Choline kinase activity along the rabbit nephron

Choline kinase catalyzes the phosphorylation of choline to phosphorylcholine which is thus made available for phosphatidylcholine biosynthesis. Choline kinase activity was determined in defined microdissected structures of rabbit nephron with a radiochemical microprocedure. Enzyme activity was present in all segments tested. When referred to tubular length, the highest activities were found in proximal convoluted tubules. Due to the lower protein content of distal structures these segments exhibited higher enzyme activities when referred to micrograms of protein. From this distribution pattern it was concluded that all nephron segments are able to use extracellular choline for phosphatidylcholine biosynthesis.     

Branched-chain amino acid aminotransferase along the rabbit and rat nephron

The activity of branched-chain amino acid aminotransferase (EC 2.6.1.42) is reported for four or five different segments of the rat and rabbit nephron as well as for patches from the papilla. In the rat the levels ranged 40-fold, from a high in the thick ascending limb of Henle to a low in the proximal convoluted tubule. The peak activity is far above that reported for most other parts of the body. Maximum activity was located also in the thick ascending limb in the rabbit, but the level was only one-third as high as in the rat. It is postulated that ammonia liberated by this amino transferase, in cooperation with glutamate dehydrogenase, could diffuse readily into the adjacent proximal straight tubule where all of the renal glutamine synthase and the highest level of alanine aminotransferase are located. Thus alanine and glutamine could be produced when the ammonia was not needed to neutralize excess acidity.     

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.     

Flow-activated transport events along the nephron

PURPOSE OF REVIEW: It is well recognized that an increase in glomerular filtration rate leads to an increase in fluid, Na+ and HCO3- absorption in proximal tubules; however, underlying mechanisms of this modulation have not been delineated. This review provides an update of flow-activated transport events along the nephron. Transporters, flow-sensors and secondary messengers that may modulate flow are also discussed. RECENT FINDINGS: We have demonstrated that both NHE3 and H-ATPase activities are modulated by axial flow in mouse proximal tubules in vitro. Experimental data and modeling calculations provide strong evidence that brush-border microvilli function as flow sensors in the proximal tubule. In addition, AngII receptor localization is regulated by flow in cultured proximal tubule cells, and flow induces eNOS translocation in TAL. SUMMARY: Flow-modulated NHE3 activity is the regulatory mechanism for GTB. It is independent of neuron and systemic hormonal regulation, but requires the intact actin cytoskeleton to transmit the signal of altered axial flow sensed by brush-border microvilli. Unanswered questions include the identification of specific signaling transduction mechanisms and second messengers in response to flow. Whether the Na+/2Cl-/K+-cotransporter in TAL is flow-activated, and whether a divalent cation, Ca2+ and Mg2+ transport, can be regulated by flow is unknown.     

Distribution of pyruvate carboxylase along the rat nephron: an immunological and enzymatic study

Antibodies against purified rat-kidney-cortex pyruvate carboxylase were raised in rabbits. These polyspecific antibodies recognize pyruvate carboxylase enzymes alone without cross reactivity with other carboxylases as detected by immunoblotting. The abundance of pyruvate carboxylase in the various renal fractions was measured by ELISA and its activity by the fixation of [14C]CO3H-. The results were corroborated by a combination of immunocytochemistry and transmission electron microscopy. A good correlation was found between the enzymatic activity and the quantity of enzyme contained in each fraction. The kidney-cortex pyruvate carboxylase was primarily located in proximal tubules, in accord with its important role in gluconeogenesis.     

Diuretics and salt transport along the nephron

The clinical use of diuretics almost uniformly predated the localization of their site of action. The consequence of diuretic specificity predicts clinical application and side effect, and the proximity of the sodium transporters, one to the next, often dictates potency or diuretic efficiency. All diuretics function by inhibiting the normal transport of sodium from the filtrate into the renal tubular cells. This movement of sodium into the renal epithelial cells on the apical side is facilitated by a series of transporters whose function is, in turn, dependent on the adenosine triphosphate (ATP)-dependent Na-K cotransporter on the basolateral side of the cell. Our growing understanding of the physiology of sodium transport has spawned new possibilities for diuretic development.     

Effects of hydrochlorothiazide on Na-K-ATPase activity along the rat nephron

Na-K-ATPase activity was determined in seven nephron segments of five-week-old, spontaneously hypertensive rats (SHR) with or without continuous hydrochlorothiazide (HCTZ) treatment for seven days. For comparison, the effects of HCTZ treatment on Na-K-ATPase activity in the nephron segments of age-matched normotensive Wistar-Kyoto rats (WKY) were also determined. Na-K-ATPase activity in proximal convoluted tubule (PCT), medullary thick ascending limb (MTAL), cortical thick ascending limb (CTAL), distal convoluted tubule (DCT) and cortical collecting duct (CCD) was significantly lower in HCTZ-treated SHR compared to control (untreated) SHR. However, there was no significant difference in Na-K-ATPase activity in proximal straight tubule (PST) and medullary collecting duct (MCD) between HCTZ-treated and control SHR. HCTZ treatment also produced a significant decrease in blood pressure (BP) and creatinine clearance (CCr) in SHR. On the other hand, HCTZ treatment did not produce a significant change in Na-K-ATPase activity in PCT, PST, MTAL, CTAL and MCD, in BP or in CCr in WKY. However, HCTZ treatment produced a decrease in the enzyme activity in the DCT and an increase in the enzyme activity in the CCD in WKY. The decrease in Na-K-ATPase activity in almost all nephron segments from SHR may be due to a significant decrease in CCr produced by HCTZ. On the other hand, a decrease in Na-K-ATPase activity in the DCT with an increase in the enzyme activity in the CCD from WKY suggest that renal compensation to the natriuretic effect of HCTZ occurs by an increase in Na+ reabsorption in the CCD.     

Gene expression of prostanoid forming enzymes along the rat nephron

BACKGROUND: To obtain information about the general capability of nephron segments to elaborate prostanoids, we determined the gene expression of key enzymes for prostanoid formation. METHODS: For this goal mRNAs were assayed for cyclooxygenases-1 and -2 as well as for the synthases of prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostacyclin (PGI2) and thromboxane A2 (TXA2) in microdissected rat nephron segments by RT-PCR. RESULTS: Cyclooxygenase-1 (COX-1) mRNA was strongly expressed in all segments of the collecting ducts and to a lesser extent in glomeruli. COX-2 mRNA was found in the cortical thick ascending limb of Henle, and weaker expression also was detected in glomeruli. The lipocalin-type PGD synthase mRNA displayed a broad expression pattern in the cortex and outer medulla, including proximal convoluted tubule, thick ascending limb of Henle, distal convoluted tubule, and cortical and outer medullary collecting duct. The hematopoietic PGD synthase mRNA was restricted to the outer medullary collecting duct, and the membrane-associated PGE-synthase mRNA was exclusively expressed in the whole collecting duct system. Prostacylin-synthase mRNA was found in the whole kidney, but not in any microdissected nephron segment analyzed in this study. TXA-synthase mRNA was expressed in glomeruli. CONCLUSION: Given that the existence of cyclooxygenase in combination with the different PG-synthases is a prerequisite for the formation of prostanoids, our data suggest that PGD2 is mainly formed in the thick ascending limb and in the collecting duct, while PGE2 appears to be mainly generated by the collecting ducts. Probably no formation of PGI2 occurs within the nephron. Whether TXA2 can be formed by nephron segments remains questionable.     

Hormone-sensitive adenylate cyclase along the nephron of genetically hypophosphatemic mice.

The response of the adenylate cyclase (AC) activity to PTH and calcitonin was measured along the nephron of normal (N) and mutant hypophosphatemic (Hyp) mice of the C 57 BL/6J strain, using in vitro single tubule AC microassay. In each experiment, a Hyp mouse was paired to a N mouse from the same litter. In the presence of PTH (10 U/ml), AC activities (femtomoles cAMP per millimeter of tubule per 30-min incubation) were reduced in the proximal convoluted tubule of Hyp mice as compared to N mice in all experiments (448 +/- (SEM) 46 vs. 831 +/- 79, N = 4, P less than 0.01). Some decrease in AC response to PTH also was noted in the cortical portion of the thick ascending limb of the loop of Henle (476 +/- 70 in Hyp mice vs. 719 +/- 83 in N mice, N = 4, P = NS). The Hyp and N AC responses to PTH were similar in the "bright" and "granular" portions of the distal convoluted tubule (1524 +/- 177 in Hyp mice and 1538 +/- 228 in N mice, N = 4). The other segments tested were not responsive to PTH (except the pars recta of the proximal tubule). In the presence of salmon calcitonin (10 ng/ml), a striking 5- to 12-fold increase in AC activity of the "bright" and "granular" portions of the distal convoluted tubule was observed in each Hyp mouse as compared to its paired N control (2434 +/- 618 vs. 399 +/- 56, N = 6, P less than 0.01). The AC response to calcitonin was also increased, though to a lesser extnet (Hyp/N = 1.8) in the "light" portion of the distal tubule (590 +/- 60 in Hyp and 352 +/- 36 in N mice, P less than 0.01). Other segments of the mouse nephron were also observed to contain calcitonin-sensitive AC, but the responses were of limited magnitude only and were not statistically different in Hyp and N mice. Dose-response curves showed that the decrease of the response to PTH in the proximal tubule as well as the increase of the response to calcitonin in the distal tubule were present in Hyp mice for the whole range of hormone concentrations tested. In both structures, the apparent Km for the cyclase activation by the hormone was similar in the Hyp and its paired N mouse.     

Multiple patterns of 11 beta-hydroxysteroid dehydrogenase catalytic activity along the mammalian nephron.

The enzyme 11 beta-hydroxysteroid dehydrogenase (11 beta-OHSD) is thought to be a protective enzyme of the mineralocorticoid receptor (MR). We have previously demonstrated (Bonvalet et al, J Clin Invest 86:832-837, 1990) that 11 beta-OHSD is colocalized with MR along the rabbit nephron. In the present study, we examined whether 11 beta-OHSD is similarly located along the nephron of other mammals. Various tubular segments were microdissected from the mouse, rat, and rabbit nephron, and incubated for two hours at 37 degrees C in the presence of 11 nM [3H]-corticosterone (B). Thereafter, the respective amounts of B and [3H]-11dehydrocorticosterone (A) in the incubation solution were measured by HPLC. In the rabbit, the mouse and the rat, about 520 pmol/10 mm of B were transformed into A in tubular segments possessing MR, that is, the distal parts of the nephron (distal and collecting tubule). Differences appeared in the aldosterone-insensitive proximal tubule; in both the initial and final parts of this segment, 11 beta-OHSD activity was low (26 pmol/10 mm) in the rabbit and the mouse, and relatively high in the rat (328 pmol/10 mm). In the cortical part of the loop of Henle, where the presence of MR is still under discussion, 11 beta-OHSD activity was low in the mouse (70 pmol/10 mm), high in the rat (533 pmol/10 mm) and intermediate in the rabbit (227 pmol/10 mm). The comparison of these results with previous data obtained with immunohistochemical methods suggests that the proximal and distal nephron might express different isoforms of 11 beta-OHSD.     

Effects of aldosterone on NEM-sensitive ATPase in rabbit nephron segments

Aldosterone (aldo) treatment of animals stimulates the rate of H+ secretion in the collecting duct, a process which may involve an H+-ATPase sensitive to inhibition by NEM (N-ethylmaleimide). Therefore, we determined NEM-sensitive ATPase activity in distal nephron segments from three groups of adrenalectomized (adx) rabbits maintained on different doses of aldo (in an osmotic minipump) for seven days. Group 1 was given 1.5 micrograms aldo/100 g body wt/day, whereas groups 2 and 3 were maintained on 5 micrograms and 50 micrograms of aldo/100 g body wt/day, respectively. Aldo concentrations in the plasma of groups 1, 2 and 3 were 10.4 +/- 0.8, 70 +/- 7 and 408 +/- 133 ng/dl, respectively. There was a significant increase in NEM-sensitive ATPase activity in connecting tubule (CNT) and cortical, outer and inner medullary duct segments (CCD, OMCD and IMCD) but not in cortical thick ascending limb (CTAL) and distal convoluted tubule (DCT) in group 2 as compared to group 1. A further increase in plasma concentration of aldo (group 3) did not produce any more increase in NEM-sensitive ATPase activity in the CNT, CCD, OMCD and IMCD, but did increase the enzyme activity in the DCT. These results are consistent with the hypothesis that aldo increases H+ secretion in the connecting tubule and collecting duct segments by increasing the activity of NEM-sensitive H+-ATPase activity in these segments.     

G proteins regulate calcium channels in the luminal membranes of the rabbit nephron

The filtered calcium (Ca2+) is reabsorbed by the luminal membrane of the proximal and distal nephron. Ca2+ enters cells across apical plasma membranes along a steep electrochemical gradient, through Ca2+ channels. Regulation by various hormones implies several steps, including binding of these hormones to the basolateral membrane, interaction with G proteins, liberation of messengers, activation of kinases and finally opening of the channels at the opposite pole of the cells. In the present study, we examined whether the Ca2+ entry through the luminal membranes of proximal and distal tubules is also regulated by G proteins, by a membrane-limited process. Luminal membranes were purified from rabbit proximal and distal tubule suspensions, and their vesicles were loaded with GTPgammas or the carrier. Then, the 45Ca2+ uptake by these membrane vesicles was measured in the presence and absence of 100 mM NaCl. In the absence of Na+, intravesicular GTPgammas significantly enhanced 0.5 mM Ca2+ uptake by the proximal membrane vesicles from 0.53 +/- 0.06 to 0.72 +/- 0.06 pmol/microg/10 s (p < 0.05). In the presence of Na+, however, this effect disappeared. In the distal tubules, intravesicular GTPgammas increased 0.5 mM Ca2+ uptake in the absence (from 0.57 +/- 0.02 to 0.79 +/- 0.02 pmol/microg/10 s, p < 0.02) and in the presence (from 0.36 +/- 0.03 to 0.55 +/- 0.03 pmol/microg/10 s, p < 0.02) of Na+. The action of GTPgammas, when present, was dose dependent with a half-maximal effect at 20 microM. The distal luminal membrane is the site of two Ca2+ channels with different kinetics parameters. GTPgammas increased the Vmax value of the low-affinity component exclusively, in the presence as in the absence of Na+. Finally, Ca2+ uptake by the membranes of the two segments was differently influenced by toxins: cholera toxin slightly stimulated transport by the proximal membrane, but had no influence on the distal membrane, whereas pertussis toxin decreased the cation uptake by the distal tubule membrane exclusively. We conclude that the nature of Ca2+ channels differs in the proximal and distal luminal membranes: Ca2+ channels present in the proximal tubule and the low-affinity Ca2+ channels present in the distal tubule membranes are directly regulated by Gs and Gi proteins respectively, whereas the high-affinity Ca2+ channel in the distal tubule membrane is insensitive to any of them.