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The renin-angiotensin system in cats withchronic renal failure
J. Comp. Path. 1996Vol. 115, 239 252
The R e n i n - A n g i o t e n s i n S y s t e m in Cats with Chronic Renal Failure F. Taugner, G. Baatz* and R. Nobiling~ Department of Veterinary Pathology, Free University of Berlin, *Kleintierpraxis, Moltkestraj3e 27 A, Berlin and ?Department of Experimental Surgery, University of Heidelberg, Germany
Summary The kidneys of eight male and two female cats with subacute (clinical illness 1 3 months) to chronic (clinical illness >3 months) renal failure were examined histopathologically, electron microscopically and immunohistochemically. Semiquantitative morphometric data, obtained by measurement of the reninpositive portion of the afferent arteriole (RPP) and evaluation of the juxtaglomerular index (JGI), were compared with data from three healthy control cats. On the basis of the morphometric data, the animals with renal failure could be classified in three groups showing either a stimulated (group A), an unaltered (group B) or an inhibited (group C) renin-angiotensin system. In the three group A cats the JGI and RPP were increased (45-5-1-3'5%; 130 gm); in the four group B cats these values were comparable with those of the controls; in the three group C animals the JGI was decreased but the RPP was unaltered (11"7% + 3"2%; 56 gm). The increase in kidney renin in animals affected by chronic renal failure (CRF) may have been due to a volume depletion. Prolonged CRF seemed to result in increasing hypertrophy of renal blood vessels, leading to renal hypoxia and increasing preglomerular resistance. Reduced kidney renin status may have been caused by inhibition of renin synthesis in prolonged CRF as a result of renal ischaemia. 9 1996W.B. SaundersCompanyLimited
Introduction Chronic renal failure (CRF) in cats is a c o m m o n disease and a frequent cause of mortality in older animals. Most of the factors that initiate tubointerstitial or glomerular damage are unknown. In a period of 3 years, 2317 cats were necropsied in our department; 278 (12%) of them suffered from C R F and, of these, 46"6% had cardiac left ventricular h y p e r t r o p h y (unpublished observations). An association between left ventricular h y p e r t r o p h y and C R F is well d o c u m e n t e d for m a n and the dog (Cowgill and Callet, 1986) but only presumed in the cat. Kobayashi et al. (1990) reported that 61% of cats with C R F were hypertensive and Lesser et al. (1992) reported a figure of 73%. This led to the question o f the connecting link between CRF, hypertension and left ventricular hypertrophy. Immunohistochemical experiments in our laboratory suggested 0021-9975/96/070239+ 14 $12.00/0
9 1996W.B. SaundersCompanyLimited
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E Taugner
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t h a t the renal r e n i n - a n g i o t e n s i n system (RAS) m i g h t be the crucial link. As only sparse d a t a on the R A S o f cats are available, we investigated the u l t r a s t r u c t u r e o f the j u x t a g l o m e r u l a r a p p a r a t u s a n d the renin status o f the k i d n e y in control cats a n d cats with C R F . M a t e r i a l s and M e t h o d s
Animals Ten cats (eight male and two female), aged 1-18 years, with chronical renal failure (CRF) were investigated. The duration of clinical signs of CRF varied from 1-24 months. Three healthy cats aged 6-24 months served as controls. Necropsy, performed immediately after euthanasia, included weighing the heart and measuring the thickness of the right and left ventricular walls and septum interventriculare. Blood samples from six uraemic cats and all control animals were taken before euthanasia for the measurement of plasma urea and creatinine concentrations and haematocrit values.
Light Microscopy Selected blocks of kidney tissue were fixed in neutral buffered 4% formaldehyde solution for 48h. The renal tissue was dehydrated through graded alcohols to chloroform before embedding in paraffin wax. Sections (5 pm) were stained by the haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and elastica van Gieson methods. Interstitial fibrosis, hypertrophy of renal blood vessels, glomerular damage, thickening of basement membranes, amount and localization of inflammatory infiltrates, tubular casts and tubular dilatation were all assessed histopathologically and scored from - (absent) to + + + (severe).
Immunohistochemist~ Slices of kidney from the hilus, cut at an angle of 90 ~ to the longitudinal axis of the organ, were fixed in modified Bouin's solution containing formaldehyde 18"5% and glutaraldehyde 0"1% in 50% saturated aqueous picric acid solution for 12 h. After dehydration through graded alcohols and chloroform, tissues were embedded in paraffin wax. Sections (5 gm) were mounted on polylysin-1- (Sigma, Deisenhofen, Germany) coated slides and treated by the streptavidin biotin technique (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, USA). Renin-positive cells were demonstrated with a cross-reacting antiserum against purified renin from the mouse submandibular gland, raised in rabbits (Malling and Poulsen, 1977) and diluted 1 in 300. As a control, the specific antibody was replaced by diluted normal goat serum. For semiquantitative evaluation of the kidney renin status, two parameters were chosen: the immunohistochemical juxtaglomerular index (JGI), i.e. the number of renal corpuscles with renin-positive vascular pole expressed as a percentage of the total number of renal corpuscles per section; and measurement of the renin-positive portion of the afferent arteriole (RPP) by means of an ocular micrometer (magnification x 200). The cortical afferent vessels selected were those visible over a length of more than 100 pm from the vascular pole or up to the interlobular artery. Efferent vessels were easily excluded because of their much smaller diameter. A total of five sections per animal, taken at regular intervals of 70 pm, were evaluated. Means and their standard deviations were calculated.
Transmission Electron Microscopy (TE~d) Blocks of renal cortical tissue were fixed in Karnovsky's solution. After rinsing overnight in 0" 1 M cacodylate buffer and postfixation in 1% osmium tetroxide for 2 h, tissues were dehydrated through graded alcohols and rinsed in propylene oxide
Renin-Angiotensin System in Cats
Fig. 1.
241
The bulk of renin (arrows) in control cats is located in the distal portion of the afferent arterioles. GL, glomerulus; ILA, interlobular arteriole. Cat no, 2. ABC technique, x 113.
before embedding in Epon 812 (Serva, Heidelberg, Germany). Ultrathin sections were cut on a Reichert OM U2 ultramicrotome, mounted on nickel grids, stained with lead citrate and uranyl acetate and examined with a Zeiss EM 10A electron microscope operated at 80 kV. Three juxtaglomerular apparatuses (JGAs) per cat were examined.
Immunocytochemistry Renal tissue was fixed in buffered 1% glutaraldehyde solution for 48 h. After dehydration, blocks of tissue were embedded in LR-White soft grade (London Resin Co.). After incubation with a 0"02 M glycine solution, thin sections were exposed for 1 h to the specific antiserum diluted 1 in 20. After rinsing in phosphate buffered saline (PBS), thin sections were incubated with Protein-A-Gold (Biocell, Cardiff, UK) for 2 h and then stained with 0"02% uranyl acetate solution followed by lead citrate. Control reactions in which specific antibody was replaced by PBS were carried out on separate grids.
Results
Control Cats Most renin-positive cells in the kidneys o f the control cats (nos 1 3) were located in the distal, j u x t a g l o r n e r u l a r p o r t i o n o f the afferent arteriole (Fig. 1). Ultrastructurally, n u m e r o u s s e c r e t o r y granules were seen in the m e d i a cells
242
Fig. 2.
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The media cells of the afferent arteriole in control cats contain numerous secretory granules of different electron density. N, nucleus; GL, glomerulus; G, granule. Cat no. 3. TEM. • 6700.
of the juxtaglomerular portion of the afferent arteriole (Fig. 2). Immunocytochemically, specific labelling for renin was observed in these granules (Fig. 3). In addition, some interlobular arteries and mesangial cells close to the glomerular tuft (Fig. 4) contained renin-positive cells in control cats 1 and 3. Values for t h e J G I and the RPP were 28"9 _+ 1-1% and 56 gm, respectively (Table 1).
Cats A2fected by CRF The length of the RPP differed markedly from control values (cats 1-3) in only three of the 10 animals with CRF (nos 4-13). The JGI, in contrast, was more variable. Animals with CRF could be classified in three groups (A,B,C) with JGIs of 8" 7-15 %, 22"4-29" 5 % or 42 49 %, showing significantly different mean values of 11 "7 __ 3"2%, 25"4_+ 3"2% and 45"5 + 3"5%, respectively (Table 1). The outstandingly high RPP values of 98 gin, 119 gm and 175 gm coincided with the highest JGIs of 42%, 45"5% and 49%, respectively. In addition to altered J G I and RPP values, other indications of stimulation or inhibition of t h e J G A were found. Thus, in the stimulated animals, in addition to increased RPP values and pronounced immunolabelling, frequent, scattered immunoreactive cells were found upstream in the interlobular arteries (Fig. 5). O n the
Renin-Angiotensin
Fig. 3. Fig. 4.
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Control cat epithelioid cell showing immunoreaction for renin. Protein-A-Gold technique. Cat no. 2. TEM. x13300. Mesangial cells in vicinity of glomerular tuft may contain renin (arrowheads) in control cats. Cat no. 2. AA, afferent arteriole; GL, glomerulus. ABC technique, x 500.
other hand, immunolabelling of afferent arterioles was weak in cats with low J G I (Fig. 6) and the media cells contained only a few secretory granules, which resembled secondary lysosomes (Fig. 7). Renin-positive cells in the vicinity of the glomerular stalk could be found in all three groups. In some cases the peripheral mesangium also contained renin-positive cells (Fig. 8). Clinical and post-mortem findings are summarized in Table 2. The average age of the animals in group A was lower than that of those in groups B and C. Cats in which the RAS was stimulated also had urea and creatinine
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Table 1 Juxtaglomerular index (JGI) and renin-positive portion of the afferent arteriole (RPP) for control (healthy) cats and those with chronic renal failure (CRF) Cat no.
JGI
Mean J G I ( • SD)
)Cumberof glomeruli counted per animal
RPP (#m)
Mean RPP
Number of afferent arterioles counted per an imal
Controls 1 2 3
27"8 29'9 29'1
28-9• l ' l
975 2012 1892
56 56 56
56
33 37 21
4* 5* 6*
49"0 42"0 45-5
45"5 • 3'5
1039 1062 1089
175 98 ll9
130 • 39
27 37 23
7I8? 9{ l0 t
26.2 29-5 23-3 22-4
903 782 954 1346
56 42 56 77
ll + 12+
11.4 8"7 15"0
754 711 1068
77 42 49
Cats w i t h CRF
13++
25.4•
11"7•
58 • 14
56 • 23
25 28 29 34 19 29 16
* Group A; ~ Group B; ~ Group C.
concentrations which were lower than those in animals of group C. Seven out of the 10 cats with CRF showed left ventricular hypertrophy combined with hypertrophy of the papillary muscle. No correlation between the functional state of the RAS and left ventricular hypertrophy was observed. The histopathological findings are shown in Table 3. In nine animals (nos 5 13) interstitial nephritis was diagnosed; in one cat (no. 4), the diagnosis was segmental membranoproliferative glomerulonephritis. From the lesion grades shown in Table 3 it seemed likely that there was a relationship between low kidney renin content (expressed as JGI) and severe interstitial fibrosis and hypertrophy of the renal vessels. The other histopathological parameters showed no clear correlation with the functional state of the renal RAS. All control procedures gave negative results. Discussion
The juxtaglomerular apparatus represents a structural and functional unit composed of the afferent and efferent arteriole, macula densa and the Goormaghtigh cells (Golgi, 1889; Goormaghtigh, 1937, 1939; Barajas, 1972). The specific task of the J G A consists in synthesis and secretion of renin in the epithelioid cells. Epithelioid cells are myo-endocrine cells. Stimulation of the RAS leads to metaplastic transformation of smooth muscle cells to epithelioid cells (Goormaghtigh, 1939, 1945; Barajas and Latta, 1963; Cantin et al., 1977a); conversely, epithelioid cells may be retransformed into smooth muscle cells as a result of inhibition of the RAS (Hart, 1967; Taugner et al., 1988). Consequently, morphometric evaluations of the renin-positive cells and the length of the renin-positive portion of the afferent arteriole reflect the degree
Renin-Angiotensin System in Cats
245
Fig. 5.
In cats with renin angiotensin system (RAS) stimulation, the renin-positive portion of the afferent arteriole (RPP) is elongated and the interlobular arteries contain scattered renin-positive cells (arrows). Cat no. 4. GL, glomerulus; AA, afferent arteriole; ILA, interlobular arteriole. ABC technique, x 250.
Fig. 6.
A cat with RAS inhibition immunolabelled for renin. The media cells of the afferent arteriole contain only a few renin-positive cells (arrowhead). Cat no. 13. GL, glomerulus; MD, macula densa; AA, afferent arteriole. Cat no. 11. ABC technique, x 500.
246
Fig, 7.
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Taugner e t
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The media cells of cats with RAS inhibition contain only a few secretory granules (arrows), resembling secondary lysosomes (inset). LAA, lumen of afferent arteriole. Cat no. 11. TEM. x1650. Inset x9720.
of stimulation of the RAS. The JGI represents a morphological equivalent of the kidney renin content. A prolongation of RPP reflects the recruitment of epithelioid cells as a result of metaplastic transformation. For most experiments on stimulation or inhibition of the RAS, mice and rats have been used. The intrarenal distribution of renin has been described for several species (Goormaghtigh, 1939; Nairn et al., 1959; Hartroft, 1963; Vikhert and Serebovskaya, 1964; Faarup, 1967, 1968; Sutherland, 1970; Matsuhashi et al., 1977; Taugner et al., 1979; Kon et al., 1986). However, little is known about the intrarenal distribution of renin and the morphology of the JGA in cats. By means of microdissection, Faarup (1967, 1968) investigated the renin content in different segments of the JGA. In a histochemical study, the JG count was assessed in several species, including cats (two animals) (Matsuhashi et al., 1977). In an immunohistoehemical study, Kon et al. (1986) compared animals of 14 species, including one cat. In 1"8% of the JGAs investigated they found renin-positive mesangial cells, whereas most of the renin is located in the distal portion of the afferent arteriole. Interspecies
247
R e n i n - A n g i o t e n s i n S y s t e m in C a t s
Fig. 8.
T h e m e s a n g i a l cells in the vicinity of the g l o m e r u l a r tuft and also the p e r i p h e r a l m e s a n g i u m contain renin. GL, glomerulus; AA, afferent arteriole (arrowhead); BC, B o w m a n n ' s capsule. C a t no. 9. ABC technique. • 500. Inset x 1250.
Table 2 Clinico-pathological findings in cats with CRF
Cat no.
Age
Sex
Findings
#ears) Urea (rag %)
Creatinine (rag %)
Haematocrit (%)
Left ventricular hypertrophy
4* 5* 6*
3 6 1
m c m c m
79 72 ll0
2"2 1'9 2'9
32 36 45
+ + + + -
7~ 8~ 9~ l0 t
13 10 4 14
m c fc f c m c
N N N N
N N N N
N N N N
+ + + + + +
11~ 12~ 13~
18 6 12
m c m c m c
144 574 382
2"4 11"5 9"2
37 37 33
+ + + + + + +
m, Male; f, female; c, castrated; N, not done; - , absent; + + + , severe. * G r o u p A; "~G r o u p B; + G r o u p C.
248
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Table 3 in the kidneys
Observation
with
CRF
Findings in cat no. 4*
Interstitial fibrosis Hypertrophy of media of RA Glomerular damage Thickening of basement membrane Pelvic infiltrate Cortical infiltrate Tubular casts Tubular dilatation
of cats
5*
--
+
+ +++ +
+ ++ --
6*
7"~
++ + +++ ++
++ ++ +++ ++
-
++
+
++
+ + +
++ --
++ _ +
++ _
8t~ ++ +++ ++ +++ -
++ +++ ++
9"~ ++ + ++ +
l0 t + -+++ ++
11;
12;
13;
++ ++ ++ ++
++++++ ++++++ +++ ++ ++++++ _ ++ ++ +++ +++ ++++++
++
-
_
++ --
+ + +
++ +
RA, renal artery; , absent; + + +, severe. * G r o u p A; "~ G r o u p B; ~ G r o u p C .
variations, especially those of a quantitative nature, are difficult to evaluate, as pointed out by Taugner and Hackenthal (1989). The experimental methods in the present study were the same as those used by Hackenthal et al. (1987), which made possible a direct comparison of results. Our control cats had a m e a n J G I of 28"9_ 1-1% and an RPP of 56 gin. These values are lower than those found previously in pigs. The J G I and RPP values in cats were slightly higher than those found previously in mice and much higher than those of guinea-pigs and Chinese hamsters (Hackenthal et al., 1987). As in other species, epithelioid cells were located in cats predominantly in the distal, juxtaglomerular portion of the afferent arteriole, but renin-positive cells were also found upstream, especially where the afferent arteriole arises from the interlobular artery, and in the interlobular artery itself. These results are in contradiction to the studies of Faarup (1968), who did not detect epithelioid cells by means of microdissection in the interlobular artery. Like Kon et al. (1986), we found renin-positive cells in the glomerular mesangium in the vicinity of the glomerular stalk. Little is known about spontaneously occurring pathological changes in the RAS of animals. Bovee et al. (1989) investigated the plasma renin activity (PRA) in one dog suffering from essential hypertension. In the present study, the cats with CRF could be divided into three groups (A,B,C). In group A, the stimulation level was markedly elevated, as indicated by the high J G I (45"5 + 3-5%) and increased RPP. Animals with RAS stimulation also showed an increase of the renin-positive cells in the interlobular arteries. This finding is in agreement with investigations of Amat et al. (1981), who found increasing numbers of renin-positive cells in the interlobular arteries of human patients with segmental renal hypoplasia. In cats with RAS stimulation, not only the mesangmm in the vicinity of the glomerular stalk but also the centrilobular mesangium contained renin-positive cells. In group B cats, the J G I and RPP values were comparable with those of the controls, but increased numbers of renin-positive cells in the mesangium and interlobular arteries indicated chronic stimulation of the RAS. In group C cats, the J G I was markedly
Renin-Angiotensin System in Cats
249
decreased (11"7 + 2"5%); the RPP values were not significantly different from those of the controls but immunoreactivity was remarkably low. In accordance with the immunohistochemical results, electron microscopy showed that the juxtaglomerular media cells contained only a few renin granules, which were of low electron density. Only in cats with RAS inhibition could we observe granules reminiscent of secondary lysosomes. Taking into account the lysosomal character of renin granules (Cantin et aL, 1977b; Taugner et aL, 1985) and the fact that in rats and mice no renin-negative granules could be found (Taugner and Metz, 1986), it seems likely that the "lysosomal" organelles were renin granules in which intracellular breakdown of the secretory product had taken place. In all three animals (nos 11 13) with RAS inhibition, the interlobular arteries and the glomerular mesangium contained renin-positive cells. This suggests that changes in the degree of stimulation, or miscellaneous effects caused by renal insufficiency, are of importance. Most h u m a n renal disorders are characterized by marked stimulation of the JGA, whereas inhibition is exceptional (Waldherr, 1989). Decrease of immunoreactivity in our animals with CRF is therefore difficult to explain. In chronic diabetes mellitus and in the hyporeninaemia-hypoaldosteronismus syndrome, hypoplasia o f t h e J G A has been described (Williams, t986; Sommers and Melamed, 1990). The hyporeninaemia-hypoaldosteronismus syndrome is characterized by slight renal insufficiency, elevated serum potassium concentrations, low PRA and decreased plasma aldosterone values. The mean age of the cats with RAS inhibition (group C) was much higher (12 years) than in the other two groups. The occurrence of a hyporeninaemia-hypoaldosteronismus syndrome seems possible in view of preliminary results indicating that plasma renin activity and plasma aldosterone values in cats with chronic uraemia are depressed (unpublished observations). In the course of CRF an initial stimulation of the RAS seems to be followed by inhibition. The cause of such a change can be deduced by correlating clinical, biochemical and histopathological data with the morphological renin status. Tables 2 and 3 indicate an inverse relationship between renin status on the one hand and degree of interstitial fibrosis and hypertrophy of renal artery walls on the other. End-stage kidneys, resulting in very high serum urea and serum creatinine concentrations, seem to enhance RAS inhibition. It seems likely that with increasing renal ischaemia the decrease of renal perfusion (i.e. stimulation of the baro receptor) leads to RAS inhibition as a consequence of renal hypoxia, resulting in generalized inhibition of synthesis. The morphometric data from group B cats were comparable with those from the controls. However, some of the findings, such as the numerous renin-positive cells in the interlobular arteries and reninpositive peripheral mesangium cells, indicate initial RAS stimulation that changed to inhibition as CRF developed. This is supported by the occurrence of moderate interstitial fibrosis and moderate hypertrophy of renal artery walls. It remains unclear why RAS inhibition in human CRF is diagnosed only infrequently. As renal insufficiency in cats often remains undetected by the owner over a long period, treatment is often given only at an advanced stage of illness (Baatz, personal communication). Perhaps in man the symptoms
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of polydipsia and polyuria are noticed more readily, leading to earlier treatment. Granular cells in the centrilobular mesangium, suspected to synthesize and secrete renin, were reported in cats by Dunihue and Boldosser (1963), but transformation ofmesangial cells into epithelioid cells remained in doubt. This doubt increased when immunospecific methods failed to detect transformed epithelioid cells in the peripheral mesangium of any of the animal models investigated. The electron-dense granules in the mesangium of a routine lupus model described by D r o m m e r et al. (1989) lacked immunohistochemical proof. Baraj as et aL (1976) and Taugner (1989) found granulated cells in the glomerular stalk of rats, but not in the centrilobular mesangium. All observations mentioned above arose from experiments based on drastic stimulation of the RAS (Dunihue and Boldosser, 1963; Barajas et al., 1976; Taugner, 1989). The fact that only in cats may the centrilobular mesangium contain epithelioid cells can at present be explained only by interspecies differences. Hence, the stimuli for renin synthesis within the glomerulus of cats would be more pronounced than in other species. Possibly the mesangial cells of the cat are better equipped for metaplastic transformation into granulated cells than those of other species.'
Acknowledgments The authors thank Mrs M. Harlacher and Mrs B. Buschenhagen for excellent technical assistance. The control animals were provided by the Institute for Veterinary Anatomy and Histology, Free University of Berlin, Germany. References Amat, D., Camilleri, J. P., Vuong, N. P., Bariety, J., Corvol, P. and Menard, J. (1981). Renin localization in segmental renal hypoplasia. Immunohistochemical demonstration in two cases. VirchowsArchiv A, 390, 193 204. Barajas, L. (1972). Anatomical considerations in the control of renin secretion. In: Control of Renin Secretion, T. A. Assaykeen, Ed., Plenum Press, New York, pp. 1-16. Bara]as, L. and Latta, H. (1963). A three dimensional study of the juxtaglomerular apparatus in the rat. Laboratory Investigation, 12, 257 269. Bara]as, L., Wang, P., Bennett, C. M. and Wilburn, R. L. (1976). The renal sympathetic system and juxtaglomerular cells in experimental renovascular hypertension. Laboratory Investigation, 35, 574-587. Bovee, K. C., Littman, M. P., Crabtree, B. J. and Aguirre, G. (1989). Essential hypertension in a dog. Journal of the American Veterinary Medical Association, 195, 81-86. Cantin, M., Araujo-Nascimento, M. F., Benchimol, S. and Desormeaux, Y. 1977a). Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries, and arterioles of the ischemic (endocrine) kidney. American Journal of Pathology, 87, 581-602. Cantin, M., Desormeaux, Y. and Benchimol, S. (1977b). On the lysosomal function of juxtaglomerular granules. Beitrdge Pathologie, 161, 310-327. Cowgill, L. D. and Callet, A. J. (1986). Systemic hypertension. In: Current Veterinary Therapy, 9th Edit., R. W. Kirk, Ed., W. B. Saunders, Philadelphia, pp. 360-364. Drommer, W., Jassim, A. M. and Kaup, F.-J. (1989). Elektronenmikroskopische Struktur der mesangial-proliferativen Glomerulonephritis (MesPGN) beim Tier. Deutsche 7~erarztliche Wochenschrift, 96, 157-240. Dunihue, F. W. and Boldosser, W. G. (1963). Observations on the similarity of mesangial to juxtaglomerular cells. Laboratory Investigation, 12, 1228-1240.
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Taugner, R., Metz, R. and Rosivall, L. (1988). Macroautophagic phenomena in renin granules. Cell and 77ssue Research, 251, 229 231. Vikhert, A. M. and Serebovskaya, Y. A. (1964). Localization of renin in kidneys of healthy people and animals. Federation Proceedings, 23 (Translation supplement), 178 182. Waldherr, R. (1989). Pathology of the human juxtaglomerular apparatus. In: The Juxtaglomerular Apparatus, R. Taugner and E. Hackenthal, Eds, Springer-Verlag, Berlin, Heidelberg, pp. 241 254. Williams, G. H. (1986). Hyporeninemic hypoaldosteronism. New England Journal of Medicine, 314, 1041 1042.
Received, November 22nd, 1995] Accepted, May 7 th, 1996 J
The R e n i n - A n g i o t e n s i n S y s t e m in Cats with Chronic Renal Failure F. Taugner, G. Baatz* and R. Nobiling~ Department of Veterinary Pathology, Free University of Berlin, *Kleintierpraxis, Moltkestraj3e 27 A, Berlin and ?Department of Experimental Surgery, University of Heidelberg, Germany
Summary The kidneys of eight male and two female cats with subacute (clinical illness 1 3 months) to chronic (clinical illness >3 months) renal failure were examined histopathologically, electron microscopically and immunohistochemically. Semiquantitative morphometric data, obtained by measurement of the reninpositive portion of the afferent arteriole (RPP) and evaluation of the juxtaglomerular index (JGI), were compared with data from three healthy control cats. On the basis of the morphometric data, the animals with renal failure could be classified in three groups showing either a stimulated (group A), an unaltered (group B) or an inhibited (group C) renin-angiotensin system. In the three group A cats the JGI and RPP were increased (45-5-1-3'5%; 130 gm); in the four group B cats these values were comparable with those of the controls; in the three group C animals the JGI was decreased but the RPP was unaltered (11"7% + 3"2%; 56 gm). The increase in kidney renin in animals affected by chronic renal failure (CRF) may have been due to a volume depletion. Prolonged CRF seemed to result in increasing hypertrophy of renal blood vessels, leading to renal hypoxia and increasing preglomerular resistance. Reduced kidney renin status may have been caused by inhibition of renin synthesis in prolonged CRF as a result of renal ischaemia. 9 1996W.B. SaundersCompanyLimited
Introduction Chronic renal failure (CRF) in cats is a c o m m o n disease and a frequent cause of mortality in older animals. Most of the factors that initiate tubointerstitial or glomerular damage are unknown. In a period of 3 years, 2317 cats were necropsied in our department; 278 (12%) of them suffered from C R F and, of these, 46"6% had cardiac left ventricular h y p e r t r o p h y (unpublished observations). An association between left ventricular h y p e r t r o p h y and C R F is well d o c u m e n t e d for m a n and the dog (Cowgill and Callet, 1986) but only presumed in the cat. Kobayashi et al. (1990) reported that 61% of cats with C R F were hypertensive and Lesser et al. (1992) reported a figure of 73%. This led to the question o f the connecting link between CRF, hypertension and left ventricular hypertrophy. Immunohistochemical experiments in our laboratory suggested 0021-9975/96/070239+ 14 $12.00/0
9 1996W.B. SaundersCompanyLimited
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E Taugner
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t h a t the renal r e n i n - a n g i o t e n s i n system (RAS) m i g h t be the crucial link. As only sparse d a t a on the R A S o f cats are available, we investigated the u l t r a s t r u c t u r e o f the j u x t a g l o m e r u l a r a p p a r a t u s a n d the renin status o f the k i d n e y in control cats a n d cats with C R F . M a t e r i a l s and M e t h o d s
Animals Ten cats (eight male and two female), aged 1-18 years, with chronical renal failure (CRF) were investigated. The duration of clinical signs of CRF varied from 1-24 months. Three healthy cats aged 6-24 months served as controls. Necropsy, performed immediately after euthanasia, included weighing the heart and measuring the thickness of the right and left ventricular walls and septum interventriculare. Blood samples from six uraemic cats and all control animals were taken before euthanasia for the measurement of plasma urea and creatinine concentrations and haematocrit values.
Light Microscopy Selected blocks of kidney tissue were fixed in neutral buffered 4% formaldehyde solution for 48h. The renal tissue was dehydrated through graded alcohols to chloroform before embedding in paraffin wax. Sections (5 pm) were stained by the haematoxylin and eosin (HE), periodic acid-Schiff (PAS) and elastica van Gieson methods. Interstitial fibrosis, hypertrophy of renal blood vessels, glomerular damage, thickening of basement membranes, amount and localization of inflammatory infiltrates, tubular casts and tubular dilatation were all assessed histopathologically and scored from - (absent) to + + + (severe).
Immunohistochemist~ Slices of kidney from the hilus, cut at an angle of 90 ~ to the longitudinal axis of the organ, were fixed in modified Bouin's solution containing formaldehyde 18"5% and glutaraldehyde 0"1% in 50% saturated aqueous picric acid solution for 12 h. After dehydration through graded alcohols and chloroform, tissues were embedded in paraffin wax. Sections (5 gm) were mounted on polylysin-1- (Sigma, Deisenhofen, Germany) coated slides and treated by the streptavidin biotin technique (Vectastain ABC Kit; Vector Laboratories, Burlingame, CA, USA). Renin-positive cells were demonstrated with a cross-reacting antiserum against purified renin from the mouse submandibular gland, raised in rabbits (Malling and Poulsen, 1977) and diluted 1 in 300. As a control, the specific antibody was replaced by diluted normal goat serum. For semiquantitative evaluation of the kidney renin status, two parameters were chosen: the immunohistochemical juxtaglomerular index (JGI), i.e. the number of renal corpuscles with renin-positive vascular pole expressed as a percentage of the total number of renal corpuscles per section; and measurement of the renin-positive portion of the afferent arteriole (RPP) by means of an ocular micrometer (magnification x 200). The cortical afferent vessels selected were those visible over a length of more than 100 pm from the vascular pole or up to the interlobular artery. Efferent vessels were easily excluded because of their much smaller diameter. A total of five sections per animal, taken at regular intervals of 70 pm, were evaluated. Means and their standard deviations were calculated.
Transmission Electron Microscopy (TE~d) Blocks of renal cortical tissue were fixed in Karnovsky's solution. After rinsing overnight in 0" 1 M cacodylate buffer and postfixation in 1% osmium tetroxide for 2 h, tissues were dehydrated through graded alcohols and rinsed in propylene oxide
Renin-Angiotensin System in Cats
Fig. 1.
241
The bulk of renin (arrows) in control cats is located in the distal portion of the afferent arterioles. GL, glomerulus; ILA, interlobular arteriole. Cat no, 2. ABC technique, x 113.
before embedding in Epon 812 (Serva, Heidelberg, Germany). Ultrathin sections were cut on a Reichert OM U2 ultramicrotome, mounted on nickel grids, stained with lead citrate and uranyl acetate and examined with a Zeiss EM 10A electron microscope operated at 80 kV. Three juxtaglomerular apparatuses (JGAs) per cat were examined.
Immunocytochemistry Renal tissue was fixed in buffered 1% glutaraldehyde solution for 48 h. After dehydration, blocks of tissue were embedded in LR-White soft grade (London Resin Co.). After incubation with a 0"02 M glycine solution, thin sections were exposed for 1 h to the specific antiserum diluted 1 in 20. After rinsing in phosphate buffered saline (PBS), thin sections were incubated with Protein-A-Gold (Biocell, Cardiff, UK) for 2 h and then stained with 0"02% uranyl acetate solution followed by lead citrate. Control reactions in which specific antibody was replaced by PBS were carried out on separate grids.
Results
Control Cats Most renin-positive cells in the kidneys o f the control cats (nos 1 3) were located in the distal, j u x t a g l o r n e r u l a r p o r t i o n o f the afferent arteriole (Fig. 1). Ultrastructurally, n u m e r o u s s e c r e t o r y granules were seen in the m e d i a cells
242
Fig. 2.
F.
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The media cells of the afferent arteriole in control cats contain numerous secretory granules of different electron density. N, nucleus; GL, glomerulus; G, granule. Cat no. 3. TEM. • 6700.
of the juxtaglomerular portion of the afferent arteriole (Fig. 2). Immunocytochemically, specific labelling for renin was observed in these granules (Fig. 3). In addition, some interlobular arteries and mesangial cells close to the glomerular tuft (Fig. 4) contained renin-positive cells in control cats 1 and 3. Values for t h e J G I and the RPP were 28"9 _+ 1-1% and 56 gm, respectively (Table 1).
Cats A2fected by CRF The length of the RPP differed markedly from control values (cats 1-3) in only three of the 10 animals with CRF (nos 4-13). The JGI, in contrast, was more variable. Animals with CRF could be classified in three groups (A,B,C) with JGIs of 8" 7-15 %, 22"4-29" 5 % or 42 49 %, showing significantly different mean values of 11 "7 __ 3"2%, 25"4_+ 3"2% and 45"5 + 3"5%, respectively (Table 1). The outstandingly high RPP values of 98 gin, 119 gm and 175 gm coincided with the highest JGIs of 42%, 45"5% and 49%, respectively. In addition to altered J G I and RPP values, other indications of stimulation or inhibition of t h e J G A were found. Thus, in the stimulated animals, in addition to increased RPP values and pronounced immunolabelling, frequent, scattered immunoreactive cells were found upstream in the interlobular arteries (Fig. 5). O n the
Renin-Angiotensin
Fig. 3. Fig. 4.
System in Cats
243
Control cat epithelioid cell showing immunoreaction for renin. Protein-A-Gold technique. Cat no. 2. TEM. x13300. Mesangial cells in vicinity of glomerular tuft may contain renin (arrowheads) in control cats. Cat no. 2. AA, afferent arteriole; GL, glomerulus. ABC technique, x 500.
other hand, immunolabelling of afferent arterioles was weak in cats with low J G I (Fig. 6) and the media cells contained only a few secretory granules, which resembled secondary lysosomes (Fig. 7). Renin-positive cells in the vicinity of the glomerular stalk could be found in all three groups. In some cases the peripheral mesangium also contained renin-positive cells (Fig. 8). Clinical and post-mortem findings are summarized in Table 2. The average age of the animals in group A was lower than that of those in groups B and C. Cats in which the RAS was stimulated also had urea and creatinine
244
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Table 1 Juxtaglomerular index (JGI) and renin-positive portion of the afferent arteriole (RPP) for control (healthy) cats and those with chronic renal failure (CRF) Cat no.
JGI
Mean J G I ( • SD)
)Cumberof glomeruli counted per animal
RPP (#m)
Mean RPP
Number of afferent arterioles counted per an imal
Controls 1 2 3
27"8 29'9 29'1
28-9• l ' l
975 2012 1892
56 56 56
56
33 37 21
4* 5* 6*
49"0 42"0 45-5
45"5 • 3'5
1039 1062 1089
175 98 ll9
130 • 39
27 37 23
7I8? 9{ l0 t
26.2 29-5 23-3 22-4
903 782 954 1346
56 42 56 77
ll + 12+
11.4 8"7 15"0
754 711 1068
77 42 49
Cats w i t h CRF
13++
25.4•
11"7•
58 • 14
56 • 23
25 28 29 34 19 29 16
* Group A; ~ Group B; ~ Group C.
concentrations which were lower than those in animals of group C. Seven out of the 10 cats with CRF showed left ventricular hypertrophy combined with hypertrophy of the papillary muscle. No correlation between the functional state of the RAS and left ventricular hypertrophy was observed. The histopathological findings are shown in Table 3. In nine animals (nos 5 13) interstitial nephritis was diagnosed; in one cat (no. 4), the diagnosis was segmental membranoproliferative glomerulonephritis. From the lesion grades shown in Table 3 it seemed likely that there was a relationship between low kidney renin content (expressed as JGI) and severe interstitial fibrosis and hypertrophy of the renal vessels. The other histopathological parameters showed no clear correlation with the functional state of the renal RAS. All control procedures gave negative results. Discussion
The juxtaglomerular apparatus represents a structural and functional unit composed of the afferent and efferent arteriole, macula densa and the Goormaghtigh cells (Golgi, 1889; Goormaghtigh, 1937, 1939; Barajas, 1972). The specific task of the J G A consists in synthesis and secretion of renin in the epithelioid cells. Epithelioid cells are myo-endocrine cells. Stimulation of the RAS leads to metaplastic transformation of smooth muscle cells to epithelioid cells (Goormaghtigh, 1939, 1945; Barajas and Latta, 1963; Cantin et al., 1977a); conversely, epithelioid cells may be retransformed into smooth muscle cells as a result of inhibition of the RAS (Hart, 1967; Taugner et al., 1988). Consequently, morphometric evaluations of the renin-positive cells and the length of the renin-positive portion of the afferent arteriole reflect the degree
Renin-Angiotensin System in Cats
245
Fig. 5.
In cats with renin angiotensin system (RAS) stimulation, the renin-positive portion of the afferent arteriole (RPP) is elongated and the interlobular arteries contain scattered renin-positive cells (arrows). Cat no. 4. GL, glomerulus; AA, afferent arteriole; ILA, interlobular arteriole. ABC technique, x 250.
Fig. 6.
A cat with RAS inhibition immunolabelled for renin. The media cells of the afferent arteriole contain only a few renin-positive cells (arrowhead). Cat no. 13. GL, glomerulus; MD, macula densa; AA, afferent arteriole. Cat no. 11. ABC technique, x 500.
246
Fig, 7.
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Taugner e t
aL
The media cells of cats with RAS inhibition contain only a few secretory granules (arrows), resembling secondary lysosomes (inset). LAA, lumen of afferent arteriole. Cat no. 11. TEM. x1650. Inset x9720.
of stimulation of the RAS. The JGI represents a morphological equivalent of the kidney renin content. A prolongation of RPP reflects the recruitment of epithelioid cells as a result of metaplastic transformation. For most experiments on stimulation or inhibition of the RAS, mice and rats have been used. The intrarenal distribution of renin has been described for several species (Goormaghtigh, 1939; Nairn et al., 1959; Hartroft, 1963; Vikhert and Serebovskaya, 1964; Faarup, 1967, 1968; Sutherland, 1970; Matsuhashi et al., 1977; Taugner et al., 1979; Kon et al., 1986). However, little is known about the intrarenal distribution of renin and the morphology of the JGA in cats. By means of microdissection, Faarup (1967, 1968) investigated the renin content in different segments of the JGA. In a histochemical study, the JG count was assessed in several species, including cats (two animals) (Matsuhashi et al., 1977). In an immunohistoehemical study, Kon et al. (1986) compared animals of 14 species, including one cat. In 1"8% of the JGAs investigated they found renin-positive mesangial cells, whereas most of the renin is located in the distal portion of the afferent arteriole. Interspecies
247
R e n i n - A n g i o t e n s i n S y s t e m in C a t s
Fig. 8.
T h e m e s a n g i a l cells in the vicinity of the g l o m e r u l a r tuft and also the p e r i p h e r a l m e s a n g i u m contain renin. GL, glomerulus; AA, afferent arteriole (arrowhead); BC, B o w m a n n ' s capsule. C a t no. 9. ABC technique. • 500. Inset x 1250.
Table 2 Clinico-pathological findings in cats with CRF
Cat no.
Age
Sex
Findings
#ears) Urea (rag %)
Creatinine (rag %)
Haematocrit (%)
Left ventricular hypertrophy
4* 5* 6*
3 6 1
m c m c m
79 72 ll0
2"2 1'9 2'9
32 36 45
+ + + + -
7~ 8~ 9~ l0 t
13 10 4 14
m c fc f c m c
N N N N
N N N N
N N N N
+ + + + + +
11~ 12~ 13~
18 6 12
m c m c m c
144 574 382
2"4 11"5 9"2
37 37 33
+ + + + + + +
m, Male; f, female; c, castrated; N, not done; - , absent; + + + , severe. * G r o u p A; "~G r o u p B; + G r o u p C.
248
F. Taugner
Histopathological
findings
et al.
Table 3 in the kidneys
Observation
with
CRF
Findings in cat no. 4*
Interstitial fibrosis Hypertrophy of media of RA Glomerular damage Thickening of basement membrane Pelvic infiltrate Cortical infiltrate Tubular casts Tubular dilatation
of cats
5*
--
+
+ +++ +
+ ++ --
6*
7"~
++ + +++ ++
++ ++ +++ ++
-
++
+
++
+ + +
++ --
++ _ +
++ _
8t~ ++ +++ ++ +++ -
++ +++ ++
9"~ ++ + ++ +
l0 t + -+++ ++
11;
12;
13;
++ ++ ++ ++
++++++ ++++++ +++ ++ ++++++ _ ++ ++ +++ +++ ++++++
++
-
_
++ --
+ + +
++ +
RA, renal artery; , absent; + + +, severe. * G r o u p A; "~ G r o u p B; ~ G r o u p C .
variations, especially those of a quantitative nature, are difficult to evaluate, as pointed out by Taugner and Hackenthal (1989). The experimental methods in the present study were the same as those used by Hackenthal et al. (1987), which made possible a direct comparison of results. Our control cats had a m e a n J G I of 28"9_ 1-1% and an RPP of 56 gin. These values are lower than those found previously in pigs. The J G I and RPP values in cats were slightly higher than those found previously in mice and much higher than those of guinea-pigs and Chinese hamsters (Hackenthal et al., 1987). As in other species, epithelioid cells were located in cats predominantly in the distal, juxtaglomerular portion of the afferent arteriole, but renin-positive cells were also found upstream, especially where the afferent arteriole arises from the interlobular artery, and in the interlobular artery itself. These results are in contradiction to the studies of Faarup (1968), who did not detect epithelioid cells by means of microdissection in the interlobular artery. Like Kon et al. (1986), we found renin-positive cells in the glomerular mesangium in the vicinity of the glomerular stalk. Little is known about spontaneously occurring pathological changes in the RAS of animals. Bovee et al. (1989) investigated the plasma renin activity (PRA) in one dog suffering from essential hypertension. In the present study, the cats with CRF could be divided into three groups (A,B,C). In group A, the stimulation level was markedly elevated, as indicated by the high J G I (45"5 + 3-5%) and increased RPP. Animals with RAS stimulation also showed an increase of the renin-positive cells in the interlobular arteries. This finding is in agreement with investigations of Amat et al. (1981), who found increasing numbers of renin-positive cells in the interlobular arteries of human patients with segmental renal hypoplasia. In cats with RAS stimulation, not only the mesangmm in the vicinity of the glomerular stalk but also the centrilobular mesangium contained renin-positive cells. In group B cats, the J G I and RPP values were comparable with those of the controls, but increased numbers of renin-positive cells in the mesangium and interlobular arteries indicated chronic stimulation of the RAS. In group C cats, the J G I was markedly
Renin-Angiotensin System in Cats
249
decreased (11"7 + 2"5%); the RPP values were not significantly different from those of the controls but immunoreactivity was remarkably low. In accordance with the immunohistochemical results, electron microscopy showed that the juxtaglomerular media cells contained only a few renin granules, which were of low electron density. Only in cats with RAS inhibition could we observe granules reminiscent of secondary lysosomes. Taking into account the lysosomal character of renin granules (Cantin et aL, 1977b; Taugner et aL, 1985) and the fact that in rats and mice no renin-negative granules could be found (Taugner and Metz, 1986), it seems likely that the "lysosomal" organelles were renin granules in which intracellular breakdown of the secretory product had taken place. In all three animals (nos 11 13) with RAS inhibition, the interlobular arteries and the glomerular mesangium contained renin-positive cells. This suggests that changes in the degree of stimulation, or miscellaneous effects caused by renal insufficiency, are of importance. Most h u m a n renal disorders are characterized by marked stimulation of the JGA, whereas inhibition is exceptional (Waldherr, 1989). Decrease of immunoreactivity in our animals with CRF is therefore difficult to explain. In chronic diabetes mellitus and in the hyporeninaemia-hypoaldosteronismus syndrome, hypoplasia o f t h e J G A has been described (Williams, t986; Sommers and Melamed, 1990). The hyporeninaemia-hypoaldosteronismus syndrome is characterized by slight renal insufficiency, elevated serum potassium concentrations, low PRA and decreased plasma aldosterone values. The mean age of the cats with RAS inhibition (group C) was much higher (12 years) than in the other two groups. The occurrence of a hyporeninaemia-hypoaldosteronismus syndrome seems possible in view of preliminary results indicating that plasma renin activity and plasma aldosterone values in cats with chronic uraemia are depressed (unpublished observations). In the course of CRF an initial stimulation of the RAS seems to be followed by inhibition. The cause of such a change can be deduced by correlating clinical, biochemical and histopathological data with the morphological renin status. Tables 2 and 3 indicate an inverse relationship between renin status on the one hand and degree of interstitial fibrosis and hypertrophy of renal artery walls on the other. End-stage kidneys, resulting in very high serum urea and serum creatinine concentrations, seem to enhance RAS inhibition. It seems likely that with increasing renal ischaemia the decrease of renal perfusion (i.e. stimulation of the baro receptor) leads to RAS inhibition as a consequence of renal hypoxia, resulting in generalized inhibition of synthesis. The morphometric data from group B cats were comparable with those from the controls. However, some of the findings, such as the numerous renin-positive cells in the interlobular arteries and reninpositive peripheral mesangium cells, indicate initial RAS stimulation that changed to inhibition as CRF developed. This is supported by the occurrence of moderate interstitial fibrosis and moderate hypertrophy of renal artery walls. It remains unclear why RAS inhibition in human CRF is diagnosed only infrequently. As renal insufficiency in cats often remains undetected by the owner over a long period, treatment is often given only at an advanced stage of illness (Baatz, personal communication). Perhaps in man the symptoms
250
F. Taugner et al.
of polydipsia and polyuria are noticed more readily, leading to earlier treatment. Granular cells in the centrilobular mesangium, suspected to synthesize and secrete renin, were reported in cats by Dunihue and Boldosser (1963), but transformation ofmesangial cells into epithelioid cells remained in doubt. This doubt increased when immunospecific methods failed to detect transformed epithelioid cells in the peripheral mesangium of any of the animal models investigated. The electron-dense granules in the mesangium of a routine lupus model described by D r o m m e r et al. (1989) lacked immunohistochemical proof. Baraj as et aL (1976) and Taugner (1989) found granulated cells in the glomerular stalk of rats, but not in the centrilobular mesangium. All observations mentioned above arose from experiments based on drastic stimulation of the RAS (Dunihue and Boldosser, 1963; Barajas et al., 1976; Taugner, 1989). The fact that only in cats may the centrilobular mesangium contain epithelioid cells can at present be explained only by interspecies differences. Hence, the stimuli for renin synthesis within the glomerulus of cats would be more pronounced than in other species. Possibly the mesangial cells of the cat are better equipped for metaplastic transformation into granulated cells than those of other species.'
Acknowledgments The authors thank Mrs M. Harlacher and Mrs B. Buschenhagen for excellent technical assistance. The control animals were provided by the Institute for Veterinary Anatomy and Histology, Free University of Berlin, Germany. References Amat, D., Camilleri, J. P., Vuong, N. P., Bariety, J., Corvol, P. and Menard, J. (1981). Renin localization in segmental renal hypoplasia. Immunohistochemical demonstration in two cases. VirchowsArchiv A, 390, 193 204. Barajas, L. (1972). Anatomical considerations in the control of renin secretion. In: Control of Renin Secretion, T. A. Assaykeen, Ed., Plenum Press, New York, pp. 1-16. Bara]as, L. and Latta, H. (1963). A three dimensional study of the juxtaglomerular apparatus in the rat. Laboratory Investigation, 12, 257 269. Bara]as, L., Wang, P., Bennett, C. M. and Wilburn, R. L. (1976). The renal sympathetic system and juxtaglomerular cells in experimental renovascular hypertension. Laboratory Investigation, 35, 574-587. Bovee, K. C., Littman, M. P., Crabtree, B. J. and Aguirre, G. (1989). Essential hypertension in a dog. Journal of the American Veterinary Medical Association, 195, 81-86. Cantin, M., Araujo-Nascimento, M. F., Benchimol, S. and Desormeaux, Y. 1977a). Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries, and arterioles of the ischemic (endocrine) kidney. American Journal of Pathology, 87, 581-602. Cantin, M., Desormeaux, Y. and Benchimol, S. (1977b). On the lysosomal function of juxtaglomerular granules. Beitrdge Pathologie, 161, 310-327. Cowgill, L. D. and Callet, A. J. (1986). Systemic hypertension. In: Current Veterinary Therapy, 9th Edit., R. W. Kirk, Ed., W. B. Saunders, Philadelphia, pp. 360-364. Drommer, W., Jassim, A. M. and Kaup, F.-J. (1989). Elektronenmikroskopische Struktur der mesangial-proliferativen Glomerulonephritis (MesPGN) beim Tier. Deutsche 7~erarztliche Wochenschrift, 96, 157-240. Dunihue, F. W. and Boldosser, W. G. (1963). Observations on the similarity of mesangial to juxtaglomerular cells. Laboratory Investigation, 12, 1228-1240.
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Received, November 22nd, 1995] Accepted, May 7 th, 1996 J