PflfigersArch. 370, 155-163 (1977) PfliJgers Archiv European Journal of Physiology

�9 by Springer-Verlag 1977

The Early Phase of Experimental Acute Renal Failure I. Intratubular Pressure and Obstruction

JUNE MASON, CHRISTOPH OLBRICHT, TOSHIKAZU TAKABATAKE, and KLAUS THURAU, with the technical assistance of SIGRID PATZ

Physiologisches Institut, Universit/it Mgnchen, Pettenkoferstrasse 12, D-8000 Mgnchen 2, Federal Republic of Germany

Summary. Tubular obstruction in acute renal failure, postulated to cause the restricted excretory function, is suggested by raising intratubular pressure, to lower effective filtration pressure and diminish urine output. To examine the applicability of the obstruction hypo- thesis to the pathogenesis of experimental acute renal failure, proximal intratubular pressure and renal function were measured after renal insults of different origins and severity. Obstruction in acute renal failure kidneys should manifest itself as an increase in intratubular pressure for a least 12 h, for within this time period following ureteral occlusion, elevated pressures were found to reflect obstruction. The con- sistent existence of raised proximal intratubular pres- sure in acute renal failure kidneys could not be detected; ischaemic and nephrotoxic models were found in which no rise in intratubular pressure could be demonstrated. The oliguric nature of acute renal failure kidneys could not be verified; ischaemic and nephrotoxic models were found in which urine output was either normal or enhanced. Only for methaemo- globin induced renal failure were raised intratubular pressure, oliguria and casts concurrent. It is concluded that obstruction is not a consistent feature of experi- mental acute renal failure and that the obstruction hypothesis may be specifically applicable to only a few models, which include haeme pigment and folic acid induced renal failure.

Key words. Acute renal failure - Proximal intra- tubular pressure - Glomerular filtration rate - Urine flow rate - Ureteral occlusion.

INTRODUCTION

The inability of the kidneys to fully excrete the products of nitrogen metabolism in acute renal

failure has been attributed to the operation of various mechanisms, which either prevent the formation of sufficient filtrate or impede the excretion of its con- stituents. One of these mechanisms, postulated to explain the inability of the kidney to form an adequate filtrate, has suggested that the primary defect is a blockage of the tubules, either from outside by interstitial compression or from within by casts and debris, resulting in a rise of intratubular pressure and a reduction of effective filtration pressure [38]. Although many investigations have led to the con- clusion that an obstructive phase in acute renal failure is not consistently apparent [19, 35,40,29, 36, 47,18], others have indicated that a primary obstruc- tive phase may be evident [45,4,46]. However, a systematic investigation of the role of obstruction across the spectrum of different types of experimental acute renal failure, particularly in the earliest phase, is not available.

The objective of the present investigation was to determine to what extent an obstruction hypothesis could be made to account for reduced renal function in the early phase of acute renal failure. To examine the applicability of this theory, a variety of experimen- tal models of acute renal failure were selected for investigation in the early stages of functional impair- ment. The previously existing examples of haeme pigment, ischaemic and nephrotoxic induced acute renal failure were extended to include new variations, demonstrating both moderate and more severe renal damage. Since the obstruction hypothesis requires that tubules are blocked and, consequently, predicts that at least initially, intratubular pressures are raised and that glomerular filtration rate and urine output are diminished, proximal intratubular pressure, poly- fructosan clearance and urine flow rate were measured and compared in normal and acute renal failure kidneys. To determine within what time period raised intratubular pressure is reliably indicative of

156 Pflfigers Arch. 370 (1977)

obstruction, proximal intratubular pressure was determined at various time intervals following com- plete ureteral occlusion.

METHODS

Preparation of the Animals. All experiments were performed on male Sprague Dawley rats (Moss, Munich), which had been fed on a standard diet (Altromin). The animals, except for those in one group, had free access to tap water until immediately before the experiment commenced. Anaesthesia was achieved by intra- peritoneal injection of 90-100 mg/kg BW of inactin (Promonta), given as a 10 ~ aqueous solution. The trachea, right jugular vein and right femoral artery were cannulated with polyethylene catheter. A continuous infusion of 0.3--0.5 ml/h/100 g BW of either a modified Ringer's solution (115 mM NaC1, 4raM KC1, 2.5 mM CaCt2, 25 mM NaHCO3) or 10 ~ polyfructosan (Inutest, Laevosan) in 0.9 ~ NaCI was given via the jugular catheter. Blood pressure was measured from the femoral artery using a pressure transducer and was monitored continuously. Rectal temperature was deter- mined throughout and maintained at 37-38~ by means of a warming plate built into the operation table. The preparation of the left kidney for micropuncture was effected through a flank incision situated just below the last rib. After dissection from the perirenal fat, the kidney was placed, dorsal uppermost, into a lucite cup, which was then attached to the operation table. The kidney was immobilized within the cup using oil-soaked-cotton wool, and paraffin oil, preheated to 37-38~ was dropped on to the surface. The ureter was catheterized near the pelvis. Micropuncture was performed within the period commencing 1 h after the kidney had been placed in the cup and terminating 6 h after the onset of anaesthesia.

Determination of Renal Function. In those animals receiving poly- fructosan infusion, the clearance was determined throughout the period of micropuncture. Repeated blood samples were taken from the femoral artery and timed urine collections were made into preweighed tubes at 1/a - l-h intervals. Plasma and urine poly- fructosan concentrations were determined using the anthrone photometric method [20] and urine volume was obtained by weighing.

Experimental Models. The ureteral obstruction experiments were performed in 4 groups of animals, in which the left ureter was ligatured twice, half way between the bladder and renal pelvis through a mid-line incision, under light Nembutal anaesthesia.

The animals were allowed to recover consciousness, were supplied with food and water and were subsequently reanaesthetized for micropuncture preparation. The kidneys were examined during the obstructed phase, 5 - 6 , 8 - 9 , 11-12 and 2 4 - 2 5 h following occlusion and 15-60 min following the release of obstruction by cutting open the ureter.

The acute renal failure experiments were performed on 10 groups of animals, comparible in body weight and ranging from 198--318 g. The nature of the noxa, the dose and time it was administered, the number of animals involved and the period in which the investigation was performed are summarized in Table 1. One group represented normal renal function and served as control, 2 groups illustrated haeme pigment induced acute renal failure, 3 groups demonstrated ischaemic renal injury and 4 groups represented nephrotoxic renal damage. Haeme pigment induced renal failure was produced by intravenous injection of purified methaemoglobin (Sigma Chemicals) under light ether anaesthesia as 0.2g/ml in 0.9~ NaC1. Renal ischaemia was achieved by clamping the left renal artery with a weakly sprung clip. Nephro- toxic renal damage was produced by administration of uranyl nitrate or mercuric chloride dissolved in 0.9 ~ NaC1, so that the dose per kg BW was contained within 1.0 ml and each animal received 0.1 ml/100 g BW subcutaneously.

Measurement of Intratubular Pressure. Micropuncture was per- formed with the aid of a double stereo microscope at a magnifica- tion of x 160 one micromanipulator (Leitz, Wetzlar) and fibre optic illumination. Patent proximal tubules were selected randomly and intratubular pressure was measured by the Landis technique [34], with a Ganer manometer and short-shafted micropipettes, 9 - 1 1 ~ OD, fitIed with 0.5 or 1.0~ lissamine green solution. Control values of intratubular pressure, for comparison with those obtained after various periods of ureteral obstruction, were obtained by pooling all the measurements from normal kidneys. Control values for comparison with those from acute renal failure kidneys, were obtained by pooling only those values from normal kidneys investigated in the same experimental series. The different experimental series consisted of: normal and 45 min ischaemic kidneys; normal, 0.5 g/kg methaemoglobin, 75 rain ischaemic, 10 mg/kg uranyl nitrate and 4.7 mg/kg mercuric chloride kidneys; normal, 0.75 g/kg methaemoglobin, 60 rain ischaemic, 15 mg/kg uranyl nitrate kidneys.

Statistical Evaluation of the Data. The data obtained from each experimental group were pooled, expressed as mean +_ SD but tested for statistical significance with the Wilcoxon test for unrelated samples. Populations were considered to be significantly different

Table 1. The experimental procedure used to produce models of acute renal failure

Experimental model Dose of noxa No. of animals Time of noxa Period of investigation (n) (h before anaesthesia) (h after noxa)

Normal kidneys

Methaemoglobin"

Renal ischaemia

Uranyl nitrate

Mercuric chloride

26

0.50 g/kg i.v. 6 1 - 3 3 - 8 0.75 g/kg i.v. 7 ! 5 - 1 8 17-24

45 min 31 1 - 4 60 rain 8 1 - 4 75 min 8 1 - 4

I0 mg/kg s.c. 9 6-- 18 8 - 2 4 15 mg/kg s.c. 7 22-- 27 24 - 33

4.7 mg/kg s.c. 8 i - - 4 3 - 1 0 6.0 mg/kg s.c. 8 3 - 6 5--12

a Dehydrated for 31 40 h and given access to water following methaemoglobin administration

J. Mason et al. : The Early Phase of Acute Renal Failure ~57

from one another when the two-tailed probability was equal to or less than 0.05.

g0 �9

RESULTS

Renal Function ~ I ~ The parameters of renal function measured, for each ~ animal averaged over the experimental period, are =

O

summarized in Table 2. = Polyfructosan clearance was significantly reduced ~

compared to normal kidneys in every model of acute renal failure. Urine flow rate was significantly differ- 8

e~

ent from that of normal kidneys in 0.5 g/kg methaemo- globin and 4.7 mg/kg mercuric chloride kidneys, in which it was reduced and in 15 mg/kg uranyl nitrate kidneys, in which it was increased. Considering also g, the experiments in which urine was collected but poly- fructosan clearance not measured, many cases of -~ enhanced urine output were apparent. In approxi- s mately 50 % of the 45, 75 % of the 60, 25 % of the 75 rain ischaemic and 25 % of the 6.0 mg/kg mercuric g chloride kidneys this was the case. Urine to plasma polyfructosan concentration ratios were significantly .a: reduced compared to normal kidneys in 0.5 mg/kg methaemoglobin kidneys, in 45, 60 and 75 rain ischaemic kidneys, in 15 mg/kg uranyl nitrate kidneys r and in 6.0 mg/kg mercuric chloride kidneys.

Intratubular Pressure

The individual values of proximal intratubular pres- sure in normal and acute renal failure kidneys, presented as a population frequency distribution, are depicted in Figures I and 2.

In the 0.5 g/kg methaemoglobin kidneys, which were characterised by mildly dilated proximal tubules and the presence of mobile purplish casts in very many distal and some late proximal tubules, pressure was significantly raised by 61%. However, in approxi- mately one third of the population, pressure was in the normal range and in all the kidneys examined pressure tended to normalize as the experiment progressed. In the 0.75 g/kg methaemoglobin kidneys, which showed tubules of normal diameter and no proximal and only a few distal tubular casts, pressure was statistically higher than that of the controls, but only by 7 ~ .

In the 45 rain ischaemic kidneys, whose proximal tubules were mostly of normal diameter, a few being dilated, pressure was not significantly different from the control values. Although about three quarters of the values fall within the normal range, the clear existence of tubules with both subnormal and super- normal pressures is apparent. In the 60 rain ischaemic kidneys, in which all proximal and distal tubules were

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158 Pflttgers Arch. 370 (1977)

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Fig. 1. Proximal intratubular pressure, PITP, plotted as a frequency populat ion histogram, for normal kidneys and methaemoglobin and ischaemic models of acute renal failure. For each group, the mean _+ SD and number of measurements is given. * denotes statistically significant difference from the corresponding control values, given on the left. + indicates that measurements were confined to patent tubules only

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Fig. 2. Proximal intratubular pressure, PITP, plotted as a frequency populat ion histogram, for normal kidneys and uranyl nitrate and mercuric chloride models of acute renal failure. For each group, the mean _+ SD and number measurements is given.* denotes statistically significant difference from the corresponding control values, given on the left

J. Mason et al. : The Early Phase of Acute Renal Failure 159

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dilated, pressure was significantly higher than the controls, the increase representing 50 %. In the 75 min ischaemic kidneys, which consisted of proximal tubu- les ranging from collapsed to dilated, pressure in the patent tubules was significantly higher than the con- trols, the rise representing 64 %.

In the 10 mg/kg uranyl nitrate model, pressure was significantly lower than the controls, the decrease being to 80 %. Virtually no nephron was found which had a pressure of greater than 14 mm Hg, most values were within and approximately one eighth were below the normal range. In the 15 mg/kg uranyl nitrate kidneys pressure was not significantly different from the controls.

In the 4.7 mg/kg mercuric chloride kidneys pres- sure was not significantly different from controls. In 6.0 mg/kg mercuric chloride kidneys, in which proximal tubules appeared extended, pressure was significantly higher than in the corresponding controls, the increase representing 37 %.

The individual values of proximal intratubular pressure for normal kidneys and kidneys during and immediately following various periods of ureteral occlusion, given as a population frequency distribu- tion, are depicted in Figure 3. Dm'ing the 5 - 6 , 8 - 9 and / 1 -12 -h periods following ureteral occlusion, proximal tubules were dilated and pressures were significantly higher than those of the controls by 94, 79 and 42 %, respectively. After 24 -25 h obstruction, tubules ranged from dilated to totally collapsed and pressure in the patent tubules remained significantly increased by 21 ~. Following the release of the 5 - 6,

8 - 9 and l l - 1 2 - h periods of ureteral obstruction, proximal tubular diameters decreased and pressures fell significantly to 50, 47 and 58 ~ of the obstruction values, respectively. After release of the 24 -25 h obstruction, the number of collapsed tubules increased and pressure in the remaining patent tubules fell significantly to 73 % of the obstruction value. The post- obstruction intratubular pressures obtained following release of 8 - 9 , 11-12 and 24 -25 h of obstruction were significantly lower than the control values, the decrease being to 84, 83 and 88 %, respectively.

DISCUSSION

The determination of renal function in acute renal failure kidneys demonstrated that polyfructosan clear- ance was significantly reduced in all experimental models. Since extensive leakage is not a feature of any of these models (to be published), this confirms that filtration rate was lowered. Urine to plasma con- centration ratios fell in the methaemoglobin, ischaemic and both higher dose nephrotoxic models, showing that tubular reabsorption was restricted. Urine flow rate, however, was only significantly depressed in 0.5 g/kg methaemoglobin and 4.7 mg/kg mercuric chloride kidneys. In 15 mg/kg uranyl nitrate kidneys, urine output was significantly raised and in all the ischaemic models and in the 6.0 mg/kg mercuric chloride kidneys, enhanced urine output was seen fre- quently. Were obstruction in acute renal failure kidneys sufficient to compromise glomerular function, a corre- lation between filtration rate and urine output would

160 Pfl~gers Arch. 370 (1977)

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U urine to plasma polyfructosan ratio, ~ -, and the calculated linear

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be expected, the more extensive the obstruction, the lower the filtration rate and urine output. As shown in Figure 4, using the data obtained from the various models of acute renal failure examined in the present experiments, no correlation could be found between polyfructosan clearance and urine flow rate, but a significant positive correlation could be demonstrated between polyfructosan clearance and the urine to plasma polyfructosan concentration ratio, indicating that the low filtration rate of acute renal failure is characterised rather by the inability of the kidney to perform its reabsorptive function than by �9 In many reported instances of experimental acute renal failure, �9 is not only absent but polyuria is prevalent. Up to some hours following 3 h partial renal ischaemia [9,10], 30 -60ra in [41] or 60rain [45, 46] total renal ischaemia, urine output was more than doubled and was still raised after 24 h [45, 13] or 3 days [49]. A few hours after 5 or 10 ml/kg 50 % glycerol, urine flow rate was very high [13,11] and

was not reduced up to 48 h [17]. Following mercuric chloride administration to frogs [37] and rats [6,15], urine production was increased or unchanged [14, 48]. In rats, early after and until 48 h following I0 mg/kg uranyl nitrate subcutaneously [18], or soon after 15 or 25 mg/kg given intravenously [8], urine output was appreciably raised and in dogs, 48 h after 10 mg/kg uranyl nitrate given intravenously, urine flow rate was unchanged, despite a reduction in filtration rate [16,43]. Following 250mg/kg folic acid, injected intravenously in rats, there was an �9 of 4-h duration followed by polyuria lasting for 8 - 9 days [42]. The present results, together with the above mentioned reports in the literature, suggest that �9 liguria is not a necessary consequence of acute renal failure in an early stage and that an explanation for diminished urine output in this phase may be inappro- priate.

Examination of the values of intratubular pressure obtained in acute renal failure kidneys revealed them to be significantly elevated in 0.5 and 0.75 g/kg methaemoglobin, 60 and 75 min ischaemic and 6.0 rag/ kg mercuric chloride kidneys, but unchanged in 45 rain ischaemic, 4.7 mg/kg mercuric chloride and 15 mg/kg uranyl nitrate kidneys and significantly reduced in 10 mg/kg uranyl nitrate kidneys. Similar reports regarding the variability of proximal intratubular pressure in various forms of experimental acute renal failure are plentiful. With 2 g/kg methaemoglobin, pressure was raised initially and normal after 3 h [40], whereas 24 h after 0.5 g/kg, pressure was raised [29]. With 10 ml/kg 50% glycerol, pressure was low [35, 51,52] or unchanged up to 4 h [47] raised until 16 h [47], or still reduced at 24 h [35,51,52] and normal at 48 h [52, 36]. Shortly after 60 rain renal ischaemia, pressure was substantially increased [45,4,46] but was either normal [4,12] or still raised [46,49] days later and immediately after 3 h partial ischaemia pressures were normal [9,10]. Some hours after 10 mg/kg uranyl nitrate subcutaneously, in rats, pres- sure was normal, as it was 48 h later [18] and when given intravenously in dogs, pressure was normal at 48 h [43] but with 15 or 25 mg/kg intravenously, in rats, pressure was raised at 2 h and normal at 6 h [8]. With mercuric chloride, 12 mg/kg intramuscularly in rats, resulted in pressures which were normal for 8 h and subsequently reduced [19] and 4.7 mg/kg subcutaneously gave normal pressures at 24 h [14]. Following 250 mg/kg folic acid intravenously, pres- sures were substantially raised after 10min and remained elevated at 24 h [26]. After injection of 1.4-1.6 mg/kg potassium dichromate subcutaneously, pressures were normal at 7 h and 7 days [7]. Both the present results and those reported by others confirm that proximal intratubular pressure is not a parameter

J. Mason et al. : The Early Phase of Acute Renal Failure J61

which remains constant throughout the course of acute renal failure and that its behaviour changes from model to model. Since it appears that for every method employed to induce acute renal failure there is a dosage and a period of time in which impaired renal function accompanies either normal or sub- normal intratubular pressure, on these occasions, even were a tubular blockage to be present, a reduction in effective filtration pressure can only be explained by changes in resistance of the glomerular capillaries.

The results of the experiments in which proximal intratubular pressure was monitored prior to and following the release of various periods of ureteral occlusion demonstrate that for at least 12 h, obstruc- tion is reflected by a rise in intratubular pressure, which decreases as soon as the ureteral occlusion is released. The pressures obtained both during and subsequent to obstruction diminish with the time of occlusion, leading to the presence of collapsed tubules after 24 h (present results, [24]) which increase in number after release of obstruction, indicating that the nature of the filtration defect changes with time. Similar conclusions have been reached previously [30, 3], when it was noted that following 24 h obstruc- tion, proximal intratubular pressure was subnormal and glomerular capillary pressure was depressed, suggesting glomerular arteriolar vasoconstriction. However, the observation that following 24 h of ob- struction proximal intratubular pressure is reduced, may be specific only to unilateral ureteral occlusion. Single obstructed tubules still have modestly elevated pressures after 24 h, although glomerular capillary pressure is diminished [3]. With complete bilateral ureteral occlusion, after 24 h, proximal tubular pres- sures are still substantially raised and return to normal levels following release of obstruction [31,53] and with partial bilateral ureteral occlusion, pressures are elevated after 2 - 4 weeks [50]. It seems certain that acute elevations in ureteral pressure are associated with an increase in proximal intratubular pressure [22,2,1] but it seems that for longer periods, if obstruction is present in one kidney, it will be expressed by a raised pressure for at least 12 h and if present in both kidneys, it will be indicated by elevated pressure for at least 24 h.

All models of acute renal failure examined, whether with unilateral involvement, as in the ischaemic models, or with bilateral involvement, as with the methaemoglobin and nephrotoxic models, were in- vestigated within the period in which obstruction is reflected by raised intratubular pressure, except for the 15 mg/kg uranyl nitrate model, in which pressures beyond the 24 h period did not differ from those obtained at 24 h. Thus a role for obstruction during this early phase of acute renal failure is only impli-

cated for both methaemoglobin models, the 60 rain and the high pressure tubules of the 75 min ischaemic kidneys and the 6.0 mg/kg mercuric chloride kidneys. Since raised intratubular pressure has been consistently observed in all examples of methaemoglobin induced renal failure, an obstructive phase seems to be indi- cated for this model and as increased pressures are found only sporadically amongst the ischaemic and nephrotoxic induced renal failures, an obstructive phase in these models seems not necessarily to accom- pany reduced renal function.

For those models where an obstructive component seems consistently indicated from the pressure meas- urements, other evidence supports this conclusion. After administration of haemoglobin or methaemo- globin, the impairment of renal function commences with the arrival of the pigment in the kidneys and progressive return of function is observed as the pig- ment is excreted [5, 27, 39]. In pigment nephropathies induced directly, by methaemoglobin, or indirectly, with glycerol, the appearance of pigmented, tubular casts coincides with the rise in intratubular pressure [35,40,47,25, present results] and by the time the casts have disappeared, pressure has normalized [35, 40, 36, 51, present results]. After folic acid, the arrival of the noxa in the kidney is concurrent with the increase in intratubular pressure [42,26]. In haeme pigment or folic acid injected animals, an initial oli- guric phase is present [40,27, present results, 42] and manipulation of urinary pH [5,26] or induction of diuresis [29, 28, 26], both of which enhance excretion of the noxa, are associated with an improvement in renal function. Thus, at least in the initial phase of haeme pigment or folic acid induced acute renal failure, a classical oliguric obstructive period is well substantiated.

For the remaining models of acute renal failure, where a specific role of obstruction is not indicated because of the only occasional incidence of raised intratubular pressure, the occurrence of increased pressure is still too frequent to be dismissed. From the present experiments, within each model, excepting methaemoglobin, increasing the dose of noxa resulted in a further decrease of filtration rate and rise of pres- sure, implying that for experimental failure of mod- erate severity, increasing functional impairment brings with it an increasing tendency for the tubules to be obstructed. However, the common coincidence of polyuria and raised intratubular pressure in the 60 min, 75 min ischaemic and 6.0 mg/kg mercuric chloride kidneys of the present experiments and the ischaemic [45, 46, 49], nephrotoxic [8] and postblock- age phase of folic acid renal failure [42, 26] reported elsewhere is confusing. One explanation may be that acute renal failure kidneys, owing to a reabsorption

162 Pflfigers Arch. 370 (1977)

defect, display the same characteristics as no rma l kidneys in diuresis. In the latter, admin i s t r a t ion of m a n n i t o l or diuretics produces a high ur ine flow, invar iab ly associated with high in t r a tubu la r pressure [23, 32, 33,21], resul t ing f rom increased flow through the high resistance par t of the nephron . Were the pr imary func t iona l defect of acute renal failure to be a reduct ion in tubu la r reabsorpt ion , polyur ia could be ant ic ipated and high pressure tubules may simply reflect a diuretic state. The concur ren t existence of tubu la r casts and raised tubu la r pressure has only been conf i rmed in the 60 m i n ischaemic kidney [45] and the demons t r a t i on that debris and cylinders are readily mobi le and require only small dr iving forces to induce their passage a long the tubule [14] and out of the collecting duct [44], may suggest that such casts only accumula te when fi l t rat ion rate is already low, and are symptomat ic rather than causal of a f i l t rat ion deficiency.

Accord ing to the present state of knowledge, tubu la r obs t ruct ion , causing a rise in tubu la r pressure and, therefore, a reduct ion in effective f i l t rat ion pres-

sure, does init iate the f i l t rat ion deficiency of those models, such as haeme p igment and folic acid renal failure, where the noxa is filtered and accumulates wi thin the tubules. However, this phase is t ransi tory, and after the d isappearance of the tubu la r blockage a func t iona l defect still exists, no longer accounted for by obs t ruc t ion [40, 29]. F o r ischaemic and nephro- toxic models, the m a n y examples showing no evidence of obst ruct ion, indicate that this is no t the pr imary, ini t ia t ing defect and that obst ruct ion, if present, cont r ibutes to a renal failure already in existence.

Acknowledgement. This investigation was supported by the Deutsche Forschungsgemeinschaft.

Part of these results have been presented previously at the following meetings: European Colloquium, Nephron Physiology. Mechanism and Regulation, Royaumont 1974; Frfihjahrstagung der Deutschen Physiologischen Gesellschaft, Hannover 1974; X Symposium der Gesellschaft ffir Nephrologie, Innsbruck 1974; XXVI International Congress of Physiological Sciences, New Dehli 1974; VI International Congress of Nephrology, Florence 1975.

The polyfructosan used, Inutest, was generously donated by Laevosan, Linz, Austria.

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Received November 29, 1976