Phosphate reabsorption in juxtamedullary nephron terminal segments

Summary Adult Munich Wistar rats undergoing mild salt diuresis (NaCl 20 g·l^–1, 0.1 ml·min^–1) were injected with tracer doses of³H-Inulin and³²P-sodium phosphate in thin descending and ascending limbs of Henle's loop, collecting ducts accessible at the surface of the papilla and early distal superficial tubules. Kidneys were prepared for simultaneous papillary microinjection and urinary flow collection. Expressed in percent of the amounts injected, unidirectional phosphate reabsorption fluxes were 5±1% and 3±1% for injections into early distal superficial tubules and collecting ducts, respectively. By contrast, the flux was 21.7±3% for injections into either the descending or ascending thin limbs of juxtamedullary nephrons. We conclude from these results that in the rat, a significant amount of phosphate is reabsorbed by the juxtamedullary distal tubules and/or the subsequent arcades connecting the juxtamedullary distal tubules to the collecting ducts.     

Role of calcium in the decline of phosphate reabsorption during phosphate loading in acutely thyroparathyroidectomized rats

In acutely thyroparathyroidectomized (TPTX) rats infusion of inorganic phosphate leads-after saturation of the reabsorptive system-to a continuous decline of the reabsorptive capacity. The present study is designed to define parameters which are responsible for this phenomenon. Animals are infused a phosphate solution (100 mmol/l, pH 7.4, 0.2 ml/min·kg BW) and a phosphate concentration in plasma of about 5.5 mmol/l is maintained for up to 7 h. While glomerular filtration rate as well as plasma sodium concentration remain virtually constant, plasma potassium concentration initially decreases within the first 4 h of the experiment, but increases again after that period. Phosphate reabsorption declines by some 30%, plasma calcium concentration falls from 1.87±0.03 to 1.11±0.05 mmol/l. The animals develope a mixed respiratory and metabolic alkalosis with a plasma pH of 7.54±0.01, a carbon dioxide tension of 4.66±0.13 kPa (35±1 mm Hg) and a plasma bicarbonate concentration of 28.2±1.3 mmol/l. Phosphaturia leads to osmotic diuresis, to an increase of urine pH and titrable acid. When the elevated plasma pH is normalized phosphate reabsorption continues to decline. When the elevated plasma bicarbonate is lowered no further significant change of phosphate reabsorption is found. When, however, the decreased plasma calcium is restored by infusion of calcium chloride a significant increase of phosphate reabsorption up to control values is demonstrated (P<0.01). From these data we conclude that hypocalcaemia in phosphate loaded TPTX rats is the crucial parameter leading to the decline of renal phosphate reabsorption. This study was supported by “Dr. Legerlotz Stiftung” Part of this work was presented at the international workshop on phosphate and other minerals in Madrid, July 13–15, 1977     

Effect of calcium,furosemide and chlorothiazide on net volume reabsorption and basolateral membrane potential of the distal tubule

In the distal tubule of the isolated kidney of Amphiuma net volume reabsorption (split-oil droplet method) and basolateral membrane potential ( _b ) were measured. Luminal perfusion solution could be changed rapidly from 108 mmol·l^–1 NaCl plus 0.1 mmol·l^–1 calcium to solutions containing 103 or 97 mmol·l^–1 NaCl plus 3.6 or plus 7.2 mmol·l^–1 calcium. Furthermore, 10^–4 mol·l^–1 furosemide or chlorothiazide were applied luminally. (1) Addition of 7.2 mmol·l^–1 calcium hyperpolarized _b from –73.4 mV to –108.3 mV and inhibited net volume reabsorption. (2) Similarly, when furosemide was injected, _b was hyperpolarized and net volume reabsorption reduced. Application of both high calcium and furosemide further inhibited volume reabsorption. (3) The effects of chlorothiazide were similar to those of furosemide. However, when both high calcium and chlorothiazide were administered _b and volume reabsorption were almost normalized. (4) The data are consistent with the hypothesis that calcium and the diuretics interfere primarly with chloride uptake across the luminal membrane and thus reduce sodium chloride transport. When chlorothiazide in the presence of high luminal calcium almost normalized chloride transport, it is likely that its effects were by stimulating calcium transport and thus increasing intracellular calcium activity.Supported by Deutsche Forschungsgemeinschaft (Wi 328)The paper was presented, in part, at the XXVIIth Int. Congr. Physiol. Sci., Paris 1977, Proc. Vol. 13:304     

Effect of glucagon on magnesium renal reabsorption in the rat

The recent localization, in the rat, of a glucagon-sensitive adenylate cyclase in these segments where the bulk of calcium and magnesium is reabsorbed suggests an effect of this hormone on calcium and magnesium tubular transport. Renal tubular handling of calcium and magnesium as well as of sodium and phosphate was therefore studied by clearance methods in anesthetized rats, either intact or thyroparathyroidectomized (TPTX), infused with glucagon at a rate of 25 ng·min^–1/100 g bw just after a priming dose of 2.5 g. The hormone administration resulted in a significant decrease of absolute and fractional magnesium excretion (from 16.3±0.7% to 9.7±1.7% for intact rats and from 20.9±1.8% to 6.9±1.0% for TPTX rats), associated with the well-known increase in sodium and phosphate fractional excretion. Moreover, a small and transient decrease of calcium fractional excretion was observed concomitantly with a decrease of plasma calcium concentration. The significant increase in magnesium absolute reabsorption, observed whatever the filtered load and independently of PTH and calcitonin, may be an evidence for a direct tubular effect of glucagon.     

Single nephron filtration,luminal flow and tubular fluid reabsorption along the proximal convolution and the pars recta of the rat kidney as influenced by luminal pressure changes

Summary Intratubular pressures were measured in free flow and after blockade of tubular flow at different distances from the glomerulum in the kidney of Wistar rats. Free flow pressure wasffp=13.3 ±2.5 Torr and stop flow pressuresfp=41.7±3.8 Torr. With increasing distance of the blockade from the glomerulum the intratubular pressure decreased being 22.4±2.1 Torr, when the tubule was blocked at the end of the pars recta. In a second series single nephron filtration rate (gfr) and late proximal flow rates were measured at different intratubular pressures. Free flowgfr _f was 26.5±5.9 nl/min and . The difference of these flow rates divided by tubular length results in a local reabsorption rate ofC=2.9±0.9 nl/min·mm in the proximal convolution. In the pars recta local reabsorption rate was 1.0±0.3 nl/min·mm. In the proximal convolutionC increased with increasing intratubular pressure: ΔC/Δitp=(2.7±1.2)·10⁻² nl/min·mm·Torr. Filtration was in disequilibrium in these animals under all conditions examined, hydraulic filtration conductance wasK=1.2±0.4 nl/min·Torr. Modified methods have been used for intratubular pressure and for flow rate measurements in order to reduce experimental procedure. It is shown, that fractional reabsorption, calculated on the basis of pressure measurements, is a good approximation to results usually obtained by inulin measurements.     

Handling of oxalate by the rat kidney

Uptake ofd- andl-glucose, and fructose by purified brush border membrane vesicles isolated from human small intestine was studied using a rapid filtration technique. The uptake ofd-glucose by the vesicles was osmotically sensitive and represented transport into an intravesicular space and not binding to the membranes. Transport of both,d- andl-glucose was inhibited by phlorizin. Uptake ofd-fructose into the brush border vesicles was not stimulated by sodium. In the presence of a sodium gradientd-glucose was taken up 5 times faster thanl-glucose. The amount ofd-glucose transported into the vesicles in the presence of a sodium gradient was transiently higher than the amount ofd-glucose taken up at equilibrium (overshoot).d-Glucose transport was stimulated only by a sodium gradient; other monovalent cations had no effect. In the presence of a sodium gradientd-glucose transport was increased by the lipophilic anion thiocyanate and decreased by the nearly impermeable anion sulfate as compared with uptake ofd-glucose in the presence of a sodium chloride gradient. This indicates an influence of the electrical membrane potential on the sodium coupled non-electrolyte transport.     

Hormonal control of distal nephron function

Summary The distal tubule and collecting tubules are important control sites of fluid and electrolyte excretion. In our presentation we consider the cell mechanisms of transport of sodium and potassium ions and the effects of several hormones. Aldosterone and antidiuretics stimulate potassium secretion directly, and the available evidence strongly suggests that this effect involves the principal cell population. Epinephrine inhibits potassium secretion at sites beyond the distal tubule. In addition to such direct effects, secondary factors such as hormone-induced changes in flow rate along the distal tubule and changes in the plasma potassium level play an important modifying role. Several examples are presented to demonstrate that interaction of several control components uncouples potassium secretion from distal flow rate and tends to stabilize urinary potassium excretion during changes in sodium and water balance.Work in the authors laboratory was supported by NIH Grant No AM-17433     

Amino acid reabsorption in the rat nephron

The concentration of nine endogenous free L-alpha-amino acids (ALA, LEU, ILE, PHE, TYR, LYS, GLU, PRO, GLY) and of taurine were determined simultaneously along the nephron of the rat kidney using free-flow micropuncture techniques without altering plasma amino acid concentration or kidney function. The amount of each amino acid was determined after dansylation (14C-labelled dansyl-chloride) in the micropuncture sample followed by thinlayer chromatography. The main site of reabsorption is the proximal tubule. After 15-20% of the proximal tubule length the bulk of reabsorption has taken place (18.9 plus or minus 3.4% S.E. of the filtered load remaining). Net reabsorption continues to a small but significant extent along the distal nephron (disal tubule and collecting duct). Reabsorption of taurine is less rapid (% remaining of filtered load at the early proximal tubule 37.0 plus or minus 4.6%). The transtubular concentration ratio of all amino acids except taurine follows a homogeneous course. Under the experimental conditions of this study no distction with respect to different systems of reabsorption "neutral", "basic", "acidic", "imino-glycine") could be made.     

Influence of calcium and ionophore 23187 on tubular phosphate reabsorption

In previous studies it has been demonstrated that a decline of plasma calcium concentration accounts for the decrease of phosphate reabsorption in thyroparathyroidectomized (TPTX) rats undergoing phosphate loading.Microinfusion studies were performed in TPTX rats in order to discriminate between a systemic effect of calcium an a direct renal effect.Thyroparathyroidectomized animals were infused with a phosphate solution continuously. When plasma calcium concentration fell below 1.30 mmol/l, proximal convoluted tubules were microinfused with a phosphate tracer solution for 42 min. After 18 min a calcium chloride-containing solution was applied superficially (superfused) to the area of the microinfused tubule. This elevation of peritubular calcium concentration led to an immediate increase of phosphate reabsorption up to 12% of the microinfused phosphate load within 24 min.In another series of experiments, the calcium specific ionophore A 23187 — a substance which is known to increase intracellular calcium — was superfused on the microinfused tubule. This resulted again in an increase of fractional phosphate reabsorption of about 15% after 24 min. In contrast, when calcium chloride-free as well as ionophore-free solutions were superfused fractional phosphate reabsorption decreased (7%).From these data we conclude that 1. calcium has a direct renal effect on phosphate reabsorption in the absence of parathyroid hormone and 2. intracellular calcium appears to be a major parameter in the regulation of renal phosphate transport under these conditions.This study was supported by Dr. Legerlotz StiftungParts of this study were presented at the fall meeting of the Nephrologische Gesellschaft in Bonn, 1977 and at the spring meeting of the Deutsche Physiologische Gesellschaft in Göttingen, 1978     

Regulation of magnesium reabsorption in DCT

The distal convoluted tubule (DCT) is the shortest segment of the nephron and consists of an early (DCT1) and late part (DCT2). Here, several transport proteins, like the thiazide-sensitive NaCl cotransporter (NCC) and the epithelial magnesium (Mg²⁺) channel (TRPM6), are exclusively expressed. This makes the DCT the major site of active transcellular Mg²⁺ reabsorption determining the final excretion in the urine. Following the Mg²⁺ influx via the apically localized TRPM6, intracellular Mg²⁺ diffuses to the basolateral membrane where it is extruded to the blood compartment via still-unidentified Mg²⁺ transporters. Recent years have witnessed multiple breakthroughs in the field of transcellular Mg²⁺ reabsorption. Epidermal growth factor and estrogen were identified as magnesiotropic hormones by their effect on TRPM6 activity. Intracellularly, receptor of activated protein kinase C 1 and adenosine triphosphate were shown to inhibit TRPM6 activity through its α-kinase domain. Furthermore, dysregulation or malfunction of transcellular Mg²⁺ reabsorption in DCT has been associated with renal Mg²⁺ wasting. Mutations in TRPM6 are responsible for hypomagnesemia with secondary hypocalcemia. A defect in the γ-subunit of the Na⁺/K⁺-adenosine triphosphatase causes isolated dominant hypomagnesemia resulting from renal Mg²⁺ wasting. Moreover, in Gitelman’s syndrome, mutations in NCC also result in impaired transcellular Mg²⁺ reabsorption in DCT. This review highlights our recently obtained knowledge concerning the molecular regulation of transcellular Mg²⁺ reabsorption.     

Correlation between fluid reabsorption and proximal tubule ultrastructure during development of the rat kidney

Parallel functional and ultrastructural studies were performed in maturing rats in order to elucidate factors determining the development of proximal tubular fluid reabsorption. Three groups of hydropenic animals, which were 22 to 24, 28 to 32 and 40 to 45 days old, were studied. Nephron function was evaluated at the single nephron level by micropuncture technique. The ultrastructure of the developing proximal tubules was analysed by morphometric techniques following fixation of single nephrons. Kidney weight, proximal convoluted tubule length and diameter increased during postnatal development. SNGFR increased from 2.98 to 8.57 and to 20.5 nl/min in respective group of rats whereas proximal tubular fluid reabsorption Jv (a) increased from 0.15 to 0.22 and 0.34 μm³μm^_2-s ^_1. Parallel to the functional development the relative area of lateral and basal cell membrane increased, resulting in a constant relationship between net fluid reabsorption and the lateral and basal cell membrane area during the fourth postnatal week and then only a slight increase in this relation during the further development. The results suggest that net fluid transport during hydropenia is determined by the amount of available lateral and basal cell membranes where the transporting enzyme for sodium is located.     

Stationary microperfusion study of phosphate reabsorption in proximal and distal nephron segments

Summary Micropuncture studies demonstrate phosphate reabsorption in proximal tubules and between the late proximal and early distal convoluted tubule accessible to micropuncture. To further define the sites of phosphate reabsorption, the stationary microperfusion technique was applied to proximal and distal nephron segments. Phosphate reabsorption was evaluated in superficial loops of proximal tubules, descending segments beyond late proximal tubules accessible to micropuncture, ascending segments up to the point of micropuncture in the distal tubule, and superficial loops of distal tubules of thyroparathyroidectomized rats. Microperfusates of 1.3 or 2.6 nl (100 mmol/l mannitol, 100 mmol/l NaCl,³²P-phosphate and³H-inulin) were injected and then withdrawn after contact times of 2–108 s. Phosphate recovery relative to that of inulin was determined. A steep exponential decline of phosphate recovery (R) with increasing contact time (t) was observed in the superficial proximal tubule and descending segments. The slopes of the logarithmic regressions (¹⁰logR)/t, ±SEM) were: –1.68±0.33 and –1.21±0.24 min^–1 in superficial proximal tubules and descending segments respectively. In contrast, no significant decline in phosphate recoveries (–0.02±0.04 and +0.11±0.10 min^–1) was apparent in the ascending segments and distal tubule. It is concluded that phosphate is reabsorbed in the proximal convoluted tubule and adjacent descending segments of the superficial nephron and that there is no significant phosphate reabsorption in distal convoluted tubules and adjacent ascending segments.Study carried out during a one year fellowship at the Mayo Clinic. Present address: Institut für Physiologie, Fritz-Pregl-Strasse 3, A-6020 Innsbruck, Austria     

The distal nephron is preferentially infiltrated by inflammatory cells in acute interstitial nephritis

Summary In acute interstitial nephritis (AIN), mononuclear cells invade the tubules (tubulitis). The segmental localization of tubulitis is not precisely known. To clarify this question, formalin-fixed kidney biopsy specimens from 13 patients with AIN were studied stereologically by identifying cortical tubules with segment-specific markers. The periodic acid-Schiff reaction, peanut lectin, and antibodies against Tamm-Horsfall protein and epidermal cytokeratins all applied to the same section were used to identify the proximal tubules (PTs), distal convoluted tubules, distal straight tubules, and the cortical collecting system (connecting tubules and cortical collecting ducts), respectively. Morphometrically, an estimate of the relative volume of the inflammatory cell infiltrates within each category of tubular segments was obtained. Inflammatory cells were infrequently found in PTs (1.2%) but were frequently localized in distal tubules and the cortical collecting system (7.6%). There was no difference in the amount of the inflammatory cell infiltrate within these segments. Re-examination of an electron microscopic study of AIN carried out in this laboratory revealed that mononuclear cells were rarely seen in convoluted PTs but were frequently observed in straight PTs and all segments distal to them. The observations indicate that it is the distal nephron which is primarily affected by inflammatory cell infiltration in AIN.     

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.     

Localization of kallikrein-like activity along a single nephron in rabbits

In order to investigate the presence of renal kallikrein, the localization of kallikrein-like proteolytic activity along a single nephron was determined in rabbits. Single nephrons were dissected into 8 segments under a microscope. Activity was fluorometrically measured with two different substrates (benzyl-l-arginine ethyl ester: BAEE and prolyl-phenylalanylarginine-methylcoumarin amide: MCA). Proteolytic activity could be detected in the early (S1), the middle (S2), and the terminal (S3) portions of the proximal tubule and in the granular portion of the distal tubule (DCTg). With MCA, the specific activity in S1, S2, S3 and DCTg was 0.77±0.08, 0.28±0.10, 0.13±0.05, and 0.27±0.05 pmoles/g/min, respectively. The activity in DCTg was inhibited by aprotinin but that in the proximal tubules was not inhibited. No activity was found in the glomerulus, the thick ascending limb of Henle's loop, the bright portion of the distal tubule, and the light portion of the cortical collecting tubule. The inhibition of the activity by aprotinin in DCTg suggests that intrarenal kallikrein could be localized only in DCTg.This research was presented in part at the 1st Asian Pacific Congress of Nephrology, Tokyo, 1979     

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

To test prolactin (PRL) action on osmoregulation in mammals, we evaluated in the rat the effect of this hormone on a major enzyme in renal regulation of water and electrolyte: renal Na–K-ATPase. Enzyme activity was determined by cytochemistry in medullary ascending limb (MAL) and distal convoluted tubule (DCT) from rats treated either by bromocriptine, or by PRL. Three hours after a bromocriptine injection (0.1 mg/100 g IP) a significant decrease of Na–K-ATPase activity is observed in both MAL (80% of control values,p<0.001) and DCT (78%,p<0.01). Reciprocally, a significant (p<0.001) increase in enzyme activity is induced 3 h after a single PRL injection (140 g/100 g IM), in both segments (MAL: 165%, DCT: 172% of control activities) and persists 6 h after the injection (MAL: 130%, DCT: 118%). Na–K-ATPase activity was correlated to plasma PRL levels (r=0.78 in DCT,r=0.89 in MAL). A direct effect of PRL on the tubule is suggested by results from experiments in which PRL, at various concentrations, is added in vitro on renal slices before Na–K-ATPase activity measurements. The increase in Na–K-ATPase activity exhibits a log-dose dependency with PRL concentration (p<0.01) and is still observed when AVP antagonist is added before PRL incubation, ruling out the possible role of AVP contamination of PRL. These results suggest a direct effect of PRL on renal Na–K-ATPase in MAL and DCT.     

Effect of hypertonic urea and mannitol on distal nephron permeability

Summary The paracellular pathway permeability is known to increase in perfused amphibian kidneys if the luminal fluid is made hyperosmotic with mannitol or urea. To investigate whether luminal hypertonicity increases paracellular pathway permeability in the mammalian nephron, early rat distal tubules were micropunctured and perfused through one micropipette with either isosmotic saline (IS), hyperosmotic urea (HU) or hyperosmotic mannitol (HM) solutions. A second micropipette was placed down-stream in the same tubule and test solutions of 30 nl of a mixture of¹⁴C-inulin and³H-mannitol or of³H-inulin and¹⁴C-urea were injected. Similar intratubular injections of tracers were performed in a second group of rats undergoing diuresis induced either by infusing intravenously saline alone (VS) or receiving saline plus 0.4 M urea (VU). In the latter group (VU) luminal urea concentration was increased without the tubular lumen being made hyperosmotic to its peritubular fluid.Urinary inulin recovery was essentially complete and unaffected by experimental procedures. Difference between mannitol recoveries in isosmotic saline and hyperosmotic urea perfusions IS-HU was 2.6±0.8% (P<0.001). Difference in urea recoveries IS-HM was 4.1±5.1% (P>0.40), IS-HU was 13.9±5.3% (P=0.015) and, VS-VU=17.0±3.4 (P<0.001). Therefore, elevated luminal urea concentration increased tracer mannitol and also tracer urea permeability, both in the presence and absence of tubular hyperosmolarity. Electron microscopic observations showed changes in geometry of tubular junctional complexes compatible with the observed increase in permeability.Supported in part by grants from the Regional Program for Scientific and TechnologicalDevelopment from OEA and from CONICIT to Guillermo Whittembury and of NIH, NSF and American Heart Association to Gerhard H. Giebisch