Acta Physiol Scand 1979, 106: 93-95
Glomerular capillary pressure in the rat Validation of pressure measurementthrough corticotomy K. HEYERAAS TBNDER and K. AUKLAND University of Bergen, Institute of Physiology, htadveien 19,5000 Bergen, Norway
In recent years many studies have been reported on capillary pressure measured by direct puncture of superficial glomeruli in rats (for ref. see Tucker & Blantz 1977). However, uncertainties still remain as to how representative the punctured glomeruli are, because (1) superficial glomeruli accessible to micropuncture are found only in special rat strains (Brenner et al. 1971, Kallskog et al. 1975) (2) the pressure in the few accessible glomeruli might differ from that in the great majority of deep glomeruli not visible on the kidney surface. To get around these problems we recently introduced corticotomy, a procedure in which a lense-shaped slice of the cortex is cut out, exposing a large number of glomeruli at different depths of the cortex (Aukland et al. 1977). Only indirect evidence was presented to show that this admittedly traumatic procedure does not change the glomerular pressure. More direct evidence is presented in the present study, showing that the pressure measured in exposed glomeruli does not differ from those measured through the intact renal capsule. Attempts were also made at vital staining with Alcian Blue to make more glomeruli visible and thereby accessible to transcapsular measurements. Experiments were performed on two strains of female Wistar rats, weight 15CL235 g. One strain was obtained from K. E. Mellegaard, Ejby, Denmark (W-Me), and the other strain was the mutant “Munich strain” from S. Ivanovas GMBH & Co, Kisslegg im Allgau, W-Germany (W-Munich). The animals were fed standard rat pellets. Food, but not water, was withdrawn in the afternoon the day before the experiment. All rats were anesthetized with inactin (Promonta, Hamburg), given as a single i.p. injection of 120 mg/kg b.w. The rats were tracheotomized and placed on a heated operating table, oesophageal temperature being controlled at 36.5-37.5”C. A femoral artery and a vein were catheterized for arterial pressure recording and for continuous saline infusion ( 1 ml/h). In W-Me rats, a
polyethylene tube (PE 10) was placed in the upper abdominal aorta via a femoral artery for infusion of Alcian Blue. The left kidney was prepared for micropuncture as previously described (Aukland et al. 1977) and glomerular capillary pressure (PG)was measured through the intact renal capsule (PcT=transcapsular). The kidney was thereafter corticotomized by cutting off a 1-2 mm thick lenseshaped slice with a diameter of about 5 mm (Aukland et al. 19771, and glomerular pressure measured through the corticotomy (Pee). The depth of the punctured glomeruli relative to kidney surface varied from 0.1 to 0.9 mm, with almost 70% of the punctured glomeruli located 0.1 to 0.3 mm under the capsule. Micropuncture was performed with glass pipettes with sharpened tip of 1-2.5 p m diameter. Measurements were obtained by means of servocontrolled counter pressure (Wiederhielm et al. 1964; Aukland et al. 1977). From 2 to 20 superficial glomeruli could be observed on the intact dorsal aspect of the kidney in
Original recordings of arterial ( P A )and glomerular capillary pressure measured through intact capsule ( P G T ) and through corticotomy (Pee). Left: Location of micropipettes in corticotomized kidney.
Fig. 1 .
Actlr Physiol Scond 106
94
K . Heyeraas Tsnder and K . Aukland
Table 1. Clomerirlur cupillnry preysirre ( m m H g ) nieasrrred throirgh intuct ctrpsirle (P(:IJ tirid tlrroiigh corticotorny (Per). Meun +S.E. n=number of animals, Number of glomeruli in parentheses. P,=mean aortic pressure, AB=Alcian Blue Strain
n
PGT
PUC
PA
W-M0 W-M0, after AJ3 injection W-Munich
14 4 14
47.3f2.2 (20) 47.4k3.6 ( 5 ) 49.1k1.6 (21)
46.3k2.4 (12) 52.0rt6.5 (4) 50.3f 1.8 (17)
110.9k2.6 (32) 97.7f4.8 (9) 107.2k2.6 (38)
all W-Munich and in 13 of the W-M0 rats. In 10 W-M0 rats, which had no detectable surface glomeruli, bolus injection of 0.1-0.2 ml Alcian Blue 8GX, Gurr (0.5-1 % in saline solution) was given in the abdominal aorta. Satisfactory measurements were obtained both in 1-3 superficial glomeruli before corticotomy and in I 4 glomeruli in the corticotomy in 9 out of 14 W-Munich and in 11 out of 17 W-M0 rats. In the remaining 11 rats, only PcT or Pcc measurements were obtained. Original recordings O f P c T and Pcr in the same kidney are shown in Fig. I . As evident from Table 1, capillary pressure in glomeruli punctured through the intact kidney capsule showed no difference from pressure in glomeruli in the corticotomy. The individual paired measurements of PCTand Pcc from a total of 11 W-M0 rats (32 glomeruli) and 9 W-Munish rats (32 glomeruli) are shown in Fig. 2. In 2 W-M0 rats, measurements of PCT were repeated after corticotomy. In these rats, PUTwas 60 and 41 mmHg before corticotomy, compared to 54 and 40 mmHg after corticotomy. In accordance with previous results (Aukland et al. 1977), P c showed no correlation to spontaneous intra- and interindividual variations in aortic pressure. 1.a. injection of Alcian Blue in 10 W-Me rats visualized up to 15 glomeruli on the intact kidney surface. However, a sudden shrinkage, discolouring of the kidney and a gradual decline in P A were observed in some rats, especially after larger doses. In the 4 rats included in Table 1, no such signs of toxicity were observed, and glomerular pressure seemed to be unchanged. The absolute glomerular capillary pressures obtained in the present study need some comments: The average Pc observed in W-Munich rats agrees fairly well with those reported for this strain by other investigators (Tucker & Blantz 1977). HOWever, the similar average Per of 46.3k2.4 mmHg in W-M0 rats is about 12 mm lower than what we have
previous reported in purportedly the same strain. The use of only females in the present study would not seem to explain the difference (Aukland el al. 1977). Since we are not aware of any change in methodology, the difference is probably due to differences among batches of rats. In fact, the batch of W-M0 rats used in the present study had nearly as many visible glomeruli as the W-Munich rats, and many more than we previously observed in Wistar rats from Msllegaard. While the supplier excludes the possibility of "contamination" from the inbred Ivanovas' Munich strain, it is well known that there are large variations within the socalled Wistar strain, which is not genetically well defined (S. Erichsen, Statens Institutt for Folkehelse, Oslo. Personal communication). The present study strongly supports our previous
80
70 60
F E.
0 Lo
50 40
30 20 10 0
0
10
20
30
40 50 PC18mmHg
60
70
80
Fig. 2. Paired measurements of glomerular pressure
punctured through intact kidney capsule (Pti,.) and through corticotomy (Pw). 0 . Wistar-Mollegaard rats; A. Wistar-Munich rats.
Glomerular capillary pressure
95
impression that corticotomy does not greatly influ- REFERENCES ence glomerular capillary pressure. Admittedly, AUKLAND, K., HEYERAAS TaNDER, K. & N E B , G. 1977. Capillary pressure in deep and superficial with only one or two exceptions, the glomeruli glomeruli of the rat kidney. Acta Physiol Scand punctured in the corticotomy were too deep to be 101: 41-27. visible through the intact capsule, and might in BRENNER, B. M., TROY, J. L. & DAUGHARTY, T. theory have had a different pressure before corM. 1971. The dynamics of glomerular ultrafiltration in ticotomy. The previous finding of similar PGC at the rat. J Clin Invest 50: 1776-1780. various depths of the cortex makes this unlikely. In KALLSKOG, 0.. LINDBOM, L. L., ULFENDAHL, H. R. & WOLGAST, M. 1975. Kinetics of the glomerular any case, we have shown that “control” pressures ultrafiltration in the rat kidney. An experimental study. measured in corticotomy agree well with transcapActa Physiol Scand 95: 293-300. sular measurements, which would therefore seem TUCKER, B. J. & BLANTZ, R. C. 1977. An analysis of the determinants of nephron filtration rate. Amer J to give a good estimate of average glomerular capilPhysiol232 (6): F477-F483. lary pressure. However, the observations do not WIEDERHIELM, C. A., WOODBURY, J. W., KIRK, S. exclude the possibility of different responses to & RUSHMER, R. F. 1964. Pulsatile pressures in the physiological or pharmacological stimuli in deep microcirculation of frog’s mesentery. Amer J Physiol and superficial glomeruli. 207: 173-176.
Glomerular capillary pressure in the rat Validation of pressure measurementthrough corticotomy K. HEYERAAS TBNDER and K. AUKLAND University of Bergen, Institute of Physiology, htadveien 19,5000 Bergen, Norway
In recent years many studies have been reported on capillary pressure measured by direct puncture of superficial glomeruli in rats (for ref. see Tucker & Blantz 1977). However, uncertainties still remain as to how representative the punctured glomeruli are, because (1) superficial glomeruli accessible to micropuncture are found only in special rat strains (Brenner et al. 1971, Kallskog et al. 1975) (2) the pressure in the few accessible glomeruli might differ from that in the great majority of deep glomeruli not visible on the kidney surface. To get around these problems we recently introduced corticotomy, a procedure in which a lense-shaped slice of the cortex is cut out, exposing a large number of glomeruli at different depths of the cortex (Aukland et al. 1977). Only indirect evidence was presented to show that this admittedly traumatic procedure does not change the glomerular pressure. More direct evidence is presented in the present study, showing that the pressure measured in exposed glomeruli does not differ from those measured through the intact renal capsule. Attempts were also made at vital staining with Alcian Blue to make more glomeruli visible and thereby accessible to transcapsular measurements. Experiments were performed on two strains of female Wistar rats, weight 15CL235 g. One strain was obtained from K. E. Mellegaard, Ejby, Denmark (W-Me), and the other strain was the mutant “Munich strain” from S. Ivanovas GMBH & Co, Kisslegg im Allgau, W-Germany (W-Munich). The animals were fed standard rat pellets. Food, but not water, was withdrawn in the afternoon the day before the experiment. All rats were anesthetized with inactin (Promonta, Hamburg), given as a single i.p. injection of 120 mg/kg b.w. The rats were tracheotomized and placed on a heated operating table, oesophageal temperature being controlled at 36.5-37.5”C. A femoral artery and a vein were catheterized for arterial pressure recording and for continuous saline infusion ( 1 ml/h). In W-Me rats, a
polyethylene tube (PE 10) was placed in the upper abdominal aorta via a femoral artery for infusion of Alcian Blue. The left kidney was prepared for micropuncture as previously described (Aukland et al. 1977) and glomerular capillary pressure (PG)was measured through the intact renal capsule (PcT=transcapsular). The kidney was thereafter corticotomized by cutting off a 1-2 mm thick lenseshaped slice with a diameter of about 5 mm (Aukland et al. 19771, and glomerular pressure measured through the corticotomy (Pee). The depth of the punctured glomeruli relative to kidney surface varied from 0.1 to 0.9 mm, with almost 70% of the punctured glomeruli located 0.1 to 0.3 mm under the capsule. Micropuncture was performed with glass pipettes with sharpened tip of 1-2.5 p m diameter. Measurements were obtained by means of servocontrolled counter pressure (Wiederhielm et al. 1964; Aukland et al. 1977). From 2 to 20 superficial glomeruli could be observed on the intact dorsal aspect of the kidney in
Original recordings of arterial ( P A )and glomerular capillary pressure measured through intact capsule ( P G T ) and through corticotomy (Pee). Left: Location of micropipettes in corticotomized kidney.
Fig. 1 .
Actlr Physiol Scond 106
94
K . Heyeraas Tsnder and K . Aukland
Table 1. Clomerirlur cupillnry preysirre ( m m H g ) nieasrrred throirgh intuct ctrpsirle (P(:IJ tirid tlrroiigh corticotorny (Per). Meun +S.E. n=number of animals, Number of glomeruli in parentheses. P,=mean aortic pressure, AB=Alcian Blue Strain
n
PGT
PUC
PA
W-M0 W-M0, after AJ3 injection W-Munich
14 4 14
47.3f2.2 (20) 47.4k3.6 ( 5 ) 49.1k1.6 (21)
46.3k2.4 (12) 52.0rt6.5 (4) 50.3f 1.8 (17)
110.9k2.6 (32) 97.7f4.8 (9) 107.2k2.6 (38)
all W-Munich and in 13 of the W-M0 rats. In 10 W-M0 rats, which had no detectable surface glomeruli, bolus injection of 0.1-0.2 ml Alcian Blue 8GX, Gurr (0.5-1 % in saline solution) was given in the abdominal aorta. Satisfactory measurements were obtained both in 1-3 superficial glomeruli before corticotomy and in I 4 glomeruli in the corticotomy in 9 out of 14 W-Munich and in 11 out of 17 W-M0 rats. In the remaining 11 rats, only PcT or Pcc measurements were obtained. Original recordings O f P c T and Pcr in the same kidney are shown in Fig. I . As evident from Table 1, capillary pressure in glomeruli punctured through the intact kidney capsule showed no difference from pressure in glomeruli in the corticotomy. The individual paired measurements of PCTand Pcc from a total of 11 W-M0 rats (32 glomeruli) and 9 W-Munish rats (32 glomeruli) are shown in Fig. 2. In 2 W-M0 rats, measurements of PCT were repeated after corticotomy. In these rats, PUTwas 60 and 41 mmHg before corticotomy, compared to 54 and 40 mmHg after corticotomy. In accordance with previous results (Aukland et al. 1977), P c showed no correlation to spontaneous intra- and interindividual variations in aortic pressure. 1.a. injection of Alcian Blue in 10 W-Me rats visualized up to 15 glomeruli on the intact kidney surface. However, a sudden shrinkage, discolouring of the kidney and a gradual decline in P A were observed in some rats, especially after larger doses. In the 4 rats included in Table 1, no such signs of toxicity were observed, and glomerular pressure seemed to be unchanged. The absolute glomerular capillary pressures obtained in the present study need some comments: The average Pc observed in W-Munich rats agrees fairly well with those reported for this strain by other investigators (Tucker & Blantz 1977). HOWever, the similar average Per of 46.3k2.4 mmHg in W-M0 rats is about 12 mm lower than what we have
previous reported in purportedly the same strain. The use of only females in the present study would not seem to explain the difference (Aukland el al. 1977). Since we are not aware of any change in methodology, the difference is probably due to differences among batches of rats. In fact, the batch of W-M0 rats used in the present study had nearly as many visible glomeruli as the W-Munich rats, and many more than we previously observed in Wistar rats from Msllegaard. While the supplier excludes the possibility of "contamination" from the inbred Ivanovas' Munich strain, it is well known that there are large variations within the socalled Wistar strain, which is not genetically well defined (S. Erichsen, Statens Institutt for Folkehelse, Oslo. Personal communication). The present study strongly supports our previous
80
70 60
F E.
0 Lo
50 40
30 20 10 0
0
10
20
30
40 50 PC18mmHg
60
70
80
Fig. 2. Paired measurements of glomerular pressure
punctured through intact kidney capsule (Pti,.) and through corticotomy (Pw). 0 . Wistar-Mollegaard rats; A. Wistar-Munich rats.
Glomerular capillary pressure
95
impression that corticotomy does not greatly influ- REFERENCES ence glomerular capillary pressure. Admittedly, AUKLAND, K., HEYERAAS TaNDER, K. & N E B , G. 1977. Capillary pressure in deep and superficial with only one or two exceptions, the glomeruli glomeruli of the rat kidney. Acta Physiol Scand punctured in the corticotomy were too deep to be 101: 41-27. visible through the intact capsule, and might in BRENNER, B. M., TROY, J. L. & DAUGHARTY, T. theory have had a different pressure before corM. 1971. The dynamics of glomerular ultrafiltration in ticotomy. The previous finding of similar PGC at the rat. J Clin Invest 50: 1776-1780. various depths of the cortex makes this unlikely. In KALLSKOG, 0.. LINDBOM, L. L., ULFENDAHL, H. R. & WOLGAST, M. 1975. Kinetics of the glomerular any case, we have shown that “control” pressures ultrafiltration in the rat kidney. An experimental study. measured in corticotomy agree well with transcapActa Physiol Scand 95: 293-300. sular measurements, which would therefore seem TUCKER, B. J. & BLANTZ, R. C. 1977. An analysis of the determinants of nephron filtration rate. Amer J to give a good estimate of average glomerular capilPhysiol232 (6): F477-F483. lary pressure. However, the observations do not WIEDERHIELM, C. A., WOODBURY, J. W., KIRK, S. exclude the possibility of different responses to & RUSHMER, R. F. 1964. Pulsatile pressures in the physiological or pharmacological stimuli in deep microcirculation of frog’s mesentery. Amer J Physiol and superficial glomeruli. 207: 173-176.
