Br. J. exp. Path. (I984) 65, I8I-I90

Spontaneous hypertension and hypertensive renal disease in the fawn-hooded rat M.H.M. Kuijpers' and E. Gruys2 'Unilever Research Laboratorium, Vlaardingen, The Netherlands and 2Institute of Veterinary Pathology, State University of Utrecht, Utrecht, The Netherlands

Received for publication 8 July I983

Summary. The fawn-hooded (FH) rat, a strain characterized by a platelet storage-pool disease, developed focal and segmental glomerulosclerosis at the age of 2-3 months (males) and approximately 6 months (females). Male animals died spontaneously at I I-I 3 months, and females at I 5 months of age, both with overt malignant nephrosclerosis. During the first half year of life focal glomeruli showed depositions of IgG, IgA, IgM, C3 and fibrinogen in a segmental pattern and mainly in mesangial areas. Mesangial IgG and IgA were already demonstrable at the age of 5 weeks. On electron microscopy no electron-dense deposits suggestive of immune complexes were found. Mean arterial blood pressure in 5.5-month-old male FH rats was increased compared with that of matched Wistar rats. One-year-old FH rats had severe hypertension. The presumed relationship between the hypertension, the renal lesions and the blood platelet defect is discussed. Keywords: fawn-headed rat, hypertension, glomerulosclerosis, malignant nephrosclerosis

The fawn-headed (FH) rat, derived from the of age. Post-mortem examination of these German brown rat, has a haemorrhagic rats revealed severe nephrosclerosis and tendency caused by a platelet storage-pool generalized arteriolosclerosis, suggestive of deficiency. The dense granules of the blood hypertensive disease. We have now investi- platelets contain reduced amounts of ADP, gated whether or not FH rats develop hyper- ATP and serotonin (Tschopp & Zucker tension, and studied the development of I972), resembling the storage-pool disease renal lesions with age. in man. These dense granules are normally extruded during the platelet-release reaction responsible for platelet adhesion and aggre- gation. Moreover, these rats are hypercho- Material and methods lesterolaemic and prone to infections. Animals. The original nucleus of FH rats was Kreisberg & Karnovsky (I978) reported obtained from Institut fur biologisch-medi- the occurrence of focal glomerulosclerosis in zinische Forschung AG, Fiillinsdorf, Switzer- male FH rats, beginning at 6 months of age land, through Dr Tschopp. A colony of FH and affecting all animals at the age of I 2 rats is maintained in our laboratory by months. In our laboratory, male FH rats died random mating. All animals were kept under at I0-I 3 months and females at I 5 months standard laboratory conditions with free Correspondence: M.H.M. Kuijpers, Pathology Section, Unilever Research Laboratorium Vlaardingen, PO Box I14, 3130 AC Vlaardingen, The Netherlands. 182 M.H.M. Kuijpers & E. Gruys access to water and food. They were housed sections were stained with uranyl acetate individually in stainless steel cages and and lead citrate and investigated using a received a diet of Muracon pellets (Trouw, Philips 201 electron microscope. Putten, Netherlands). Wistar rats of the Cpb:WU strain, specific pathogen-free Immunofluorescence (IF) studies. Fresh renal obtained at weaning from the Institute for tissue was frozen in isopentane, cooled to its Breeding of Laboratory animals, TNO Zeist, coagulation point with liquid nitrogen and Netherlands, were used as controls. subsequently stored at -80C. Cryostat sec- tions were fixed in acetone for Io min at Blood pressure. Systolic and diastolic arterial - 20°C and incubated with antisera by the blood pressures were measured by cannula- indirect method. Goat antisera against rat tion of the carotid artery under Nembutal IgA, IgG, IgM, IgE, fibrinogen and C3, and (sodium pentobarbital, Abbott; 50 mg per kg fluorescein-isothiocyanate (FITC) labelled body weight intraperitoneal) anaesthesia, rabbit antigoat serum, all from Nordic Im- using a Statham P-23 db pressure trans- munological Laboratories, Tilburg, Nether- ducer with a Hellige Program i9 amplifier lands, were used. Control sections of each and a Kipp BD 9 recorder. Rats were allowed were incubated with phosphate-buf- to stabilize I 5 min after surgical preparation. fered saline (PBS) and FITC labelled rabbit anti-goat serum. The degree and extent of Light microscopy (LM). Tissues were pre- fluorescence found in the glomeruli were served in a I0% buffered formalin solution graded from 0 to 4 (o = negative, 4 = maxi- but kidneys were preserved in Dubosq-Brazil mum intensity). solution. Paraffin sections were stained with Harris' haematoxylin and azophloxin, and Experiment I. Mean arterial blood pressure of Masson's trichrome stain. Kidney sections six male FH rats aged 5.5 months was were additionally stained with periodic acid compared with that of matched Wistar rats. Schiff-stain (PAS) for neutral glycoproteins In addition, blood pressure in three (basement membranes and mesangial I5-month-old FH rats was compared with matrix), Martius scarlet and blue (MSB) and that of two matched Wistar rats. methenamine silver for basement membrane and fibrin staining, phosphotungstic acid- Experiment I. Four groups ofsix male FH rats haematoxylin (PTAH) for fibrin, and with were killed at the age of 2, 3, 4 and 5 months Congo red for amyloid. Renal histopatholo- respectively for LM, IF and EM studies of gic changes were characterized and graded kidneys. In addition, renal lesions of male using coded slides on a semi-quantitative and female FH rats, varying in age from I9 scale ranging from o to 4: o = normal; days to 1 5 months, were investigated by LM, I = mild; 2 = moderate; 3 = moderately IF and EM. From these animals several other severe; 4= severe changes. organs were examined for pathological Liver and adrenals were stained with changes. Prussian blue for iron and with long Ziehl- Neelsen for lipofuscin. Results Electron microscopy (EM). Fresh renal tissue was fixed by immersion in a 3% glutaralde- Blood pressure hyde solution buffered with o. i M cacodylate Mean arterial blood pressures under Nembu- and post-fixed in I% osmium tetroxide. After tal anaesthesia of s 5-month-old male FH embedding in Araldite, i-2-4um sections rats (I48 + 4 7 mmHg) was significantly were stained with toluidine blue and studied increased compared with those ofWistar rats by LM to select areas for EM. Ultrathin of the same age (I 20.5 ± 6.5 mmHg) Hypertension and renal disease in the fawn-hooded rat I83 (P

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Fig. i. Glomerulosclerosis in a 5-month-old male FH rat. Segmental increase in mesangial matrix and capsular adhesions. MSB. x 300.

Fig. 2. Malignant nephrosclerosis in an i i-month-old male FH rat. Haematoxylin-Azophloxin. x 750. I86 M.H.M. Kuijpers & E. Gruys male rats showed increased mesangial the immunofluorescent picture. An attempt matrix, thickening and sometimes splitting was made to quantify glomerular deposits; ofthe glomerular basement membrane and a the results are shown in Table I. Focal local or diffuse loss of epithelial foot pro- deposition of IgG, IgA, IgM, C3 and cesses. In some podocytes and in mesangial fibrinogen was observed in affected glomer- cells numerous electron-dense droplets, uli. IgE was not detected. The deposits con- grouped together in the cytoplasm were sisted of finely to coarsely granular or seen. The material present in these droplets clumped material located in a segmental was homogeneous or clumped and sur- pattern along the glomerular capillary walls rounded by an electron-lucent band followed and/or in mesangial areas (Fig. 4). Occa- by an electron-dense membrane. These drop- sionally, the deposits were mainly located lets most likely correspond with the PAS- and around the vascular pole or in peripheral PTAH-positive hyalin droplets identified by capillary loops. Fibrinogen was often found LM. Cytoplasmic vacuoles and pockets were outlining crescent-like one part ofBowman's found in many podocytes and they often capsule, presumably in places of adhesions occupied a major portion of the podocyte. between the glomerular tuft and Bowman's Some granular dispersed material was seen capsule. in these epithelial pockets. No electron-dense Focal coarse droplets in proximal tubular deposits suggestive of immune complex ac- cells and tubular casts stained positively for cumulation were found. Foam cells showed immunoglobulins, C3 and fibrinogen. cytoplasmic, rounded, transparent vacuoles. Dilated tubules often stained diffusely with IgA, IgG and fibrinogen. In a few animals Immunofluorescence studies. In 3-week-old FH interstitial mononuclear cells stained posi- rats no glomerular or tubular deposits tively for IgA or IgM. End-stage animals had occurred. At 5 weeks of age a finely granular entirely sclerotic kidneys with glomerular deposition of IgA and IgG in the glomerular and tubular deposits ofimmunoglobulins, C3 mesangium was seen in some glomeruli (Fig. and fibrinogen and similar tubular casts. 3). Later on there were great variations in Control sections incubated with PBS and

Fig. 3. Immunofluorescent staining for IgG in a male FH rat aged 5 weeks. Mesangial localization ofIgG. x 300. Hypertension and renal disease in the fawn-hooded rat I87

Fig. 4. Immunofluorescent staining for IgG in a male FH rat aged 5 months. Mesangial and pericapillary deposits of IgG. x 300.

FITC-labelled anti-goat serum were always tic activity and haemosiderin storage. The negative. number of megakaryocytes had increased. Splenic arteries were whorled and showed Histopathology of other laminated concentric thickening ofthe walls organs with luminal narrowing. Striking histopathological changes in several Pancreatic vessels and mesenteric vessels organs occurred in i-year-old male and also showed hyalin and hyperplastic arterio- female FH rats. losclerosis, and were surrounded by inflam- Myocardial fibres showed swelling, loss of matory cells. In some cases fibrinoid degene- striation, and lysis. The interstitium showed ration of mesenteric arterial walls was sur- oedema, areas with fibrosis and diffuse pro- rounded by extensive inflammatory tissue liferation of fibroblasts and histiocytic cells. containing many eosinophils and was identi- Myocardial vessel walls were thickened and fied as periarteritis nodosa. surrounded by mononuclear cells and in- Lymph nodes showed follicular hyper- creased connective tissue. In a few cases focal plasia, and medullary cords were engorged mineralization of degenerated cardiac with plasma cells. Reticulum cells and sinu- muscle as well as of vessel walls was seen. soidal macrophages showed erythrocyto- Pulmonary congestion wascommon, often phagia and haemosiderin storage. Syncytia accompanied with haemosiderin-laden and of macrophages with pale cytoplasm were erythrophagous alveolar macrophages. In a present in the paracortex. few cases haemoglobin crystals had evoked a The hepatocytes contained exceptionally granulomatous reaction. Pulmonary arteries large amounts of lipofuscin pigment. In the showed muralhypertrophy, fragmentation of Kupffer cells iron pigment was detected. smooth muscle cell nuclei and occasionally In animals that had died spontaneously inflammatory infiltration involving theentire several organs showed signs of diffuse intra- vessel wall. vascular coagulation, which was most The spleen showed extensive haemopoie- obvious in the adrenals. I88 M.H.M. Kuijpers & E. Gruys mesangial staining for immunoglobulins Discussion and complement in the FH rat may have The renal, myocardial and vascular lesions evolved via several mechanisms. When observed in FH rats that had died spon- mesangial function is impaired, deposits, taneously are suggestive of accelerated whether derived from immune complexes or hypertensive disease. The blood pressure resulting from glomerular leakage of plasma values of FH rats in the present study were proteins, will persist. It has been proposed markedly increased compared with those of that mesangial immune deposits reflect Wistar rats. These values, however, were mesangial dysfunction (Romen & Morath obtained by the direct technique under Nem- 1979). Also mesangial immune deposits are butal anaesthesia, which lowers blood pres- implicated in the pathogenesis of glomeru- sure. A subsequent study in which the blood losclerosis (Michels et al. I980; Romen & pressure was monitored by the tail-cuff Morath I979). method in conscious animals confirmed that Impaired systemic clearance of macromo- FH rats develop severe spontaneous hyper- lecules due to a defective or saturated reticu- tension, with values of 220-270 mmHg loendothelial system (Barcelli et al. I98I; systolic in the end phase (to be published). Cruse et al. I982) will cause mesangial The observed hypertension may be respon- overload and thus may also contribute to the sible for the high incidence of malignant development of glomerulosclerosis. A satu- nephrosclerosis in our colony of FH rats. rated reticuloendothelial system may be the The early renal lesions, found from 2 result of persistent infection or continuous months onwards, consisted of focal segmen- exposure to an endogenous antigen as in the tal glomerulosclerosis, occasionally accom- autoimmune diseases, resulting in circulat- panied with hyalinosis. Glomerulosclerosis ing immune complexes. Under certain cir- has also been observed in hypertensive men cumstances circulating immune complexes (Habib I973; Katz et al. I979). On the other affect glomerular structure and function, hand, idiopathic focal glomerulosclerosis in and cause serum sickness-like disease. Our man is often accompanied by hypertension EM studies, however, and those by Kreisberg (Habib I973). It is of interest to know & Karnovsky (I9 78) revealed no electron- whether the glomerulosclerosis observed in dense deposits indicative of glomerular im- young FH rats is a result of hypertension or mune complexes. Preliminary investigations whether the glomerular lesions are the un- for anti-ds-DNA antibodies in FH rat serum derlying cause of the hypertension. by the Chritidia Luciliae test (Aarden et al. Immunofluorescence examination of glo- I975) were also negative (not reported meruli revealed mesangial and pericapillary here). immune deposits. Interestingly, mesangial A second pathogenetic mechanism caus- immune deposits were already present in FH ing protracted functional overload of the rats at 5 weeks of age when there were no mesangium leading to glomerulosclerosis is visible changes at the light microscopic level. an increased permeability of the glomerular These deposits may be derived from glomeru- capillary walls. Hypertension is one factor lar immune complexes or from leakage of known to enhance this permeability, and plasma proteins. Pericapillary deposits are finally results in (Olivetti et al. normally shifted to the mesangium and will I98I). The presence of immunoglobulins, gradually disappear when adequate mesan- C3 and fibrinogen in the casts and in the gial function is present. The mesangium droplets in proximal tubular cells observed participates in the clearance of glomerular on IF examination ofFH rat kidneys suggests immune complexes and other macromole- a non-selective proteinuria. The pericapill- cules (Lee & Vernier I980; Michael et al. ary glomerular deposits may similarly result I980; Olivetti et al. I98I). This increased from leakage of several plasma proteins Hypertension and renal disease in the fawn-hooded rat 189 through the glomerular basement mem- elevation of the blood pressure per se may brane rather than representing immune produce endothelial injury. In hypertensive complexes. human patients with renal disease increased The glomerular lesions in FH rats morpho- urinary beta-thromboglobulin, another logically resemble the spontaneous lesions platelet release product, was found, indicat- described for ageing Sprague-Dawley and ing an increased platelet activation in vivo Wistar rats (Berg I 96 5; Bolton et al. 19 76; (Anderton et al. I980; Coccheri & Fiorentine Elema & Arends I 9 75). Unlike the lesions in I97I). Similar mechanisms may be opera- these rat strains, the lesions in FH rats occur tive in the hypertensive FH rat. at a very early age, affecting more animals and progressing to end-stage renal disease resulting in a markedly restricted life-span. It Acknowledgements is not known whether the glomerulosclerosis The authors gratefully appreciate the expert in the other rat strains is accompanied by assistance ofA.J. van de Kooij for histopatho- hypertension. The renal disease of the FH rat logical preparations, J.F. Quadt for measur- strain bred in our laboratory seems to be ing direct blood pressure and J.S.W. Kleinek- even more severe than that described by oort for supervising animal care. Part of this Kreisberg & Karnovsky (I 9 78). study was presented at the 29th Annual For the spontaneously hypertensive rat Meeting of the European Society for Veterin- (SHR rat) end-stage nephrosclerosis and ary Pathology in Bremen, Germany on 27 severe proteinuria have been reported (Man- May I980. A summary was published in dal et al. I 9 78; Nagaola et al. I 9 72; Van Liew Berl. Munch. Tierartzl. Wschr. 94, 39, I98I & Feld 1980). It is likely that, as in SHR rats, (abstr.). the pathological changes in FH rat kidneys, heart and blood vessels as well as proteinuria are due to the hypertension. The renal References lesions may contribute to a further elevation AARDEN L.A., DE GROOT E.R. & FELTKAMP T.E.W. of the blood pressure. (I 9 75) Immunology of DNA III. Crithidia luci- No data are available on a possible rela- liae: a simple substrate for the detection of tionship between the platelet pathology and anti-dsDNA with the immunofluorescence the development of hypertension in FH rats. technique. Ann. N.Y. Acad. Sci. 254, 505-515. A great number of studies in man and rat ANDERTONJ.L., FANANAPAZIR L. & DAWESJ. (I980) Urinary beta-thromboglobulin in essential have revealed the involvement of platelets in hypertension. Br. J. Haemat. 44, 307-3II. glomerular injury (Catell & Mehotra, I980; BARCELLI U., RADEMACHER R., Ooi Y.M. & Ooi B.S. Duffy et al. I 9 70; Kincaid-Smith I 972; Man- (I98I) Modification of glomerular immune dal et al. I978; Miller et al. I980). Only complex deposition in mice by activation of the Parbtani & Cameron (1979) reported reticuloendothelial system. J. clin. Invest. 67, depressed platelet serotonin levels in various 20-27. forms ofhuman renal disease, both immune- BERG B.N. (I965) Spontaneous , with proteinuria, hyperglobulinaemia in the rat. complex mediated and of non-immunopath- Proc. Soc. exp. Biol. Med. 119, 417-420. ogenesis. Premature release of serotonin BOLTON W.K., BENTON F.R., MACLAY J.G. & STUR- content of platelets may occur via immune GILL B.C. (I976) Spontaneous glomerular scler- adherence (Brown I 976; Dixon & Rosse osis in aging Sprague-Dawley rats: I. Lesions 19 77; Zahavi & Marder I 9 74), or via acti- associated with mesangial IgM deposits. Am. J. vation within altered renal microvascula- Path. 85, 277-300. or other BROWN D.L. (1976) Platelets in immunological ture, sites of vascular injuries (Duffy reactions. In Platelets in Biology and Pathology et al. 1970; Kincaid-Smith I972; Miller et al. Research Monographs in Cells and Tissue Phy- 1980), the degranulated platelets remaining siology. Ed. J.L. Gordon. Amsterdam: North- in the circulation (O'Brien I978). Chronic Holland. pP. 313-324. g90 M.H.M. Kuijpers & E. Gruys CArrEL V. & MEHOTRA A. (I980) Focal mesangial Immunofluorescence microscopy. III. Effect of proliferative in the rat caused by Habu heparin Microvasc. Res. i6, 373-390. venom: the effect of antiplatelet agents. Br. J. MICHAEL A.F., KEANE W.F., RAIJ L., VERNIER R.L. & exp. Path. 6i, 310-314. MAUER S.M. (I980) The glomerular mesan- COCCHERI S. & FIORENTINE P. (197I) Platelet gium. Kidney Int. 17, 141-I54. adhesiveness and aggregation in hypertensive MICHELS L.D., DAVIDMAN M. & KEANE W.F. (I980) patients. Acta med. scand. (Suppi.) 525, The effect ofchronic mesangial immune injury 273-275. on glomerular function. 1. Lab. clin. Med. 96, CRUSE J.M., LEWIS R.E., LOCKARD V.G. & MOHAM- 396-407. MAD A. (1982) Immune complex formation and MILLER K., DRESNER I.G. & MICHAEL A.F. (I980) resolution in systemic erythomatosus Localization of platelet antigens in human (SLE)-prone and non SLE-prone mice. Exp. mol. . Kidney Int. i8, 472-479. Path. 36, i-i8. NAGAOKA A., KIKUcHI K., KAWAJI H., MATSUO T. & DIXON R.H. & ROSSE W.F. (I977) Mechanism of ARAMAKI Y. (I 9 72) Life span, hematological complement-mediated activation of human abnormalities, thrombosis and other macrosco- blood platelets in vitro. Comparison of normal pical lesions in the spontaneously hypertensive and paroxysmal nocturnal hemoglobinuria rats. In Spontaneous Hypertension. Its Patho- platelets. J. clin. Invest. 59, 360-368. Genesis and Complications Ed. K. Okamoto. DUFFY J.L., CINQUE T., GRISHMAN E. & CHURG J. Tokyo, Japan: Igaku Shoin Ltd. pp. 149-I59. (1970) Intraglomerular fibrin, platelet aggre- O'BRIEN J.R. (1978) 'Exhausted' platelets con- gation and subendothelial deposits in lipoid tinue to circulate. Lancet ii, I 3 I6-I 3 I 7. nephrosis. J. clin. Invest. 49, 251-258. OLIvETri G., KITHIER K., GIACOMELLI F. & WIENER ELEMA J.D. & ARENDS A. (I 9 75) Focal and segmen- J. (I98I) Glomerular permeability to endo- tal glomerular hyalinosis and sclerosis in the geneous proteins in the rat. Effects of acute rat. Lab. Invest. 33, 554-56I. hypertension. Lab. Invest. 44, I27-137. HABIB R. (I973) Focal glomerular sclerosis (Edi- PARBTANI A. & CAMERON J.S. (I979) Platelet and torial). Kidney Int. 4, 355-36I. plasma serotonin concentrations in glomeru- KATZ S.M., LAVIN L. & SWARTZ C. (I 9 79) Glomeru- lonephritis I. Thromb. Res. 15, I09-I25. lar lesions in benign essential hypertension. ROMEN W. & MoRATH R. (I9979) Diffuse glomeru- Archs Path. lab. Med. 103, 199-203. losclerosis-a dysfunction of the mesangium? KINCAID-SMITH P. (I972) Coagulation and renal A morphometric study of the rat's kidney. disease (Editorial Review). Kidney Int. 2, Virchows Arch. B. Cell. Path. 31, 205-210. I83-190. TSCHOPP T.B. & ZUCKER M.B. (1972) Hereditary KREISBERG J.I. & KARNOVSKY M.J. (1978) Focal defect in platelet function in rats. Blood 40, glomerular sclerosis in the fawn-hooded rat. 217-226. Am. 1. Path. 92, 637-652. VAN LIEW J.B. & FELD L.G. (I980) Proteinuria in LEE S. & VERNIER R.L. (I 980) Immunoelectron hypertension. In Contributions to i 9 microscopy ofthe glomerular mesangial uptake Eds G.M. Berlyne et al. Basel: Karger. pp. and transport ofaggregated human albumin in I48-154. the mouse. Lab Invest. 42, 44-58. ZAHAVI J. & MMwER V.J. (I 9 74) Acquired 'storage MANDAL A.K., OLEINICK S.R., JAMES T.M., WISE pool disease' of platelets associated with circu- W., LONG H., NORDQUIST J.A., BELL R.D., YUNICE lating antiplatelet antibodies. Am. J. Med. 56, A.A. & PARKER D. (I978) Glomerular throm- 883-890. bosis in spontaneously hypertensive rat. II.