Stimulation of tubular reabsorption of magnesium and calcium by antidiuretic hormone in conscious rats

The effect of antidiuretic hormone on urinary electrolyte excretion was investigated by clearance techniques in conscious rats in metabolic cages. Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) were studied in the absence of exogenous ADH (control group = C,n=4), and after several weeks of continuous dDAVP infusion (period A) followed by discontinuation of dDAVP (period B) (experimental group = E,n=6). dDAVP, a non-pressor antidiuretic analogue to ADH, induced 1) a high urine concentration (2,645±44 (SEM) in group E vs 131±6 mosmol/kg H₂O in group C),P<0.001; 2) no significant change in plasma osmolality (288±2 vs 297±7 mosmol/kg H₂O respectively) and in plasma concentration of major electrolytes, Na, K, Cl, Mg, and Ca; 3) a large decrease in urinary excretion of calcium and magnesium and no change in other electrolyte or total osmolar excretion. Fractional excretions in rats of groups C and E during period A were, respectively, for Na: 0.59±0.03 (SEM) and 0.51±0.33% (NS), for Ca: 2.92±0.62 and 0.34±0.05% (P<0.001) and for Mg: 7.75±0.83 and 1.38±0.28% (P<0.001). After treatment discontinuation, plasma osmolality in group E rose to 304±2 mosmol/kg H₂O (P<0.01 compared to period A) with slight increases in plasma Na and Cl concentrations. Urine osmolality fell below, and urine flow rate rose above values observed in the control group. Fractional excretion of Ca and Mg rose to values seen in DI rats (3.30±0.37%, NS, for Ca) or above (26.95±0.65%,P<0.001, for Mg), with no change in other solute fractional excretion. Other works, from our and other's groups have shown that 1) long-term exposure to dDAVP induces a marked hypertrophy of the epithelium of the thick ascending limb of Henle's loop in its medullary part (MTAL) and 2) dDAVP induces an increase in Ca and Mg tubular reabsorption between end proximal and early distal sites of micropuncture. Taken together, these results suggest that the effects of ADH on divalent cation fractional excretion, seen in the present study, probably results from an increased Ca and Mg voltage-dependent reabsorption in the MTAL. This reabsorption is linked to the increased salt transport induced in this segment, both by a direct effect of ADH and by an indirect effect resulting from the increased solute delivery to the MTAL in the concentrating kidney.     

Effect of indomethacin in vivo and PGE2 in vitro on MTAL Na-K-ATPase of the rat kidney

We have previously shown that prostaglandin synthesis inhibition in rats which reduces urinary excretion of PGE₂ and sodium, is associated with increased Na-K-ATPase activity in renal medulla. To further characterize this interaction studies were performed in isolated segments of medullary thick ascending limb of Henle's loop (MTAL) in rats. The effect of pretreatment with indomethacin in vivo and incubation with PGE₂ in vitro on MTAL Na-K-ATPase activity was studied. Pretreatment of rats with indomethacin increased Na-K-ATPase of the MTAL from 37.2±2.0×10⁻¹¹ mol/mm/min in controls to 62.7±2.2 (p<0.001) while Mg-ATPase was only slightly decreased. Incubation of MTAL Na-K-ATPase from indomethacin pretreated rats with increasing concentration of PGE₂ in vitro dose dependently inhibited MTAL Na-K-ATPase activity with no effect on Mg-ATPase. Baseline Na-K-ATPase was 62.7±2.2 in MTAL from indomethacin pretreated rats and decreased to 36.9±1.4 (p<0.001) with 1 uM of PGE₂, to 26.5±2.3 (p<0.001) with 10 uM PGS₂ and to 22.0±1.0 (p<0.001) with 100 uM PGE₂. 100 uM PGE₂ in the incubation medium inhibited MTAL Na-K-ATPase of intact rats from 37.2±2 to 21.3±1.2 (p<0.001) and completely abolished the indomethacin induced increase in MTAL Na-K-ATPase. The results of this study show stimulation of MTAL Na-K-ATPase by pretreatment with indomethacin in vivo and, direct inhibition of MTAL Na-K-ATPase by PGE₂ in vitro. These findings suggest that the increase in MTAL Na-K-ATPase induced by pretreatment with indomethacin may stem directly from PGE₂ depletion. These observations are consistent with direct inhibitory effect of PGE₂ on Na reabsorption in the MTAL.     

Influence of chronic ADH treatment on adenylate cyclase and ATPase activity in distal nephron segments of diabetes insipidus Brattleboro rats

The medullary thick ascending limb (MAL), but not the medullary collecting tubule (MCT), has been shown to have an impaired adenylate cyclase (AC) responsiveness to ADH and a selective hypoplasia in Brattleboro diabetes insipidus (DI) rats. Since chronic ADH administration has been found to increase epithelium volume and basolateral membrane surface area in MAL but not in MCT, we investigated whether chronic ADH infusion would affect the hormone-sensitive AC and the Na–K-ATPase activity — two markers of the basolateral membrane — in single microdissected portions of thick ascending limb and collecting tubule in DI rats. Results indicate that 1. in MAL of ADH-treated rats, AC resposes to in vitro AVP and glucagon and Na–K-ATPase activity increased to the same extent as did epithelium volume (60–80%); 2. changes in the other segments were independent of any morphological alteration. In the cortical thick ascending limb, AVP and glucagonsensitive AC decreased by 30–40% whereas Na–K-ATPase activity did not change. In the collecting tubule, AC response to in vitro AVP was not altered by ADH-treatment but glucagon-sensitive AC dropped by 50% and Na–K-ATPase activity doubled, independently of any variation in plasma aldosterone and glucagon levels. These results show that, in the MAL, the ADH-induced variations in enzyme activity are a reflection of the enlargement of the basolateral membrane surface area. Further studies are needed to clarify the origin of enzymatic alterations in the other segments.     

Collecting duct function in liver cirrhotic rats with early sodium retention

Liver cirrhosis is a chronic disease associated with sodium retention due to increased tubular sodium reabsorption. However, the exact tubular site of increased sodium reabsorption in uncertain. We have recently demonstrated selective hypertrophy of the inner stripe of the outer medulla (ISOM) in rats with liver cirrhosis induced by common bile duct ligation (CBL). The present study was designed in order to measure Na-K-ATPase activity in the two major tubular segments located in the ISOM: the thick ascending limb of henles (MTAL) and the collecting ducts (OMCD) in CBL rats. Sham-operated rats were used as controls. In addition, the natriuretic response to amiloride (0.2 mg kg(-1) h(-1) i.v) was examined in conscious, chronically instrumented rats during conditions where amiloride-induced volume losses were replaced continuously using a servo-controlled i.v. volume replacement system. For 4-5 weeks after CBL, cirrhotic rats showed sodium retention relative to control rats without any sign of ascites. Plasma levels of sodium and aldosterone were normal, but plasma vasopressin was increased. Effective renal plasma flow was significantly increased, whereas glomerular filtration rate (GFR) and renal lithium handling were normal. The CBL rats showed a blunted natriuretic response to amiloride (DeltaFE(Na): 1.17 +/- 0.15% vs. 1.65 +/- 0.13%; P < 0.05). In rats with CBL, Na-K-ATPase activity per mm tubular length was decreased in the OMCD and unchanged in the TAL segment. These results suggest that increased tubular sodium reabsorption in liver cirrhotic rats with early sodium retention is localized in segments proximal to the collecting ducts.     

Effects of antidiuretic hormone on electrolyte reabsorption and secretion in distal tubules of rat kidney

The effects of (1-desamino-8-d-arginine) vasopressin (dDAVP) on water and electrolyte transport in the distal tubule were investigated by micropuncture. Since, in addition to antidiuretic hormone, parathyroid hormone, calcitonin and glucagon stimulate the adenylate-cyclase system in this nephron segment, experiments were performed on hormone-deprived rats, i.e. homozygous DI Brattleboro rats with reduced levels of endogenous parathyroid hormone, calcitonin and glucagon. Along the distal tubule, dDAVP enhanced water, Cl, Na and Ca reabsorption and sharply increased net K secretion. Phosphate transport was left unchanged and Mg reabsorption was not significantly altered by dDAVP between the early and late distal tubule. Antidiuretic hormone also slightly increased water filtration rate in the superficial nephron, which rose in proportion to whole kidney glomerular filtration rate. It is concluded that, in rats: 1) antidiuretic hormone stimulates water, NaCl and Ca absorption and enhances K secretion along the distal tubule and 2) the tubular effects of dDAVP on electrolyte transport in the loop and distal tubule are responsible for decreasing Mg and Ca urinary excretion.     

Ingestive behaviors of the rat deficient in vasopressin synthesis (Brattleboro strain). Effect of chronic treatment by dDAVP

Spontaneous manipulator and locomotor activities, food and fluid intake have been recorded from rats suffering from a genetic lack of central vasopressin (VP) synthesis (Brattleboro strain, DI), their heterozygous litter mates (HZ) or Long Evans (LE) rats. The daily patterns of activities did not differ, except for their drinking behavior. This was mainly associated with food intake during the dark period with LE rats but was distributed equally during light and dark periods with DI rats. HZ rats showed a behavioral heterogeneity, some of them following the daily pattern of LE rats, and others, that of DI rats. The daily feeding pattern was identical in the three genotypes but the selection between two isocaloric contrasted diets was different. When they were fed ad lib, HZ and DI rats consumed less carbohydrate than LE rats, the protein intake being unchanged. On the contrary, when the DI rats were only fed during the dark period, they ate more carbohydrate than LE rats. The peripheral infusion of a V2 AVP agonist (dDAVP) restored a normal hydric balance in DI rats but failed to modify the diet selection. These data show that in the rats, the lack of central VP synthesis disturbs both the selection of diets and the efficiency of the satiety signals. These disturbances were unchanged by the peripheral VP treatment which suggested the direct involvement of the central release of the neuropeptide.     

Papillary plasma flow in rats

Papillary plasma flow (PPF) was measured by the albumin accumulation technique in rats of the Brattleboro strain with or without diabetes insipidus (DI and HZ respectively) and in Wistar rats. Measurements were also performed in DI rats receiving antidiuretic hormone for 30 min or 5 days and in dehydrated Wistar rats. PPF in HZ control and Wistar control rats was similar to previously published measurements. In contrast PPF was significantly higher in DI rats (461±26l/min·g versus 263±28 in HZ) and decreased significantly after acute ADH administration. It returned to control values after prolonged ADH administration (262±40). Plasma flow entering the papilla was inversely correlated with urine osmolality up to 1000 mosmol/kg H₂O. Further increases in urine concentration (dehydration of Wistar rats) did not modify further PPF (255±28 versus 270±16 in non dehydrated Wistar). PPF might be influenced indirectly by ADH or prostaglandins and seems to depend on the osmotic environment of the papilla up to a certain limit. The factors which maintain PPF at a given minimum level with further increases in urine concentration are not known.     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.     

Effects of osmolality and antidiuretic hormone on prostaglandin synthesis by renal papilla

Prostaglandin (PG) production by the kidney is known to be reduced both in vivo and in vitro in rats with hereditary diabetes insipidus (DI), totally lacking ADH. Exogenous ADH restores normal PG excretion in these rats. On the other hand, osmolality in vitro, and urine flow rate in vivo have been shown to influence PG synthesis rate. In order to determine whether the decreased PG synthesis of DI rats is due to the lack of antidiuretic hormone itself or to low tissue osmolality, we studied in vivo and in vitro PG production in DI rats in which urine osmolality had been raised either with ADG (infused by Alzet minipumps), or without ADH (by dehydratation) and in control DI rats.PGE₂ and PGF₂ were measured by radioimmunoassay in the urines and in supernatants of papillary homogenates incubated at 37°C for 15–120 min. ADH administration and dehydration led to similar urine osmolalities (900–1,000 mosmol/kg H₂O versus 150 in controls). However, only ADH administration but not dehydration increased PG urinary excretion (×5,P<0.001) and subsequent in vitro papillary synthesis (×1.6,P<0.01). These results show that antidiuretic hormone increases PG-synthesis of the renal papilla directly and not through its effects on papillary osmolality.     

Identification of two arginase isoenzyme activities along the nephron of Meriones shawi

 Conflicting theories on the existence of several renal arginase isoenzymes remain in debate. Because the activity of arginase is high in two embryologically different nephron segments of the Meriones shawi kidney, namely the cortical (CPST) and medullary (OSPST) proximal straight tubule and the outer medullary collecting duct (OMCD), we postulate that these nephron segments may contain different isoforms. Isolated nephron segments were dissected from collagenase-treated kidneys. Tubules were permeabilized with Triton X-100 (0.25%) and incubated with increasing Arg concentrations to characterize the arginase activity. The results were as follows: (1) in OMCD, one arginase isoform (E₁), characterized by a high Arg affinity (1.160 mM), was present; (2) in CPST, two arginase isoforms were discovered – one, E₁, had a similar K _m (1.407 mM) to that found in OMCD whereas the other (E₂) had a low affinity for Arg (K _m =18.8 mM); and (3) in OSPST, two isoenzymes were present – E₁ which had a K _m of 1.478 mM and the second isoform that we named E₂ which had a K _m of 9.07 mM. In addition, arginase located in CPST and OMCD was strongly inhibited by Orn and Lys. The K _i value for Lys varied between 1.635 and 2.288 mM. Therefore, this work demonstrates that two arginase isoforms are present in the kidney of Meriones shawi. Isoform E₁ is present in the proximal tubule and the collecting duct whereas isoform E₂ is restricted to the proximal tubule. Received: 13 August 1998 / Accepted: 25 September 1998     

Atrial natriuretic peptide and its mRNA in heart and brain of vasopressin-deficient Brattleboro rats

To understand the secretion and synthesis of atrial natriuretic peptide we measured immunoreactive atrial natriuretic peptide from plasma, heart tissues and brain areas, and ANP mRNA was determined from heart auricles and ventricles of vasopressin-deficient Brattleboro rats (DI) and from desmopressin treated Brattleboro rats (DI + DDAVP). Long-Evans rats (LE) served as controls. DI + DDAVP rats were given for 3 days sc. injections of 0.5/g l-desamino-8-D-arginine vasopressin in 1 ml. saline twice a day. The rats were housed in single metabolic cages and urinary output and water intake were measured daily. All the body and organ weight parameters were similar in the three groups when the rats were killed. No change was seen in the plasma ANP level between the groups. The right ventricle of DI + DDAVP rats had significantly (P < 0.05) higher concentration of ANP than LE rats (15.8 + 4.4 vs. 3.4 + 0.6 ng mg“¹ tissue). The left ventricle of DI and DI+DDAVP had significantly (P < 0.05) lower amounts of ANP mRNA than LE rats (0.5 ± 0.2 vs. 1.3 + 0.2 and 0.5 + 0.1 vs. 1.3 + 0.2 arbitrary units). In the hypothalamus, the ANP concentration was significantly (P < 0.05) lower both in DI and in DI + DDAVP rats than in LE rats (9.3 ±1.3 vs. 14.5 ±±1.6 and 6.1+0.6 vs. 14.5 ± 1.6 pg mg^-1 tissue). To conclude, although the water intake and urinary output of DI rats were changed towards normal with desmopressin treatment, the heart ventricular and hypothalamic ANP did not parallel the change. Desmopressin increased the ANP concentration in the right ventricle of DI rats. Thus the correction of the complete vasopressin deficiency-does not appear to associate with synthesis or release of atrial natriuretic peptide in heart or hypothalamus.     

PGE2-induced inhibition of AVP-dependent cAMP accumulation in the OMCD of the rat kidney is cumulative with respect to the effects of α2-adrenergic and A1-adenosine agonists,insensitive to pertussis toxin and dependent on extracellular calcium

The accumulation of cyclic adenosine 3,5-phosphate (cAMP) elicited by antidiuretic hormone (arginine vasopressin, AVP) in the medullary collecting tubule (OMCD) microdissected from the rat kidney is inhibited by different factors: the A₁ agonist of adenosine (-)-N ⁶-(R-phenylisopropyl) adenosine (PIA), an ₂-adrenergic agonist clonidine (CLO), and prostaglandin E₂ (PGE₂). The negative regulation elicited by PGE₂ was further characterized by measuring summation of inhibition with other inhibitors, by testing the effect of pertussis toxin and by studying the part played by extracellular calcium. Inhibitors were used at concentrations inducing maximum effects. The simultaneous addition of 0.3 M PGE₂ with either 0.1 M PIA or 1 M CLO led to an inhibition of the response to AVP (80.0±3.5%, SEM, N=7 and 92.6±0.8%, N=5, respectively) greater than those elicited by each agent alone. In contrast, PIA and CLO added together induced an inhibition similar to that due to CLO alone. The action of PGE₂ in combination with either PIA or CLO corresponded to a partial summation fitting with the values calculated by assuming a cumulative inhibition. Preincubation of OMCD samples with pertussis toxin (100 ng/ml or 1 g/ml) relieved the inhibitory effects of CLO and PIA but did not affect the action of PGE₂. PGE₂-induced inhibition was prevented in a calcium-free medium [0 Ca²⁺+0.1 mM [ethylene-bis (oxyethylenenitrilo)] tetraacetate (EGTA)]: values were 67.0 ±2.1% and 5.8±8.7% (± SEM) in 2 mM Ca²⁺ and 0 Ca²⁺ medium, respectively, N=7. When applied to Fura-2-loaded OMCD, 0.3 M PGE₂ increased intracellular calcium concentration ([Ca²⁺]_i) with a peak phase (in 2 mM or 0 Ca²⁺ medium) followed by a plateau phase (observed only in 2 mM Ca²⁺ medium). It is concluded that: (1) in the rat OMCD, PGE₂, PIA and CLO act on the same AVP-sensitive cell, (2) PGE₂ induces a cumulative inhibition on the cAMP level when combined with other inhibitors by a mechanism insensitive to pertussis toxin, (3) the presence of extracellular calcium is a prerequisite condition to observe PGE₂-induced inhibition, and (4) the inhibition by PGE₂ might be linked to its capacity of increasing [Ca²⁺]_i.     

The activation of protein kinase C prevents PGE2-induced inhibition of AVP-dependent cAMP accumulation in the rat outer medullary collecting tubule

Previous studies have demonstrated that prostaglandin E₂ (PGE₂) inhibits arginine vasopressin-(AVP)dependent adenosine 3,5-cyclic monophosphate (cAMP) accumulation in microdissected rat outer medullary collecting tubules (OMCD), by a mechanism unrelated to the inhibition of cAMP synthesis. The potential role of the activation of protein kinase C (PKC) to explain the negative regulation elicited by PGE₂ was investigated in this study. Single OMCD samples were pre-incubated (10 min, 30°C) in the presence or absence of either activators of PKC, phorbol 12-myristate 13-acetate (PMA), 1-oleoyl-2-acetyl-glycerol (OAG), dioctanoylglycerol (DOG) or an inhibitor of PKC, staurosporine (SSP). These compounds were present also with the agonists tested during the incubation period (4 min, 35°C). In contrast to PGE₂, activators of PKC did not decrease AVP-dependent cAMP accumulation (mean ±SEM): 1nM AVP=47.1±6.8 fmol · mm^–1· 4 min^–1; AVP + 0.3 M PGE₂=20.1±2.7, P<0.01 versus AVP; AVP + 10 nM PMA=42.0±4.7, NS versus AVP; AVP + 50 g/ml OAG=44.1±4.8. NS versus AVP, N= 5 experiments. However, 10 nM PMA prevented PGE₂-induced inhibition: AVP + PGE₂= 44.2±3.5% of the response to AVP and 90.3±3.2% without and with PMA respectively, N= 16. Similar results were obtained with either 50 g/ml OAG or 25 g/ ml DOG (AVP + PGE₂ + OAG=92.9±6.6% of the response to AVP, N= 8; AVP + PGE₂ + DOG=94.1 ±5.3%, N= 7). OAG, DOG, PMA or PMA + PGE₂ had no intrinsic agonist activity in the rat OMCD and the addition of an inactive phorbol ester did not prevent PGE₂-induced inhibition. SSP, 50 nM or 0.1 M, did not affect the inhibition due to PGE₂ but abolished the reversion by PMA of PGE₂-induced inhibition. A similar regulation was observed on forskolin-(FK)dependent cAMP accumulation: 5 M FK + 0.3 M PGE₂= 37.7±6.2% of the response to FK; FK + PGE₂ + 10 nM PMA=89.5±6.7%; FK + PGE₂ + PMA + 0.1 M SSP=43.1±7.9%, N= 4. The inhibition induced by an ₂-adrenergic agonist, clonidine 1 M, was not blocked by the activation of PKC. In fura-2-loaded OMCD samples, 10nM PMA decreased by 63.3±5.0% and by 57.2±7.1% the peak and plateau phases, respectively, of the increase in intracellular calcium concentration ([Ca²⁺]_i) obtained with PGE₂ when compared to control responses in the same tubules (n=12) and did not affect the increase in [Ca²⁺]_i induced by 0.1 mM carbachol. It is concluded that: (1) in the rat OMCD the activation of PKC by PMA or analogues of diacylglycerol did not reproduce PGE₂-induced inhibition of AVP- or FK-dependent cAMP accumulation, but prevented specifically this inhibitory action; and (2) this reversion might be the consequence of the effect of PKC activation which impaired the rises in [Ca²⁺]_i induced by PGE₂.     

Dopamine is metabolised by different enzymes along the rat nephron

The purpose of this study was to determine the basal levels of dopamine (DA) and to examine the enzymes involved in DA metabolism in different microdissected nephron segments from rat kidneys. Segments were incubated with DA (50 nM) or DA plus monoamine oxidase (MAO) or catechol-O-methyl transferase (COMT) inhibitors. Basal DA levels were higher in the proximal convoluted tubule (PCT, 10.8±3.7 pg/mm) and in the medullary collecting duct (MCD, 10.9±4.0 pg/mm) than in the medullary thick ascending limb of Henles loop (MTAL, 4.9±0.9 pg/mm) (P<0.05). The percentage of exogenously added DA that was not metabolised was similar in both PCT (67±13%) and MCD (65±5%) and lower in MTAL (35±7%), suggesting that MTAL is a major site of DA metabolism. Inhibition of MAO (pargyline 1 mM) significantly increased the basal content of DA and the percentage of the added non-metabolised DA (to 95±10%) in PCT but had no effect on MTAL or MCD. Conversely, inhibition of COMT (nitecapone or Ro-41-0960, both 1 mM) slightly increased the basal levels of DA only in MTAL, whereas the percentage of added DA not metabolised rose to 97±10% in MTAL and to 91±15% in MCD. COMT inhibition had no effect in PCT. In conscious rats pargyline (50 mg/kg) increased urinary DA from 680±34 to 1,128±158 ng/d/100 g BW (P<0.01) while nitecapone (40 mg/kg) produced a slight non-significant increment. Our results show that DA is present all along the rat nephron and that renal DA is metabolised continuously and predominantly by MAO in proximal segments, and by COMT in the more distal ones.     

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°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±2.3; 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. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [¹²⁵I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [¹²⁵I] insulin binding sites may correspond to physiological insulin receptors.Abbreviations used [¹²⁵I] Insulin [¹²⁵I] Tyr A¹⁴ human insulin - PCT proximal convoluted tubule - PR pars recta - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT outer medullary collecting tubule     

Renal and hepatic expression of the ammonium transporter proteins, Rh B Glycoprotein and Rh C Glycoprotein

A family of ammonium transporter proteins was recently identified. Members of this family, Rh B Glycoprotein (RhBG) and Rh C Glycoprotein (RhCG) are expressed in the kidney and the liver, important tissues for ammonium metabolism. Immunohistochemical studies demonstrate basolateral RhBG immunoreactivity in the connecting segment (CNT) and collecting ducts, but not in the proximal tubule or the loop of Henle. Colocalization with thiazide sensitive cotransporter and carbonic anhydrase II confirms expression in the CNT, initial collecting tubule (ICT), and throughout the collecting duct. Colocalization with AE1 and pendrin demonstrates expression is greatest in A-type intercalated cells in the cortical collecting duct (CCD), outer medullary collecting duct (OMCD) and inner medullary collecting duct (IMCD), present in the CCD principal cell, and not detectable in either pendrin-positive CCD intercalated cells or in non-intercalated cells in the OMCD and IMCD. RhCG immunoreactivity has a similar axial distribution as RhBG. However, RhCG immunoreactivity is apical, and is detectable in all CCD and outer stripe of OMCD cells. The liver, a second organ involved in ammonia metabolism, also expresses both RhBG and RhCG. Basolateral RhBG immunoreactivity is present in the perivenous hepatocyte, but is not present in either the periportal or mid-zonal hepatocyte. Hepatic RhCG mRNA is expressed at lower levels than RhBG, and RhCG protein is detected in bile duct epithelium. These findings indicate that RhBG and RhCG are involved in at least two organs that transport ammonia, and that they are located in sites where they are likely to mediate important roles in ammonia transport.     

Salt and water intake in Brattleboro rats with hypothalamic diabetes insipidus

Brattleboro rats lacking vasopressin have an elevated plasma osmolality and a stimulated renin-angiotensin system relative to Long-Evans rats (LE). The current studies were performed to elucidate the factors controlling water and salt intake in the Brattleboro rat with diabetes insipidus (DI). DI and LE rats were given the choice of water and saline solutions ranging from 0.1-1.0% to assess palatability, dialyzed with isotonic glucose to test for sodium appetite after sodium depletion, and infused intracranially with an angiotensin II analogue (saralasin) to assess the role of angiotensin II in spontaneous salt and water intake. DI rats exhibited spontaneous salt intake which was not significantly different from LE rats and increased their intake of 3.0% NaCl following sodium depletion, although less reliably than LE rats. A significant proportion of those DI rats not developing a sodium appetite showed attenuation of their diabetes following dialysis. No evidence for involvement of angiotensin II in spontaneous salt and water intake was found.     

The effect of prostaglandin E2 and ADH on diffusional water permeability in the collecting duct of an isolated rat papilla

The effect of prostaglandin on diffusional water permeability has been studied in collecting ducts in an isolated rat papilla. PGE₂ increased water permeability. The effect was significant at a concentration of 10^–8 mol l^–1 and was maximal with a concentration of 10^–6 mol l^–1. The maximal increment of 0.94±0.10 (SEM) m s^–1 was approximately half that produced by maximal stimulation with antidiuretic hormone (2.18±0.12 m s^–1).A concentration of 10^–8 mol l^–1 produced an increase in basal water permeability and 25 unit ml^–1 ADH, which without PGE₂ present gave a similar increase, had no incremental effect. ADH 100 unit ml^–1 increased permeability to a value similar to that observed in the absence of PGE₂. Thus PGE₂ and ADH both increase water permeability but the increments are not additive.Indomethacin in a concentration that inhibited prostaglandin production altered the response of the collecting duct to ADH. The dose response curve was shifted to the left and the maximal increase in water permeability and the lowest dose at which a response occurred took place at concentrations less than 1/2 those required in its absence.Prostaglandins influence the action of ADH and it is likely that in life they regulate and modulate the change in water permeability induced by anti-diuretic hormone.     

Micropuncture study on the effects of lithium on proximal and distal tubule function in the rat kiney

Micropuncture studies were performed in rats infused with LiCl to induce stable plasma lithium concentrations of 2–3 mEq/l, or with an equivalent amount of NaCl. In free flow experiments LiCl reduced proximal tubule fractional reabsorption of sodium and potassium. Reduced reabsorption of bicarbonate, as reflected by a decrease in TF/P_cl, was also observed. Proximal fractional reabsorption of chloride, however, was not affected. The TF/P_In at the end proximal tubule was 2.6±0.2 (mean ±SEM) in controls and 2.1±0.1 in experimental animals (P<0.025). In the distal portions of the nephron lithium treatment caused a fall in fractional reabsorption of water and sodium, while potassium secretion was stimulated in the distal tubule.Previous studies have indicated that lithium influences antidiuretic hormone stimulated water transport in the collecting duct. These experiments demonstrate that lithium also affects the transport of water and electrolytes in multiple nephron segments, including the proximal and distal convolution.     

Morphologic heterogeneity along the rat inner medullary collecting duct

The qualitative and quantitative morphologic features of the cells lining the inner medullary collecting duct (IMCD) in the outer (IMCD1), middle (IMCD2) and inner (IMCD3) segments were investigated. Kidneys of male rats were fixed by in vivo vascular perfusion with glutaraldehyde and processed for light microscopy and scanning and transmission electron microscopy. The IMCD1 consisted of both principal cells and intercalated cells similar to those present in the outer medullary collecting duct. The principal cells were covered with small microvilli and a single cilium. Most of the IMCD2 and the entire IMCD3 contained one cell type (IMCD cell). When compared with the principal cells, the IMCD cells were taller, had fewer basal infoldings and a lighter staining cytoplasm containing numerous free ribosomes and small electron-dense cytoplasmic bodies in the basal region. The luminal surface was covered with prominent microvilli, but had no cilia. Morphometric analysis demonstrated that the surface density of apical and basal plasma membranes decreased from IMCD1 to IMCD3. However, because of an overall increase in tubule volume from IMCD1 to IMCD3, there were no significant differences in the absolute area of apical or basal membranes between the three segments. In contrast, the absolute area of lateral membranes increased significantly from IMCD1 to IMCD3. This study demonstrates that the IMCD1 consists of principal cells and intercalated cells similar to those in the outer medullary collecting duct, whereas the cells in most of the IMCD2 and the entire IMCD3 appear to represent a distinct and separate cell type which we choose to call the IMCD cell. Thus, both morphologic and functional heterogeneity appear to exist along the IMCD.