) receptor that shares sequence homology with a cullin multigene family but not with the AVP receptors. To characterize the VACM-1 receptor, we examined its tissue-specific expression using Northern blot, RT-PCR, and immunostaining analyses. Northern blot hybridization identified a 6. 4-kb cRNA species that was expressed in the rabbit kidney medulla, brain, heart, and ovaries. In human tissue, VACM-1 mRNA is a larger (7.5 kb) cRNA found in the kidney, brain, heart, placenta, and skeletal muscle. VACM-1-specific RT-PCR products were detected in mRNA from rabbit kidney medulla, brain, heart, and mesenteric arteries. No expression of VACM-1 could be detected in rabbit aorta, gastrointestinal tract, or liver. Coimmunostaining with anti-VACM-1 antibodies (Ab) and a specific vascular endothelial cell marker, CD31 monoclonal Ab, localized VACM-1 expression to the vasculature in specific tissues. We identified the kidney cells expressing VACM-1 receptor by coimmunostaining with the following monoclonal Ab, which recognize epitopes in specific segments of the nephron: rct-30 Ab, reactive against the cortical and medullary collecting tubule (CT) cells; mr-omct Ab, reactive against the mitochondria-rich cells of the outer medullary CT; and an Ab specific against the loop of Henle segment. These studies indicated that the VACM-1 receptor is expressed only in the medullary CT. Kidney coimmunostaining with anti-VACM-1 and CD31 Ab identified VACM-1-receptor expression in glomeruli and medullary vascular bundles. These results demonstrate that the novel VACM-1 receptor, expressed in many organs, is localized to the endothelial cells. In the kidney, it is also expressed in the medullary CT cells. Thus VACM-1 may be involved in the regulation of endothelial permeability and water transport in the CT.

Activation of protein kinase C stimulates cAMP phosphodiesterase in rat renal collecting tubule.

The present study was undertaken to evaluate whether protein kinase C (PKC) affects adenosine 3',5'-cyclic monophosphate phosphodiesterase (cAMP-PDIE) activity in microdissected rat renal medullary collecting tubules (MCT). Phorbol 12-myristate 13-acetate (PMA, 10(-8) M), an activator of PKC, significantly stimulated cAMP-PDIE activity in intact MCT (29.5 +/- 1.5 to 38.3 +/- 3.7 fmol.min-1.mm-1, P < 0.05) but not in the proximal straight tubule [4.7 +/- 0.8 vs. 4.8 +/- 0.8, not significant (NS)] or the medullary ascending limb of Henle's loop (20.5 +/- 2.1 vs. 22.4 +/- 2.8, NS). PMA-stimulated cAMP-PDIE activity was reversed by PKC inhibitors, staurosporine (10(-8) M) and calphostin C (10(-8) M), but not by the cyclooxygenase inhibitor, indomethacin (5 x 10(-6) M). 1,2-Dioctanoyl-sn-glycerol (50 micrograms/ml), a synthetic analogue of diacylglycerol that stimulates PKC, also increased cAMP-PDIE activity in broken-cell preparations from MCT. This stimulation was also suppressed by staurosporine (10(-8) M) and calphostin C (10(-8) M). The stimulatory effect of PMA on cAMP-PDIE activity was lost with rolipram (10(-4) M), a type IV PDIE inhibitor, whereas it was preserved with N-(6-amino-hexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) (10(-4) M), a calmodulin inhibitor, or vinpocetin (10(-4) M), a direct inhibitor of type I PDIE. From these results, we suggest that activation of PKC specifically stimulates rolipram-sensitive cAMP-PDIE, but not the calmodulin-sensitive isozyme, in rat MCT.

Expression of Na(+)-K(+)-ATPase alpha- and beta-subunits along rat nephron: isoform specificity and response to hypokalemia.

The activity of Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase), the sodium pump, which drives active Na+ reabsorption along the nephron, varies over an order of magnitude, depending on the nephron segment, and activity is increased in the outer medullary collecting tubule (MCT) during hypokalemia. The aims of the present study were to assess abundance of sodium pump alpha 1- and beta 1-subunits in dissected nephron segments of the rat by immunoblotting, to determine if alpha 2- or alpha 3-protein could be detected in the collecting tubules, as suggested by Barlet-Bas et al. (C. Barlet-Bas, E. Arystarkhova, L. Cheval, S. Marsy, K. Sweadner, N. Modyanov, and A. Doucet. J. Biol. Chem. 268: 11512-11515, 1993) for rabbit, and to determine if alpha 1 and beta 1 were increased in MCT by hypokalemia. Tubules from the rat were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12-100 mm/lane), blotted, and probed with subunit-specific antisera. alpha 1 and beta 1, detected in all tubule segments assayed, were highest in cortical and medullary thick ascending limbs and proximal convoluted tubule (PCT), lower in the MCT and barely detectable in proximal straight tubule. In the cortical collecting tubule (CCT), alpha 1 abundance was equivalent to that in PCT, whereas beta 1 and enzymatic activity were both less than one-half of that in PCT. After 2 wk of a K(+)-deficient diet, alpha 1- and beta 1-subunit levels in MCT increased 3.4 +/- 0.6- and 11.7 +/- 4.0-fold, respectively, associated with a 5-fold increase in activity. alpha 2 and alpha 3 were not detected in the CCT or MCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Collecting duct changes in potassium depletion: effects of ACE inhibition

Potassium depletion is associated with a hyperreninemia that may be responsible for some of the renal hemodynamic and functional changes observed in K-deficient states. The present study was designed to evaluate whether interruption of the renin-angiotensin system with enalapril alters the collecting duct changes observed in K depletion. Adrenalectomized male Sprague-Dawley rats were allocated to either a normal (NK) or low-K diet (LK), and they either received enalapril or vehicle for 3 wk. Na:K pump activity (pmol.mm-1.h-1) in microdissected cortical collecting (CCT) and medullary collecting tubules (MCT) was determined at 21 days after group allocations. K depletion had a minimal effect on CCT outer diameter. In contrast, a marked hypertrophy was observed in the MCT diameter (91% increase, P < 0.001) that was significantly attenuated by enalapril treatment (56% increase, P < 0.001 vs. LK). An increase in Na:K pump activity was observed with LK, in the CCT from 497 +/- 47 to 1,089 +/- 83 (P < 0.001) and in the MCT from 489 +/- 36 to 1,396 +/- 45 pmol.mm-1.h-1 (P < 0.01). In K-replete rats, enalapril had no effect on Na:K pump activity in either CCT or MCT. Enalapril administration during LK had no effect on the increase in Na:K pump activity in the CCT (1,023 +/- 75 pmol.mm-1.h-1, P < 0.001), not different from LK alone. In the MCT, however, enalapril reduced the increment in Na:K pump activity induced by LK (1,116 +/- 39 pmol.mm-1.h-1, less than the change with LK alone).(ABSTRACT TRUNCATED AT 250 WORDS)

H-K-ATPase in distal renal tubular acidosis: urinary tract obstruction, lithium, and amiloride

In previous studies we suggested that urinary tract obstruction and chronic administration of lithium or amiloride were models of "voltage-dependent" distal renal tubular acidosis (DRTA). Subsequently, differences among these three models suggested that the pathogenesis was far more complex than we originally proposed. A recent study showed that H-adenosinetriphosphatase (H-ATPase) activity was decreased in all three experimental models. In the current experiments we examined the effect of 24-h unilateral ureteral obstruction (UUO) and chronic administration of amiloride and lithium on collecting tubule H-K-ATPase, the other renal H-ATPase enzyme. In the obstructed kidney, cortical collecting tubule (CCT) H-K-ATPase activity was enhanced by 73 +/- 10.0%, whereas the enzyme activity in medullary collecting tubule (MCT) was decreased by 67 +/- 5.4%. In the normal contralateral kidney, activities of H-ATPase, H-K-ATPase, and Na-K-ATPase were increased by approximately 30% in both CCT and MCT. Following amiloride (3 mg.kg-1.day-1 x 3 days ip), rats had normal acid-base status, slight hyperkalemia, and markedly elevated plasma aldosterone levels. Both CCT and MCT H-K-ATPase activities in amiloride-treated rats were unchanged. After LiCl (4 meq.kg-1.day-1 x 3 days ip), rats developed mild metabolic acidosis and had normokalemia and normal aldosterone status. CCT H-K-ATPase activity in lithium-treated rats was decreased by 64 +/- 8.8%, whereas the enzyme activity in MCT remained unchanged. Lithium in vitro (30 meq/l) inhibited CCT, but not MCT, H-K-ATPase activity, whereas amiloride had no effect on the enzyme activity. (ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical and Scanning Electron Microscopic Study of Cytokeratin Distribution in the Collecting Tubule of the Rat Kidney

Cytokeratin distribution in the collecting tubules (CTs) of the rat kidney was studied by immunohistochemistry and scanning electron microscopy (SEM). After preparation of sections from formalin-fixed, paraffin-embedded tissue for immunohistochemistry, the remaining tissue was prepared for SEM. The cut surfaces of the tissue were examined by SEM and compared with sections stained with anti-cytokeratin antibody. The immunostained sections revealed positive staining along the entire CTs. However, in addition to diffusely stained cells, unstained and partially stained cells were seen. The latter were not distributed in inner medullary CTs, whereas the diffusely stained cells were observed in cortical, outer medullary and inner medullary CTs. This immunohistochemical heterogeneity of cytokeratin reactivity was prominent in the outer medullary and cortical CTs. From this comparative study of immunostained sections and SEM specimens, it was concluded that the heterogeneously stained cells correspond to intercalated cells, whereas the diffusely stained cells represent most principal cells. These results suggest that the difference in cytokeratin density among CT cells may represent different functional states, at least in intercalated cells.

Control of bicarbonate transport in collecting tubules from normal and remnant kidneys

Bicarbonate transport in the rabbit cortical collecting tubule (CCT) and outer medullary collecting tubule (MCT) in vitro was studied under two types of conditions that were anticipated to alter distal tubule bicarbonate transport: 1) reduction of renal mass, and 2) acid and base loading in vivo. Bicarbonate secretion (both total and acetazolamide sensitive) and bicarbonate reabsorption (studied separately) in CCT and bicarbonate reabsorption in the MCT were not different between tubules from normal and remnant kidneys. The control or conditioning of the separate processes of bicarbonate secretion and bicarbonate reabsorption was also studied in CCT from normal and remnant kidneys. Bicarbonate secretion was not increased by base-loading animals with either normal or remnant kidneys. In contrast, bicarbonate secretion was consistently decreased by acid loading (studied in CCT from remnant kidneys). Bicarbonate reabsorption in the CCT was not altered by acid or base loads given to animals with normal kidneys. And bicarbonate reabsorption in MCT was not increased by acid loading of animals with remnant kidneys. These studies demonstrate that bicarbonate transport (and its conditioning by acid or base loads in vivo) in both CCT and MCT in vitro is not altered by reduction of renal mass in rabbits. The predominant conditioning effect of acid or base loads in vivo is for acid loads to inhibit CCT bicarbonate secretion.

AVP-sensitive cAMP production is dependent on calmodulin in both MTAL and MCT.

Vasopressin (AVP) plays a key role in maximal urine concentration by stimulating NaCl reabsorption in the medullary thick ascending limbs of Henle (MTAL) and by increasing water permeability in the medullary collecting tubules (MCT). These effects of AVP in MTAL and MCT are mediated by activation of adenylate cyclase. Because effects of high ambient Ca2+ on AVP-sensitive adenosine 3',5'-cyclic monophosphate (cAMP) production are quite different in MTAL and MCT, we examined whether the Ca2+-calmodulin system is involved differently in AVP-sensitive cAMP production in MTAL and MCT of mouse kidney using two dissimilar calmodulin inhibitors, trifluoperazine (TFP) and W-7. TFP and W-7 inhibited AVP-sensitive cAMP production in both nephron segments in a dose-dependent manner with maximal inhibition of both agents being greater than 90%. A half-maximal inhibition by TFP and W-7 was about 45, 100 microM in MTAL and about 40, 40 microM in MCT, respectively. The inhibitory effect of W-5, a chemically similar to W-7 but less potent calmodulin inhibitor, was significantly less than that of W-7 in both nephron segments. TFP and W-7 but not W-5 also inhibited glucagon-sensitive cAMP production in MTAL. W-7 inhibited forskolin-sensitive cAMP production but the inhibition by W-5 was significantly less than that by W-7 in MTAL and MCT. Results suggest that AVP-sensitive cAMP production is MCT.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory guanosine triphosphate-binding protein-mediated regulation of vasopressin action in isolated single medullary tubules of mouse kidney.

Vasopressin (AVP) plays a key role in maximal urine concentration by stimulating NaCl reabsorption in the medullary thick ascending limbs of Henle (MAL) and by increasing water permeability in the medullary collecting tubules (MCT). These effects of AVP in MAL and MCT are mediated by cAMP. Alpha 2-adrenergic stimulation in MCT, and high ambient Ca2+ and PGE2 in MAL inhibit AVP-dependent cAMP production and thereby modulate urine concentration. The present study was undertaken to clarify the mechanisms underlying the inhibition of AVP-dependent cAMP production by these agents using microdissected mouse MAL and MCT. Preincubation of MCT and MAL with 1 microgram/ml pertussis toxin for 3 and 6 h, respectively, resulted in ADP-ribosylation of an approximately 41-kD protein, which was presumably an alpha subunit of the inhibitory GTP-binding protein Gi. Epinephrine, 10(-6) M, via alpha 2-adrenergic stimulation, inhibited AVP-dependent cAMP production in MCT. Preincubation of MCT for 3 h with pertussis toxin abolished the inhibition of AVP-dependent cAMP production by epinephrine. High ambient Ca2+ and PGE2 both inhibited AVP-dependent cAMP production in MAL. Preincubation of MAL for 6 h with pertussis toxin abolished the inhibition by high ambient Ca2+ and attenuated the inhibition by PGE2. Preincubation of MCT or MAL with pertussis toxin for 1 h was ineffective in ADP-ribosylation and did not modify the inhibition of AVP-dependent cAMP production by these agents in both nephron segments. Our data suggest that the inhibition of AVP-dependent cAMP production by alpha 2-adrenergic stimulation in MCT, and by high ambient Ca2+ and adrenergic stimulation in MCT, and by high ambient Ca2+ and PGE2 in MAL, is mediated, at least in part, through activation of Gi.

Insulin receptors along the rat nephron: [125I] insulin binding in microdissected glomeruli and tubules.

Binding of [125I] Tyr A14 human insulin ([125I] insulin) was measured at 4 degrees C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10(-18) mol [125I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5 +/- 0.3; proximal convoluted tubule (PCT), 12.6 +/- 0.6; pars recta (PR), 4.0 +/- 2.6; thin descending limb (TDL), 0.6 +/- 0.2; thin ascending limb (TAL), 0.6 +/- 0.2; medullary thick ascending limb (MAL), 0.8 +/- 0.1; cortical ascending limb (CAL), 2.1 +/- 0.1; distal convoluted tubule (DCT), 5.6 +/- 1.1; cortical collecting tubule (CCT), 3.2 +/- 0.3 and outer medullary collecting tubule (MCT), 2.3 +/- 0.1. Specific [125I] insulin binding to glomeruli and tubule segments was time and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [125I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

CAMP-binding proteins in medullary tubules from rat kidney: effect of ADH

Little is known of the regulatory steps in the cellular action of vasopressin (AVP) on the renal epithelium, subsequent to the cAMP generation. We studied cAMP-binding proteins in the medullary collecting tubule (MCT) and the thick ascending limb of Henle's loop (MTAL) microdissected from the rat kidney by use of photoaffinity labeling. Microdissected tubules were homogenized and photoaffinity labeled by incubation with 1 microM 32P-labeled 8-azido-adenosine 3',5'-cyclic monophosphate (N3-8-[32P]-cAMP); the incorporated 32P was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Both in MCT and MTAL preparations, the analyses showed incorporation of N3-8-[32P]cAMP into two bands (Mr = 49,000 and Mr = 55,000) that comigrated with standards of the cAMP-dependent protein kinase regulatory subunits RI and RII. In MCT, most of the 32P (80%) was incorporated into RI, whereas in MTAL the 32P incorporated into RI and RII was equivalent. When freshly dissected MCT segments were incubated with 10(-12)-10(-6) M AVP, the subsequent photoaffinity labeling of RI with N3-8-[32P]cAMP was markedly diminished in a dose-dependent manner compared with controls. Our results suggest that cAMP binds in MCT and MTAL to regulatory subunits RI and RII of cAMP-dependent protein kinase. However, in MCT the dominant type of cAMP-dependent protein kinase appears to be type I. The outlined procedure is suitable to indirectly measure the occupancy of RI by endogenous cAMP generated in MCT cells in response to physiological levels (10(-12) M) of AVP.(ABSTRACT TRUNCATED AT 250 WORDS)

In situ phosphorylation of proteins in MCTs microdissected from rat kidney: effect of AVP.

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein phosphorylation is considered a key step in the cellular action of vasopressin (AVP) to regulate water permeability in collecting tubules. However, the proteins serving as a substrate(s) for phosphorylation in undisrupted cells have not yet been identified. In the present study, we developed a method for investigation of in situ phosphorylation of microdissected segments of medullary collecting tubules (MCT) from rat kidney. Incubation of microdissected MCT segments with low concentrations of saponin, "semipermeabilization," increased permeability of the membrane for ATP but did not allow leakage of macromolecules such as lactate dehydrogenase. This treatment also did not cause major disruption of cell structure, or impairment of AVP-sensitive adenylate cyclase. Incubation of semipermeabilized MCT with gamma-[32P]ATP resulted in incorporation of 32Pi into two major protein bands [band "A" of apparent molecular mass (Mr) approximately equal to 66 kDa, and band "B" of Mr approximately equal to 45 kDa] detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent autoradiography. Similar incubation of tubules disrupted by hyposmotic solutions and a stronger detergent Triton X-100 resulted in 32Pi incorporation into multiple protein bands. Incubation of MCT with 1 microM AVP resulted in increased 32Pi radioactivity in band A and decreased 32Pi radioactivity in band B. These findings demonstrate a novel method for identification of endogenous protein substrate(s) for cAMP-dependent protein kinase and other protein kinases and phosphatases that are probably involved in post-cAMP steps in the cellular action of AVP in the intact cells of collecting tubules.

Isolation by monoclonal antibody of intercalated cells of rabbit kidney

We produced a monoclonal antibody, gamma G6, that reacts only with one cell type in the connecting (CNT) and collecting tubules (CT) of rabbit kidney. The gamma G6 antibody-reactive cells revealed carbonic anhydrase activity, showing one of the characteristics of intercalated (IC) cells. Using immunoelectron microscopy, we demonstrated that IC cells in cortical CT consist of the gamma G6 antibody-reactive and non-reactive cells, whereas all IC cells in medullary CT were reactive with the gamma G6 antibody. We used a cell sorter to enrich this cell type from the isolated kidney cell suspension. When we measured hormone-sensitive adenylate cyclase (ACase) activities of the sorted cells, the presence of parathyroid hormone (PTH) and isoproterenol (ISO) almost doubled ACase activities when compared with the basal values; however, no additive effect of PTH and ISO was observed. They showed no calcitonin-sensitive ACase and negligible arginine vasopressin (AVP)-sensitive ACase. We suggest that the IC cells recognized by the gamma G6 monoclonal antibody possess a receptor(s) for PTH and/or ISO but not for AVP in the CNT and CT, although it remains to be clarified whether the reactivities to PTH and ISO in these cells originate from single or dual cells.

Potassium transport by medullary collecting tubule of rabbit: effects of variation in K intake.

These studies examine the effect of dietary K content on the rate efflux coefficient (KK) assessed both isotopically (42K) and by net K flux in segments dissected from the inner stripe of the outer medullary collecting tubule (MCT). Female New Zealand White rabbits were given either a K-replete diet (KCl diet) or one of two K-restricted diets with similar (142 meq K/kg diet) K content [basic (Na-deplete) diet or NaCl (Na-replete) diet]. After 7 days, renal K excretion (mmol/24 h) varied with K intake: KCl diet, 13.8 +/- 3.0; basic diet, 4.36 +/- 0.12; NaCl diet, 4.13 +/- 0.48. To determine whether alterations in KK of MCT could contribute to these changes in urinary K excretion, the MCT from the three groups of rabbits was perfused in vitro and volume reabsorption and KK were measured. The KK of tubules from the K-replete control group (KCl diet) was 82 +/- 14 nm/s but was significantly greater in tubules from either K-restricted group: 224 +/- 49 nm/s (basic diet group) or 193 +/- 34 nm/s (NaCl diet group). Net K flux in the presence of a 15 mM lumen-to-bath K gradient confirmed a significantly greater K reabsorptive flux and net rate coefficient in either K-restricted group (KNetK = 179 +/- 77 nm/s for the NaCl diet, KNetK = 170 +/- 27 nm/s for the basic diet) than the K-replete control group (KNetK = 12.0 +/- 18 nm/s for the KCl diet).(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations of enzymatic activities along rat collecting tubule in potassium depletion

This study was designed to correlate morphological alterations induced in rat collecting tubule by potassium depletion with changes in the activity of enzymatic markers of the cell basolateral membrane. Results show the following responses. 1) Potassium depletion induced a huge and progressive hypertrophy of the outer medullary collecting tubule (MCT). Hypertrophy was paralleled by enhancements of vasopressin- and forskolin-dependent adenylate cyclase (AC) activities. Glucagon-sensitive AC was also increased, but with a different kinetics, whereas isoproterenol-dependent AC was only modestly stimulated. 2) In cortical (CCT) and papillary collecting tubules, AC response to hormones did not change. The concentrating defect of K-deprived rats, therefore, does not appear to result from an intrinsically defective adenylate cyclase system in any portion of the collecting tubule. Decreased AC response of the medullary thick ascending limb to vasopressin and glucagon, observed after 3-5 wk of K depletion, might account, at least in part, for reduced hypertonicity of medullary tissue. 3) Na+-K+-ATPase activity fell in CCT, probably in relation to decreased K secretion. Conversely, in MCT, Na+-K+-ATPase rose much more than tubular volume. The physiological significance of this latter observation remains to be established.

Angiotensin I converting enzyme and kinin–hydrolyzing enzymes along the rabbit nephron

Angiotensin I converting enzyme (ACE) and kininase activities were measured in various segments of the rabbit nephron. ACE was determined with tritiated hippuryl-glycylglycine as substrate. Lysyl-bradykinin (LBK) hydrolysis (kininase activity) was measured by radioimmunoassay. ACE was only found in the glomerulus and in the two parts of proximal tubule: the convoluted proximal tubule and the pars recta (PR). It was distributed along a concentration gradient which increased from the glomerulus to PR. Kininase activity was found in both proximal and distal parts of the nephron. Besides intense LBK-hydrolyzing activity in the proximal tubule, a kininase activity was also found in the medullary collecting tubule (MCT). Kininase activity in the glomerulus and the proximal tubule was completely inhibited by chelating agents. Captopril inhibited this activity only in the PR and at high concentrations (above 10(-7) M). These results indicate that several types of enzymes other than ACE hydrolyze kinins in the glomerulus and in the proximal tubule. The contribution of ACE to kinin hydrolysis appears only minimal. The kininase activity found in MCT was different from ACE and other proximal tubule kininases because it was not inhibited by chelating agents. This kininase may play a physiological role in inactivating the kinins formed by kallikrein at or beyond the connecting tubule.

PGE2, PGF2 alpha, 6-keto-PGF1 alpha, and TxB2 synthesis along the rabbit nephron.

The capacity of synthesis of prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-PGF1 alpha, and thromboxane (TxB2) along the rabbit nephron was determined. A sensitive enzyme immunoassay was applied to isolated nephron segments, from the glomerulus to the terminal collecting tubule. The three prostaglandins and thromboxane (PG) were measured on the same samples after incubation in the presence of arachidonic acid. In the glomerulus, PGE2 synthesis (29.4 +/- 3.3 pg X glomerulus-1 X 30 min-1) represented 60% of the sum of the four PGs. PGF2 alpha and 6-keto-PGF1 alpha represented 22 and 17%, respectively, and TxB2 1.4%. The contribution of each PG to tubular synthesis was different, since at least 90% of PG synthesis consisted of PGE2. In the medullary collecting tubule (MCT), by far the major tubular site of PG synthesis, it was 809.6 +/- 140.8 pg X mm-1 X 30 min-1 for PGE2, 17.7 +/- 7.2 for PGF2 alpha, 8.3 +/- 1.9 for 6-keto-PGF1 alpha and 0.24 +/- 0.08 for TxB2. These relative proportions were roughly respected all along the tubule. Values were much lower in the convoluted and straight portions of the proximal tubule (proximal convoluted tubule: PGE2 8.2 +/- 2.0, PGF2 alpha 0.4 +/- 0.06, 6-keto-PGF1 alpha 0.26 +/- 0.04, TxB2 0.017) and the medullary and cortical thick ascending limb of the loop. They increased regularly along the distal structures of the tubule (light portion of the cortical collecting tubule (CCT1): PGE2 228.3 +/- 20.4, PGF2 alpha 4.34 +/- 0.6, 6-keto-PGF1 alpha 1.8 +/- 0.3, TxB2 0.22 +/- 0.07).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of temperature, ouabain and diuretics on the cell sodium and potassium contents of isolated rat kidney tubules.

Cell Na+ and K+ contents were measured by flame photometry in single pieces of rat medullary thick ascending limb (MAL) and medullary collecting tubules (MCT) after an overnight incubation at various temperatures. Below 8 degrees C, MCT samples were no more able to sustain high-K+, low-Na+ cell concentrations, and sodium progressively replaced cell potassium as the temperature decreased. The loss of potassium and the accompanying accumulation of sodium by MCT cells occurred at lower or higher temperature when amiloride (20 mumol/l) or ouabain (1 mmol/l) was present respectively in the incubation medium compared to that observed on control non-treated MCT. In contrast, MAL samples maintained normal cation gradients across their membrane at all temperatures, including 0 degree C, even in the presence of ouabain. However, MAL cells lost nearly all their potassium which was replaced by sodium when they were incubated in K+-free solution. These Na+-loaded, K+-depleted MAL cells restored high-K+ and low-Na+ contents similar to those of control samples when they were further incubated for 1 h at 0 degree C in presence of 5 mmol/l external potassium. Even in the presence of 1 mmol/l ouabain and at 0 degree C, a restoration of almost normal cation gradients occurred provided that Na+-loaded MAL were incubated for 24 h after potassium addition to the external medium. The results are discussed in relation to the respective effects of low temperatures on the passive and active components of the cation balance in the cells of the two nephron segments.

Dynamics of nucleotides in distal nephron of mice with nephrogenic diabetes insipidus

In mice with hereditary nephrogenic diabetes insipidus (NDI), the high activity of cAMP-phosphodiesterase (cAMP-PDIE) in medullary collecting tubules (MCT) prevents the increase in cAMP content in response to vasopressin [Arg8]vasopressin (AVP). Even when the cAMP response to AVP is partly corrected by cAMP-PDIE inhibitor 1-methyl-3-isobutylxanthine (MIX), under all tested conditions the cAMP levels in MCT of NDI mice remained much lower than in controls (B. A. Jackson, R. M. Edwards, H. Valtin, and T. P. Dousa, J. Clin. Invest. 66: 110-122, 1980). In the present study, we explored which factors may account for this defect. We determined contents of ATP, nicotinamide adenine dinucleotide (NAD), and the levels of cAMP in MCT and in medullary thick ascending limb of Henle's loop (MAL) microdissected from control and NDI mice. In the presence of 1 microM AVP and 0.05 mM MIX, the cAMP levels accumulated in MCT of NDI mice were four times lower compared with controls, but the levels of ATP and NAD were not different. ATP levels in MAL of NDI mice were slightly (delta -23%) lower than in MAL from controls, and in distal convoluted tubules (DCT) of NDI mice the ATP levels were also decreased (delta -49%). Although AVP alone had little effect on cAMP levels in mouse MAL in the presence of 0.1 mM forskolin, the AVP elicited a 20-fold increase of cAMP of both the control and NDI mice. Addition of 0.1 mM forskolin further increased the cAMP accumulation in MCT incubated with AVP.(ABSTRACT TRUNCATED AT 250 WORDS)