Balance between tubular flow rate and net fluid reabsorption in the proximal convolution of the rat kidney

Summary In the present study it is demonstrated that reabsorptive functions of the proximal tubule during partial renal artery clamping display significant differences when compared to spontaneous GFR variations. During moderate clamping fractional fluid reabsorption was 69.8% and fell during severe clamping to 55.2%. Proximal tubular passage time increased progressively with the degree of clamping. This indicates that radius does not change as a function of GFR, a finding supported by microphotography of the renal surface. During moderate arterial clamping proximal reabsorptive rate was correlated to the tubular cross-sectional area, but in the face of severe clamping this relationship was abolished. We conclude, therefore, that mechanisms other than tubular geometry participate in regulating proximal tubular reabsorption, when GFR is altered by arterial constriction.Supported by the Deutsche Forschungsgemeinschaft and the US-Department of the Army, through its European Research Office.     

A direct evaluation of the Gertz hypothesis on single rat proximal tubulesin vivo: Failure of the tubular volume to be the sole determinant of the reabsorptive rate

Summary Studies have been undertaken to directly test the predictions of the Gertz hypothesis on single proximal tubules of the rat kidneyin vivo. By altering the resistance to flow at the end of single proximal tubules, either by means of external obstruction by a glass device or by decompression secondary to fenestration of the last segment, marked changes in transit time and tubular radius were induced. In decompressed nephrons the reabsorptive rate did not decrease in spite of significant reductions in transit time and radius, while in obstructed proximal tubules a fall in reabsorptive rate occurred in the face of large increases in radius and transit time. Accordingly, this direct evaluation of single proximal tubular function supports the conclusion that geometric factors cannot determine the reabsorptive rate in the manner suggested by Gertz. Further, the prolongation of contact time during furosemide diuresis does not appear to significantly contribute to the constancy of fractional reabsorption. An alternate mechanism, which may determine reabsorptive rate, is suggested.Supported by a Medical Research Fellowship of the Medical Research Council of Canada.     

Influence of luminal diameter and flow velocity on the isotonic fluid absorption and36Cl permeability of the proximal convolution of the rat kidney

Summary In the first experimental series proximal convolutions of the rat kidney were perfused with a modified Ringer solution and the isotonic fluid absorption was measured. In a second series the tubule was perfused with equilibrium solution which contained³⁶Cl and the chloride permeability was determined. By the recollection method each individual tubule was perfused twice either at constant luminal diameter but different perfusion rates (10:30 or 6:16 nl/min) or at constant perfusion rates but different luminal diameters (20:30 ). The perfusate was recollected at two different sites which were at least 500 distant from the infusion site.The isotonic fluid absorption as well as the³⁶Cl permeability was unchanged when the tubule was distended from 20–30 . Both, however, increased about 20% when the perfusion rate was increased 3-fold.The data led to the following conclusions: 1. It is unlikely that there is a flow reactor type dependence of proximal tubular transport on flow rate. 2. The tubular distension cannot be responsible for the glomerulo-tubular balance. 3. It is more advantageous to relate permeability data of the rat nephron to tubular length. 4. In microperfusion experiments non steady sampling does not affect transepithelial fluxes per unit tubular length, provided that the pump delivery is constant.This work was presented in part at the 38th Meeting of the Deutsche Physiologische Gesellschaft in Erlangen, September 1970.     

Correlation between luminal hydrostatic pressure and proximal tubular fluid reabsorption in the rat kidney

Summary Split-drop experiments were performed to evaluate the effect of changes in luminal hydrostatic pressure on net fluid reabsorption in proximal convoluted tubules of the rat kidney. While hydrostatic pressure in control droplets averaged 28.9±1.03 mm Hg, it increased to a mean of 65.2±3.3 mm Hg during pressure elevation and fell to 10.8±1.04 mm Hg during pressure reduction. In paired measurements in identical tubules net fluid absorption changed from a control value of 2.96±0.14 nl/min·mm to 3.88±0.14 nl/min·mm when luminal pressure was elevated. During pressure reduction net fluid absorption fell from a control of 2.98±0.09 nl/min·mm to 2.26±0.13 nl/min·mm (P<0.001). This dependency of fluid absorption upon hydrostatic pressure was not greatly affected by the finding that microphotography overestimated the true intradroplet volume by 31% during control and by 30.2% and 50% during elevated and reduced pressure respectively. From the relation between the changes of net absorption and luminal hydrostatic pressure an apparent hydraulic conductance of 0.04 nl/min·mm Hg was estimated.     

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.     

The role of bicarbonate and other buffers on isotonic fluid absorption in the proximal convolution of the rat kidney

Summary The fluid reabsorption from the proximal convolution of the rat kidney was measured with the Gertz shrinking droplet technique. Simultaneously, the peritubular capillaries were perfused with artificial solutions. In some experimental series, fluid from the shrinking droplet was withdrawn and analysed for Cl^–, Na⁺, and osmolality so that the transtubular transport of Na⁺, Cl^–, and HCO ₃ ^– could be calculated. Capillary perfusate in some experiments was also withdrawn and its pH was measured. The following results were obtained: 1. With increasing concentration of HCO ₃ ^– in the capillary perfusate, the transtubular water, sodium, chloride, and bicarbonate reabsorption increased. 2. The sulfonamide buffers sulfamerazine and glycodiazine (Redul^®), which easily penetrate the tubular wall, could, in equimolar concentrations, substitute totally for the bicarbonate buffer in promoting isotonic fluid absorption. 3. Butyrate, propionate, and acetate were also effective; pyruvate, lactate, and paraaminohippurate, however, were not. 4. The effect of HCO ₃ ^– and glycodiazine on isotonic absorption was shown to depend exclusively on the concentration of the buffer anion and not on the concentration of undissociated acid or pH. From these data it is suggested that for proximal isotonic absorption of water, sodium, and chloride, the reabsorption of buffer anions via H⁺ secretion and nonionic diffusion may be essential. The H⁺ secretion or the buffer anion absorption across the luminal cell wall may secondarily influence the active Na⁺ transporting mechanism located at the basal cell site either by a luminal H⁺–Na⁺ exchange mechanism or by a lyotropic effect which would increase the Na⁺ permeability of the luminal cell site. Thereby more Na⁺ would be delivered to the Na⁺ pumping site and the rate of Na⁺ pumping would be augmented.     

Stimulation of proximal tubular sodium reabsorption by ile5 angiotensin II in the rat kidney

A direct dose-dependent stimulation or inhibition by val⁵-angiotensin II of sodium reabsorption in the rat proximal nephron has been shown using stationary microperfusion combined with perfusion of the peritubular capillaries. Since the circulating form of the hormone in the rat has been suggested to be the ile⁵-analogue these experiments have been repeated using the ile⁵-peptide at peritubular concentrations covering the normal physiological range (10^–10–10^–13M). Comparison of the results with the previously published data shows no significant difference between the actions of these two analogues. At the concentrations tested both caused significant stimulation of sodium reabsorption with a maximum effect at 10^–11M.     

Effect of isotonic volume expansion on proximal tubular reabsorption of Na and fluid in the developing rat kidney1

Young rats (aged 22–24 days) and adult rats (aged 40–42 days) were studied during hydropenia (HP) and during volume expansion (VE) in order to clarify the role of the proximal tubule of the immature kidney in the blunted natriuretic response seen in young mammals during VE. The position of the last accessible site for micropuncture of the proximal tubular segment was determined. The disadvantages of using lissamine green as a marker of different tubular segments were investigated. Tubular function was ascertained by micropuncture of superficial proximal nephrons. Measurements of tubular length were made from latex casts of the proximal tubule. No side-effects of lissamine green were detected, when small quantities were used (20–30 μl) and at least 20 min elapsed between the infusions of the dye and tubular samplings. The last accessible proximal tubule available for micropunction was found to be similarly located in young and adult rats. Fractional reabsorption during HP remained constant during development. An equivalent degree of VE induced an increase in tubular load in both age groups, but it was more marked among younger rats. Absolute proximal reabsorption in both young and old rats in HP paralleled that of the tubular load. Fractional reabsorption, however, decreased slightly during VE but to the same extent in both age groups. This indicates a great flexibility in the immature proximal tubule under various tubular loads although it had been thought that this part of the nephron was in the later stages of development. The results imply that the proximal tubule does not create the blunted sodium response in the immature kidney during VE.     

The relation between the reabsorption of urea and of water in the distal tubule of the rat kidney

Summary Micropuncture studies on rats with hereditary hypophysial diabetes insipidus were undertaken to examine the effects of water and osmotic diuresis and dietary protein on renal tubular urea handling.The results show that in a massive water diuresis the urea present at end of the proximal tubules is practically quantitatively exreted in the final urine. In osmotic diuresis an ever increasing amount of urea reaches the end of the proximal tubule, sinceTF/P _urea of 1.31±0.16 in water diuresis remains constant in osmotic diuresis, whilstTF/P _inulin falls from 2.41±0.21 in water diuresis to values lower than 2.00 in osmotic diuresis. In this case fractional amounts of urea of the same order as in the proximal tubule were measured in the distal convolution and in the final urine. A value of 0.99±0.17×10^–4 mm/sec for the distal tubular urea permeability was obtained by direct measurement at high plasma and high intratubular urea concentrations and did not change significantly after pretreatment on low or high protein diet (1.00±0.20 and 1.05±0.23×10^–4 mm/sec, respectively). From these results we conclude: 1. The magnitude of the urea excretion in a massive diuresis is primarily determined by the water reabsorption in the distal nephron. 2. The urea permeability of the distal convolution is independent of the concentration thus excluding the suggested possibility that there exists a reabsorption mechanism for urea autonomous of water reabsorption. 3. The fall in fractional urea excretion in animals on low protein diet is due entirely to mechanisms located in the collecting duct.Supported by the Deutsche Forschungsgemeinschaft.     

Influence of intratubular pressure on proximal tubular compliance and capillary diameter in the rat kidney

Tubular compliance is the response of tubular diameter to changes in intratubular pressure [7]. Proximal tubular compliance was determined directly by measurements of tubular diameter and pressure and indirectly using a mathematical model of tubular fluid flow based on measurements of the hydraulic pressure gradients along the tubule under free flow conditions and during an induced pressure reduction at the end of the proximal tubule. The two independent methods yielded similar values for compliance. Proximal tubular complicance was found to depend upon the intratubular pressure: tubular compliance was significantly higher (P<0.001) when the intratubular pressure was reduced below normal (1.0 m cm H₂O^–1) than when the pressure was increased above the control value (0.4 m cm H₂O^–1) Almost identical compliance values were measured in sodium pentobarbital and inactin anaesthetized rats (P>0.8).Intratubular pressure changes resulted in inverse changes in the diameters of the adjacent capillaries, suggesting that the peritubular capillaries are distensible structures.     

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.     

Effect of SH-, NH2- and COOH-site group reagents on the transport processes in the proximal convolution of the rat kidney

Summary The effects of site group reagents were tested on the following transport processes of the proximal convolution. Isotonic Na⁺ absorption, evaluated by the shrinking droplet procedure, histidine and glucose transport, evaluated by measuring the respective transtubular concentration difference at zero substance and water net flux. The test substances were applied either by continuous microperfusion of the peritubular capillaries or by luminal perfusion prior to the transport tests or by addition to the luminal test solution.The SH reagents (0.2 mM) N-ethylmaleimide,p-chloromercuribenzoate (pCMB) 3,6-bis-(acetatomercurimethyl)dioxane and Mersalyl (Salyrgane) caused 50% inhibition of the isotonic Na⁺ absorption in approximately 1.5 min when applied to the capillary perfusate. The same effect was reached in 2–3 min by 0.2 mMp-chloromercuriphenylsulfonate, benzamido-4-iodo-acetylstilbene-2,5-disulfonate and 2,2-dihydroperoxy-2,2-dibutylperoxide. However, the large molecular SH reagentspCMB-dextran T10 and benzoxanthene-3,4-dicarboxylic-N-iodoacetyloligoprolyl-2-aminoethylimid, did not inhibit the isotonic Na⁺ absorption. If an inhibitory effect was observed on the Na⁺ transport its onset was faster, when the substance was applied from the blood site than when it was given from the tubular lumen. Because SH reagents inhibit the isotonic Na transport faster when applied from the blood side, and because SH reagents with MW up to 690 are inhibitory whereas larger ones with MW over 1700 are not, it seems that they exert their inhibitory action on SH groups located a) predominantly on the blood side and b) deep within the membrane and not at the surface.Histidine- and glucose transport was inhibited only when the sodium transport was inhibited considerably.The oxygen consumption of teased kidney slices is not inhibited by 0.2 mMpCMB or Mersalyl within 10 min, but it is inhibited considerably by 1 mM of these substances in the same period of incubation time.The COOH reagents N,N-carbonyl-diimidazole and N-ethyl-N-(3-dimethyl-aminopropyl)carbodiimid (10 mM) and the NH₂ reagents 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid, 2 Na⁺ (SITS) (1 mM) as well as danslychloride (applied from the lumen at 5 mM in paraffin oil) did not inhibit the isotonic Na⁺ absorption.     

Active Ca2+ reabsorption in the proximal tubule of the rat kidney

Summary Using the stop flow microperfusion technique with simultaneous capillary perfusion the rate of active Ca²⁺ reabsorption was evaluated by measuring the static head electrochemical potential difference as well as the permeability of the tubular wall for Ca²⁺ ions. Under control conditions the active Ca²⁺ transport was calculated to be 3.35×10^–13 mol/cm·s. It declined toward zero if the ambient Na⁺ was replaced by choline or lithium. Parallel experiments in the golden hamster showed that active Ca²⁺ transport, vanished completely if active Na⁺ transport was blocked by ouabain (1 mM). These data indicate that the active Ca²⁺ reabsorption from the proximal tubule depends on the active reabsorption of Na²⁺ presumably via a Na⁺–Ca²⁺ countertransport at the contraluminal cell membrane. The static head electrochemical potential difference of Ca²⁺ is the same in late and early proximal tubules. It is also not affected by the presence of acetazolamide (10^–4 M) by the absence of bicarbonate or glycodiazine buffer or by the absence or presence of phosphate (2 mM).     

Different correlations between plasma protein concentration and proximal fractional reabsorption in the rat during acute and chronic saline infusion

Summary The effect of acute and chronic saline infusion on filtration and reabsorption in the proximal convolution was investigated in micropuncture experiments on rats. In both acute and chronic infusion single nephron filtration rate and total kidney GFR was increased at equal rates.During acute saline infusion there was a reduction of both fractional reabsorption (from 60.5 to 41.9%) and plasma protein concentration (from 5.8 to 5.2 g-%). During chronic infusion of about 70% of BW/day for 9–17 days there was only a slight decrease in proximal fractional reabsorption from 57.1 to 52.2%, whereas plasma protein concentration was much more reduced (from 5.9 to 3.6 g-%).It could be shown that the decrease in plasma protein was accompanied by an even greater reduction of plasma oncotic pressure. We conclude that the variation of peritubular protein concentration and oncotic pressure cannot influence proximal fractional reabsorption under physiological conditions.Supported by the Deutsche Forschungsgemeinschaft.     

Phosphate transport in the proximal convolution of the rat kidney

Proximal inorganic phosphate (P _i ) transport was evaluated using the standing droplet method with simultaneous microperfusion of the peritubular blood capillaries.In chronic parathyroidectomized (PTX) rats addition of 3 M of the Ca²⁺ ionophore A 23187 to the luminal perfusate had no effect on the P _i transport, although the isotonic fluid reabsorption was reduced by 20%. When the Ca²⁺ concentration in the perfusates was raised from 1.5 mM to 3.0 mM the reabsorption did not change significantly. But when Ca²⁺ was omitted from the perfusates the P _i reabsorption dropped by 19%, and when 2 mM EDTA were added to the perfusates P _i transport decreased by 35%.The influx of P _i from the interstitial space and from the cell into the phosphate-free luminal perfusate did not change, when the perfusates were Ca²⁺-free, but it increased by 23% in the presence of 2 mM EDTA.The data indicate that 1. a rise in intracellular Ca²⁺ above normal is not a factor which modifies basal P _i transport i.e. when P _i transport is independent of the action of parathyroid hormone. 2. A reduction of extracellular Ca²⁺ concentration from normal toward zero reduces P _i transport without changing the paracellular leak permeability for P _i . 3. With EDTA the paracellular leak permeability for P _i is increased, thus causing an even greater reduction in net P _i transport than with Ca²⁺-free solutions alone.     

Active sulfate reabsorption in the proximal convolution of the rat kidney: Specificity,Na+ and HCO 3 − dependence

Using the standing droplet technique in the proximal convolution and simultaneous microperfusion of the peritubular capillaries, the decrease in luminal sulfate concentration with time and the zero net flux transtubular concentration difference of sulfate ( ) at 45 s was determined — the latter being taken as a measure of the rate of active sulfate reabsorption. Starting with 0.5 mmol/l sulfate in both perfusates the value of 0.35 mmol/l was approached exponentially with a half value time of 4.3 s. The values in the early proximal and late proximal convolution did not deviate from each other. If the Na⁺ concentration in the perfusates was reduced, the approached zero and extrapolated to a slightly negative value (c _i>c _o). When 1 mmol/l ouabain was added to the perfusates decreased by 66% (the latter experiments were performed in the golden hamster which is more sensitive to ouabain than the rat). 1 mmol/l thiosulfate diminished by 68% and 1 mmol/l molybdate by 24%. Omitting or replacing bicarbonate by HEPES or glycodiazine reduced the sulfate reabsorption significantly, while acetazolamide (0.1 mmol/l) and increasing the CO₂-pressure from 4.66 to 14.0 kPa (i.e. 5–15% CO₂) had no effect. SITS 1 mmol/l had no effect on sulfate reabsorption.The data indicate that the sulfate reabsorption is driven by a Na⁺ gradient and inhibited by thiosulfate and molybdate, i.e. molecules which have a similar tetrahedral molecule structure. The sulfate reabsorption depends in an undefined manner on the presence of bicarbonate ions.