Kidney ultrastructural lesions in dogs experimentally infected with Dirofilaria immitis (Leidy, 1856)

Veterinary Parasitology 113 (2003) 157–168

Kidney ultrastructural lesions in dogs experimentally infected with Dirofilaria immitis (Leidy, 1856) E.C. Paes-de-Almeida a,∗ , A.M.R. Ferreira a , N.V. Labarthe b , M.L.R. Caldas a , J.W. McCall c a

Departamento de Patologia, Universidade Federal Fluminense (UFF), Rua Marquˆes do Paraná, 303, HUAP, 4th Floor, Centro, Niterói, Rio de Janeiro CEP 24030-210, Brazil b Departamento de Patologia e Clinica Veterinaria, Universidade Federal Fluminense (UFF), Rua Vital Brazil Filho, 64, Santa Rosa, Niterói, Rio de Janeiro CEP 24230-340, Brazil c Department of Medical Microbiology and Parasitology, College of Veterinary Medicine, University of Georgia, Athens, GA 30602, USA Received 22 March 2002; received in revised form 6 December 2002; accepted 10 January 2003

Abstract Kidneys of 16 beagles with experimentally induced heartworm (Dirofilaria immitis) infections and 4 heartworm-na¨ıve dogs were studied by light and electron microscopy. The infections were induced either by subcutaneous injection of infective larvae or by the transplantation of adult parasites, and infection periods varied from 111 to 818 days and 365 to 923 days, respectively. One control group of heartworm-na¨ıve dogs and four groups of heartworm-infected dogs, which were divided according to the type and the length of infection, were used. In the infected dogs, thickening of the glomerular basement membrane (GBM), the presence of dense deposits in the GBM, and foot process effacement were the most frequent lesions observed. In some dogs, electron dense deposits were seen in the GBM and the mesangium and/or enlargement of the mesangial matrix could be characterized. The longer the infection period, the thicker the GBM and the more common the occurrence of foot process effacement. In general, these alterations were more evident in animals that had been infected for more than 1 year, had high microfilaremia, and had 14 or more parasites in the main pulmonary artery and its branches. The presence of dense deposits suggests that the pathogenesis of kidney disease in dirofilariasis is associated with deposits of immune complexes in the membrane. The finding of ultrastructural changes in dogs with early prepatent infections

∗ Corresponding author. Present address: Elan Cardozo Paes de Almeida, Rua Alonso Faria, 1249, Ze Garoto, Sao Gonçalo, Rio de Janeiro CEP 24440-370, Brazil. Fax: +55-21-2712-8385. E-mail address: [email protected] (E.C. Paes-de-Almeida).

0304-4017/03/$ – see front matter © 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0304-4017(03)00020-7

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suggests that immature heartworms, as well as microfilariae and possibly adult worms, contribute to the glomerulonephropathy. © 2003 Elsevier Science B.V. All rights reserved. Keywords: Dirofilaria immitis; Canine heartworms; Dirofilariasis; Ultrastructural; Kidneys

1. Introduction Dirofilariasis is a disease caused by Dirofilaria immitis, which is readily transmitted among certain mammals by mosquitoes, but most commonly infects dogs (Bain and Chabaud, 1986). It is a disease that affects many organs, including the lung, heart, liver, and kidney (Rawlings, 1986; Lombard, 1987). The most important damage is found in the pulmonary arteries, where adult parasites locate much of the time (Lombard, 1987). The presence of adult worms in the right ventricle of the heart and the pulmonary arteries mechanically damage the endothelium eliciting an inflammatory reaction which ends in villous arteritis and, consequently, functional and morphological changes in the pulmonary tissues (Lombard, 1987; Rawlings and Calvert, 1989). Right ventricle congestive heart insufficiency, subsequent to progressive pulmonary artery disease, develops in some infected animals (Rawlings, 1986). Kidney damage, such as glomerulonephritis is described in dogs with heartworm disease (Klei et al., 1974; Simpson et al., 1974; Casey and Splitter, 1975; Shirota et al., 1979; Abramowsky et al., 1981; Aikawa et al., 1981; Grauer et al., 1987; Paes-de-Almeida et al., 2001); however, kidney failure and uremia generally are not seen (Osborne et al., 1981). Functional alterations in the glomerular basement membrane (GBM) (Buoro and Atwell, 1983) may lead to proteinuria, which is commonly associated with D. immitis infections (Barsanti, 1977; Osborne et al., 1981; Buoro and Atwell, 1983; Grauer et al., 1987; Ludders et al., 1988). Several kinds of kidney damage are described in dogs infected with D. immitis: (1) immune-mediated glomerulonephropathy (Rawlings, 1986; Sutton, 1988), (2) glomerulosclerosis (Simpson et al., 1974; Grauer et al., 1989), (3) chronic interstitial nephritis (Shirota et al., 1979; Paes-de-Almeida et al., 2001) and (4) amyloidosis (Drazner, 1978; Rawlings, 1986). The most common ultrastructural changes are seen in the GBM, such as thickening (Klei et al., 1974; Casey and Splitter, 1975; Aikawa et al., 1981; Rawlings, 1986; Grauer et al., 1987; Sutton, 1988) and electron dense deposits (Abramowsky et al., 1981; Aikawa et al., 1981; Grauer et al., 1987). In the mesangium, there is an increase in the number of mesangial cells (Klei et al., 1974; Simpson and Jackson, 1985; Rawlings, 1986) and size of the matrix (Simpson and Jackson, 1985), in addition to the presence of electron dense deposits (Casey and Splitter, 1975; Abramowsky et al., 1981; Aikawa et al., 1981). Another lesion that can be clearly observed is foot process effacement of the epithelial cells (Osborne et al., 1981; Simpson and Jackson, 1985; Grauer et al., 1987). The pathogenesis of kidney damage is widely discussed and polemic. Microfilaria and adult worm antigens (Nakagaki et al., 1990, 1993) are reported to have an important role in the process through immune-mediated mechanisms (Klei et al., 1974; Casey and Splitter, 1975; Abramowsky et al., 1981; Aikawa et al., 1981; Grauer et al., 1987, 1988). To better clarify kidney pathophysiology in dirofilariasis, it is necessary to detail the glomerular injury

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as well as to study the role(s) of microfilariae, immature heartworms, and adult worms in the pathogenesis. Thus, the objective of the present paper is to identify and describe, by ultrastructural observation, kidney damage in D. immitis-infected dogs with various worm burdens, infection periods, and microfilarial counts.

2. Materials and methods Twenty dogs (eight females, eight intact males, and four castrated males), ranging in age from 5 to 21 months were used. The experimental inoculation procedures, adult heartworm transplantation procedures, blood collection and analysis, postmortem examinations, and recovery and enumeration of the parasites at necropsy were performed at TRS Labs Inc., Athens, GA, USA. Four beagles were infected by IV transplantation of worms via the right jugular vein: 3 dogs received 18 five-month-old worms and 1 dog received 35 fully grown adult worms (Dzimianski et al., 1989). Twelve beagles were experimentally infected by SC injection in the inguinal area with 50 (5 animals) or 400 (7 animals) infective, third-stage larvae (L3 ), as previously described (McCall, 1981). Heartworm-infected dogs were allocated to 4 groups of 3–5 dogs each, based on type of infection (SC-induced or IV transplantation of worms), size of the inoculum (50 or 400 L3 ; 18 or 35 worms transplanted), age of the inoculum (5-month-old or fully grown adult worms), and/or infection period. Four heartworm-na¨ıve female dogs, ages 33 months, were used as controls. The animals’ lives were terminated, under controlled conditions, between 111 and 923 days after infection. Before euthanasia, a blood sample was collected in EDTA from each dog to make a microfilarial count (Dzimianski et al., 1989). Euthanasia and postmortem examinations were performed as described previously (McCall et al., 1996). At necropsy, the heart and lungs were thoroughly examined by opening the main pulmonary artery and its branches, and the recovered parasites were counted. The kidneys were removed, cut longitudinally into equal parts, and kept in 10% buffered formalin until they were processed and examined in the Histopathology Laboratory of the Pathology Department at the Universidade Federal Fluminense. For electron microscopic processing, a small section of kidney cortex (area smaller than 1 cm2 from a 1 mm thick section) was removed and then washed in sucrose buffer, fixed in 2.5% glutaraldehyde solution, and postfixed in 1% osmium tetroxide and cacodylate buffer. Afterwards, the tissue samples were dehydrated with different concentrations of alcohol, placed in propylene oxide, and then embedded in Araldite 502. The resin was prepared in propylene oxide by mixing dodecynyl succinic anhydride (DDSA) and DMP 30 (n-dibutyl phthalate) in rising concentrations. Semi-thin sections of 1 ␮m thickness were stained with 1% toluidine blue, and ultra-thin sections of 60 nm thickness were contrasted with uranyl acetate for 20 min and lead citrate for 5 min (Hayat, 1989). The kidney cortex samples were examined using a Zeiss-EM-109 electron microscope. The main findings were identified as follows: GBM focal and segmental or focal and global thickening, GBM vacuolation, dense deposits in the GBM, foot process effacement, expansion of mesangial matrix, and microfilaria in the capillary lumen. These findings were graded as absent or present.

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3. Results All of the 16 infected dogs had heartworms in the heart and lungs and associated vessels at necropsy, while the control dogs were free of heartworms (Table 1). The heartworms recovered from Group B dogs were immature and the infections were of short duration (111 days), while heartworms from all other infected dogs were fully grown and the infections were of relatively long duration (>325 days). The dogs in Groups A and D had similar worm burdens, which were low to moderate, but the infection period for Group D was longer (365–923 days) than that for Group A (325 days). Some dogs in Groups B and C had relatively heavy worm burdens, but the infection period for Group B was only 111 days, whereas that for Group C ranged from 419 to 818 days. With the exception of Dogs 3A Table 1 Groups of animals according to type and length of infection, with microfilarial counts and numbers of adult worms recovered at necropsy Animal Number

Sexa

Age at necropsy (months)

Microfilarial count/20 ␮l

Infection period (days)

Total number worms at necropsy

Group A (50 L3 SC) 1A M 2A M 3A M 4A F 5A F

20 18 18 18 20

37 68 0 16 0

325 325 325 325 325

10 11 18 5 2

Group B (400 L3 SC)b 1B N 2B N 3B N 4B N

14 14 14 14

0 0 0 0

111 111 111 111

74 108 5 33

Group C (400 L3 SC) 1C M 2C M 3C M

22 35 35

325 488 230

419 818 818

57 71 32

Group D (IV adult worm) 1Dc F 33 M 20 2Dc 3Dc M 20 F 39 4Dd

383 258 301 111

365 365 365 923

16 14 15 15

– – – –

– – – –

– – – –

Group E (control) 1E F 2E F 3E F 4E F a

33 33 33 33

F, female; M, male; N, neutered male. Immature worms in the pulmonary lobar arteries and arterioles. c Eighteen adult worms. d Thirty-five adult worms. b

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Fig. 1. Transmission electron micrograph of a normal glomerulus. Control dog (3E)—glomerular basement membrane (GBM) (–) and foot processes of podocytes (PP) normal (6600× negative).

and 5A, all dogs with infections of more than 111 days duration had microfilariae. Group A dogs showed lower microfilaremia when compared to dogs of other groups (Table 1) No ultrastructural damage was noted in glomeruli of any of the heartworm-naive control animals (Group E) (Fig. 1). Group A dogs showed no meaningful ultrastructural evidence of glomerular lesions (Table 1, Fig. 2). In this group, microfilariae frequently were found in glomerular capillaries, peritubular areas, and interstitial blood vessels, even in the absence of any damage. Animals infected for more than 1 year showed interstitial nephritis (Groups C and D), while dogs with shorter infection periods (Groups A and B) did not. The most frequent glomerular damage in infected dogs included: (1) focal and segmental and/or focal and global GBM thickening, (2) GBM vacuolation, (3) deposits in the GBM, (4) focal and segmental or diffuse foot process effacement, and (5) expansion of the mesangial matrix (Table 2). These damages were noted in all infected groups, except Group A, but were more common in dogs infected for prolonged periods (>1 year) and with high microfilarial counts (Groups C and D). In one Group B animal (1B), expansion of the mesangial matrix was clear. Another Group B animal (3B) showed focal and segmental foot process effacement and had dense deposits in the mesangium (Fig. 3). Damage in the basal capillary membrane was similar in Dogs 1C and 2C. These were characterized as irregular and severe thickening of the GBM (with projection of the membrane between the foot processes), and including at the same time, thickening of the lamina rara interna (LRI) and lamina rara externa (LRE) (Fig. 4). There were also electron lucent areas within the LRE, i.e. deposits in reabsorption (Fig. 5). Diffuse foot process effacement was also found associated with these GBM lesions (Fig. 4).

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Table 2 Ultrastructural lesions found in the kidneys of dogs experimentally infected with D. immitis Animal GBM thickening

GBM GBM electron vacuolation dense deposits

Foot processes effacement (focal/segmental or diffuse)

Mesangial cell proliferation

Mesangial matrix increase

Absent Absent Absent Absent Absent

Absent Absent Absent Absent Absent

Absent Absent Absent Absent Absent

Absent Absent Absent Absent Absent

Present Present Present Present Present

Absent Absent Absent Absent Absent

Group B (400 L3 SC) 1B Absent Absent 2B Absent Absent Absent 3Bb Absent

Absent Absent Absent

Absent Absent Absent

Absent Absent Present (focal/segmental) Absent

Present Present Present

Present Absent Absent

Present

Absent

Focal/ segmental Group A (50 L3 SC) 1A Absent 2Aa Absent 3A Absent 4A Absent 5A Absent

4B

Absent

Global

Absent

Absent

Group C (400 L3 SC) 1C Absent Present Present 2Cc Present Absent Present 3C Present Absent Present

Absent

Present Present Present

Present Present Present (focal/segmental)

Absent Present Present

Present Present Absent

Group D (IV adult worm) Present Present 1Db Absent Absent Absent 2Dd Present

Present Absent

Present Present

Absent Absent

Present Present

Present Absent

Absent Absent Absent Absent

Absent Absent Absent Absent

Absent Absent

Absent Absent

Present Present (focal/segmental) Absent Present

Group E (heartworm-na¨ıve control) 1E Absent Absent Absent 2E Absent Absent Absent 3E Absent Absent Absent 4E Absent Absent Absent

Absent Absent Absent Absent

Absent Absent Absent Absent

3Db 4D

Absent Present

Absent Absent

a

Capillary collapse, GBM irregularities focal. Mesangial deposits. c Increased mesangial cells. d Mesangial interposition in GBM, capillary collapse. b

Glomerular basement membrane alterations, similar to those described for Dogs 1C and 2C, were observed in Dog 3C. However, these were focal and segmental and milder. Focal and segmental foot process effacement also occurred in these areas. Focal /segmental or global GBM thickness alterations were observed in two of the three dogs in Group D, which were infected for 1 year or longer. In Dog 1D, GBM lesions were also similar in degree to those described for Dogs 1C and 2C. In these, however, there was no clear involvement in the LRI (Fig. 6). Enlargement of the mesangial matrix, associated with the presence of dense deposit and foot process effacement, was also observed

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Fig. 2. Transmission electron micrograph of a normal glomerulus. 1A dog—glomerular basement membrane (–), podocytes (P), foot processes of podocytes (PP), endothelial cell (E), mesangium (M) (10,500× negative).

Fig. 3. Transmission electron micrograph of a glomerulus. 3B dog—focal and segmental foot processes effacement (PP), presence of electron dense deposits in the mesangium (star) (10,500× negative).

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Fig. 4. Transmission electron micrograph of a glomerulus. 1C dog—irregular thickening of the glomerular basement membrane (GBM), including lamina rara interna (LRI) and lamina rara externa (LRE) with the projection of the GBM between the foot processes (PP). Lamina densa (LD) and podocytes (P) (10,500× negative).

Fig. 5. Transmission electron micrograph of a glomerulus. 1C dog—deposits in reabsortion into the lamina rara externa LRE (star) (10,500× negative).

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Fig. 6. Transmission electron micrograph of a glomerulus. 1D dog—thickening of the glomerular basement membrane (GBM), lamina rara externa (LRE), with deposits in reabsortion (star) (18,000× negative).

(Fig. 7). These latter lesions were not observed in Dog 2D, which, however, showed focal and segmental foot process effacement, circumferential mesangial interposition (Fig. 8) and capillary collapse. In Dog 3D, mesangial electron/dense deposits were seen and an enlarged mesangium was evident. The most important lesions in Dog 4D were thickening of focal and segmental GBM and foot process effacement (Fig. 9).

Fig. 7. Transmission electron micrograph of a glomerulus. 1D dog—enlargement of the mesangial matrix (M), associated with presence of dense deposit (thick arrow) (6600× negative).

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Fig. 8. Transmission electron micrograph of a glomerulus. 1D dog—focal and segmental foot processes effacement (PP) and mesangial interposition in glomerular basement membrane (GBM) (star) (10,500× negative).

Fig. 9. Transmission electron micrograph of a glomerulus. 4D dog—thickening and duplication of the glomerular basement membrane (GBM) (star), with mesangial interposition (M) and foot processes effacement (PP) (4400× negative).

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4. Discussion and conclusions Our ultrastructural study of the kidneys of dogs with experimentally induced heartworm (D. immitis) infections revealed glomerular lesions similar to those described in previous studies on dogs with spontaneous or experimental dirofilariasis (Klei et al., 1974; Simpson et al., 1974; Casey and Splitter, 1975; Shirota et al., 1979; Abramowsky et al., 1981; Aikawa et al., 1981; Osborne et al., 1981; Simpson and Jackson, 1985; Rawlings, 1986; Grauer et al., 1987; Lombard, 1987; Ludders et al., 1988). The results of the current study reaffirm that the main kidney damage in D. immitis-infected dogs is membrano-proliferative glomerulonephritis (Simpson et al., 1974; Grauer et al., 1987; Ludders et al., 1988; Nakagaki et al., 1990; Paes-de-Almeida et al., 2001). The most frequent kidney damage was: (1) thickening of GBM, (2) vacuolation in GBM, (3) deposits in GBM, (4) foot process effacement, and (5) proliferation of mesangial cells. These lesions were observed mainly in dogs with infections of at least 1 year duration and with high microfilarial counts. Lesions found in the GBM, along with foot process effacement of the glomerular epithelial cells, change the selective semi-permeability of the glomeruli, which may result in various degrees of proteinuria (Sutton, 1988). These findings support other reports which suggest that the heartworm parasite elicits glomerulonephropathy by immune complexes (Abramowsky et al., 1981; Aikawa et al., 1981; Grauer et al., 1987) and that the microfilariae have an important function in the pathogenesis of glomerulonephritis (Klei et al., 1974; Casey and Splitter, 1975; Abramowsky et al., 1981; Aikawa et al., 1981). Other studies suggest that adult worms also contribute antigens to these immune complexes (Nakagaki et al., 1990, 1993). Unfortunately, these previous papers do not indicate whether or not the dogs had been microfilaremic earlier or whether the infections were naturally acquired or experimentally induced (Osborne et al., 1981; Wolfsheimer and Werner, 1983). All dogs infected in the present study, even those in Group B, which did not develop microfilaremia, showed kidney lesions, mainly mesangial cell proliferation. Thus, our observation of glomerular lesions in two of the four amicrofilaremic dogs in Group B, which had only sexually immature worms, suggests that immature heartworms, as well as microfilariae and possibly older adult worms, are responsible for the glomerulonephritis. The most severe glomerular and interstitial lesions were observed in the dogs with high microfilarial counts and long infection periods. Although glomerular damage may be elicited by antigens released by immature (young adult) heartworms, fully grown adult worms, and microfilariae, the presence of microfilariae seems to exacerbate the condition. References Abramowsky, C.R., Powers, K.G., Aikawa, M., et al., 1981. Dirofilaria immitis. 5. Immunopathology of filarial nephropathy in dogs. Am. J. Pathol. 104, 1–12. Aikawa, M., Abramowsky, C.R., Powers, K.G., 1981. Dirofilariasis. IV. Glomerulonephropathy induced by Dirofilaria immitis infection. Am. J. Trop. Med. Hyg. 30, 84–91. Bain, O., Chabaud, A.G., 1986. Atlas de larves infestantes de filaires. Trop. Med. Parasitol. 3, 301–340. Barsanti, J.A., 1977. Serum and urine proteins in dogs infected with Dirofilaria immitis. In: Otto, G.F. (Ed.), Proceedings of the Heartworm Symposium, vol. 77. American Heartworm Society, Atlanta, GA, pp. 53–56. Buoro, I.B.J., Atwell, R.B., 1983. Urinalysis in canine dirofilariasis with emphasis on proteinuria. Vet. Rec. 112, 252–253.

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