Pfliigers Arch. 347, 223--234 (1974) 9 by Springer-Verlag 1974)

Renal Blood Flow after Temporary Ischemia of Rat Kidneys* * * Renal V e n o u s O u t f l o w a n d C l e a r a n c e T e c h n i q u e s Georg M. E i s e n b a c h , B a r b a r a Kitzlinger, a n d Michael S t e i n h a u s c n I. Physiologisches Institut der Universit~t Heidelberg, Germany Received August 2, 1973

Summary. Total renal blood flow (RBF-dir) and total renal resistance were determined in acutely uninephrectemized rats by measuring the renal venous outflow after cathetcrizing the renal vein. Renal vein catheterization and the procedure involved caused an initial fall in C-IN and C-PAH followed by a steady level over an experimental period of 2--3 h. Absolute and fractional water excretion increased approx. 9-fold during that time. Mean RBF-dir was 14.2 4-0.67 (/V = 13) ml/min • kg BW • 1 kidney. In another series of animals acute renal damage was induced by subjecting the kidneys to a 60 min period of temporary ischemia 3 days prior to use. RBF-dir decreased 10~ total renal resistance increased ll~ neither parameter being significantly different from controls. The clearance of inulin and PAH, the extraction ratio of PAtt, and the urine flow rate were depressed to about 10~ of control (P < 0.001). A considerable discrepancy was found between data obtained by clearance methods and RBF-dir after ischemia: The ratio RPFPAH/RPF-dir was 1.05 under control conditions and was significantly depressed to 0.47 (P < 0.005) after isehemia. -- These results indicate that a general increase in resistance of the vasa afferentia alone cannot be responsible for the oliguric phase. At least two important factors are involved in the cause of oliguria 3 days after temporary ischemia: Backdiffusion of tubular fluid through the damaged tubular epithelium and a decrease in GFR.--Clearancc methods are not considered to be reliable determinants of GFR and RPF in renal failure after temporary ischemia. Key words: Kidney -- Renal Blood Flow -- Acute Renal ~ailure -- Renal Resistance -- Clearance Techniques -- Renal Venous Outflow -- Temporary Ischemia of the Kidney. Two m a i n hypotheses for t h e e x p l a n a t i o n of the pathogenesis of the oligo-anurie phase after e x p e r i m e n t a l acute r e n a l damage have been proposed i n the p a s t : 1. Cessation of GFI~ due to a decrease i n filtration pressure caused b y a general increase in resistance i n t h e vasa afferentia possibly m e d i a t e d b y the local m a c u l a - d e n s a - r e n i n - a n g i o t e n s i n mechanism. 2. Backdiffusion of t u b u l a r fluid t h r o u g h the d a m a g e d t u b u l a r epithelium. I n recent studies, we r e p o r t e d t h a t after m e r c u r y poisoning [35] as well as after t e m p o r a r y isehemia [13] of the r a t kidney, baekdiffusion of * Supported by the Deutsche l%rschnngsgemeinschaft (SFB 90). ** Presented in part at the V. Internat. Congress of Physiol. Sci., Mexico 1972. 16"

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t u b u l a r fluid is evident, since the t u b u l e s were f o u n d to be permeable to i n u l i n a n d lissamine green. This s t u d y was u n d e r t a k e n i n order to assess the h e m o d y n a m i c changes occurring in the k i d n e y after t e m p o r a r y ischemia. T o t a l r e n a l blood flow was d e t e r m i n e d directly b y measuring the renal venous outflow i n the r a t k i d n e y a n d b y c o n v e n t i o n a l clearance methods. I t was f o u n d t h a t t o t a l renal blood flow a n d t o t a l r e n a l resistance were n o t significantly different from control, b u t the clearance of i n u l i n a n d P A H , the e x t r a c t i o n ratio of P A H , a n d the u r i n e flow rate were depressed to a b o u t 10~ of control. This is i n c o m p a t i b l e with t h e hypothesis t h a t oliguria is solely due to a general increase i n resistance of the vasa afferentia, a n d lends strong s u p p o r t to the baekdiffusion theory. This s t u d y also furnishes i n f o r m a t i o n on the comparison b e t w e e n directly a n d i n d i r e c t l y m e a s u r e d renal blood flow u n d e r control a n d pathological conditions, since i n u l i n a n d P A I t p a r a m e t e r s were d e t e r m i n e d s i m u l t a n e o u s l y with the r e n a l venous outflow m e a s u r e m e n t s .

Methods 43 Wistar rats of both sexes weighing 250--400 g were used. They had free access to Altromin standard diet and tap water. The animals we,re anaesthetized with Inactin 1 (100 mg/kg BW, i.p.) and placed on a thermostatically controlled heated table. A tracheal cannula and two catheters in a jugular vein were inserted. Arterial blood pressure in a carotid artery was recorded throughout the experiment using a Statham transducer and the 4-channel Hellige ttellcoscript. The left kidney was exposed through a flank incision and carefully freed from surrounding tissue. The kidney capsule remained untouched. The left ureter was catherized for clearance measurements. The left renal artery and vein were carefully dissected up to their origins from the aorta and vena cava inf., resp. The renal vein and artery were separated from each other and two silk threads placed around the renal vein. The renal artery was then clamped with a small bulldog clamp and the renal vein tied off close to the vena cava inf. A small incision was made in the renal vein and a silastic catheter (medical grade tubing, 0.03"ID; 0.065"0D) tied in. Immediately thereafter the renal arterial clamp was removed. This procedure took 45--200 see. hTotincluded in this study are animals with periods of ischemia longer than 200 sec. Shortly before the renal venous catheter was tied in, the animals received 1 ml saline containing 500 USPU/ml heparin 2. Thoughout the experiment the animals were given 8 ml/h • kg BW saline containing inulin and para-amino hippuric acid (PAH) creating a plasma concentration of inulin of approx. 100 rag/100 ml and PAH concentrations between 2--5 mg/100 ml. In oliguric animals the amount infused was adjusted accordingly. The renal venous effluent was reinfused simultaneously into a jugular vein via an extracorporeal circuit. The extracorporeal circuit (Fig. 1) consisted of silastic tubings connecting the renal vein with a droplet counting device, and the reservoir with the jugular vein. The reservoir was mounted approx. 12 cm below the kidney. The openings of the venous catheter were kept constant. The output of the droplet counting device was 1 We wish to thank Promonta, Hamburg, for generously supplying free preparations. 2 Liquemin, Roche.

Renal Blood Flow after Temporary Ischemia of R a t Kidneys t

225

LEFT

}torn C.A.

RECORDER R.B.F. . . . .

Q

b to J,V.

4---

Fig. 1. Extracorporeal circuit. Renal venous outflow was collected in a reservoir (b) passing a droplet counting device (a) the o u t p u t of which was recorded simultaneously with arterial blood pressure in a carotid artery (C.A.). The blood in the reservoir was reinfused into a jugular vein (J.V.) using a peristalsis pump. The p u m p speed was adjusted in such a way t h a t t h e blood level in t h e reservoir remained unchanged

recorded on the 4-channel Hellcoscript, thus permitting the calculation of total renal resistance. The renal venous outflow was collected in the reservoir a n d pumped into the jugular vein using a persistalsis pump. The p u m p was adjusted in such a way t h a t the blood level in t h e reservoir remained unchanged. The reservoir a n d the tubings were surrounded b y water, kept a t 37 ~ C, thermostatically controlled. The dead space of the extracorporeal circuit was 1.4 ml and t h a t of the reservoir 10 ml. Prior to the eatherization of the renal vein, 2 ml saline containing 500 U S P U / m l heparin were p u t into t h e circuit, t h e n the reservoir was filled to the 10 ml m a r k with blood from a litter mate, harvested shortly before. The donor animal was anaesthetized with I n a c t i n a n d given 5 ml saline containing 500 U S P U / m l heparin. After tying off b o t h kidneys t h e animal was bled t h r o u g h a carotid artery. The m e a n hematocrit of the donor blood was 42.3% . The droplet counting device was repeatedly calibrated with blood of rats, kept a t 37 ~ C; there were no changes in the droplet/ml ratio in a physiological range of hematocrit nor at different flow rates. I n the course of the experiment 5 - - 7 ml blood from the donor animal were added to the extracorporeal circuit over a period of 120--180 rain. Clearances of inulin a n d P A H were performed according to s t a n d a r d techniques. Inulin concentrations were determined according to Fiihr et al. [15], P A H concentrations according to B r a t t o n a n d Marshall [10] in the modification of Deetjen et al. [12]. Clearance periods were 20--30 rain intervals with midpoint blood samples being w i t h d r a w n from a femoral artery. Blood samples from the reservoir were t a k e n simultaneously. Proximal t u b u l a r transit time of Lissamine green was determined according to Steinhausen [33] b y injecting a bolus of 0.04--0.07 ml of a 10 g/100 ml reerystallized Lissamine green solution. I n b o t h series (control group a n d ischemia group) t h e animals h a d their opposite (right) kidney removed 90 rain prior to renal venous catherization. I n the following, these animals are referred to as acutely uninephrectomized.

G.M. Eisenbach st aL

226

Temporary isehemia was produced as described previously El3]. 3 days prior to the experiment the left renal artery was clamped for a 60 min period under slight anaesthesia (Nembutal, 30--40 mg/kg BW, i.p.). Total renal resistance was calculated as follows: T R R = MAPB/RBF, whereas MAPB = BPai~st -}- 1/s (BPsyst -- BPdiast) ; R B F = RBF-dir = renal venous outflow. Renal venous pressure was considered to be zero. In calculating R P F with clearance methods (Cx -- fr)/Ex urine flow rate was not taken into account; neither were the clearance data corrected for plasma water content. All parameters have been calculated ~o the same 30 min intervals. The data is given on a body weight basis (kg) and for one kidney. Statistical analyses were performed according to Snedecor [32]. All values represent the mean and the standard error of the mean.

Results T o t a l r e n a l b l o o d flow was d e t e r m i n e d b y m e a s u r i n g t h e v e n o u s o u t f l o w o f t h e l e f t k i d n e y ( R B F - d i r ) . T h i s was d o n e in c o n t r o l a n i m a l s a n d

C N

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1 II

II

V/GFR

4

II II II

9'0 60 3'0 R'VC 3'0 d0 9'0 150 150rain Fig.2. The clearance of inulin (C-IN), the clearance of P A H (O-PAH), hematocrit (Hct), fractional (V/G.F.R), and absolute ( I?) water excretion as a function of time before and after renal venous catheterization (R VC) in control animals

Renal Blood Flow after Temporary Isohemia of Rat Kidneys o.

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Fig. 3. Relationship of renal blood flow (gBF-dir, ordinate) and 1/proximal tubular transit time (pTT, abscissa) in control animals. Y = 1.23 X + 3.96; r = 0.85; Sx, y ~ 1.75; n = 29

in animals in which renal failure of the left kidney was produced b y subjecting the left kidney to a 60 min period of temporary isehemia 3 days prior to use [13]. All animals had the opposite (right) kidney removed 90 rain prior to renal venous eatherization (RVC). RBF-dir in 13 acutely uninephrectomized control animals stabilized approx. 30 rain after RVC to a steady level of 14.2 4- 0.67 ml/min • kg BW • 1 kidney, which lasted over an experimental period of 2 h and more. I n seven acutely uninephreetomized animals of the isehemia group, RBF-dir was 13.2 4-0.82 m l / m i n • B W • 1 kidney. The means are not significantly different. Fig. 2 demonstrates the effects of RVC and the procedures involved upon kidney function (disseetion of the renal artery and vein, the short but necessary clamping of the renal artery, and the administration of foreign blood from a donor rat). The clearance of inulin and P A H deereased to a new and steady level which was well maintained over an experimental period of 2.5 h and more ( e - I N 4.02 -t-0.59 (N = 10) and 2.294-0.36 ( N = 1 0 ) ml/min• BW• C-PAH 10.94-2.2 (N = 5) and 7.3 4- 0.8 (N ~- 5) ml/min • kg BW • 1 kd, resp.). Hematocrit fell gradually from a mean of 47.2 4-1.4 (N = 8)0/0 before RVC to a mean of 42.1 4-1.9 (N = 8)0/0 after RVC due to the somewhat lower hematoerit of the blood from the donor animal and the saline in the extraeorporeal circuit. Fractional water excretion was 0.740/0 before RVC and increased gradually approx. 9-fold. Absolute water excretion

228

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Fig.4. Comparison of functional parameters of control animals (open bars) and animals after ischemia (closed bars) plotted as a function of time (rain), time zero being renal venous catheterization. RBF-dir renal venous outflow; TRR total renal resistance; lCP_F-PAH renal plasma flow, determined with the clearance and extraction ratio of PAH; Hct hematocrit; C-1N clearance of inulin; C - P A H clearance of PAH; E - P A H extraction ratio of PAH; l? urine flow rate

increased to t h e s a m e e x t e n d . P r o x i m a l t u b u l a r t r a n s i t t i m e i n c r e a s e d f r o m 9 . 4 4 - 1 . 0 (N ---- 16) sec to 14.4=~1.0 (N = 16) see. Fig. 3 shows a linear r e l a t i o n s h i p b e t w e e n R B F - d i r a n d t h e reciprocal of p r o x i m a l t u b u l a r t r a n s i t t i m e in c o n t r o l animals. This suggests t h e

Renal Blood Flow after Temporary Isehemia of Rat Kidneys

229

Table 1. Comparison of clearance methods and directly determined renal plasma flow under control conditions and after isehemia. Comparison of extraction ratio of inulin and filtration fraction. M :]: SEM, n = number of measurements Control

~[schemia

RPF-PAH RPF-dir

1.051 • 0.034 (n = 32)

P < 0.001

0.470 • 0.109 (n = 23)

RPF-in RPF-dir

0.916 q- 0.074 (n = 36)

P < 0.001

0.239 =~ 0.044 (n = 18)

E-in 0.998 -4- 0.026 (n = 17) C-IN/RPF-dir

same relationship between R B F and GFI~, at least in the superficial cortex and is presumably caused b y a slight change in the afferent artcriolar resistance. Fig.4 shows the comparison of functional parameters of control kidneys vs. kidneys subjected to t e m p o r a r y ischemia 3 days before. All data is obtained simultaneously in 5 control and 6 experimental animals, resp. R B F - d i r - - m e a s u r e d as renal venous outflow--was about 90~ of control, the difference is not significant. Total renal resistance (given in arbitrary units) showed a slight increase during the first h after RVC and reached a steady state thereafter under control conditions as well as following ischemia. The latter group exhibited a ll~ increase in the mean, the difference is not significant. Hematocrit was not different from controls throughout the experiment. The clearance of inulin and P A I l , the extraction ratio of PAIl, and urine flow rate were significantly decreased to approx. 10~ of controls (P < 0.001). Fractional water excretion was significantly increased in the ischemia group (P < 0.005). Renal plasma flow, calculated with the clearance and extraction ratio of PAH, was smaller after ischemia. This difference was significant in the later stages of the experiment when the steady state was reached. However, the renal plasma flow (PAH) was significantly different, too, from the simultaneously determined renal plasma flow taken b y means of the renal venous outflow technique in the same group of animals. The same was true for RPF-inulin vs. RPF-dir. Table 1 shows the ratio R P F - P A H / R P F - d i r under control conditions and after ischemia. This ratio was 1.05 under control conditions and was not different from unity, but was significantly different from 0.47 (P < 0.001) found in the ischemia group. Similar data was obtained using inulin as the marker substance. The filtration fraction, calculated with C-IN/RPF-dir, was 0.24 =k 0.04 under control conditions, a figure which is

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not different from 0.23 ~=0.02, the extraction ratio of inulin. This indicates an excellent agreement between two different measurements revealed under control conditions using different methods. Discussion Findings of recent micropuncture studies on the pathogenesis of the oligo-anurie phase after experimental acute renal damage have been interpreted differently. As shown by Bank et al. [6] and Biber et al. [8], we demonstrated in previous studies that in the mercury model [34], as well as in the isehemia model [13], baekdiffusion of tubular fluid through the damaged tubular epithelium does occur. Backdiffusion was demonstrable 24 h up to 7 days after the onset the lesion. This contrasts with the statements of Oken and his coworkers [14, 24, 25], Ruiz-Guinazu et al. [26], and others [7, 18, 20] who interpreted their findings as oliguria due exclusively to a cessation of GFI~, caused by a general increase in resistance of the afferent arteriols. This would severely decrease total renal blood flow and increase total renal resistance. As pointed out by Smith [30] and by others (s. ref. in [29]) and demonstrated in this study, the direct Fiek principle applied to determine renal plasma flow under pathological conditions is apt to be inaccurate, particularly after acute renal damage, for a variety of reasons, e.g. : low urine flow rate, possible storage of marker substances [4], small arterio-venous concentration differences of the marker substances, escape of marker substances by way of renal lymph flow and metabolism of PAI-I [23], etc. In this study, the measurement of the renal venous outflow has been utilized to determine renal blood flow. The suitability of this procedure has been established in studies on the dog [5, 19, 28] and other species, but rarely in the rat [27]. This method has the advantage of being very accurate, simple to apply [21], independent of the functional state of the kidney, and allows measurements on the renal venous blood to be done. In our hands, renal venous catherization resulted in a stable preparation for a fairly long experimental period with respect to C-IN, C-PAl=I, and RBF-dir. Total renal blood flow of 14.2 ml/min • kg BWX 1 kidney, and found in this study to be approx. 3.5 ml/min X g KW, is in close agreement with figures of total renal blood flow of the rat kidney derived with the inert gas wash-out method using Xenon [3]. The most striking findings in this study are that there was only an insignificant decrease in total renal blood flow as well as an insignificant increase in total renal resistance 3 days after renal failure was induced by

Renal Blood Flow after Temporary Isehemia of Rat Kidneys

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temporary ischemia. In the same kidneys a severe decrease, in the clearance of inulin and PAIt and in E-PAH and urine flow rate, was found. These findings emphasize our earlier statement that under these circumstances the clearance of inulin does not represent the actual GFR but a fraction thereof according to the degree of backdiffusion of this substance. Oliguria cannot be explained solely on the basis of a cessation of GRF mediated by a general increase in afferent arteriolar resistance. This is contrary to the recently published observation of Chedru et al. [11] who state that 24 h after the injection of glycerol in waterdrinking rats, renal cortical blood flow (hydrogen wash-out method) was decreased to 24~ of control. Ayer et al. [3], using the same model and the Xenon wash-out method, found that R B F had decreased to 450/0 of control 7 days after glycerol injection in still azotemic rats. The answer to this discrepancy must remain open at this time. I t is conceivable that the ischemia model and the glycerol model are different and that the time course of study is important. Following the equations given by Gomez [17] and taking glomerular hydrostatic capillary pressure as 50 mm Hg (unpublished observations using the stop-flow pressure technique [16])one can calculate the distribution of resistances to blood flow within the nephron, at least in the superficial cortex. Glomerular capillary pressure of 50 mm Hg is somewhat higher than t h a t reported b y Brenner et al. [10] and Andreucci et al. [2] and lower than the figures given by others [16, 20]. In this way, 540/0 of the total renal resistance is occupied by the preglomerular resistance [10]. Since there is no precise information available on the pressure in the glomerular capillaries in the ischemia model, all discussions about changes in resistance or distribution of resistances are necessarily speculative. In functional studies, it was concluded that within the autoregulatory range of blood pressure, changes in total renal resistance [36], as well as in the distribution of resistances within different parts of the kidney cortex, are most likely due to changes in the afferent resistance [1], the efferent arteriolar vascular tone being more or less unchanged. Assuming the increase in resistance found in this study is due to a change in preglomerular resistance alone, the glomerular hydrostatic pressure dropped some two $o four mm tIg. This drop in glomerular capillary pressure cannot explain the severe decrease in C-IN to approx. 10~ of control and is more likely explained by the escape of inulin by way of backdiffusion. I f there were a significant fall in efferent resistance, masking a substantial increase in afferent resistance, hydrostatic pressure in peritubular capillaries should be markedly increased. In methemoglobinuric renal failure the measurements of Ruiz-Guinazu et al. [26] suggest no decrease in efferent resistance. Observations on single welling points and

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peritubular capillaries in the isehemia model revealed a tendency of vessel diameter to be decreased [34]. I f this is indicative of decreased pressure in these vessels a decrease in efferent resistance is not likely. I f there is no change in efferent artcriolar vascular tone, the findings of Chedru et al. [11] t h a t "during the recovery phase after glycerol injection the measured values of G F R were lower than the predicted ones and t h a t in animals with B U N values greater t h a n 50 mg/100 ml the 'filtration fraction' were lowest", are most likely explainable b y a loss of inulin b y way of baekdiffusion. The findings presented in this study (no decrease in RBF-dir despite a severe fall in C-IN) could also be explained b y shunts operating as described b y Trueta et al. [37] or by transglomerular shunts. Since we found t h a t the blood flow rate in single peritubular capillaries after ischemia was not significantly different from controls [35], this possibility seems to be remote. Also, to our knowledge, there is no evidence in the literature for transglomerular shunts operating in the superficial cortex [22]. I n summary, the results of this study demonstrate t h a t in renal failure--3 days after oliguria was induced by t e m p o r a r y isehemia of the k i d n e y - - t o t a l renal resistance was only insignificantly increased despite a severe fall in C-IN and urine flow rate. The conclusions to be drawn arc t h a t a general increase in the afferent resistance cannot be solely responsible for oliguria in the ischemia model. At least two important factors are operating: Backdiffusion of tubular fluid and a decrease in GFR. Finally, this study shows t h a t the conventional clearance methods for the determination of G F R and R P F are not applicable under these circumstances. We thank Miss Rita Klein, Mr. l~udolf Dussel, and Mr. Heinz Braun for excellent technical assistance and Mrs. Elizabeth Applebaum for help in preparing the English manuscript. 1. 2.

3. 4. 5.

References Abe, Y.: Intrarenal blood flow distribution and autoregulation of renal blood flow and gIomerular filtration rate. Jap. Circular. J. 10, 1163--1173 (1971) Andreueci, V.E., Herrera-Acosta, J., Rector, F. C., Seldin, D.W.: Effective glomerular filtration pressure and single nephron filtration rate during hydropenia, elevated ureteral pressure, and acute volume expansion with isotonic saline. J. clin. Invest. 50, 2230--2234 (1971) Ayer, G., Grandchamp, A., Wyler, T., Truninger, B. : Intrarenal hemodynamics in glycerol-induced myohemoglobinurie acute renal failure in the rat. Circular. Res. 29, 128--135 (1971) Bs P., Fekete, A., Forgacs, J.: Quantitative considerations on the storage of clearance substances in the kidney. Clin. Sci. 26, 345--350 (1964) B~lint, P., Fekete, A., Hs L., Harza, T.: Postischemie renal failure and intrarenal distribution of blood flow. Acta physiol. Acad. Sci. hung. 86, 203--214 (1969)

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6. Bank, N., Mutz, B. F., Aynedjian, Y.: The role of "leakage" of tubular fluid in annria due to mercury poisoning. J. clin. Invest. 46, 695--704 (1967) 7. Barenberg, R.L., Solomon, S., Papper, S., Anderson, R.: Clearance and micropuncture study of renal function in mercuric chloride treated rats. J. Lab. clin. IVied.42, 473--484 (1968) 8. Biber, T. U.L., Mylle, M., Baines, A.S., Gottschalk, C.W., Oliver, J. R., MacDowell, M. C.: A study by mieropuncture and microdissection of acute renal damage in rats. Amer. J. Med. 44, 664--705 (t968) 9. Bratton, A. C., Marshall, E. K. : A new coupling component for sulfanilamide determination. J. biol. Chem. 128, 537--550 (1939) 10. Brenner, B. M., Troy, J. L., Daugharty, T.M.: The dynamics of glomerular ultrafiltration in the rat. J. clin. Invest. 50, 1776--1779 (1971) 11. Chedru, M.F., Baethke, R., Oken, D . E . : Renal cortical blood flow and glomerular filtration in myohemoglobinurie acute renal failure. Kidney Internat. 1, 232--239 (1972) 12. Deetjen, P., Sonnenberg, H. : Der tubul~re Transport yon p-Aminohippursaure. Mikroperfusionsversuche am Einzelnephron der Rattermiere in situ. Pflfigers Arch. ges. Physiol. 285, 35--44 (1965) 13. Eisenbach, G.M., Steinhausen, M.: Micropuneture studies after temporary ischemia of rat kidneys. Pflfigers Arch. 348, 11--25 (1973) 14. Flanigan, W. J., Oken, D. E.: Renal micropuncture study of the development of anuria of the rat with mercury-induced acute renal failure. J. clin. Invest. 44, 449--457 (1965) 15. Fuehr, J., Kaczmarczyk, J., Kriittgen, C.D.: Eine einfache Mcthode zur Inulinbestimmung fiir Nieren-Clearance-Untersuchungen bei Stoffwechselgesunden und Diabetikern. Klin. Wsehr. 83, 729--730 (1955) 16. Gertz, K. H., Brandis, M., Braun-Schubert, G., Boylan, J. W.: The effect of saline infusion and hemorrhage on glomerular filtration pressure and single nephron filtration rate. Pfliigers Arch. 810, 193--205 (1969) 17. Gomez, D.M.: Evaluation of renal resistances, with special reference to changes in essential hypertension. J. clin. Invest. 30, 1143--1155 (1951) 18. Henry, L.N., Lane, C.E., Kashgarian, M.: Micropuncture studies of the pathophysiology of acute renal failure in the rat. Lab. Invest. 19, 309--314 (1968) 19. Hinshaw, L. B.: Autoregulation in normal and pathological states including shock and ischemia. Circular. 1%es.~9, Suppl. 1, 46--50 (1971) 20. Jaenike, J . R . : Micropuncture study of methemoglobin-induced acute renal failure in the rat. J. Lab. clin. Med. 73, 459--468 (1969) 21. Kramer, K., Loehner, W., Wetterer, E.: Methods of measuring blood flow. In: Handbook of Physiology, Circulation, Washington, D.C. Amer. Physiol. Soc. 1963, sect. 2, vol. 2, pp. 1277--1324. 22. Ljungquist, A., Wagermark, J.: The adi'energie innervation of intrarenal glomerular and extraglomerular circulatory routes. Nephron 7, 218--229 (1970) 23. Ms M., Girndt, J., Ms G., Ochwadt, B.: Die Metabolisierung yon p-Aminohippurat in Nieren yon normalen Ratten und Ratten mit experimentellem Goldblatt-Hochdruck. Pflfigers Arch. 383, 156--165 (1972) 24. Oken, D.E., Arce, M.L., Wilson, D . R . : Glycerol-induced hemoglobinuric acute renal failure in the rat. I. Micropuncture study of the development of anuria. J. clin. Invest. 45, 724--735 (1966) 25. Oken, D. E., DiBona, G. F., McDonald, F. D.: Micropuncture studies of the recovery phase of myohemoglobinuric acute renal failure in the rat. J. clin. Invest. 49, 730--737 (1970)

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