Effect of mesangiolysis on autoregulation of renal blood flow and glomerular filtration rate in rats BJARNE GUNNAR
M. IVERSEN, FRED I. KVAM, HORVEI, WILMA BAGCHUS,
KNUT JORIS
MATRE, GROND,
LARS M@RKRID, AND JARLE OFSTAD Renal Research Group, Medical Department A, University of Bergen, N-5021 Bergen, Norway; and Department of Pathology, State University of Groningen, 9713 EZ Groningen, The Netherlands Iversen, Bjarne M., Fred I. Kvam, Knut Matre, Lars Msrkrid, Gunnar Horvei, Wilma Bagchus, Joris Grond, and Jarle Ofstad. Effect of mesangiolysison autoregulation of renal blood flow and glomerular filtration rate in rats. Am.
We studied RBF autoregulation 1 h, and GFR and RBF autoregulation 30 h, after infusion of anti-Thy l-l antibodies intravenously in Wistar rats. In addition, we demonstrated in vitro that the angiotensin II-induced J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F361F366, 1992.-Interlobular arteries and afferent arterioles are acute mesangial cell contraction was abolished by antiinvolved in autoregulation of renal blood flow (RBF) and Thy l-l antibodies. The main finding was that autoregglomerular filtration rate (GFR). The question of whether the ulation of RBF seemed unaffected and GFR autoregulal-l contractile mesa&al cells are also involved in autoregulation tion was restricted after the injection of anti-Thy was investigated in Wistar rats. Autoregulation of RBF was antibodies.
examined before and 1 h after infusion of antithymocyte (antiThy l-l) antibodies, and both RBF and GFR autoregulation were examined 30 h after the infusion of antibodies. Mesangial cell destruction was present 30 h after the infusion of antibodies. The angiotensin II-induced contraction of isolated glomeruli (70% of control volume, P < 0.001) was abolishedafter the glomeruli had been exposedto anti-Thy l-l in vitro. RBF, as well asthe lower limit of RBF autoregulation, were not different from control 30 h after the infusion (82 t 5 vs. 79 * 4 mmHg, P > 0.10). Autoregulation of GFR wasmaintained in the control group but was restricted in the experimental group (autoregulatory index: 0.71 =t 0.42 for left kidney, 0.02 t 0.35 for control; P < 0.05). The afferent arteriolar diameter was unchanged 30 h after the infusion of antibodies (17.8 t 0.8 vs. 17.6 t 0.4 pm, P > 0.10). One hour after infusion of the antibodies, RBF autoregulation was normal. It is concludedthat mesangialcells do not seemto be involved in RBF autoregulation, but may in part influence autoregulation of GFR during pressure reduction. mesangium;afferent arterioles; anti-Thy l-l antibodies WHEN RENAL PERFUSION PRESSUREis reducedin
normotensive euvolemic rats, renal blood flow (RBF) and glomerular filtration rate (GFR) are kept constant by autoregulation of pre- and postglomerular vascular resistances until a lower pressure limit of 80 mmHg (7) . On the upstream side of the glomerulus both the interlobular artery and the afferent arterioles are involved in autoregulation (5, 11). The intention of the present study was to investigate whether the contractile mesangial cells embedding the glomerular hilar vessels participate in autoregulation as a third precapillary resistance segment in addition to the afferent arteriole and the interlobular artery. For this purpose we used the recently established antithymocyte (anti-Thy l-l) glomerulonephritis rat model (3, 4). Injection of anti-Thy l-l antibodies induces a complement-dependent acute necrosis of the mesangial cells, accompanied by derangement of the glomerular structure. Proteinuria, proliferation of glomerular cells, and invasion of cells from the circulating blood follow after 1 day or more (3). This model may thus offer the possibility to study autoregulation in rat kidneys where the contractile property of the mesangium is eliminated.
METHODS The experiments were carried out in male Wistar rats (Mol: Wist; Mollegaard Breeding Center, Denmark) with body weights of ~300 g. The animals were fed rat chow containing 35 mmol sodium and 230 mmol potassiumper kilogram chow; three animals were kept in each cage. Hemodynamic
Study
The animals were kept without food overnight before the study but had free accessto water. They were anesthetizedwith an intraperitoneal injection of pentobarbital sodium,50 mg/kg body wt, suppliedwith extra doses(l-2 mg iv) when necessary, and placed on a thermostat-regulated heating plate to keepthe body temperature constant at 37OC.The trachea was cannulated, and polyethylene (PE) catheters (Portex, Hythe, UK) were introduced into the left femoral vein for infusions and into the left femoral artery for blood pressuremeasurements. The arterial catheter was connected to a pressuretransducer (SE Labs EMI, Middlesex, UK) and a Hewlett-Packard 7700 recorder (Hewlett-Packard, CO). A special-purposeultrasound Doppler flow probe was designedfor measurementof RBF. The flow probe was madeof silicone rubber, and the internal diameter was 0.8 mm, giving sufficient contact with the renal artery without causingstenosis.The lo-MHz piezoelectric crystal waslarge enoughto sonify the whole vessellumen, permitting accurate mean velocities to be recorded.The flow probe wasconnectedto a lo-MHz pulsedDoppler meter (Alfred; Vingmed Sound A/S, Horten, Norway). The ultrasound Doppler probes were calibrated in vitro using fresh renal artery from the samebatch of animals as usedfor the experiments. Catheters were connected to the distal part of the artery and to the aorta. The vessel with the probe was placed in a bath with Ringer solution at 37OC.Human bank blood with hematocrit (Hct) value of ~45 waspumpedthrough the artery by meansof a roller pump and collected in a graded cylinder. Three ultrasound Doppler probeswere calibrated. The relation between recqrded mean velocity and flow rate gave correlation coefficients of 0.979-0.994 after linear regression analysisin the flow range 0.5-10 ml/min. With assumptionof a corresponding wall thickness during in vivo measurements, the linear regressionequation obtained wasusedin converting velocity data into flow rate. A screw clamp was placed on the aorta above the renal arteries for adjustment of the renal perfusion pressure. Sys-
036306127/92 $2.00 Copyright 0 1992 the American Physiological Society
F361
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
F362
MESANGIUM
140-
[INFUSION]
AND RBF-GFR
AUTOREGULATION
Controls
120s
Anti-Thy
80
l-l
2 E loonu a x
70
t t t **
80-
60
1 II
I 0
1 10
I 20
I 30
I 40
I 50
g sz j k 100 s 5 60
I 60
90
-
80
-
fY
TIME (min) Fig. 1. Mean blood pressure (MAP) during and 50 min after infusion of anti-Thy l-l antibodies and control serum. *P < 0.05. tP < 0.01.
70 60
.-c f : E zi ii 0
6-
44/
’
1
60 Anti-Thy
l-l
5-
4-
0 2 m
3-
G z
*-
l-
1
0
I
I
80
90
I
100
1
n
110
120
(mmHg) Fig. 3. RBF autoregulation before (0) and 1 h after (0) infusion of anti-Thy l-l antibodies (top) and before and 1 h after infusion of control serum (bottom). PERFUSION
PRESSURE
rate of Ringer acetate. Clearance determinations were performed in three lo-min periods, i.e., 1) at control perfusion pressure,2) at a pressure10 mmHg above the lower pressure limit of RBF autoregulation, and 3) at a perfusion pressureof 60 mmHg. The animals rested 15 min between each clearance period for achievement of the new steady-state situation with reducedperfusion pressure. Autoregulatory index was calculated according to Semple and dewardener (15).
&
7r
70
~
lb
210
;o
LIO
s’o
s’o
TIME (mid Fig. 2. Renal blood flow (RBF) during and 50 min after infusion of anti-Thy l-l and control serum. $P < 0.001. tP < 0.01. * P < 0.05.
Infusion
of
Antibodies
The anti-Thy l-l antibodieswere infused through a femoral vein catheter. The duration of the infusion was 10 min, and the doseof antibodies (immunoglobulin G) was 1,000pg/2 ml Ringer acetate in all rats. Normal mouseserum was used as control.
temic blood pressure and RBF were recorded continuously during infusion of anti-Thy l-l antibodiesand for the following 50 min; the valueswere read every 5 min, and the averagevalue for each lo-min period was calculated. For measurementof GFR, both ureters were cannulated (PE- Measurement of Glomerular Contraction 10).Thereafter an intravenous infusion of Na-[ ““I]iothalamate Rat glomeruli were isolated by a modification of the method (Amersham Laboratories, Buckinghamshire, UK) in Ringer acetate was started. Na-[ 1251]iothalamate(5 &i) in 2 ml of describedby Krackower and Greenspoon(9). After the glomerRinger acetate solution was given as a priming dose followed uli had been washedin saline, four samplesof a suspensionof by a continuous infusion of 0.5 &i/ml Ringer acetate delivered glomeruli in salinewere incubated for 15min at 37°C asfollows: at a rate of 8 ml/h by a Harvard infusion/withdrawal pump 1) control suspensionof glomeruli, 2) glomeruli + angiotensin (model971, Harvard Apparatus). The animals were allowed to II (low6M) (ll), 3) glomeruli + anti-Thy l-l (10 pg/ml), and rest for 15 min to obtain a steady-state situation. Urine for 4) glomeruli + anti-Thy l-l + angiotensin II. Rat plasma,200 clearancemeasurementswas collected from both ureters using pi/ml, was added to the suspensionbefore incubation as a preweighedglasscapillary tubes. Urine wascollected in lo-min source of complement. After the incubation, 200 ~1 of the periods. The urine volume, determined from weight, was cor- suspensionwas transferred to a Burkner counting chamber, rected for density measured with a gravimeter (Uricon-N, and the diameter of 75 glomeruli were measuredby use of an Atago, Japan). In the middle of each period, 0.05 ml blood was eyepiecemicrometer. The largest diameter of the glomerular sampled from the right femoral artery in heparinized glass tuft in isolated intact glomeruli was measured.All measurecapillary tubes for determination of Hct and counting of 1251. ments were done blindly. lzsIwascounted in duplicate samplesof urine and plasma(1282 Compugamma;LKB-Wallace, Turku, Finland). Clearancewas Measurement of Afferent Arteriolar Diameter (DAJ calculated as [(cpm in urine)/(cpm in plasma)] x urine volume wherecpm is counts per minute. The arterial Hct wasmeasured The animals were prepared for DAA measurementby an every 10 min; stability was obtained by adjusting the infusion intraperitoneal injection of pentobarbital asdescribedabove.A Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
MESANGIUM
Table
AND RBF-GFR
1. Renal vascular resistance during infusion of anti-Thy
F363
AUTOREGULATION
l-l antibodies Time, min
n
0
10
20
30
50
60
1,800
20.4k2.0 24.324.1 NS
20.4f2.0 23.lk3.7 NS
21.4+2.0 17.4zt3.1 NS
40
RVR, ml. mm-‘. mmHg-’ 2O.Ok2.1 20.1*2.0 20.3h1.8 Control 6 20.3k2.0 19.9+1.9 47.5211.3 31.8-cg.O 29.4rt5.3 Anti-Thy l-l 6 20.5zh2.8 184.5&28.2 co.002 NS P NS 0.10) (Fig. 3, mouseserumwere usedas controls. Table 1). As RBF was still slightly but significantly less
Subacutestudy. In sevenrats RBF and GFR autoregulation were examined 30 h after infusion of anti-Thy l-l. In the control group (6 animals), RBF and GFR autoregulation were examined 30 h after infusion of normal mouseserum. DAA measurement.DAA was measuredin the following three groups: 1) six rats 20-30 min after start of anti-Thy l-l
than control (6.1 rt 0.8 vs. 4.9 z!z0.8 ml/min;
P < 0.05),
some of these kidneys might nevertheless have been slightly vasoconstricted (Figs. 1 and 2). One hour after the antibodies were infused, neither the lower pressure limit of RBF autoregulation nor the RVR at this pressure were different from control values [83 f 5 vs. 81 +- 4 infusion, 2) six rats 30 h after infusion of anti-Thy l-l, and 3) six control rats infused with normal mouseserum and studied mmHg (P > 0.10) and 20.4 f 2.0 vs. 23.1 It: 3.7 ml. 30 h after the infusion. min-l . mmHg-‘, respectively] (Fig. 3, Table 1). Thirty hours after the infusion of antibodies, neither MAP, RBF, RVR, nor DAA were different from control Statistical Methods [105 +- 5 vs. 113 + 5 mmHg (P > O.lO), 6.8 f 0.6 vs. 5.9 (P > O.lO), 21.2 + 2.0 vs. 17.4 + 3.1 mlThe results are means+ SE. One-way analysis of variance + 0.5 ml/min (P > O.lO), and 17.8 & 0.8 vs. 17.6 f 0.4 wasperformed amongthe groups,and where significant differ- min-’ -mmHg-’ and the ability to autoreences were found, Student’s t test was used. P c 0.05 was pm (P > O.lO), respectively], gulate RBF was not significantly changed (Fig. 4). The consideredto be statistically significant. Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
MESANGIUM
AND RBF-GFR
AUTOREGULATION
F365
Fig. 8. Electron micrograph (x4,500) showing severe mesangiolysis (*) 30 h after infusion of anti-Thy l-l antibodies.
lower pressure limit of autoregulation was 82 + 5 mmHg in the antibody-infused group vs. 79 I~I 4 mmHg in the control group (P > 0.10). The GFR 30 h after the infusion of anti-Thy l-l antibodies was significantly increased in both kidneys (2.1 + 0.2 for left and 1.7 +- 0.3 for right kidney vs. 1.4 rt 0.2 ml/min for control; PC 0.05). During systemic blood pressure reduction to 92 mmHg, GFR autoregulation in the control group was perfect (autoregulatory index, 0.02 & 0.35). GFR autoregulation was restricted in the antibody-infused group (autoregulatory index both kidneys, 0.71 + 0.42; significantly different from controls, P < 0.05). At pressures below 92 mmHg, GFR autoregulation was absent in both groups (Fig. 4). Contractions of isolated glomeruli during incubation with angiotensin II and anti-Thy l-l antibodies, alone or in combination, are shown in Fig. 5. Angiotensin II reduced the glomerular diameter significantly (176 + 5 vs. 156 + 4 pm; P < 0.001). The calculated volume reduction of the glomeruli was -70%. This effect was abolished after preincubation with anti-Thy l-l antibodies. Light microscopy showed destruction of the intraglomerular mesangial area 30 h after infusion of antibodies (Fig. 6). In some glomeruli, connections between adjacent capillary loops and also fusion of capillaries with aneurysm formation could be seen. No mesangial destruction was observed 60 min after the antibody infusion, with the exception of scattered apoptotic bodies in the mesangial cells. Electron microscopy showed mesangiolysis in some
areas of the glomeruli 1 h after infusion of antibodies (Fig. 7). The afferent arteriole and macula densa were normal. Thirty hours after infusion of antibodies, subtotal mesangiolysis was seen with marked dilation and aneurysm formation of the glomerular capillaries (Fig. 8). The afferent arterioles and the macula densa were also normal after 30 h. DISCUSSION
Intact RBF autoregulation after injection of anti-Thy l-l was the main finding in this study. The abolished glomerular shrinking when anti-Thy l-l antibodies were applied in combination with angiotensin II in vitro, as well as the mesangial cell damage observed by microscopical examination, strongly indicate that active contraction or relaxation of the mesangial cells were absent both in the acute experiments and when RBF and GFR autoregulation were studied 30 h after the antibody infusion. Taken together, this indicates that mesangial cells do not participate in autoregulation of RBF in the rat. Studies of immunologically induced mesangial cell damage by others support the assumption that these cells were nonfunctional after the injection in our study (4). The glomerular damage observed in our study corresponds with that reported in the study by Bagchus et al. (4) where antibodies from the batch applied in the present study were used in a corresponding dose. The antiThy l-l glomerulonephritis has been shown to depend
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F366
MESANGIUM
AND RBF-GFR
on complement activation induced by an antigen-antibody reaction on the mesangial cell membrane (3, 4). Adler al. (1) have demonstrated that complement activation induces production of oxygen radicals in mesangial cells and suggests that these antibodies may cause mesangial cell injury. This damage may thus influence the membrane function necessary for the normal contractile function of the mesangial cells, as observed in the in vitro study, and may also explain the mesangial cell necrosis in vivo. An immediate renal vascular reaction similar to that observed in this study has been observed in the passive Heymann nephritis, another complement-dependent glomerulonephritis with intraglomerular complement activation (14). The role played by the different vascular resistance segments during autoregulation cannot be ascertained without micropuncture. The normal RVR and the normal DA* observed 30 h after the injection indicate, however, that the preglomerular resistances acted normally during autoregulation in these experiments. This was probably the case in the acute experiments as well, because both RVR and RBF were almost normalized before the autoregulation was recorded following the injection. An eventual persistence of the afferent arteriolar constriction, observed during the blood pressure drop 20 min after the injection in these experiments, would be expected to reset the lower pressure limit of RBF autoregulation to the right (7). This implies that the normal lower pressure limit of RBF autoregulation in the acute experiments was not due to resetting of autoregulation to a lower pressure range secondary to changes of the systemic pressure (7). In this study the efferent arteriole is of particular interest, because th-is vessel has been reported to be surrounded by mesangial cells assumed to have a constrictive function (13). Furthermore, the postglomerular resistance has been observed to increase during lowering of the perfusion pressure in some studies (8, 12). Elimination of this postglomerular increase of the vascular resistance would be expected to decrease the lower pressure limit of RBF autoregulation by reducing the minimal total RVR and also to increase the corresponding pressure limit of GFR autoregulation by decreasing the glomerular filtration pressure. The normal RBF autoregulation after the infusion thus argues against a postglomerular effect of anti-Thy l-l, but a minor effect on the postglomerular resistance may explain the effect on GFR autoregulation 30 h after the infusion of anti-Thy l-l antibodies. A similar separation between RBF and GFR autoregulation, as observed in our experiments, has been found in the rat and dog kidney during treatment with saralasin or during converting enzyme inhibition, possibly caused by a postglomerular effect (2). However, the gross changes of glomerular capillary anatomy due to mesangiolysis makes an interpretation of the GFR measurement in the context of autoregulation questionable as long as the filtering area and also the permeability of the capillary wall are unknown.
AUTOREGULATION
In conclusion, the present study indicates that the mesangial cell does not play a decisive part in RBF autoregulation but may in part influence the autoregulation of GFR during pressure reduction. The experimental protocol including stepwise perfusion pressure reduction is supposed to engage both the myogenic and tubuloglomerular feedback mechanisms of vascular resistance control in the kidney. Autoregulation of RBF by these mechanisms thus seems to be independent of mesangial cell function. Address for reprint requests: B. M. Iversen, Medical Dept., Haukeland Hospital, N-5021 Bergen, Norway. Received 7 January 1991; accepted in final form 1 October 1991. REFERENCES 1. Adler, S., P. J. Baker, R. J. Johnson, R. F. Ochi, P. Pritzi, and W. G. Couser. Complement membrane attack complex stim-
ulates production of reactive oxygen metabolites by cultured rat mesangial cells. J. Clin. Invest. 77: 762-767, 1986. 2. Arendshorst, W. J., and W. F. Finn. Renal hemodynamics in the rat before and during inhibition of angiotensin II. Am. J. Physiol. 233 (Rend Fluid Electrolyte Physiol. 2): F290-F297,1977. 3. Bagchus, Bakker.
W. M.,
P. J. Hoedemaeker,
J. Rozing,
and
W. W.
Acute glomerulo-nephritis after intravenous injection of monoclonal anti-thymocyte antibodies in the rat. Immunol. L&t.
12: 109-110,1986. 4. Bagchus, W. M., P. J. Hoedemaeker, Bakker. Glomerulonephritis induced
antibodies. A sequential histological the rat. Lab. Invest. 55: 680-687, 1986.
J. Rozing,
and
W. W.
by monoclonal anti-Thy 1.1 and ultrastructural study in
K. J., and K. Aukland. Interlobular arterial resistance: influence of renal arterial pressure and angiotensin II. Kidney
5. Heyeraas,
Int. 31: 1291-1298,1987. 6. Iversen, B. M., L. Merkrid,
and J. Ofstad. Afferent arteriolar diameter in DOCA-salt and two-kidney one-clip hypertensive rats.
Am. J. Physiol. 245 (Renal F762,1983. 7. Iversen, B. M., I. Sekse,
blood flow autoregulation J. Physiol. 1987. 8. Klllskog,
252 (Rend
Fluid
Electrolyte
Physiol.
14): F755-
and J. Ofstad. Resetting of renal in spontaneously hypertensive rats. Am.
Fluid
Electrolyte
G., L. 0. Lindbom,
Physiol.
H. Ulfendahl,
21): F480-F486, and M. Wolgast.
Hydrostatic pressures within the vascular structures in the rat kidney. Pfluegers Arch. 363: 205-210, 1976. 9. Krackower, C. A., and S. A. Greenspoon. Localization of the nephrotoxic antigen within the isolated renal glomerulus. Arch. Puthol. 10. Markrid,
51: 629-633,1951. L., J. Ofstad,
and
Y.
Effect of steric of microspheres in the
Willassen.
restriction of the intracortical distribution dog kidney. Circ. Res. 39: 608-615, 1976.
J., B. M. Iversen, L. Msrkrid, and I. Sekse. Autoregulation of renal blood flow (RBF) with and without participation of afferent arterioles. Actu Physiol. &and. 130: 25-32, 1987.
11. Ofstad,
12. Robertson, C. R., W. M. Deen, J .L. Troy, and B. M. Brenner. Dynamics of glomerular ultrafiltration in the rat. III. Hemodynamics and autoregulation. Am. J. Physiol. 223: 1191-1200,1972. 13. Schnabel, E., W. Kriz, and M. Steinhausen. Outflow segment
of the efferent arteriole of the rat glomerulus investigated by in vivo and electron microscopy. Rend Physiol. 10: 318-326, 1987. 14. Sekse, I., B. M. Iversen, R. Matre, and J. Ofstad. The acute effect of passive Heymann nephritis on renal blood flow and glomerular filtration rate in rats. Nephron 53: 364-372, 1989. 15. Semple, S. J. G., and H. E. DeWardener. Effect of increased renal venous pressure on circulatory autoregulation of isolated dog kidneys. Circ. Res. 7: 643-648, 1959. 16. Sraer, J. D., J. Sraer, R. Ardaillou, and 0. Mimoune. Evidence of renal glomerular receptors for angiotensin II. Kidney Int. 6: 241-246,1974.
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M. IVERSEN, FRED I. KVAM, HORVEI, WILMA BAGCHUS,
KNUT JORIS
MATRE, GROND,
LARS M@RKRID, AND JARLE OFSTAD Renal Research Group, Medical Department A, University of Bergen, N-5021 Bergen, Norway; and Department of Pathology, State University of Groningen, 9713 EZ Groningen, The Netherlands Iversen, Bjarne M., Fred I. Kvam, Knut Matre, Lars Msrkrid, Gunnar Horvei, Wilma Bagchus, Joris Grond, and Jarle Ofstad. Effect of mesangiolysison autoregulation of renal blood flow and glomerular filtration rate in rats. Am.
We studied RBF autoregulation 1 h, and GFR and RBF autoregulation 30 h, after infusion of anti-Thy l-l antibodies intravenously in Wistar rats. In addition, we demonstrated in vitro that the angiotensin II-induced J. Physiol. 262 (Renal Fluid Electrolyte Physiol. 31): F361F366, 1992.-Interlobular arteries and afferent arterioles are acute mesangial cell contraction was abolished by antiinvolved in autoregulation of renal blood flow (RBF) and Thy l-l antibodies. The main finding was that autoregglomerular filtration rate (GFR). The question of whether the ulation of RBF seemed unaffected and GFR autoregulal-l contractile mesa&al cells are also involved in autoregulation tion was restricted after the injection of anti-Thy was investigated in Wistar rats. Autoregulation of RBF was antibodies.
examined before and 1 h after infusion of antithymocyte (antiThy l-l) antibodies, and both RBF and GFR autoregulation were examined 30 h after the infusion of antibodies. Mesangial cell destruction was present 30 h after the infusion of antibodies. The angiotensin II-induced contraction of isolated glomeruli (70% of control volume, P < 0.001) was abolishedafter the glomeruli had been exposedto anti-Thy l-l in vitro. RBF, as well asthe lower limit of RBF autoregulation, were not different from control 30 h after the infusion (82 t 5 vs. 79 * 4 mmHg, P > 0.10). Autoregulation of GFR wasmaintained in the control group but was restricted in the experimental group (autoregulatory index: 0.71 =t 0.42 for left kidney, 0.02 t 0.35 for control; P < 0.05). The afferent arteriolar diameter was unchanged 30 h after the infusion of antibodies (17.8 t 0.8 vs. 17.6 t 0.4 pm, P > 0.10). One hour after infusion of the antibodies, RBF autoregulation was normal. It is concludedthat mesangialcells do not seemto be involved in RBF autoregulation, but may in part influence autoregulation of GFR during pressure reduction. mesangium;afferent arterioles; anti-Thy l-l antibodies WHEN RENAL PERFUSION PRESSUREis reducedin
normotensive euvolemic rats, renal blood flow (RBF) and glomerular filtration rate (GFR) are kept constant by autoregulation of pre- and postglomerular vascular resistances until a lower pressure limit of 80 mmHg (7) . On the upstream side of the glomerulus both the interlobular artery and the afferent arterioles are involved in autoregulation (5, 11). The intention of the present study was to investigate whether the contractile mesangial cells embedding the glomerular hilar vessels participate in autoregulation as a third precapillary resistance segment in addition to the afferent arteriole and the interlobular artery. For this purpose we used the recently established antithymocyte (anti-Thy l-l) glomerulonephritis rat model (3, 4). Injection of anti-Thy l-l antibodies induces a complement-dependent acute necrosis of the mesangial cells, accompanied by derangement of the glomerular structure. Proteinuria, proliferation of glomerular cells, and invasion of cells from the circulating blood follow after 1 day or more (3). This model may thus offer the possibility to study autoregulation in rat kidneys where the contractile property of the mesangium is eliminated.
METHODS The experiments were carried out in male Wistar rats (Mol: Wist; Mollegaard Breeding Center, Denmark) with body weights of ~300 g. The animals were fed rat chow containing 35 mmol sodium and 230 mmol potassiumper kilogram chow; three animals were kept in each cage. Hemodynamic
Study
The animals were kept without food overnight before the study but had free accessto water. They were anesthetizedwith an intraperitoneal injection of pentobarbital sodium,50 mg/kg body wt, suppliedwith extra doses(l-2 mg iv) when necessary, and placed on a thermostat-regulated heating plate to keepthe body temperature constant at 37OC.The trachea was cannulated, and polyethylene (PE) catheters (Portex, Hythe, UK) were introduced into the left femoral vein for infusions and into the left femoral artery for blood pressuremeasurements. The arterial catheter was connected to a pressuretransducer (SE Labs EMI, Middlesex, UK) and a Hewlett-Packard 7700 recorder (Hewlett-Packard, CO). A special-purposeultrasound Doppler flow probe was designedfor measurementof RBF. The flow probe was madeof silicone rubber, and the internal diameter was 0.8 mm, giving sufficient contact with the renal artery without causingstenosis.The lo-MHz piezoelectric crystal waslarge enoughto sonify the whole vessellumen, permitting accurate mean velocities to be recorded.The flow probe wasconnectedto a lo-MHz pulsedDoppler meter (Alfred; Vingmed Sound A/S, Horten, Norway). The ultrasound Doppler probes were calibrated in vitro using fresh renal artery from the samebatch of animals as usedfor the experiments. Catheters were connected to the distal part of the artery and to the aorta. The vessel with the probe was placed in a bath with Ringer solution at 37OC.Human bank blood with hematocrit (Hct) value of ~45 waspumpedthrough the artery by meansof a roller pump and collected in a graded cylinder. Three ultrasound Doppler probeswere calibrated. The relation between recqrded mean velocity and flow rate gave correlation coefficients of 0.979-0.994 after linear regression analysisin the flow range 0.5-10 ml/min. With assumptionof a corresponding wall thickness during in vivo measurements, the linear regressionequation obtained wasusedin converting velocity data into flow rate. A screw clamp was placed on the aorta above the renal arteries for adjustment of the renal perfusion pressure. Sys-
036306127/92 $2.00 Copyright 0 1992 the American Physiological Society
F361
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
F362
MESANGIUM
140-
[INFUSION]
AND RBF-GFR
AUTOREGULATION
Controls
120s
Anti-Thy
80
l-l
2 E loonu a x
70
t t t **
80-
60
1 II
I 0
1 10
I 20
I 30
I 40
I 50
g sz j k 100 s 5 60
I 60
90
-
80
-
fY
TIME (min) Fig. 1. Mean blood pressure (MAP) during and 50 min after infusion of anti-Thy l-l antibodies and control serum. *P < 0.05. tP < 0.01.
70 60
.-c f : E zi ii 0
6-
44/
’
1
60 Anti-Thy
l-l
5-
4-
0 2 m
3-
G z
*-
l-
1
0
I
I
80
90
I
100
1
n
110
120
(mmHg) Fig. 3. RBF autoregulation before (0) and 1 h after (0) infusion of anti-Thy l-l antibodies (top) and before and 1 h after infusion of control serum (bottom). PERFUSION
PRESSURE
rate of Ringer acetate. Clearance determinations were performed in three lo-min periods, i.e., 1) at control perfusion pressure,2) at a pressure10 mmHg above the lower pressure limit of RBF autoregulation, and 3) at a perfusion pressureof 60 mmHg. The animals rested 15 min between each clearance period for achievement of the new steady-state situation with reducedperfusion pressure. Autoregulatory index was calculated according to Semple and dewardener (15).
&
7r
70
~
lb
210
;o
LIO
s’o
s’o
TIME (mid Fig. 2. Renal blood flow (RBF) during and 50 min after infusion of anti-Thy l-l and control serum. $P < 0.001. tP < 0.01. * P < 0.05.
Infusion
of
Antibodies
The anti-Thy l-l antibodieswere infused through a femoral vein catheter. The duration of the infusion was 10 min, and the doseof antibodies (immunoglobulin G) was 1,000pg/2 ml Ringer acetate in all rats. Normal mouseserum was used as control.
temic blood pressure and RBF were recorded continuously during infusion of anti-Thy l-l antibodiesand for the following 50 min; the valueswere read every 5 min, and the averagevalue for each lo-min period was calculated. For measurementof GFR, both ureters were cannulated (PE- Measurement of Glomerular Contraction 10).Thereafter an intravenous infusion of Na-[ ““I]iothalamate Rat glomeruli were isolated by a modification of the method (Amersham Laboratories, Buckinghamshire, UK) in Ringer acetate was started. Na-[ 1251]iothalamate(5 &i) in 2 ml of describedby Krackower and Greenspoon(9). After the glomerRinger acetate solution was given as a priming dose followed uli had been washedin saline, four samplesof a suspensionof by a continuous infusion of 0.5 &i/ml Ringer acetate delivered glomeruli in salinewere incubated for 15min at 37°C asfollows: at a rate of 8 ml/h by a Harvard infusion/withdrawal pump 1) control suspensionof glomeruli, 2) glomeruli + angiotensin (model971, Harvard Apparatus). The animals were allowed to II (low6M) (ll), 3) glomeruli + anti-Thy l-l (10 pg/ml), and rest for 15 min to obtain a steady-state situation. Urine for 4) glomeruli + anti-Thy l-l + angiotensin II. Rat plasma,200 clearancemeasurementswas collected from both ureters using pi/ml, was added to the suspensionbefore incubation as a preweighedglasscapillary tubes. Urine wascollected in lo-min source of complement. After the incubation, 200 ~1 of the periods. The urine volume, determined from weight, was cor- suspensionwas transferred to a Burkner counting chamber, rected for density measured with a gravimeter (Uricon-N, and the diameter of 75 glomeruli were measuredby use of an Atago, Japan). In the middle of each period, 0.05 ml blood was eyepiecemicrometer. The largest diameter of the glomerular sampled from the right femoral artery in heparinized glass tuft in isolated intact glomeruli was measured.All measurecapillary tubes for determination of Hct and counting of 1251. ments were done blindly. lzsIwascounted in duplicate samplesof urine and plasma(1282 Compugamma;LKB-Wallace, Turku, Finland). Clearancewas Measurement of Afferent Arteriolar Diameter (DAJ calculated as [(cpm in urine)/(cpm in plasma)] x urine volume wherecpm is counts per minute. The arterial Hct wasmeasured The animals were prepared for DAA measurementby an every 10 min; stability was obtained by adjusting the infusion intraperitoneal injection of pentobarbital asdescribedabove.A Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
MESANGIUM
Table
AND RBF-GFR
1. Renal vascular resistance during infusion of anti-Thy
F363
AUTOREGULATION
l-l antibodies Time, min
n
0
10
20
30
50
60
1,800
20.4k2.0 24.324.1 NS
20.4f2.0 23.lk3.7 NS
21.4+2.0 17.4zt3.1 NS
40
RVR, ml. mm-‘. mmHg-’ 2O.Ok2.1 20.1*2.0 20.3h1.8 Control 6 20.3k2.0 19.9+1.9 47.5211.3 31.8-cg.O 29.4rt5.3 Anti-Thy l-l 6 20.5zh2.8 184.5&28.2 co.002 NS P NS 0.10) (Fig. 3, mouseserumwere usedas controls. Table 1). As RBF was still slightly but significantly less
Subacutestudy. In sevenrats RBF and GFR autoregulation were examined 30 h after infusion of anti-Thy l-l. In the control group (6 animals), RBF and GFR autoregulation were examined 30 h after infusion of normal mouseserum. DAA measurement.DAA was measuredin the following three groups: 1) six rats 20-30 min after start of anti-Thy l-l
than control (6.1 rt 0.8 vs. 4.9 z!z0.8 ml/min;
P < 0.05),
some of these kidneys might nevertheless have been slightly vasoconstricted (Figs. 1 and 2). One hour after the antibodies were infused, neither the lower pressure limit of RBF autoregulation nor the RVR at this pressure were different from control values [83 f 5 vs. 81 +- 4 infusion, 2) six rats 30 h after infusion of anti-Thy l-l, and 3) six control rats infused with normal mouseserum and studied mmHg (P > 0.10) and 20.4 f 2.0 vs. 23.1 It: 3.7 ml. 30 h after the infusion. min-l . mmHg-‘, respectively] (Fig. 3, Table 1). Thirty hours after the infusion of antibodies, neither MAP, RBF, RVR, nor DAA were different from control Statistical Methods [105 +- 5 vs. 113 + 5 mmHg (P > O.lO), 6.8 f 0.6 vs. 5.9 (P > O.lO), 21.2 + 2.0 vs. 17.4 + 3.1 mlThe results are means+ SE. One-way analysis of variance + 0.5 ml/min (P > O.lO), and 17.8 & 0.8 vs. 17.6 f 0.4 wasperformed amongthe groups,and where significant differ- min-’ -mmHg-’ and the ability to autoreences were found, Student’s t test was used. P c 0.05 was pm (P > O.lO), respectively], gulate RBF was not significantly changed (Fig. 4). The consideredto be statistically significant. Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
MESANGIUM
AND RBF-GFR
AUTOREGULATION
F365
Fig. 8. Electron micrograph (x4,500) showing severe mesangiolysis (*) 30 h after infusion of anti-Thy l-l antibodies.
lower pressure limit of autoregulation was 82 + 5 mmHg in the antibody-infused group vs. 79 I~I 4 mmHg in the control group (P > 0.10). The GFR 30 h after the infusion of anti-Thy l-l antibodies was significantly increased in both kidneys (2.1 + 0.2 for left and 1.7 +- 0.3 for right kidney vs. 1.4 rt 0.2 ml/min for control; PC 0.05). During systemic blood pressure reduction to 92 mmHg, GFR autoregulation in the control group was perfect (autoregulatory index, 0.02 & 0.35). GFR autoregulation was restricted in the antibody-infused group (autoregulatory index both kidneys, 0.71 + 0.42; significantly different from controls, P < 0.05). At pressures below 92 mmHg, GFR autoregulation was absent in both groups (Fig. 4). Contractions of isolated glomeruli during incubation with angiotensin II and anti-Thy l-l antibodies, alone or in combination, are shown in Fig. 5. Angiotensin II reduced the glomerular diameter significantly (176 + 5 vs. 156 + 4 pm; P < 0.001). The calculated volume reduction of the glomeruli was -70%. This effect was abolished after preincubation with anti-Thy l-l antibodies. Light microscopy showed destruction of the intraglomerular mesangial area 30 h after infusion of antibodies (Fig. 6). In some glomeruli, connections between adjacent capillary loops and also fusion of capillaries with aneurysm formation could be seen. No mesangial destruction was observed 60 min after the antibody infusion, with the exception of scattered apoptotic bodies in the mesangial cells. Electron microscopy showed mesangiolysis in some
areas of the glomeruli 1 h after infusion of antibodies (Fig. 7). The afferent arteriole and macula densa were normal. Thirty hours after infusion of antibodies, subtotal mesangiolysis was seen with marked dilation and aneurysm formation of the glomerular capillaries (Fig. 8). The afferent arterioles and the macula densa were also normal after 30 h. DISCUSSION
Intact RBF autoregulation after injection of anti-Thy l-l was the main finding in this study. The abolished glomerular shrinking when anti-Thy l-l antibodies were applied in combination with angiotensin II in vitro, as well as the mesangial cell damage observed by microscopical examination, strongly indicate that active contraction or relaxation of the mesangial cells were absent both in the acute experiments and when RBF and GFR autoregulation were studied 30 h after the antibody infusion. Taken together, this indicates that mesangial cells do not participate in autoregulation of RBF in the rat. Studies of immunologically induced mesangial cell damage by others support the assumption that these cells were nonfunctional after the injection in our study (4). The glomerular damage observed in our study corresponds with that reported in the study by Bagchus et al. (4) where antibodies from the batch applied in the present study were used in a corresponding dose. The antiThy l-l glomerulonephritis has been shown to depend
Downloaded from www.physiology.org/journal/ajprenal by ${individualUser.givenNames} ${individualUser.surname} (129.016.069.049) on January 14, 2019.
F366
MESANGIUM
AND RBF-GFR
on complement activation induced by an antigen-antibody reaction on the mesangial cell membrane (3, 4). Adler al. (1) have demonstrated that complement activation induces production of oxygen radicals in mesangial cells and suggests that these antibodies may cause mesangial cell injury. This damage may thus influence the membrane function necessary for the normal contractile function of the mesangial cells, as observed in the in vitro study, and may also explain the mesangial cell necrosis in vivo. An immediate renal vascular reaction similar to that observed in this study has been observed in the passive Heymann nephritis, another complement-dependent glomerulonephritis with intraglomerular complement activation (14). The role played by the different vascular resistance segments during autoregulation cannot be ascertained without micropuncture. The normal RVR and the normal DA* observed 30 h after the injection indicate, however, that the preglomerular resistances acted normally during autoregulation in these experiments. This was probably the case in the acute experiments as well, because both RVR and RBF were almost normalized before the autoregulation was recorded following the injection. An eventual persistence of the afferent arteriolar constriction, observed during the blood pressure drop 20 min after the injection in these experiments, would be expected to reset the lower pressure limit of RBF autoregulation to the right (7). This implies that the normal lower pressure limit of RBF autoregulation in the acute experiments was not due to resetting of autoregulation to a lower pressure range secondary to changes of the systemic pressure (7). In this study the efferent arteriole is of particular interest, because th-is vessel has been reported to be surrounded by mesangial cells assumed to have a constrictive function (13). Furthermore, the postglomerular resistance has been observed to increase during lowering of the perfusion pressure in some studies (8, 12). Elimination of this postglomerular increase of the vascular resistance would be expected to decrease the lower pressure limit of RBF autoregulation by reducing the minimal total RVR and also to increase the corresponding pressure limit of GFR autoregulation by decreasing the glomerular filtration pressure. The normal RBF autoregulation after the infusion thus argues against a postglomerular effect of anti-Thy l-l, but a minor effect on the postglomerular resistance may explain the effect on GFR autoregulation 30 h after the infusion of anti-Thy l-l antibodies. A similar separation between RBF and GFR autoregulation, as observed in our experiments, has been found in the rat and dog kidney during treatment with saralasin or during converting enzyme inhibition, possibly caused by a postglomerular effect (2). However, the gross changes of glomerular capillary anatomy due to mesangiolysis makes an interpretation of the GFR measurement in the context of autoregulation questionable as long as the filtering area and also the permeability of the capillary wall are unknown.
AUTOREGULATION
In conclusion, the present study indicates that the mesangial cell does not play a decisive part in RBF autoregulation but may in part influence the autoregulation of GFR during pressure reduction. The experimental protocol including stepwise perfusion pressure reduction is supposed to engage both the myogenic and tubuloglomerular feedback mechanisms of vascular resistance control in the kidney. Autoregulation of RBF by these mechanisms thus seems to be independent of mesangial cell function. Address for reprint requests: B. M. Iversen, Medical Dept., Haukeland Hospital, N-5021 Bergen, Norway. Received 7 January 1991; accepted in final form 1 October 1991. REFERENCES 1. Adler, S., P. J. Baker, R. J. Johnson, R. F. Ochi, P. Pritzi, and W. G. Couser. Complement membrane attack complex stim-
ulates production of reactive oxygen metabolites by cultured rat mesangial cells. J. Clin. Invest. 77: 762-767, 1986. 2. Arendshorst, W. J., and W. F. Finn. Renal hemodynamics in the rat before and during inhibition of angiotensin II. Am. J. Physiol. 233 (Rend Fluid Electrolyte Physiol. 2): F290-F297,1977. 3. Bagchus, Bakker.
W. M.,
P. J. Hoedemaeker,
J. Rozing,
and
W. W.
Acute glomerulo-nephritis after intravenous injection of monoclonal anti-thymocyte antibodies in the rat. Immunol. L&t.
12: 109-110,1986. 4. Bagchus, W. M., P. J. Hoedemaeker, Bakker. Glomerulonephritis induced
antibodies. A sequential histological the rat. Lab. Invest. 55: 680-687, 1986.
J. Rozing,
and
W. W.
by monoclonal anti-Thy 1.1 and ultrastructural study in
K. J., and K. Aukland. Interlobular arterial resistance: influence of renal arterial pressure and angiotensin II. Kidney
5. Heyeraas,
Int. 31: 1291-1298,1987. 6. Iversen, B. M., L. Merkrid,
and J. Ofstad. Afferent arteriolar diameter in DOCA-salt and two-kidney one-clip hypertensive rats.
Am. J. Physiol. 245 (Renal F762,1983. 7. Iversen, B. M., I. Sekse,
blood flow autoregulation J. Physiol. 1987. 8. Klllskog,
252 (Rend
Fluid
Electrolyte
Physiol.
14): F755-
and J. Ofstad. Resetting of renal in spontaneously hypertensive rats. Am.
Fluid
Electrolyte
G., L. 0. Lindbom,
Physiol.
H. Ulfendahl,
21): F480-F486, and M. Wolgast.
Hydrostatic pressures within the vascular structures in the rat kidney. Pfluegers Arch. 363: 205-210, 1976. 9. Krackower, C. A., and S. A. Greenspoon. Localization of the nephrotoxic antigen within the isolated renal glomerulus. Arch. Puthol. 10. Markrid,
51: 629-633,1951. L., J. Ofstad,
and
Y.
Effect of steric of microspheres in the
Willassen.
restriction of the intracortical distribution dog kidney. Circ. Res. 39: 608-615, 1976.
J., B. M. Iversen, L. Msrkrid, and I. Sekse. Autoregulation of renal blood flow (RBF) with and without participation of afferent arterioles. Actu Physiol. &and. 130: 25-32, 1987.
11. Ofstad,
12. Robertson, C. R., W. M. Deen, J .L. Troy, and B. M. Brenner. Dynamics of glomerular ultrafiltration in the rat. III. Hemodynamics and autoregulation. Am. J. Physiol. 223: 1191-1200,1972. 13. Schnabel, E., W. Kriz, and M. Steinhausen. Outflow segment
of the efferent arteriole of the rat glomerulus investigated by in vivo and electron microscopy. Rend Physiol. 10: 318-326, 1987. 14. Sekse, I., B. M. Iversen, R. Matre, and J. Ofstad. The acute effect of passive Heymann nephritis on renal blood flow and glomerular filtration rate in rats. Nephron 53: 364-372, 1989. 15. Semple, S. J. G., and H. E. DeWardener. Effect of increased renal venous pressure on circulatory autoregulation of isolated dog kidneys. Circ. Res. 7: 643-648, 1959. 16. Sraer, J. D., J. Sraer, R. Ardaillou, and 0. Mimoune. Evidence of renal glomerular receptors for angiotensin II. Kidney Int. 6: 241-246,1974.
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