capillary permeability to albumin in normotensive and spontaneously hypertensive rats

Acta physiol. scand. 1977. 101. 72-83 F r o m the Department of Physiology, University of Goteborg, Sweden

Capillary Permeability to Albumin in Normotensive and Spontaneously Hypertensive Rats

BY BENGT RIPPE and BJORN FOLKOW

Received 14 March 1917

Abstract

RIPPE, B. and B. FOLKOW. Capillary permeability to albumin in normotensive and spontaneously hypertensive rats. Acta physiol. scand. 1977. 101. 72-83.

Transcapillary passage of plasma proteins is enhanced in man’s primary hypertension and it is debated whether this reflects increased permeability or merely a raised capillary pressure. To elucidate this problem, maximally vasodilated hindquarters of spontaneously hypertensive rats (SHR) and normotensive controls (NCR) were perfused in parallel at constant flow with dextran, horse serum or mixtures of the two, using labelled albumin as indicator of capillary permeability to macromolecules. By equal increases of venous pressure modest filtration was maintained during one hour, after which the edema and its albumin content were determined. - There was less edema in SHR, reflecting a slightly lower postcapillary resistance and a much higher precapillary resistance compared with NCR, which here resulted in a lower capillary pressure in SHR. In both SHR and NCR the presence of dextran slightly enhanced the capillary filtration coefficient but increased albumin permeability up to tenfold, also after antihistamine drugs. However, for each perfusate the SHR capillaries were, if anything, slightly less permeable to albumin than the NCR ones. -The results suggest that the enhanced transcapillary passage of plasma proteins in primary hypertension reflects an increased capillary pressure in some circuit(s), probably mainly skeletal muscle, resulting from the functional balance in uiuo between the pre- and postcapillary resistances.

Quantitative studies concerning design and principal hemodynamic characteristics of the different series-coupled vascular sections have been sparse in primary hypertension, particularly when compared to the situation in normotension. However, such a study was recently performed by Folkow et al. (1974) in spontaneously hypertensive rats (SHR; Okamoto 1969), in comparison to normotensive control rats (NCR). The results showed that the structural increase in wall/lumen ratio, generally characterizing large arteries and resistance vessels in primary as well as secondary hypertension (cf. Folkow et al. 1973), is largely confined to the precapillary vessels, at least in the hindquarter section of the systemic circulation. Thus, the capillary section in the preparation used seemed to be essentially unchanged in SHR, insofar as the “isogravimetric” capillary pressure and also the capillary filtration coefficient (CFC) values were the same as in NCR at maximal dilatation. The postcapillary resistance vessels appeared to be, if anything, structurally slightly wider in the SHR than in the NCR hindquarter vascular bed and they exhibited no sign of any “hyperreactivity”

12

CAPILLARY PERMEABILITY TO ALBUMIN IN NCR AND SHR 73

to constrictor or dilator agents, a response which characterizes the precapillary vessels (Folkow et al. 1974). Such observations indicate that the increased precapillary resistance in hypertension “protects” the exchange and postcapillary venous vessels from the impact of the increased arterial pressure, though the situation may later change if e.g. a failing heart leads to an increased central venous pressure which is transmitted in the retrograde direction.

It was, however, recently discussed by Parving (1975) whether capillary permeability might be increased already in early phases of essential hypertension in man, in respects that are not necessarily reflected in CFC measurements. This proposal was mainly based on the fact that already in early essential hypertension there occurs an increased transcapillary escape rate (TER) of intravenously injected labelled albumin (cf. Parving and Gyntelberg 1973, Ulrych 1973). Capillary permeability is, however, only one of the factors determining the rate of transcapillary exchange of plasma proteins, another important factor being the level of capillary pressure. Thus, there is good evidence that elevations of capillary hydrostatic pressure increase the net protein escape across the exchange vessels (e.g. Landis et al. 1932, Szabo, Magyar and Papp 1963, Haddy, Scott and Grega 1972, Lassen, Parving and Rossing 1974).

For such reasons the increase of TER, observed in subjects with essential hypertension, may simply be due to a raised capillary pressure. Ulrych (1973) has, for example, discussed whether an increased TER might ensue as a result of vasoconstriction particularly involving the venous side. However, it is in this connection of considerable relevance that muscle blood flow is commonly enhanced in essential hypertension (cf. Pickering 1968), particularly in its early “hyperkinetic” phases (cf. Julius and Shork 1971) simulating the hemodynamics in very light exercise or, perhaps better, in a mild defence reaction (cf. Folkow and Neil 1971, Julius and Esler 1975). In such situations the precapillary resistance of the muscle circuit is lowered compared with most other circuits, while venous capacitance tone is, if anything, increased. Particularly when associated with an increased arterial pressure, this must imply a substantial enhancement of capillary pressure in skeletal muscle tissue. Inevitably the Starling equilibrium in this large tissue mass is then shifted towards net filtration and with this follows an increased capillary transfer of plasma proteins, as during normal exercise. A quite recent study (Rippe, Kamiya and Folkow 1977) provides strong evidence against the often proposed view that macromolecules should mainly pass capillary walls by way of micropinocytosis in such situations.

In experiments on man it is very difficult to distinguish clearly between e.g. a slight deviation in the capillary permeability-surface characteristics (PS product) and one in mean capillary pressure, particularly when comparing mild, uncomplicated cases of essential hypertension with normotensive controls. Therefore, if primary hypertension really involves a deviation in capillary PS characteristics, the chances of revealing such a change would be better if animal models of primary hypertension are utilized instead. Since this problem is one of principal interest in the evaluation of hypertension in general, it was considered worthwhile to perform a quantitative comparison between SHR and NCR hindquarters with regard to their capillary permeability to proteins under strictly controlled experimental conditions. This was done, keeping the entire capillary bed open to flow while maintaining other hernodynamic factors as equal and constant as possible during paired SHR-NCR

74 BENGT RIPPE AND BJORN FOLKOW

perfusions with plasma and different plasma substitutes, containing labelled albumin as a tracer of transcapillary protein transfer.

Methods

Technically successful experiments were performed on 29 pairs of Wistar spontaneously hypertensive rats (SHR; Okamoto 1969) and weight matched Wistar normotensive control rats (NCR). All rats were of male sex and weighed between 290-410 g. The majority of them were 6-9 months old and hence in relatively early or medium phases of “established” hypertension. The isolated hindquarters of one SHR and one weight matched NCR were simultaneously perfused at constant rate and at essentially equal flows. Venous outflow pressures were moderately elevated but to an equal extent in the two animals, to induce a suitable capillary filtration and consequent transfer of plasma proteins.

Preparation: Initially mean arterial blood pressure (PA) was measured in the awake experimental animal uia a polyethylene cannula, inserted into the tail artery during brief ether anesthesia, utilized for pressure recordings throughout the experiment uia a Statham P23 AC transducer, writing on a Grass Polygraph. Afterwards the animals were anesthetized with Nembutal@, 3 mg/100 g administered into the tail artery cannula, simultaneously evicerated and the abdominal aorta and caval vein were freed between the renal and iliolumbar vessels. The hindquarters were otherwise completely isolated by standardized mass ligatures a t identical levels just proximal to the iliolumbar vessels, and the tail distal to the tail artery cannula and the paws were excluded so that the hindquarter preparation mainly consisted of skeletal muscle (cf. Folkow et al. 1970).

After heparinization the abdominal aortas in both rats were ligated, immediately cannulated in the distal direction and connected to a common perfusion system which was already running. The two caval veins were then ligated, immediately cannulated and connected to 30 cm long outflow tubes, after which the animals were killed. The pressure drop along the two venous outflow tubes was equal and about 5 mmHg a t a perfusion rate of 10 ml/min. Their outflow tips could be raised or lowered about 25 cm and to assure that venous outflow pressures of the two preparations were always identical the tips were linked together.

To induce moderate filtration, venous outflow pressures (Pv) were elevated to an identical extent in the two preparations, usually from 6 up to 18 mmHg. In some of the paired experiments venous pressures just proximal to the outflow tubes were measured directly oia cannulae in venous side branches and found to be identical at given levels of outflow pressure. The artificial perfusion system utilized was common to both animals, as described in detail earlier (e.g. Folkow et al. 1970).

Perfusates: It is known since long that perfusion of vascular beds with e.g. dextran affects capillary membrane characteristics, considered to be mainly due to the absence of native proteins (rf. Landis and Pappen- heimer 1963, Areekul 1969). To investigate the problem further in these comparisons of normotensive and hypertensive vascular beds, 4 different perfusates were employed, uiz. horse serum (Normal Serum, SBL, Sweden), dextran (Macrodex@-70; MV about 70.000; generously supplied by AB Pharmacia, Sweden) and two different mixtures of dextran and horse serum. All colloids were mixed with Tyrode solution and the perfusates were kept a t 38°C and oxygenated. Because of the differences in pedusate the animals were divided into 4 groups:

In group Z six matched pairs of NCR-SHR were perfused with horse serum (containing 65 g of protein per liter), diluted with Tyrode to 60 per cent of its initial concentration, i.e. a 3.9 per cent content of protein.

In group ZZeight matched pairs of NCR-SHR were perfused with 4% dextran in Tyrode. In group ZZZ eight matched pairs of NCR-SHR were perfused with dextran in Tyrode of the same con-

centration as in group 11, but with addition of 5 g/l of horse serum protein. In group ZV seven matched pairs of NCR-SHR were perfused with a mixture of dextran-Tyrode (40

g/1) and native horse serum, in the proportions of 2 : 1. The final dextran concentration was then 2.7% and the protein concentration about 2 %.

“Control” stage of perfusion: The vascular beds were initially “flushed” for 2-3 rnin by adjusting the pump to deliver 15-20 ml/min x 100 g tissue in order to wash out all traces of blood. To induce maximal dilatation, repeated doses of papaverine, 1-2 mg at a time, were given to both hindquarters via a common mixing chamber in the perfusion system until no further pressure drop occurred. This state of maximal dilatation was kept stable throughout by repeated papaverine injections or papaverine infusion.

During the subsequent 10-15 min flow was kept low, a t 3-5 ml/min x 100 g tissue, while venous outflow pressure was near to zero, as indicated by the readily visible collapse of both venous trees. The hindquarter capillary bed is then slightly absorbing, as observed earlier (Folkow et a / . 1974) and confirmed in experi-

CAPILLARY PERMEABILITY TO ALBUMIN IN NCR A N D SHR 75

ments where the hindquarters were continuously weighed. This was done to avoid initial edema formation so that the preparations would preferably have a slightly reduced interstitial fluid space. At the end of this period, flows were increased identically and varied rapidly during a few minutes in order to obtain data for constructing a flow-pressure curve for each preparation, which better characterizes the dimensions of their resistance vessels. Some filtration was thereby induced, more or less restoring the initial fluid balance, as shown from experiments where the hindquarters were continuously weighed.

Perfusion with tracer: After these initial stages of perfusion, tracer quantities (2 x 1 OW5 g/l) of lZ5J-Human Serum Albumin (Radiochemical Center, Amersham England; y-radiation energy 0.035 MeV) were added to the perfusate while the two venous outflow cannulae were raised identically to induce filtration. Flow was then kept around 9 ml/min x 100 tissue and equal in the two preparations. This period of perfusion with addition of labelled protein to the perfusate was continued for 60 min, the venous effluents being collected and, after warming and reoxygenation, recirculated once or twice. Arterial pressure was continuously checked and it was ensured that maximal vasodilatation was throughout maintained in both preparations. Thus, the entire capillary surface area was exposed to flow throughout and at moderately raised pressure levels because of the increased venous outflow pressure.

The specific activity of the perfusate was determined repeatedly (see below) and its radioactivity fell about 2-3 :h in 1 h because of the recirculations. Non-protein bound radioactivity of the perfusate was determined before and after the perfusion period as specific activity of the supernatant, following precipita- tion of the perfusate proteins with 10 ’% trichloroacetic acid. It varied from 1.0-1.8 per cent of total perfusate radioactivity but did not change in the course of a given experiment.

After about 60 min of perfusion with the tracer-containing perfusate, a rapid shift to perfusion with a tracer-free, but in other respects identical perfusate was performed. This tracer-free perfusion was stopped when the specific activity of the venous effluent was well below 3 per cent of the initial activity, which usually occurred after 7-8 min. During this final tracer-free perfusion period, the venous outflow cannulas were lowered to the level of the animals in order to minimize further filtration.

Determination of tracer transfer: After stopping the tracer-free perfusion, three separate muscle groups from each of the two hindquarters were dissected out bilaterally, namely 1) the anterior crural group (m tibialis ant., m. extensor dig. longis and m. extensor hallucis proprius); 2) m. gastrocnemius and soleus; 3) the larger part of m. quadriceps. Immediately after dissection each muscle preparation was put into a test tube. After sealing and weighing, the tube was put into a well counter, connected to a Packard Auto- gamma Spectrometer (Model 410 A), for determination of radioactivity by counting a minimum of 10 000 imp. Each muscle was then completely dried at 90-100°C for 72 h and reweighed.

Wet weights (W,) and dry weights (D) of corresponding muscle groups from 6 NCR and 6 SHR, not used for expts., were determined separately as controls. The SHR and NCR muscles did not differ mutually concerning the relationship of dry weight/wet weight. The following normal values for the various muscle groups, expressed as per cent of their wet weights ((D/W,) x loo), were determined and used as controls: 1) the anterior crural muscle group 24.7 per cent, 2) m. gastrocnemius-soleus 24.2 per cent, 3) m. quadriceps 23.6 per cent. Knowing these percentages ((D/W,) x 100) for muscles from non-perfused “control” animals, together with the dry weights and wet weights after perfusion (W,) from the muscles of the experimental groups, the edema content (W,-W,) of the “experimental” muscles, expressed in g per 100 g of tissue, could be determined. Muscle radioactivity, corrected for background activity, was then divided by the edema content (W,-W,) and by the specific activity of the perfusion medium. Correction for free iodine activity was not deemed necessary because the free iodine is supposed to be distributed extracellularly and should thus easily be washed out during the 8 min of final tracerfree perfusion.

In this way the ratio between the albumin concentration of the newly formed “edema fluid” (C,) and that of the perfusate (“plasma”) (C,) was obtained for every muscle group and animal. All the experiments, especially those of group I , were performed under conditions of moderately enhanced capillary filtration. The purpose was to create a situation in which the transcapillary albumin flux was largely filtration dependent, while absorption and net diffusion could be neglected (concerning micropinocytosis (see Rippe, Kamiya and Folkow 1977). Furthermore, escape by means of lymphatic flow could also be ignored since essentially all lymphatic vessels were blocked by ligation during preparation. The newly formed edema must then be largely equal to the filtrate formed during the experimental period.

Under these conditions the filtrate/plasma concentration ratio of albumin, C,/C,, will approach A,/A,, i.c. the ratio of “effective” pore area available to albumin to that of water. The same deduction can be made from the relation developed by Pappenheimer (1953) C,/C, = (PS + FAsIAw)/(PS + F), where C,/C, is the filtrate/ plasma concentration ratio and PS the diffusion capacity for the macro-molecule, while F is the filtration rate. At high filtration rates C,/C, will be a good approximation of A,/A,, and thus fairly

76 BENGT RIPPE AND B J ~ R N FOLKOW

“CLEARANCE” ml pwfusate/min

M r,,,=osa(n-a~~ P<OOOI , 11 .......... 0 ,

rNcR=061 (n.161 p<005

rSHR=052(n;161 p<005 ,’

0.5

00

- 10%

~ 0%

r I I I , , , , , , . 0.0 0 5 10

FILTRATION RATE, ml/min x lOOg

Fig. 1. Relationship between perfusate clearance of albumin and filtration rate for NCR and SHR in groups I (circles: 60% horse serum and 40% Tyrode) and I1 (squares: 4 % dextran in Tyrode). As seen, dextran perfusion implies a tenfold increase in albumin clearance but without any appreciable difference NCR-SHR. Further, for each of the perfusates shown in the Fig. C,/C, remains essentially constant independent of the filtration rates used, which was also the case for the other two perfusates (groups 111 and IV). - Each point represents one out of three duplicate determinations per animal, performed on three different muscle groups which varied somewhat concerning extent of edema formation.

constant. In the present experiments C,/C, could thus be used as a measure of capillary permeability to albumin. At the prevailing filtration rates there was namely a good correlation between the “plasma” clearance of albumin and filtration rate in all groups, i.e. an almost constant C,/C, (“A,/A,) was obtained.

Statistical evaluation was performed by the pairing design t-test, mean values of the six determinations of C,/C, in every SHR being compared with the corresponding NCR values. To obtain comparison between the differently perfused groups, the groups comparison t-test was utilized. At p values below 0.05 the differences were considered significant.

Results

A . Filtrate to “plasma” albumin concentration ratios (C,/C,)

1. C,l C, at different filtration rates and different “plasma” compositions: A good correlation was found between the rate of edema formation and net transcapillary outflux of albumin in all animal groups. Independent of the filtration rate (which varied between 0.1-1.0 ml/min x 100 g depending on the venous pressure level as related to the respective levels of isogravimetric capillary pressure for the different perfusates) the concentration of protein in the filtrate remained almost constant for a given perfusate. This was evident by the fact that the concentration of tracer albumin in the filtrate us. that in the perfusate did not change appreciably with increasing filtration, and this was so for both NCR and SHR (Fig. 1).


60% Horse Dextran(4Og/l) Dextran(4Og/l) 67% Dextran +05% horse +33% horse serum proteins serum ( n s )

1

g 2 0 0 +I +I

0 0 0 0

$ 2

I II

n = 7 pairs ( p < O 05)

n.

Ip

Fig. 2. Filtrate to perfusate concentration ratios of labelled albumin (C,/C,) in NCR and SHR during perfusion with diluted horse serum (group 1) or dextran-containing perfusates (groups 11-IV; see text). Each observation on a given animal represents a mean value from three duplicate determinations; for every paired group of experimental animals mean+S.E. are given in the figure. - Note that C&, increases markedly whenever the perfusate contains dextran but that the SHR capillaries are, if anything, less permeable to protein than the NCR ones for each perfusate used.

During perfusion with horse serum-Tyrode (group I) the percentage of tracer protein in filtrate versus “plasma” was as low as 5-7% in both NCR and SHR (see also Fig. 2). When 40 g/1 of dextran (Macrodex@’) in Tyrode was employed as perfusate (group ll), CF/Cp increased tenfold, being 0.70-0.75 in both NCR-SHR, but if small amounts of horse serum proteins (5 g/l) were added to this dextran perfusate (group 111), CF/Cp decreased to 0.45-0.5. However, as long as dextran was present no further reduction of CF/Cp was seen when the protein concentration was increased up to 20 g/1 (group IV).

2. C,/C, in SHR compared with NCR: Mean values of CF/Cp in the six SHR hindquarters perfused with horse serum (group I) was 0.053 1-0.008 compared with 0.067 k0.013 in the 6 matched NCR (Fig. 2), values which closely agree with findings in blood perfused cat muscle (Appelgren, Jacobson and Kjellmer 1966). Although NCR here showed a somewhat higher CF/CP than SHR in 5 expts. out of 6, the difference was not statistically significant. Anyway it seems safe to conclude that SHR does not show any higher albumin permeability thanNCR. The results are in agreement with earlier findings concerning identical capillary filtration coefficients in SHR and NCR hindquarters at maximal dilatation (Folkow et al. 1974), confirmed in other, more recent measurements (unpublished).

During perfusion with dextran alone (group 11) CF/Cp increased, as mentioned, about tenfold in both NCR and SHR, and this was so also in preparations given large amounts of an antihistaminic agent. However, under these conditions of a very marked increase in capillary membrane permeability to albumin, CF/CP was identical in SHR and NCR being 0.72 1-0.05 (mean 1-S.E.) in both. After addition of proteins to the dextran perfusate, first 5 g/1 (group 111) and then 20 g/1 (group IV) mean C,/C, values were reduced to 0.51 F0.04 in NCR and to 0.43 k0.03 in SHR (15 pairs) with no significant differences between groups

78 BENGT RTPPE AND BJORN FOLKOW

EDEMA,

tissue x h. g / l0Og

30.

20

IO-

60% Horse Dexfran(4Og/l) Dextran(4Og/l) 67% Dextron serum t05% horse +33% horse

(n.s.) n - 8 pairs n = 7 pairs

n.6pairs n=8pai rs (p<o,ol) serum proteins serum

1 (p<0.05) ( p < O 0 5 )

I II m

I T

Fig. 3. Amount of edema (mean* S.E.) formed during one hour of perfusion with the four perfusates used. Although capillary pressure was kept largely equal in all groups, 17-18 mmHg in NCR, effective filtration pressure varied between the four groups due to differences in colloid osmotic pressure; further, CFC also differed modestly with the perfusate (see text). In NCR effective filtration pressure was estimated to 10-11 mmHg in group I, and some 9, 7 and 5 mmHg, respectively, in groups 11, 111, IV, the corresponding values being 1-2 mm lower in SHR.

I11 and IV. However, here the slightly lower values in SHR differ significantly from those in NCR (Fig. 2).

B. Edema formation

1. Differences in edema formation rate in the various groups: The edema formation during 1 h of tracer perfusion varied significantly between the 4 groups (Fig. 3). The explanation is partly that effective filtration pressure, i.e. the difference between the prevailing mean hydrostatic pressure in the capillaries (Pc) and the isogravimetric one (Pci), differed between the groups depending on the perfusates used.

In NCR mean Pc was estimated to 17.710.4 mmHg during the period of raised Pv in the majority of experiments, as calculated from the prevailing PA and P, values and from the pre- to postcapillary resistance ratio (which at these distending pressures is about 3 : 1 in NCR, according to Fig. 1 in the paper by Folkow et al. 1974). This Pc value during the filtration period did not differ significantly in the various groups of NCR, as tested with variance analysis. The same calculations for SHR in the various groups (where the P, increases were always identical to the paired NCR and where the prevailing pre- to postcapillary resistance ratio is rather about 5 : 1 according to Folkow et al. 1974) resulted in a mean value for Pc of 16.6 k0.4 mmHg at the prevailing PA and P, levels. This Pc level in SHR was significantly lower than that in NCR (p <0.001), and the results by Folkow et al. 1974, as well as unpublished results with the isogravirnetriCtechnique are, in agreement with a true difference in Pc between NCR and SHR when P, and flow are equal.

From these Pc values the effective filtration pressures can be approximately estimated for the four groups of NCR and SHR. To start with group I1 (4% dextran) the Pci value is here


around 9 mmHg for both NCR and SHR, as judged from unpublished “isogravimetric” estimations using the dextran perfusate. This gives an effective filtration pressure of about 9 mmHg in NCR with the SHR value being 1 or 2 mmHg lower, which at least in part explains the different edema amounts (Fig. 3).

In group 111 (4% dextran +0.5% horse serum proteins) the edema formation was some 30-35 % lower than in group 11. This is likely to reflect the moderately higher colloid osmotic pressure of this perfusate, in association with some reduction in capillary permeability as caused by the protein addition, and independent CFC measurements show this. Hcwever, the CFC reduction is only of the order of 20-25 %, where the raised colloid osmotic pressure is likely to account for the rest of the difference between groups I1 and 111. A proper fit with the present data is obtained if the 0.5 % protein addition raised Pci from 9 to about 1 1 mmHg. If so the effective filtration pressures in group 111 would amount to about 7 mmHg in NCR and 6 mmHg in SHR (Fig. 3).

In group IV (2.7 % dextran +2% horse serum proteins) the edema formation again decreased significantly, presumably due to a further increase in colloid osmotic pressure of the perfusate. 4% dextran exerts a colloid osmotic pressure in the skeletal muscle vascular bed on only some 16 mmHg (Eliassen et al. 1974), and 2.7 per cent dextran mixed with 2% plasma proteins, which per unit weight exerts about the same colloid osmotic pressure in uivo as dextran, must imply a colloid osmotic pressure that is perhaps 2.5-3 mmHg higher than for 4 % dextran. Consequently the effective filtration pressures would here be in the order of 4-5 mmHg in NCR and 2-3 mmHg in SHR.

Finally, in group I (3.9% horse serum proteins) the colloid osmotic pressure is bound to be a few mmHg below that in group 11, being about 14-15 mmHg and resulting in a Pci around 8 mmHg for both NCR and SHR. The effective filtration pressure in NCR would then be 10-11 mmHg and 1-2 mmHg lower in SHR, i.e. somewhat higher than for group 11. However, CFC is lower (20-25%) for horse serum than for dextran, as mentioned above, so the rate of edema formation would be expected to be fairly equal in groups I and 11, as also shown in Fig. 3. - These estimations of differences in net filtration pressures with the various perfusates are admittedly approximate but are likely to be realistic as to their general order. They are necessary to explain the differences in edema formation between the four groups of experiments and also for the comparison between SHR and NCR.

2. Amount of edema in SHR and NCR: Although the paired SHR-NCR hindquarters were always exposed to identical venous pressures, the SHR hindquarters showed throughout a lower amount of edema formation than NCR during the 60 min of perfusion, as shown in Fig. 3. Though this difference was quite small and not significant in group I, it was more substantial in the dextran group I (p <0.01) and in groups I11 and IV (p <0.05) where dextran and horse serum were mixed. These differences can hardly be ascribed to any differences in CFC, since SHR and NCR here show equal values (Folkow et a/ . 1974; unpublished observations). They rather suggest first a lower mean hydrostatic pressure in SHR than in NCR as a result of differences in both pre- and postcapillary resistances (cj. Folkow et al. 1974). Second, there might be a slightly smaller leakage of macromolecules to the interstitial space in SHR (see Fig. 2) which, if anything, would maintain a somewhat higher “effective” colloid osmotic pressure in the SHR capillaries. In any case, there are

80 BENGT RIPPE AND BJORN FOLKOW

certainly no signs of any increased capillary permeability in SHR compared with NCR in the present results.

Discussion

As outlined in Introduction, studies on man indicate an increased transcapillary exchange rate of albumin (TER) in essential hypertension (Parving and Gyntelberg 1973, Ulrych 1973, Parving 1975). Whether this increased turnover rate is due to an altered capillary permeability to macromolecules in general or simply caused by an increased mean capillary pressure in some major systemic circuits, or to a combination of both, has not been analyzed in detail in hypertensive subjects. Parving has discussed the former alternative, while Ulrych seems to be more inclined towards the latter view.

It is of principal interest both concerning which type of early vascular changes that occur in primary hypertension and for the correct analysis of the hemodynamic situation in this disorder, to know whether also the capillary exchange vessels proper exhibit any early alterations in permeability. It is, for example, theoretically possible that the genetically linked deviations in cardiovascular design and function might involve a “primary” deviation also in this respect and/or that “secondary” changes may occur early in the deve- lopment.

Against such a background the present investigation was performed to study selectively albumin permeability and rate of edema formation during induced filtration in the artificially perfused, isolated and maximally dilated hindquarter vascular beds of spontaneously hypertensive and normotensive control rats (SHR, NCR). The essential result was that there are no signs of any increased capillary permeability to albumin in SHR compared with NCR, nor do they differ in capillary filtration coefficient (cf. Folkow et al. 1974). If anything, the SHR hindquarter vascular bed seems to show a slightly lower albumin permeability than that of NCR. Thus, there is no evidence of any “primary” increase of capillary permeability in this type of primary hypertension.

The capillary exchange vessels might as mentioned, also display a secondary increase in permeability, e.g. if they are in the course of hypertension frequently exposed to local pressure rises. However, such a possibility also seems to be excluded by the present findings in SHR, being in the phase of fairly well “established” hypertension. The main reason is probably the fact that the increased resistance in established hypertension is largely confined to the precapillary vessels (Folkow et al. 1974), which tends to “protect” the capillaries from pressure elevations except when the precapillary resistance for functional reasons is relatively reduced. However, even when this is the case, as normally occurs in the muscle circuit during exercise or defence reactions, the capillary membranes prove to be quite resistant, at least as long as such pressure rises are within the “physiological” range. Perhaps the most drastic example is the situation normally facing the lower limb capillaries of man while erect, though precapillary autoregulatory responses, reflex adjustments and the venous “pump” will also here protect the exchange vessels from undue pressure rises.

This by no means denies that acute, marked pressure increases may overcome the precapillary counterregulatory mechanisms, causing vascular distension, wall edema and also gross capillary leakage (Giese 1964a, b), a situation evidently not seldom met with in

CAPILLARY PERMEABlLlTY TO ALBUMIN IN NCR AND SHR 81

hypertensive crises with serious consequences particularly for the brain (e.g. Johansson 1974). The precapillary autoregulatory response then yields to the overwhelming rise in wall tension, thereby transmitting the greatly increased transmural pressure to the exchange vessels. However, the structural precapillary changes typical of hypertension here offer a relative protection, since their stronger media does not yield as easily as that of normotensive vessels (cj, Hallback, Lundgren and Weiss 1974, Johansson 1975).

In any case, the present experiments on an animal model of man’s essential hypertension provides no evidence of either any “primary” or “secondary” increases in capillary permeability in SHR, whether in terms of protein permeability or CFC (Folkow et al. 1974). It is then likely that the increased TER in essential hypertension (Ulrych 1973, Parving 1975) reflects a hemodynamic situation where instead mean capillary pressure is somewhat raised in at least some of the major systemic circuits. In fact, also young SHR show a tendency of an increased TER in the “resting” awake steady state (Rippe, Lundin and Folkow 1977). As mentioned in Introduction, muscle blood flow is commonly increased in essential hypertension and particularly so in early, “hyperkinetic” stages (cf. Julius and Shork 1974), when the hemodynamic pattern simulates that of a mild defence reaction or very light exercise The muscle vasodilatation in these situations is mainly precapillary in nature, which inevitably implies a raised capillary pressure, and therefore an increased formation of filtrate. With this follows an increased transcapillary albumin escape, apparently mainly by means of increased filtration through large pores (bulk flow), while capillary permeability per se

in all likelihood remains unchanged (cf. Arturson and Kjellmer 1964). A concomitant presence of a raised central venous pressure due to e.g. venoconstriction will, of course, further tend to enhance the filtration by a retrograde effect on the pressure level in the microvessels.

The present experiments further showed a less pronounced edema formation in SHR compared with NCR (Fig. 3), despite largely equal CFC values and isogravimetric capillary pressures (Folkow et al. 1974). Filtrate to plasma concentration ratios of albumin (C,/C,) were in most experiments slightly reduced in SHR compared to NCR (about 15 %). Therefore capillary permeability to macromolecules is, if anything, lower in SHR than in NCR. As a result the total outflux of labelled albumin into the SHR hindquarter interstitial fluid space was up to some 35% lower in SHR. This indicates that effective filtration pressure must have been throughout lower in SHR than in NCR, in extent varying with the perfusates used as outlined in Fig. 3 and essentially resulting from a different balance between pre- and postcapillary resistances in SHR, since venous outflow pressure and flow were equal. At maximal vasodilatation the SHR hindquarter vascular bed displays a substantially higher precapillary resistance than the NCR one (Folkow et al. 1974), but had this been the only difference at equal flows mean capillary pressure would have also been equal. Therefore, both the mentioned study and the present results suggest that postcapillary resistance of the hindquarter vascular bed is slightly lower in SHR than in NCR during complete vascular relaxation. However, in the in vivo situation, the superimposed neurogenic-myogenic smooth muscle tone may, of course, readjust the hemodynamic situation over a wide range when necessary.

The drastic increases in CF/Cp caused by dextran and only moderately reversed by plasma proteins in the perfusate were in these experiments utilized for comparing the permeability

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82 BENGT RIPPE AND B J ~ R N FOLKOW

properties of the capillaries over a wide range of situations. The nature of these changes in permeability is, however, per se of interest and worthy of a brief comment already here, though the problem will he dealt with in a subsequent study, designed to compare simultaneously the filtration and diffusion events (cf. Rippe and Stage 1976). The decreased capillary selectivity for macromolecules induced by dextrans has earlier been studied by Areekul in the perfused rabbit ear. At blood perfusion the reflection coefficient for the plasma colloids was 0.94 while it was only 0.3 at perfusion with 3 % dextran concentration (mol. w. around 80 OOO), increasing to around 0.5 after adding 5 g/l of pig plasma proteins to the perfusate. These values can be roughly transformed so as to correspond to the presently used sieve coefficients (C,/C,) by being subtracted from 1, and they are then closely similar to the C,/Cp values of the present study (cf. Fig. 2.). Further, preliminary results on the isolated rat hindquarter preparation show that CFC increases some 35% by shifting from horse serum perfusion to 4% dextran perfusion, i.e. from around 0.035 ml/min x 100 g (cf. Renkin and Zaun 1955) to around 0.048 ml/min x 100 g at dextran perfusion at complete vascular relaxation. These changes in permeability to pore-bound water filtration are thus small compared with the nearly tenfold changes in large molecular permeability (0.06 compared with 0.70).

On the basis of CFC determinations it has been claimed that the permeability changes induced by dextran can be almost completely reversed by the addition of proteins to the perfusate (cf. Landis and Pappenheimer 1963). In the present study the addition of even small amounts of proteins to the dextran perfusate considerably reduced CFC, but the albumin concentration in the filtrate remained fairly high even if substantial additions of plasma protein were given. Thus, the presence of dextran molecules seems to more strongly enhance capillary permeability to large molecules than that determining the filtration of water, but we will, as mentioned, come back to these problems in another study.

The mechanisms behind these actions of dextran on the capillary membrane are not understood (cf. Areekul 1969) but the influence on permeability to colloids shows some rela- tionships to that exerted by e.g. histamin or bradykinin. Although dextran is known to release histamin from mast cells in rats, infusion of even huge doses of the anithistaminic drug clemastin (TavegyP, Sandoz) in four experiments did not affect the C,/Cp values significantly. Further, in rabbits histamin is evidently not released in response to dextran, and still the present data closely simulate those found in rabbits (Areekul 1969), implying that histamin is not likely to mediate the mentioned permeability changes induced by dextran. It might represent some type of a direct action of dextran on the pores, s i n e there are reasons to believe that micropinocytosis is not involved in these processes (Rippe, Kamiya and Folkow 1977).

This study was supported by grants from the Swedish Medical Research Council (Contract No 14X-00016) and from the Medical Faculty, University of Goteborg. AB Hassle generously covered part of the expenses for a technician. The authors are grateful to Mrs Gertrud Karlsson for skilful and devoted technical assistance.

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