Signor, Department of Comparative Biomedicine and Food Science, Legnaro, Italy
Since 2008, many vets from Europe and America have joined the International Veterinary Renal Pathology Service and the European Veterinary Renal Pathology Service. The two services have been created to provide information and instruments to improve health care for dogs and cats affected by renal diseases. Also, they supply a wide information network that helps clinicians to take decisions over pets, to send samples to laboratories, to have shipment time shortened and to have an efficient schedule time that provides reports in a short time. Specific goals of this service were established a priori such as improvement of diagnostic process and outcomes of pets affected by renal diseases and the main purpose was to describe guidelines for histologic, ultrastructural, and immunofluorescence examinations of renal tissue. When performing a renal biopsy few criteria should be encountered. First, the biopsy should be considered as a procedure to improve therapy and the biopsy itself should be done safely. The tissue sample should be evaluated by expert and specialized nephropathologists to get the most informative description using all the methods required to characterize the pathological changes. The required methods for renal biopsy evaluation include special staining protocols for light microscopic (LM) evaluation, as well as for transmission electron microscopic (TEM), and immunofluorescence evaluations (IF). Generally, two techniques for sampling are considered: 1) needle core biopsy, 2) wedge biopsy. Needle biopsies can be obtained performing by laparoscopy, celiotomy, manual palpation, and ultrasound-guided needle biopsy techniques. The latter is commonly performed and generally with satisfying results.
Nephropathology is a quite unique specialization in anatomic pathology and a complete set of analysis comprising LM, IF, and TEM should be always considered. Samples for LM and TEM needs to be placed in formalin and glutaraldehyde, respectively. Whereas, samples for IF need to be stored in the transport solution (Michel’s transport media) and dispatched within 48 hours.
For LM, the renal tissue sections are usually thinner (2–3 µm thick) compared to sections obtained for other tissues in routine histopathology (5–6 µm thick). The method results important for assessment of glomerular cellularity as well as for evaluation of glomerular basement membrane (GBM) through histochemical stains. Slides are alternatingly processed with at least 5 different histochemical stains: haematoxylin-eosin (H&E), periodic Acid-Schiff (PAS), Jones methenamine silver (JMS), Masson’s trichrome (TR) and Congo red stain (CR). The H&E-stained sections are useful for assessing cellular details such as the intrinsic cellular components of glomeruli, tubules, interstitium, and vessels, as well as extrinsic cells such as inflammatory cells. The PAS and JMS stains are indicated for assessing GBM thickening. PAS stain accentuates the tubular, Bowman’s capsule, and glomerular capillary wall basement membranes. Degenerating and atrophic tubules are visualized with PAS staining showing thickened basement membranes around the collapsed degenerated tubules. The JMS stain is useful for assessing external surface of the GBM (subepithelial deposits), thickness, irregularity, small projections of GBM matrix such as spikes, holes, and double contours. The TR stain stains collagen and fibrous connective tissues (e.g., interstitial fibrosis, glomerulosclerosis) in blue. This staining is indicated to identify the presence of immune complexes.
For EM, as soon as the tissue is obtained from the kidney, it should be rapidly placed in the fixative. Afterwards, tissue is processed into plastic, trimmed, cut a 1-µm section and stained with toluidine blue. The sections required for the analysis are cut by ultramicrotome and collected on a copper grid and stained with lead citrate and uranyl acetate. Usually, one or 2 glomeruli are examined. At different magnification, capillary loops, mesangial regions, tubular interstitial areas, and vascular structures are examined. Some of the most important ultrastructural pathologic abnormalities are into the endothelial cells, GBM and visceral epithelial cells (i.e., podocytes). Additionally, EM evaluation is fundamental to detect electron-dense deposits of immune complexes (IC), which have different implications and identify different disease processes. The location of IC is also very important whether in the mesangium or capillary walls (subepithelial, intramembranous, subendothelial, or associated with mesangial cell interpositioning).
For IF examination, fresh unfixed renal specimens are embedded in OCT gel, snap-frozen in liquid nitrogen and stored at -80°C. Then, sections from snap-frozen tissue are mounted. The common use of IF labelling in veterinary nephropathology is to determine the phenotype of immune-deposits. The available antibodies are able to label canine IgG, IgM, IgA, C3 component of complement.
Recently, the World Small Animal Veterinary Association Renal Standardization Study Group (YVSAVA) has defined a diagnostic algorithm for glomerular diseases classification in dog. Using hierarchical cluster analysis, two common patterns of glomerular injury were identified and these resulted very stable creating a simplified and accurate guide for veterinary pathologists when evaluating renal biopsies. The analysis revealed 2 large categories of glomerular diseases based on the presence or absence of IC deposition: immune complexmediated glomerulonephritis (ICGN) category including membranoproliferative GN, membranous GN, and mixed type glomerulonephritis. The second category including dogs with non-ICGN: glomerular amyloidosis, focal segmental glomerulosclerosis, minimal change disease (MCD), and juvenile nephropathies (JN).
Focusing on the immune complex damage in ICGN, this is characterized by a dynamic interaction between antigen and antibody and the injury is dependent by several factors such as mechanism of formation, the site of deposition, the composition of the deposits and the amount of the deposits. The ICs may be performed from circulating antigens and antibodies and only ICs with an antigen excess tend to deposit along the capillary walls and mesangium. Aetiology of ICGN has been associated with several and inflammatory diseases, but there are many reports in which the antigen source or underlying disease is not identified and in this case, the GN is referred to as idiopathic. Furthermore, ICs may form in situ in the glomerulus, and this occurs when circulating antibodies react with endogenous glomerular antigens or with “planted” non-glomerular antigens (i.e., Dirofilariosis). The ICs can damage the glomerular structures by two mechanisms: attracting circulating inflammatory cells or activating resident glomerular cells, which release vasoactive substances, cytokines, and activators of coagulation. Nonetheless, the most important glomerular injury mediator is the complement cascade, particularly C5b-9 membrane attack complex formation. Within the membranes of glomerular cells, this complex causes activation of both glomerular epithelial cells and mesangial cells to produce inflammatory mediators, such as oxidants and proteases. The glomerulus responds to this injury by cellular proliferation, thickening of the GBM subsequent to upregulation of epithelial cell receptors for transforming growth factor and epithelial detachment (causing proteinuria) and, if the insult persists, hyalinisation and sclerosis. The glomerular visceral epithelial cells (podocyte) gives a fundamental contribution in glomerular filtration permeselectivity and they respond to injury with reversible changes such as hypertrophy, foot process effacement, cell body attenuation, and microvillus formation.