Activation of tubulo-glomerular feedback by chloride transport

Summary To define the luminal agent(s) responsible for the reduction of nephron filtration rate following increases of loop of Henle flow rate early proximal flow rate (EPFR) during loop perfusion with 17 different salt solutions were compared to the non-perfused fubules. During orthograde microperfusions a reduction of EPFR as indication of a feedback response was noted with a number of nonovalent Cl^– and Br^– salts (LiCl, KCl, NaCl, RbCl, CsCl, NH₄Cl, choline Cl, NaBr, KBr), with Na⁺ salts except Na acetate (NaHCO₃, NaNO₃, NaF, NaI, NaSCN), and with CaCl₂ and MgCl₂. These latter 2 solutions where used in a concentration of 70 mM while all other solutions had a concentration of 140 mM. During retrograde perfusion from the distal to the proximal end of the loop of Henle EPFR fell significantly with Cl^– and Br^– salts with percentage changes of EPFR ranging from –8.0 to –44.3%. In contrast, Cl^– free salts and Cl^– salts of divalent cations were associated with percentage changes of EPFR ranging from +7.1 to –6.2%, significance being reached only during perfusion with NaSCN.When furosemide (5×10^–4M) was added to NaBr or KBr a feedback response was not observed. During orthograde perfusion with NaNO₃ distal Cl^– concentrations were 44.2±5.08 mM (mean±S.E.) at a perfusion rate of 10nl/min and 59.1±3.93 mM at a rate of 40 nl/min. CaCl₂ perfusion induced a marked elevation of distal Cl^– concentrations to levels higher than 140 mM. Loop chloride handling was normal during RbCl perfusion.The magnitude of the feedback response during retrograde perfusion was not changed by lowering NaCl concentration from 140 to 60 mM, but fell when NaCl concentration was further reduced. In contrast to orthograde perfusions it was insensitive to changes in flow rate.Our results are compatible with the thesis that feedback responses depend critically upon the rate of Cl^– transport probably across the macula densa cells. Br^– ions can replace Cl^– because they appear to share a common transport pathway which can be inhibited with furosemide. Unspecificity of feedback responses during orthograde microperfusions is due to presence of Cl^– ions in the macula densa region even when solutions are initially Cl^– free. Cl^– salts of divalent cations do not elicit a feedback response because Cl^– transport is severely curtailed.Supported by the Deutsche Forschungsgemeinschaft.     

Modification of feedback influence on glomerular filtration rate by acute isotonic extracellular volume expansion

The behavior of the feedback mechanism, that causes glomerular capillary pressure and filtration rate to decrease when tubule fluid flow rate through the loop of Henle of the same nephron is increased, was examined in rats before and during isotonic extracellular fluid volume expansion. The loop of Henle was perfused from the late proximal tubule at either 10 or 40 nl/min while proximal fluid was collected to measure single nephron filtration rate (SNGFR), while proximal stop-flow pressure (P_SF) was measured, or while fluid was collected from the early distal tubule to assess reabsorption of fluid and electrolytes by the loop of Henle. During control periods increasing loop perfusion caused SNGFR to decrease 37%, P_SF to decrease 19%, and absorption of fluid, sodium and chloride by the loop of Henle to increase. After 1 h of infusion of isotonic NaCl solution the same change in loop flow causes a 19% decrease in SNGFR and an 8% decrease in P_SF. Fluid absorption by the loop of Henle did not increase with increased loop perfusion. Increases in Na and Cl absorption were similar to the increases in control periods. The smaller decreases in SNGFR and P_SF indicate that acute volume expansion decreases the sensitivity of the feedback response. The mechanism of this decrease in gain could involve interference with local generation or action of angiotensin, or a change in the composition or pressure of interstitial fluid tending to dilate the pre-glomerular resistance vessels.     

The effect of saline infusion and hemorrhage on glomerular filtration pressure and single nephron filtration rate

Summary Single nephron filtration rate (GFRs) and effective glomerular filtration pressure (EFP) measured as the difference between intratubular stop-flow (SFP) and free-flow pressures (FFP), were determined in control rats and following saline infusion or hemorrhage. Infusion of isotonic or 4% NaCl increased EFP and GFRs without significantly affecting TF/P inulin. These findings could not be related to changes in arterial blood pressure. Controlled bleeding produced a marked decrease in EFP and in GFRs, again without significant change in TF/P-inulin. In both infusion and hemorrhage the change in GFRs was disproportionately greater than the change in EFP. Analysis of the components of the filtration process suggests that elevation of EFP is attended by an increase in permeability of the filtering membrane. This sensitive dependence of GFRs upon EFP, combined with a demonstrated constancy of total kidney GFR (GFR_T) over a wide range of urine concentrations and flow rates, connotes a close regulation of EFP in this experimental animal.Supported by Deutsche Forschungsgemeinschaft.     

Micropuncture method for the determination of nephron filtration rate

Summary Two possible artifacts may explain the phenomenon that nephron GFR (N-GFR) measured by distal tubular puncture is smaller than that measured by proximal tubular puncture: a loss of the inulin-like substance used in this laboratory (polyfructosan) from the tubular lumen or unreliable distal punctures. To test these possibilities (a) known amounts of polyfructosan were injected into the proximal tubule and the percentage recovery from the distal tubule measured, (b) N-GFR was measured by distal puncture, subsequently by recollection from the same site and finally by a proximal puncture.On the average, 98.5±7.5% of the proximally injected polyfructosan was recovered from the distal tubule. This is not significantly different from 100% (p>0.1) and demonstrates that proximal tubule and loop of Henle are impermeable to polyfructosan. The ratio between the N-GFR measured by a distal puncture and that measured by subsequent recollection was 1.016±0.096 and not significantly different from 1.000 (p<0.20), demonstrating the reliability of distal tubular puncture. The mean distal N-GFR of 27.9±5.3 nl/min was significantly smaller (p<0.001) than the proximal N-GFR of 35.1±8.0 nl/min. The existence of the proximal-distal N-GFR difference thus is confirmed and two possible artifacts eliminated. The best explanation remains the operation of a tubulo-glomerular feedback mechanism.A current point of dispute is the effect of alterations in intratubular pressure (ITP) on N-GFR. Collection of tubular fluid at ITPs below the previously measured free flow pressure (FFP) resulted in a change of N-GFR of 0.45 nl/min· cm H₂O. In contrast, fluid collection at ITPs greater than the FFP resulted in a change of N-GFR of 1.48 nl/min· cm H₂O. We conclude that although N-GFR is sensitive to ITP changes in both directions, pressure decreases are of little practical importance for the determination of N-GFR whereas intratubular pressure increases are to be avoided.This work was supported by the Deutsche Forschungsgemeinschaft.On leave from the University of Melbourne as a recipient of a scholarship from the Deutscher Akademischer Austauschdienst.     

Resetting of tubulo-glomerular feedback sensitivity by dietary salt intake

Summary Tubulo-glomerular feedback participates in the regulation of superficial nephron filtration rate, SN-GFR [13,29,30,32]. In the present experiments we investigated the possibility that the feedback response is modulated by salt intake. Five groups of rats were pretreated with diets containing different intake. Five groups of rats were pretreated with diets containing different amounts of sodium chloride in either food or drinking water. Estimated NaCl intake ranged from about 0.5 to 12 mEq per day and rat. Feedback response was tested in individual nephrons by comparing SN-GFR's derived from either distal tubular segments (i.e. with the macula densa region being perfused at normal rates) or end-proximal segments (i.e. with the flow in the macula densa region being acutely reduced). In accordance with earlier observations [8, 28], proximal SN-GFR exceeded the distal value in salt-deprived and control animals by 8.7 nl/min±8.6 (P<0.05) and 7.6 nl/min±5.4 (P<0.001) respectively. In contrast, rats supplied with 6, 11, or 12 mEq Na/day showed a proximal-distal SN-GFR difference of 1.4 nl/min ±8.5 (P<0.05), 1.2 nl/min±6.7 (P>0.05), and 4.6 nl/min±8.8 (P<0.001). Thus, elevation of NaCl intake either abolishes or diminishes feedback sensitivity in the subnormal flow range. We conclude that tubulo-glomerular feedback sensitivity is reset when sodium chloride intake is altered.Work performed during tenure of an Alexander von Humboldt-Dozentenstipendium.     

Micropuncture studies on the filtration rate of single superficial and juxtamedullary glomeruli in the rat kidney

Summary Single nephron filtration rates of superficial and juxtamedullary nephrons were determined in high and low sodium rats. Single nephron GFR was calculated from TF/P inulin and tubular flow rate in superficial nephrons and single juxtamedullary GFR from corresponding data in long loops of Henle. In low sodium rats superficial nephron GFR was 23.5±6.4 (SD)×10^–6 ml/min×g KW, juxtamedullary nephron GFR was 58.2±13.6 and total kidney GFR (C _In) was 0.94±0.16 ml/min×g KW. Using these single nephron values, total kidney GFR and a total number of 30,000 glomeruli per kidney, the number of superficial and juxtamedullary glomeruli was calculated to be 23,267 and 6,733, respectively. During high sodium diet superficial nephron GFR increased to 38.1±11.3 and single juxtamedullary GFR decreased to 16.5±6.6, total kidney GFR increasing to 1.01±0.24. Calculation again revealed the same distribution of the two nephron types. End-proximal TF/P inulin in superficial nephrons was 2.36±0.36 in low sodium and 2.31±0.28 in high sodium rats. Loops of Henle TF/P inulin and intratubular flow rate were inversely related: the highest TF/P inulin values and lowest intratubular flow rates were found in the descending limb. These data quantify the distribution of superficial and juxtamedullary nephrons on a functional basis and suggest a mechanism by which the kidney adjusts sodium excretion by altering the contribution of each nephron type to total kidney GFR.Supported by the Deutsche Forschungsgemeinschaft and the U.S. Department of the Army, through its European Research Office.     

Single nephron glomerular filtration rate ratios of superficial,intercortical and juxtamedullary nephrons in rats during development

Summary In 20, 40 and 60-day-old rats the filtration rate was studied, using Baines modification of Hanssen's Na₄Fe(¹⁴CN)₆ method enabeling the determination of single nephron glomerular filtration rate (SNGFR) ratio between superficial (S), intercortical (I) and juxtamedullary (J) nephrons. The proximal tubule lengths were determined as well. A close correlation was obtained between age and S/I, I/J and S/J of proximal tubule lengths and between age and I/J, S/J¹⁴C-activities of ferrocyanide. Presented findings confirm the data on the increasing role of superficial nephrons in the course of postnatal development of rat.     

The existence of a tubulo-glomerular feedback mechanism in the Amphiuma nephron

A single nephron tubulo-glomerular feedback control of the glomerular filtration rate, which is known in mammlian animals, could be one way by which amphibians regulate the glomerular filtration rate (GFR). To investigate whether theAmphiuma means shows any sign of a tubuloglomerular feedback control, micropuncture experiments were carried out. Six different series of experiments were performed.In the first series, tubular stop-flow pressure (SFP) was measured during distal tubular perfusion with amphibian Ringer solution at a rate of 10, 25 and 50 nl/min. A significant decrease of SFP was found at the three perfusion rates compared to the controls. In the second group, single nephron glomerular filtration rate (SNGFR) was measured, while the distal tubule was perfused at 10, 25 and 50 nl/min. At a perfusion rate of 10 nl/min the SNGFR did not decrease, whereas at 25 and 50 nl/min it decreased significantly. In the third group the perfusion pipette was located in the proximal tubule and the nephron was perfused at 10, 25 and 50 nl/min, while at the same time the proximal tubular stop-flow pressure was measured. No reduction of SFP was found at a perfusion rate of 10 nl/min, while significant reductions were noted at rates of 25 and 50 nl/min. In the fourth group the SNGFR was measured in the distal tubule beyond the macula densa and in Bowman's space of the same nephron. No significant difference was found. In the fifth group, the glomerular capillary pressure (GCP) was measured before and after blockade of the tubular fluid flow. No significant difference was found between these two measurements.The sixth series deals with the changes occuring at the single nephron level by the tubulo-glomerular feedback control. The single nephron filtration fraction (FF) was determined from efferent arteriolar protein concentration with and without a feedback-induced reduction of the SNGFR. The FF values were not significantly different from one another. From these results and data from the other series, the afferent (R _aff) and efferent (R _eff) arteriolar resistances were calculated.R _eff did not change, whileR _aff increased significantly when a feedback stimulus was applied.These experiments indicate the existence of a tubuloglomerular feedback control which depresses the SNGFR and SFP by contracting the afferent arteriole.     

Reduction of the glomerular filtration rate by the efferent blood from a kidney taken from a previously dehydrated dog

Summary The two kidneys of one dog were perfused separately with the same blood pool and at a constant arterial pressure. After the control periods, one of the kidneys was replaced by another one taken from a dehydrated dog. The presence of the dehydrated kidney resulted in significant and almost immediate decreases of creatinin clearance, urine flow and sodium excretion by the contralateral kidney, without any change in the overall renal blood flow. These changes disappeared, after the removal of the dehydrated kidney. They were not observed if the dehydrated kidney was replaced either by a kidney taken from a salt-depleted, non water-deprived dog, or by a simple arterio-venous shunt. These results suggest that in the status of dehydration, the kidney releases some humoral material which might be responsible for the decrease of the measured GFR (C creat) perhaps by the way of permeability changes.     

Single glomerular filtration rate of superficial and Juxta-medullary nephrons in the rat during different types of arterial hypertension

Summary The single nephron glomerular filtration rate (SNGFR) of superficial (S) and juxtamedullary (JM) nephrons were measured using¹⁴C-ferrocyanide infusion technique in rats under 3 different states of hypertension: acute hypertensions obtained either by bilateral carotid clamping (CC) or by contralateral renal ischemia (CI), and chronic Goldblatt hypertension. The juxtaglomerular index (JGI) was determined on the experimental kidneys.During acute hypertensions the JGI was normal, the granularity being more marked for the superficial than for the deep cortex. SNGFRs were higher in the JM (CC=39.2±3.4 SE nl/min; CI=41.0±4.5) than in the S (CC=30.3±2.5 CI=30.3±3.0) nephrons. These values are not different from those of normal rats. In the untouched kidney of Goldblatt rats the JGI was equal to zero. There was a general increase in SNGFRs. This increase was more marked for the S than for the JM nephrons, and the SNGFRs equalized in these two categories of nephrons (S=73.0±8.4; JM=74,7±8.2).From these observations, it is suggested that the renin content of the juxtaglomerular apparatus may play some role in the absolute value and intrarenal distribution of SNGFRs.This work was supported in part by a grant D.G.R.S.T. No. 7172 2726.Chargé de recherches à l'INSERM.     

The effect of experimental alterations in urine concentration on nephron filtration rate

Summary Experiments were performed to clarify the cause of the dependency of juxtamedullary nephron filtration rate (JN-GFR) on the diuretic state of the animal. Using the ferrocyanide technique of de Rouffignacet. al. [5] the distribution of nephron filtration rates was determined during selective modification of final urine osmolarity by papillary superfusion with concentrated or isotonic solutions. Papillary superfusion with a 2000 mosmolar solution led to a mean urine osmolarity of 1848±78 mOsm/l. JN-GFR displayed a mean increase of 66.6% over the superficial nephron filtration rate (SN-GFR). When the superfusion fluid was isotonic urine osmolarity averaged 754±53 mOsm/l and JN-GFR increased by a mean of only 26.3% over the SN-GFR. Using the micropuncture technique it was shown that SN-GFR in a given animal was not altered when the superfusion fluid was interchanged. We conclude therefore that the change in the percentage increase in GFR from superficial to juxtamedullary nephrons is caused by a change of juxtamedullary nephron filtration rate. Since plasma ADH concentration was not altered this effect appears to be elicited by the changed medullary solute concentration rather than by a vasomotor action of the antidiuretic hormone.This work was supported by the Deutsche Forschungsgemeinschaft.     

Development of glomerular filtration rate and electrolyte and osmolal clearance in the late-embryonic and newly hatched chicken

Summary Techniques have been developed for collection of urine in embryonic and newly hatched chickens for the purpose of studying the development of renal function.The reliability of EDTA-⁵¹Cr as a substitute for inulin-¹⁴C in the determination of GFR was studied. Since inulin and EDTA-⁵¹Cr clearances in the hatched chicken averaged 1.61±0.23 (S.E.) ml/min per kg body weight and 1.58±0.27 ml/min per kg body weight, respectively, EDTA-⁵¹Cr clearance was considered a suitable measure of GFR.GFR increased significantly in the first few days after hatching. Filtration rate was 0.068±0.008 (S.E.) ml/min per g kidney weight in the embryo and increased to 0.148±0.008 ml/min per g shortly after hatching. By nine days after hatching GFR had risen to 0.290±0.015 ml/min per g, a value comparable to that reported for the adult.Clearances of sodium, potassium, chloride and total osmolyte also increased with age. When these clearances were corrected for changing glomerular filtration rates the embryonic chicks were found to excrete a greater percentage of the filtered load. These results show that adult levels of glomerulo-tubular balance are not attained until after hatching.A preliminary report of this work has already been published [3].     

Inhibitory effect of methylxanthines on feedback control of glomerular filtration rate in the rat kidney

Summary Microperfusion experiments were performed in rats to assess the effect of luminal application of theophylline and the more lipophilic 3-isobutyl-1-methylxanthine (IBMX) on feedback regulation of glomerular filtration rate. Elevation of loop of Henle flow from 0 to 40 nl/min caused a 20.3±4.5% reduction of stop flow pressure (SFP) and a 32.2±3.7% reduction of early proximal flow rate (EPFR) when the perfusate was a 140 mM NaCl solution. When theophylline was added in a concentration of 5 mM SFP fell by only 5.3±1.8% and EPFR by 7.9±3.3%, changes which were significantly smaller than in the control perfusions (P<0.01). An identical change of loop of Henle flow in the presence of IBMX in concentrations of 1 and 5 mM was associated with a 4.5±3.9% decrease and a 12.1±2.7% increase of EPFR. In orthograde perfusion experiments full inhibition of the feedback response was noted at an IBMX concentration of about 1 mM while during retrograde perfusion a concentration of 0.4 mM was sufficient to produce the same effect. This indicates that methylxanthines diffuse out of the loop of Henle to a considerable extent. Methylxanthines reduced absolute and fractional water absorption along the loop of Henle to some extent while Cl absorption rates and early distal Cl concentrations were not significantly altered. Cyclic AMP applied from the luminal side in a concentration of 10 mM did not affect the feedback response of EPFR to flow elevation from 0 to 40 nl/min. Luminal application of dibutyryl cyclic AMP at 10 mM induced a small, but significant reduction of the feedback response when tested by pairedt-test (P<0.05). Our results show that luminal application of methylxanthines strongly interfere with feedback regulation of glomerular filtration rate. It is unclear at present whether this effect is related to inhibition of cyclic nucleotide phosphodiesterase and a rise in tissue cyclic AMP levels or to interference with a mechanism involving the local action of adenosine or 5-AMP.Supported by the Deutsche Forschungsgemeinschaft     

The influence of tubulo-glomerular feedback on the autoregullation of filtration rate in superficial and deep glomeruli

Single nephron glomerular filtration rate (SNGFR) of superficial and juxtamedullary nephrons were measured at normal and reduced perfusion pressure in the left kidney of young Sprague Dawley rats. Perfusion pressure was lowered by constricting the aorta proximal to the branching of the left renal artery. The influence of the tubulo-glomerular feedback mechanism on SNGFR was quantitated by measuring SNGFR during intact and interrupted urine flow to the macula densa region. By using a modified Hanssen technique, SNGFR was measured under free-flow conditions. In other experiments, the urine flow to the distal nephron was blocked by a micropuncture technique, which was used for collection of the tubular fluid for measuring the filtration rate. All nephron populations autoregulated SNGFR from 70–80 to 130 mmHg, which was the upper limit of this investigation, when urine flow throughout the nephron was intact. The autoregulation in this pressure range was lost when tubular fluid was prevented from reaching the distal nephron. It was shown that the influence of negative feedback on SNGFR by the macula densa mechanism at normal blood pressure is greater in deep nephrons than in superficial ones.     

Serum cystatin C is a sensitive early marker for changes in the glomerular filtration rate in patients undergoing laparoscopic surgery

OBJECTIVE:

Pneumoperitoneum during laparoscopy results in transient oliguria and decreased glomerular filtration and renal blood flow. The presence of oliguria and elevated serum creatinine is suggestive of acute renal injury. Serum cystatin C has been described as a new marker for the detection of this type of injury. In this study, our aim was to compare the glomerular filtration rate estimated using cystatin C levels with the rate estimated using serum creatinine in patients with normal renal function who were undergoing laparoscopic surgery.

METHODS:

In total, 41 patients undergoing laparoscopic cholecystectomy or hiatoplasty were recruited for the study. Blood samples were collected at three time intervals: first, before intubation (T1); second, 30 minutes after the establishment of pneumoperitoneum (T2); and third, 30 minutes after deflation of the pneumoperitoneum (T3). These blood samples were then analyzed for serum cystatin C, creatinine, and vasopressin. The Larsson formula was used to calculate the glomerular filtration rate based on the serum cystatin C levels, and the Cockcroft-Gault formula was used to calculate the glomerular filtration rate according to the serum creatinine levels.

RESULTS:

Serum cystatin C levels increased during the study (T1 = T2<T3; p<0.05), whereas serum creatinine levels decreased (T1 = T2>T3; p<0.05). The calculated eGlomerular filtration rate-Larsson decreased, whereas the eGlomerular filtration rate-Cockcroft-Gault increased. There was no correlation between cystatin C and serum creatinine. Additionally, Pearson''s analysis showed a better correlation between serum cystatin C and the eGlomerular filtration rate than between serum creatinine and the eGlomerular filtration rate.

CONCLUSION:

This study demonstrates that serum cystatin C is a more sensitive indicator of changes in the glomerular filtration rate than serum creatinine is in patients with normal renal function who are undergoing laparoscopic procedures.     

Technical problems in the micropuncture determination of nephron filtration rate and their functional implications

Summary The theory of a functional coupling between distal tubular fluid composition and glomerular filtration rate implies that the blockade of flow at a proximal site should lead to a marked increase of GFR. This potential alteration of steady state GFR was studied by comparing the influence of sampling from distal or proximal sites on the filtration rate of identical nephrons. During antidiuresis an average GFR of 25.2 nl/min±7.5 S.D. was found in distal collections, while proximally collected samples gave an average GFR of 34.5 nl/min±8.4 S.D. This difference of 9.3 nl/min is highly significant (p<0.001). During saline diuresis a mean nephron GFR of 41.6 nl/min±5.0 was found by distal sampling and of 45.3 nl/min±5.4 by proximal sampling (p>0.05). The proximal-distal difference in nephron GFR is interpreted to indicate the operation of a tubulo-glomerular feedback control system. Thus, a true steady-state GFR probably cannot be obtained by proximal fluid collection.Even in the presence of high intratubular pressures and unusually short oil blocks no evidence of sample contamination by retrograde fluid flow past an injected oil block was obtained.The application of a counter-pressure to the sampling pipette which has been recommended by Gertzet al. [5] as a means to standardize fluid collections, was found to lead to abnormally high intratubular pressures. The reason for this finding appears to be an unexpectedly high and inconstant tip resistance to flow during fluid flow into the pipette.     

Filtration in surface glomeruli as regulated by flow rate through the loop of henle

Summary A new pressure transducer microperfusion system has been used to measure quantitatively SN GFR, early proximal free flow pressure and stop flow pressure of varying flow rates through the loop of Henle in the range of 0 to 50 nl/min. Perfusing the loop with an isoosmolal artificial tubular fluid at physiological flow rates SN GFR was 19.9±1.1 and 27.7±1.0 nl/min in two different strains of antidiuretic rats. SN GFR increased when loop perfusion was interrupted. The question whether a feedback control mechanism of SN GFR is operative in the rat kidney was evaluated in further experiments in which early proximal tubular pressure was measured in functionally isolated proximal convolutions in vivo under conditions of constant flow as well as stopped flow. Experiments in which perfusion rate through the loop of Henle was varied demonstrated the existence of a feedback signal which originates downstream of the late proximal convolution and which affects filtration into individual early proximal segments. This feedback mechanism exhibited an asymmetrical behaviour: Elevation of loop perfusion above the control value resulted in an early proximal pressure drop, under simulated free flow conditions as well as under stop flow conditions. In contrast lowering of perfusion rate below the predetermined physiological value had no significant effect on early proximal pressures.Index of Abbreviations FFP free flow pressure - P _a blood pressure - P _G mean glomerular capillary pressure - PCT proximal convoluted tubule - P _tub intratubular hydrostatic pressure - SFP stop flow pressure - SN GFR single nephron glomerular filtration rate - TF tubular fluid - TF/P concentration ratio between tubular fluid and plasma - V_(TF) collected volume of tubular fluid, flow rate Supported by Deutsche ForschungsgemeinschaftParts of the present work have been presented at the following meetings: Int. Symp. on Renal Handling of Sodium, Brestenberg1971; Workshop of Renal Micropuncture Techniques, Yale University 1971; Int. Congress of Nephrology, Mexico City 1972.     

Détermination du taux individuel de filtration glomérulaire des néphrons accessibles et inaccessibles à la microponction

Résumé Nous présentons dans ce travail une modification de la technique de Hanssen qui permet de la rendre quantitative.Un indicateur glomérulaire marqué (ferrocyanure de sodium¹⁴C) est perfusé à des rats anesthésiés jusqu'à l'obtention d'une concentration plasmatique stable. Puis 20 l d'une solution à 10% de ferrocyanure non marqué sont injectés rapidement au dessus de l'artère rénale gauche. Quelques secondes plus tard, le pédicule rénal gauche est lié. Le ferrocyanure contenu dans le rein est alors précipité sous forme de bleu de Prusse insoluble, puis les tubules proximaux sont disséqués après macération. De la radioactivité contenue dans le néphron entre le glomérule et le précipité de ferrocyanure non marqué (radioactivité qui correspond donc au¹⁴C ferrocyanure filtré pendant un temps déterminé) et de la concentration plasmatique de cet indicateur, on déduit le débit de filtration (fg) du néphron considéré.Les résultats obtenus chez trois rats non diurétiques sont donnés à titre d'exemple. La fg de néphrons superficiels et profonds ayant une longueur moyenne de tubule proximal de 7,6 mm et 8,9 mm respectivement a été calculée à partir de l'équation de la droite de régression décrivant la variation de la fg en fonction de la longueur du tubule proximal: elle a été trouvée égale à 29,1 nl/min pour les néphrons superficiels et 40,1 nl/min pour les néphrons profonds.La validité de la technique est attestée 1. par le fait que la filtration glomérulaire totale du rein, calculée à partir des fg, est sensiblement identique à celle mesurée quelques minutes plus tôt (C _in 2. par le fait que les valeurs des fg ainsi que les corrélations anatomo-fonctionnelles rapportées sont identiques à celles obtenues par la technique de Hanssen, 3. enfin par le fait que les fg des néphrons superficiels sont semblables à celles habituellement mesurées par microponction.Notre technique conserve tous les avantages de la méthode de Hanssen, mais elle en supprime les inconvénients liés à l'hétérogénéité de distribution dans le rein de l'indicateur marqué puisqu'ici, au lieu d'être administré en une seule injection, le ferrocyanure marqué est perfusé à l'animal; de plus, elle permet une mesure quantitative des valeurs individuelles de fg même sur des reins anuriques.

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Determination of glomerular filtration rate of individual nephrons accessible and inaccessible to micropuncture

Summary We describe a modification of Hanssen's technique. Anesthetized rats were infused with a¹⁴C–Na-ferrocyanide solution. At equilibrium, 20 l of a 10% non labelled ferrocyanide solution were injected as a short pulse above the left renal artery. A few seconds later, the left renal pedicle was tied. Ferrocyanide contained within the kidney was precipitated as insoluble Prussian blue and the proximal tubules were microdissected after maceration. From the radioactivity contained within the nephron from the glomerulus to the precipitate of non labelled ferrocyanide (radioactivity which is due to the¹⁴C filtered during a given time) and from the¹⁴C plasmatic concentration, we deduced the single glomerular filtration rate (SGFR) of the nephron.Three non diuretic rats are given as example. The SGFR of superficial and juxtamedullary nephrons was respectively equal to 29,1 nl/min and 40,1 nl/min when calculated for the mean value of the length of their proximal tubules (respectively 7.6 mm and 8.9 mm) from the equation of the regression line which described the correlation between SGFR and the length of the proximal tubule.The reliability of the technique is supported 1. by the fact that the value of the GFR of the whole kidney calculated from SGFR's was the same asC _in measured just prior, 2. by the fact that SGFR values and the anatomo-physiological correlations were identical to those obtained with the Hanssen's technique, 3. and by the fact that the SGFR of superficial nephrons agree with most of those obtained with the micropuncture techniques.Most of the criticisms of Hanssen's technique, which arise from the observation that the bolus of labelled precipitate is sometimes inhomogenously distributed from one region of the kidney to another, are not valid here because instead of being injected as a pulse, the¹⁴C-ferrocyanide is infused continuously into the animal. Moreover, our technique allows measurements of SGFR's even in non urine forming kidneys.

Docteur en médecine, Attaché de recherche à l'Institut National de la Santé et de la Recherche Médicale.     

The tubulo-glomerular feedback mechanism — a determinant for the autoregulation of the glomerular filtration rate in superficial and juxtamedullary nephrons

Summary The intrinsic myogenic hypothesis and the tubuloglomerular feedback mechanism (TGF) give the presently most cherished explanation to the autoregulation of renal blood flow and glomerular filtration rate. A series of experiments was performed on young, normohydrated rats in order to evaluate the importance of TGF as an autoregulatory factor of the single nephron glomerular filtration rate (SNGFR) in superficial and juxtamedullary nephron populations. Micropuncture techniques were applied to tubular structures of the renal surface and on the papilla for the measurement of hydrostatic pressures and SNGFR. The SNGFR was also measured with a modified Hanssen technique. A TV-technique was used to record the urine free flow rate in the loop of Henle.The net driving forces for glomerular filtration at the afferent end of the glomerular capillaries were estimated to be 19 and 47 mm Hg for superficial and juxtamedullary nephrons respectively, when the urine flow at the macula densa was zero. The SNGFR of the two nephron populations amounted to 29.6 and 84.1 nl·min^–1·g^–1 K.W., as measured with the micropuncture technique. With a modified Hanssen technique the corresponding values were 25.8 and 27.7 nl·min^–1. g^–1 K.W. (kidney weight).The SNGFR was found to be well autoregulated when the urine flow at the macula densa was intact, but not when the urine flow was interrupted.The flow rate in the loop of Henle was in free flow conditions 7.3 nl·min^–1·g^–1 K.W. which shall be compared with 19.2 nl·min^–1·g^–1 K.W. when the urine flow to the macula densa was zero.We conclude that SNGFR is mainly autoregulated by the TGF-mechanism in young, normohydrated rats at lower arterial pressures. In normal conditions TGF is highly activated for juxtamedullary nephrons, but not for the superficial ones. The high urine flow rate in the loop of Henle at reduced flow rates at the macula densa may invalidate the use of loop blockade in studies of water and solute transfer across the loop walls.     

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