Journal of Microscopy, Vol. 232, Pt 3 2008, pp. 387–394 Received: 25 January 2008; accepted: 26 May 2008

Comparison between light and electron microscopy in canine and feline renal pathology: a preliminary study F . E . S C A G L I O N E ∗ , D . C A T A L A N O ∗ , R . B E S T O N S O †, C . B R O V I D A †, A . D ’ A N G E L O ∗ , R . Z A N A T T A ∗ , S . C O R N A G L I A ‡, E . C O R N A G L I A ∗ & M . T . C A P U C C H I O ∗

∗ Department of Animal Pathology, Faculty of Veterinary Medicine, University of Torino, Via

Leonardo Da Vinci 44, 10095, Grugliasco (Torino), Italy †Private practitioner, Torino, Italy ‡Azienda Sanitaria Ospedaliera Molinette San Giovanni Battista Torino, Corso Bramante 88/90-10126 Torino, Italy

Key words. Cat, dog, histology, renal diseases, ultrastructure.

Summary The aim of this study is to compare the accuracy and clinical use of light and transmission electron microscopy in detecting the early stages of renal pathologies in domestic animals. We examined 30 samples of renal tissue from cats and dogs referred to the Veterinary Hospital of the Department of Animal Pathology for different systemic diseases. The progressions of the kidney pathologies were classified using the scheme system proposed by the International Renal Interest Society. All samples were submitted for conventional histology and ultrastructural examination. Our study shows that electron microscopy is necessary to complete the histological examinations, especially to define early stages of kidney diseases (minimal changes disease, epithelial tubular pathologies, tubular basement membrane and glomerular basement membrane changes). Electron microscopy can be more accurate in defining the level of focal lesion, and permits discrimination between different clinical and pathological alterations such as fibrillary deposits. In conclusion, transmission electron microscopy associated with clinical, histological, histochemical and immunological examinations, is an essential method for diagnosis and prognosis of renal disease. Introduction Both in human and in animal pathologies, kidney disorders are one of the most fascinating chapters, involving general and systemic pathology. In fact, renal function has a key role in the whole body homeostasis, though a kidney impairment Correspondence to: F. E. Scaglione. Tel: 0039-0116709035; Fax: 0039-011 6709031; e-mail: [email protected]  C 2008 The Authors C 2008 The Royal Microscopical Society Journal compilation 

can cause a relevant alteration in the homeostatic balance. Renal pathologies can have different causes, from vascular to metabolic causes, and inflammatory processes, most of all due to infective factor (Benderitter et al., 1988; Nieto et al., 1992; Cook & Cowgill, 1996; Stokes & Forrester, 2004). In domestic carnivores most kidney diseases are localized in the glomeruli, and are frequently due to immunocomplex deposits and not to the direct action from the pathogen noxa (Germut & Rodriguez, 1973; Lopez et al., 1996). In human medicine glomerulo-nephrites definition and classification are very complex and submitted to numerous and continuous updates (Jennette et al., 2007). Nowadays for diagnostic purposes conventional light histology and immunofluorescent techniques with ultrastructural support on biopsies are the intra-vitam gold standard. The use of more accurate diagnostic techniques shows a higher incidence of glomerulopathies in domestic animals than was known in the past. In veterinary medicine International Renal Interest Society (IRIS) has recently (Brown et al., 1999) proposed a classification for the chronic kidney diseases (CKD) in dogs and cats. This classification scheme is based on the use of serum creatinine concentration to estimate progression of kidney disease. The aims of this study are to compare light microscopy to electron transmission microscopy, in terms of differences and usefulness in clinical practice, and to develop an accurate and specific method to establish intra-vitam future diagnosis for domestic carnivore renal diseases. Material and methods We examined 30 animals referred to the Veterinary Hospital of the Department of Animal Pathology for different systemic diseases.

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There were 14 cats and 16 dogs. Male gender was prevalent (73%). Dogs were of different breeds aged from 5 to 16 years. Cats were mainly European breed aged between 1 and 18 years. Seven of 14 cats were younger than 6 years. All of them were submitted to main haematological (haemochrome, total proteins, creatinine, azotaemia, phosphorum, calcium) and urinary analysis (specific gravity, urine protein, urine protein to creatinine ratio). All had increased creatinine and azotaemia level, as would be expected in clinical feature of kidney failure (Finco & Duncan, 1976; Brown et al., 1999). Forty percentage of the samples were collected by ultrasound guided biopsy intra-vitam on animals with specific renal pathologies. Sixty percentage of the samples have been collected during autoptic examinations by a trucut device on animals that were submitted for euthanasia, at the owners’ request, using Propofol and Tanax. In the latter cases, their systemic critical conditions had caused a renal impairment. Samples were collected within 10 min post-mortem to avoid all the altered histological findings secondary to post-mortem phenomena at the glomeruli and renal tubules. Samples were then divided into two aliquots using stereomicroscopy to ensure that each aliquot was formed by at least two glomeruli. One aliquot was processed for light microscopy, the other for ultrastructural investigations. Samples processed for histological examination were fixed in 10% neutral buffered formalin (pH 7) for 24 h. Using ethanol at increasing concentration and then xilol solutions, samples were dehydrated. At this stage they were paraffin embedded and 4–5-μm sections were obtained using the microtome (Leica Microsystems, Welzlar, Germany). These sections were stained with haematoxylin and eosine, PAS, PTAH and acid fucsin orange G. To evaluate these slides we used a light microscope Leica DM LS2 (Leica Microsystems). Samples processed for ultrastructural evaluations were fixed in 2.5% gluteraldehyde phosphate (pH 7.3) and stored at 4◦ C for 24 h. After the post-fixation process (in 1% osmium for 2 h and a quick wash out in 30% acetone) the samples were dehydrated in acetone and Spurr resin embedded. From each sample, using the ultramicrotome, we obtained thin sections (0.90 μm), stained with toluidine blue, and afterwards ultrathin sections of 70 nm contrasted by uranyl acetate and Pb citrate. The grids were evaluated using a transmission electron microscope (Zeiss model EM 109 JD; Zeiss, Oberkochen, Germany). Results All the lesions observed are summarized in Tables 1 and 2. All the cases are classified using the scheme proposed by IRIS using a four-stage scale of progression from stage 1 to stage 4. In the IRIS classification, cats with plasma creatinine 5.0 mg/dL are stage 4. In dogs, animals with plasma creatinine 5.0 mg/dL are stage 4. (Elliot & Brown, 2004; Brown et al., 2006). The histological and ultrastructural features observed, respectively, in dogs and in cats are reported in Tables 1 and 2. From the analysis of the tables, it appears that there is no correlation between the clinical findings and the histological and ultrastructural features. The interstitium was evaluated first, followed by the tubules and the glomeruli. Of all the glomeruli, Bowman’s capsule, mesangium, podocytes, glomerular basement membrane (GBM) and vessels were evaluated in a systematic manner. We classify as membranous glomerulonephritis a pathology characterized by changes in the GBM; the membranoproliferative glomerulonephritis includes different forms of glomerulonephritis in which there is thickening of the glomerular capillary wall and an increase in the number of cells in the glomerular tuft (mesangial or endocapillary hypercellularity). The mesangial thickening is an increase of the mesangial matrix without hypercellularity and mesangial hypercellularity is an increase of the mesangial cells (Jennette et al., 2007). All IRIS stage 1 observed in dogs are analysed as follows. Stages from 2 to 4 and cats related findings will be reported by comparative criteria for all the single feature compared to the IRIS stage 1 in dogs. Out of the dog group, four were classified in the initial stage of the CKD. They were non-azotemic with low plasma creatinine level and without specific clinical signs of kidney disease. Age ranged from 7 to 13 years. Histologically, multifocal to diffuse chronic nephritis were present in two cases. One animal showed numerous cortical cysts in addition. Focal necrosis of the tubular epithelium were described in two cases; steatosis in other two animals. Intra-tubular hyalin casts were present in one animal. By electron microscopy tubular necrosis (two cases) and steatosis (one case) were observed within tubular epithelium. Segmental crescent (one case), diffuse thickening of the Bowman’s capsule (two cases), mesangial thickening (four cases) associated in one dog to mesangial hypercellularity and multifocal proliferative glomerulonephritis and sclerosis (one case) were detected in the glomeruli. Erythrocytes into the urinary space were also observed in one dog. Thickening of the Bowman’s capsule (one case) and calcifications (one case), erythrocytes and mononuclear cells into the urinary space (one case), mesangial thickening (two cases) and hypercellularity (one case), diffuse podocyte foot process melt (three cases), severe GBM thickening (three cases) and segmental sclerosis (one case) were reported in the glomeruli. Only three animals, ranging from 2 to 5 years, were observed with clinical signs classified as stage 2. These dogs showed a mild azotemia in absence of clinical signs. One dog showed vacuolar degeneration of the tubular epithelium. In one of the

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Table 1. Histological and ultrastructural features reported in dogs classified using the classification scheme proposed by IRIS. Legend: TBM: tubular basement membrane; BC: Bowman’s capsule and GBM: glomerular basement membrane. IRIS stage

Histology

TEM

1

Interstitium: focal/multifocal, moderate/severe chronic nephritis (2); cysts (1). Tubules: focal necrosis (2); steatosis (2); hyaline casts (1).

Interstitium:/Tubules: diffuse necrosis (2); steatosis (1).

Glomeruli: crescent (1); diffuse thickening of the BC (2); mesangial thickening (3) and hypercellularity (1); multifocal proliferative glomerulonephritis and sclerosis (1); erythrocytes into the urinary space (1).

Glomeruli: thickening (1) and calcifications (1) of the BC; erythrocytes and mononuclear cells into the urinary space (1); mesangial thickening (2) and hypercellularity (1); diffuse podocyte foot process melt (3); severe GBM thickening (3); segmental glomerular sclerosis (1).

Interstitium: multifocal severe chronic nephritis (1).

Interstitium:/Tubules: TBM thickening (1); TBM calcifications (1); vacuolar degeneration (1); steatosis (1).

2

Tubules: vacuolar degeneration (1); steatosis (1); TBM thickening (1).

3

4

Glomeruli: proliferative glomerulonephritis (3); focal thickening of the BC (1); capillary expansion with erythrocytes (1).

Glomeruli: thickening and calcifications of the BC (1); mesangial hypercellularity (2); mesangium absence (1); segmental podocyte foot process melt (2); focal thickening of GBM (2); capillary expansion with erythrocytes (1).

Interstitium: multifocal, moderate/severe chronic nephritis (2).

Interstitium: mononuclear inflammatory cells (2).

Tubules: multifocal/diffuse necrosis (3); steatosis (1) intratubular calcifications (1); BM calcifications (1); hyaline casts (1).

Tubules: necrosis (3); degeneration with multilayering of the TBM (1); TBM calcifications (1); BM indentations (1).

Glomeruli: moderate, focal/diffuse thickening of the BC (3); diffuse calcifications of the BC (1); focal sclerosis and severe/diffuse membranous glomerulonephritis (1).

Glomeruli: crescents (1); severe thickening of the BC (1); calcifications of the BC (2); mesangial thickening and hypercellularity (1); segmental podocyte foot process melt and effacement (2); segmental GBM thickening (2); degeneration/thickening of pores (1).

Interstitium: multifocal/diffuse, moderate/severe, nephritis (4); cysts (1).

Interstitium: mononuclear inflammatory cells (2).

Tubules: multifocal/diffuse necrosis (4); erythrocytes (1), hyaline casts (1); BM calcifications (1).

Tubules: necrosis (3); hyaline casts (1); calcifications of the TBM (1); TBM thickening (1).

Glomeruli: thickening of the Bowman’s capsule (3); membranoproliferative glomerulonephritis (3). focal/diffuse hyalinosis/sclerosis (2).

Glomeruli: thickening of the BC (3); calcification of the BC (1); mesangial thickening (1); podocyte foot process melt (2); thickening of the GBM (3); degeneration of pores (1); subendothelial deposits (1); diffuse glomerular sclerosis (2).

three dogs we did not detect the mesangium and in another animal the glomerular blood vessels were expanded with numerous erythrocytes into the lumen. The dogs in stage 3 (five cases), ranging from 6 to 8 years, showed moderate azotemia and moderate clinical signs typical of the first stage of advancing CKD. From the histological point of view, no particular features were reported apart from the presence of tubular basement membrane calcifications. Electron microscopy allows the observation of degeneration with multilayering, calcifications and indentations of the tubular basement membrane (one case).

The dogs in the late stage, ranging from 10 to 13 years, showed severe clinical signs typical of uremic syndrome. Histologically, any new features were described. It is interesting to observe that every dog (four cases) showed multifocal or diffuse, moderate to severe non-suppurative nephritis and focal or diffuse necrosis of the tubular epithelium. The presence of multifocal sub-endothelial deposits was observed by transmission electron microscopy (TEM) in one dog. The Table 2 describes the features reported in 14 cats classified, similar to the dogs, using the IRIS classification scheme.

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Table 2. Histological and ultrastructural features reported in cats classified using the classification scheme proposed by IRIS. Legend: TBM: tubular basement membrane; BC: Bowman’s capsule and GBM: glomerular basement membrane. IRIS stage

Histology

TEM

1

Interstitium: focal/multifocal, moderate/severe nephritis (4); suppurative necrotizing nephritis (1); cysts (1). Tubules: focal necrosis (3); diffuse steatosis (2). Glomeruli: proliferative glomerulonephritis (2).

Interstitium: mononuclear inflammatory cells (1).

2

Tubules: focal/diffuse necrosis (2); steatosis (1). Glomeruli: BC thickening (1); BC calcifications (1); mesangial thickening and hypercellularity (1); podocyte foot process melt (2); GBM thickening (1) Glomerular sclerosis (1); inflammatory cells into the blood vessels (1).

Interstitium: multifocal moderate nephritis (2) cysts (1). Tubules: steatosis (1); focal necrosis (1) hyaline casts and erythrocytes (1). Glomeruli: focal thickening of the BM (1); erythrocytes into the urinary space (1); endothelial hypertrophy (1); membranoproliferative glomerulonephritis with focal sclerosis (1).

Interstitium: mononuclear inflammatory cells (1). Tubules: steatosis (2) necrosis (2).

3

Interstitium: multifocal, moderate/severe nephritis (2). Tubules: steatosis (1); hyaline casts and erythrocytes (1). Glomeruli: proliferative glomerulonephritis (1).

Interstitium: mononuclear inflammatory cells (1). Tubules: steatosis (1); necrosis (1). Glomeruli: focal thickening of BC (1); podocyte foot process melt (1); thickening of GBM (1); inflammatory cells in the mesangium (1).

4

Interstitium: multifocal/diffuse, moderate/severe chronic nephritis (4); hyalinosis (2); cysts (1). Tubules: steatosis (1); degeneration/necrosis (2); ialine casts (2); erythrocytes (1). Glomeruli: thickening of the BC (1); proliferative glomerulonephritis (2); membranous glomerulonephritis (1); glomerular sclerosis (2).

Interstitium: mononuclear inflammatory cells (1).

Five cats were classified in stage 1 ranged from 1 to 6 years. Histologically, a suppurative necrotizing nephritis was present in one animal. Two animals showed severe steatosis of tubular cells like the dogs. TEM did not add any interesting features. Two cats were classified in stage 2 (12 and 15 years old) and other two animals were classified in stage 3 (2 and 14 years old). Histological and ultrastructural features were similar to those in stages 2 and 3 reported in dogs. Five cats were reported in stage 4, ranging from 1 to 18 years. In these animals histologically multifocal or diffuse, moderate to severe chronic nephritis were present in all cases. Interstitial hyalinosis were reported in two animals. One cat showed severe tubular steatosis. Interesting findings by TEM were the presence in one cat of tubular basement membrane degeneration with stratification such as a form of tubular regeneration and the presence in a different subject of glomerular endothelial hyperplasia. In another case, multifocal sub-endothelial deposits were detected.

Glomeruli: BC thickening (1); erythrocytes into the urinary space (1); mesangial hypercellularity (1); podocyte foot process melt (2); GBM thickening (1); pores degeneration (2); glomerular sclerosis (2).

Tubules: steatosis (2); focal necrosis (1); TBM regeneration (1). Glomeruli: focal thickening of the BC (1); mesangial thickening (1) and hypercellularity (1); podocyte foot process melt (4); GBM thickening (3); pores degeneration (4); endothelial hypercellularity (1); sub-endothelial deposits (1); glomerular sclerosis (1).

Discussion There is little existing literature on the comparison of light and electron microscopy in the veterinary renal pathology (Krohn et al., 1973; Wright et al., 1973; Wright & Cornwell, 1983; Poli et al., 1991; Ramos-Vara et al., 2004; Wakamatsu et al., 2007). The aim of this paper is to compare light and TEM, with specific regard to the importance (usefulness/accuracy) of the TEM in detecting early stages of renal pathologies. The authors evaluate the findings observed in the interstitium, the tubules and the glomeruli using histological and ultrastructural methods. With regard to interstitial diseases, all the findings we can observe (such as inflammation cells in case of interstitial acute or chronic nephritis, post-inflammatory or post-necrotic fibrosis, erythrocytes outside the blood vessels) are better defined by light microscopy because of a wider field. In these cases it is easier to determine the severity of the lesions

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Fig. 1. Dog, kidney. Tubular basement membrane: lamellar thickening, TEM 4400×.

and the inflammatory reaction distribution and to verify the presence of malformations or post-inflammatory cysts, hyalin or amyloid deposition in the cortical or medullar interstitium. In cases of tubular disease, TEM adds an important view of the tubular basement membrane, that can be compromised in early stage of renal disease and that cannot always be seen with light microscopy (thickenings, calcifications, indentations). In our cases, basal membrane alterations were frequently reported both in dogs and in cats in different stage of CKD frequently associated with tubular degeneration or necrosis. We found the presence of indentations interesting, in one male dog, 3 years old, classified in the third IRIS stage, that showed diffuse and severe necrosis of tubular epithelial cells. In this case the tubular basement membrane is characterized by numerous, irregular and slight protrusions like slight fingerings (Fig. 1). In another dog, female, 6 years old, also classified in the third IRIS stage, a diffuse lamellar thickening of the TBM was detected as a clear sign of irreversible damage. The basal membrane was morphologically altered with atypical multilayering. The regeneration of the epithelial tubular cells (polyptychial epithelium) secondary to a lesion without damage to the basal membrane was observed in one cat only by TEM. In our study tubular regenerative processes were not detected by light microscopy, even though sometimes these features could be observed also on histological sections. As a clinical consequence, a sign of regeneration is an acute and/or not severe damage without any residual sign which could lead to a complete restitutio ad integrum. Thanks to TEM it is possible to study the morphology and the metabolic status of the tubular cells, observing the mitochondria, the microvacuoles as a metabolic index and the changes in the proximal contorti tubular cells microvilla. Among dogs, two animals showed tubular steatosis and only

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Fig. 2. Cat, kidney. Membranous glomerulonephritis. H-E 400×.

one showed vacuolar degeneration of the tubular epithelium. As regards tubular steatosis in cats only three cases, classified in different stages, were considered pathological. Steatosis observed in the other cats was considered as an age-related physiological feature. TEM is even more useful in the identification of glomerular disease (Murray et al., 1971; Reinacher & Frese, 1991; Verlander, 1998). Histology in fact permits the classification of glomerular pathologies as membranous or proliferative (Figs 2 and 3), but it is difficult or impossible to establish the localization of the deposits and the proliferating cell type. TEM allows the identification of minimal lesions not easily seen by light microscopy. It is possible to spot an initial thickening of Bowman capsule, due to proliferation or crescent-increasing depositions (Fig. 4), or a minimal and initial calcium deposition (Fig. 5). The presence of erythrocytes in the urinary space was observed in two cases by light and electron microscopy. This feature is associated with qualitative defects of the glomerular filtration with damage into the GBM and haematuria similar to the presence of intratubular erythrocytes. All the glomerular diseases defined as ‘hypercellularity’ are more systematically classified with TEM, thanks to the possibility of identifying the different cell populations: mesangial, endothelial (Fig. 6), podocytes. All these different populations are responsible for different aetiological damage: endothelial proliferation will cause the vessel obliteration, an infarction and consequent glomerular sclerosis; a podocytes or a mesangial proliferation will determine a filtration defect. Sometimes ‘hypercellular’ finding at light microscopy could be associated with the use of TEM to complete different lesions, such as in some parasite infestations. TEM has proved to be useful in the differential diagnosis of mesangial thickening from basal membrane thickening and from mild glomerular sclerosis, which are difficult to

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Fig. 5. Cat, kidney. Bowman’s capsule calcification, TEM 3000×.

Fig. 3. Dog, kidney. Proliferative glomerulonephritis. acid fucsin orange G 400×.

Fig. 6. Cat, kidney. Glomerulus: endothelial cells hypercellularity, TEM 3000×.

Fig. 4. Dog, kidney. Bowman’s capsule: crescent, TEM 3000×.

distinguish with light microscopy or immunohistochemical staining. In one German Shepherd dog, 5 years old, classified in the second IRIS stage, TEM detected a global glomerular presence of empty spaces in place of the mesangial matrix. The podocytes were elongated and flattened although the pedicels were morphologically normal. No alterations of the GBM were

detected. The aetiopathogenetic mechanism of this feature is not clear at the moment. At the filtration membrane level using TEM we can define the pedicels morphology. They can be normal, melt (Fig. 7), detached or aberrant. Basal membrane can be thickened and contain deposits (Figs 8 and 9) that can be identified and classified. All these possible changes affect the renal filtration quality. Glomerular flocculi capillaries dilatation associated with podocyte pedicels fusion represents a classic feature in cases of haematuria. As a sporadic finding not associated with the podocyte changes could be the congestion due to the use of Propofol and Tanax administered in euthanasia.

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Fig. 7. Cat, kidney. Glomerulus: foot process melt, TEM 7000×.

Fig. 8. Dog, kidney. Glomerulus: electron-dense deposits, TEM 3000×.

This work did not show any significant differences between lesions in dogs and cats as expected. The similarity of the lesions does not exclude a different aetiology. As regards to different stages of IRIS the lesions are very similar in both dogs and cats. However, the severity of lesions increases from the first to the fourth stage except for interstitial damage. The distribution of the lesions could be different too. Interstitium in fact almost always shows interstitial nephritis even if the severity of this inflammation varies from moderate to severe in the same stage. An interstitial flogosis could be focal or diffuse, with or without minimal alterations of haematological and urinary exams. On the other hand severe alterations of the clinical exams are correlated to increased tubular and glomerulal damage. In human beings, nephropathology is the only anatomic pathology sub-speciality that uses TEM for routine evaluation

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Fig. 9. Dog, kidney. Glomerulus: high magnification of a deposit, TEM 7000×.

of the specimens. Generally TEM is a valuable method required for the diagnosis of some diseases (fibrillary glomerulonephritis) and may reveal a diagnosis unsuspected after light and immunofluorescence examination. TEM is not always essential in veterinary renal pathology, but provides fundamental information especially for the diagnosis of tubular epithelium, and is even more useful in identifying changes of the tubular basal membrane and of the glomerular filtration membrane. In these sites it can identify membrane thickening, aberrant stratification, alterations of pores and pedicels, classify different deposits and precisely localize them (sub-epithelial, sub-endothelial, mesangial or intramembranous). In renal pathology it has assumed a very important role because the histological and immunological evaluations cannot give precise information especially on the localization of the glomerular lesions (Shore & Moss, 2002; Vaden, 2004, 2005; Amann & Haas, 2006). In early stages of a renal disease, TEM could allow a prompt diagnosis and specific therapy (Shore & Moss, 2002). Moreover, diagnosis by TEM can be much more detailed, especially if associated with traditional immunological techniques, to evaluate the deposit composition (Wright et al., 1973). It is an accurate diagnostic technique, but it requires a huge experience in specific applications. The main limitation of TEM is a restricted field compared to the light microscope. In fact, in a small sample, some particular structures of small size could be not included even if in human pathology a small specimen containing at least one glomerulus is considered sufficient and adequate for electron microscopy (Furness, 2000; Amann & Haas, 2006; Danilewicz & Danilewicz, 2007). Moreover, by TEM the extension and distribution of a change in the tissue could be difficult to evaluate.

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Other limitations are the longer time needed for sample preparation, and the higher costs. That is why it is not used in routine veterinary diagnostic practice, only in research sectors. In conclusion, TEM used in association with clinical diagnosis and histological evaluation is an essential method for identifying early stages of renal disease in domestic animals. This would allow a prompt and correct therapeutic treatment changing the prognosis of renal diseases.

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