Detection and Classification of Novel Renal Histologic Phenotypes Using Deep Neural Networks.

With the advent and increased accessibility of deep neural networks (DNNs), complex properties of histologic images can be rigorously and reproducibly quantified. We used DNN-based transfer learning to analyze histologic images of periodic acid-Schiff-stained renal sections from a cohort of mice with different genotypes. We demonstrate that DNN-based machine learning has strong generalization performance on multiple histologic image processing tasks. The neural network extracted quantitative image features and used them as classifiers to look for differences between mice of different genotypes. Excellent performance was observed at segmenting glomeruli from non-glomerular structure and subsequently predicting the genotype of the animal on the basis of glomerular quantitative image features. The DNN-based genotype classifications highly correlate with mesangial matrix expansion scored by a pathologist (R.E.C.), which differed in these animals. In addition, by analyzing non-glomeruli images, the neural network identified novel histologic features that differed by genotype, including the presence of vacuoles, nuclear count, and proximal tubule brush border integrity, which was validated with immunohistologic staining. These features were not identified in systematic pathologic examination. Our study demonstrates the power of DNNs to extract biologically relevant phenotypes and serve as a platform for discovering novel phenotypes. These results highlight the synergistic possibilities for pathologists and DNNs to radically scale up our ability to generate novel mechanistic hypotheses in disease

Proliferative, necrotizing and crescentic immune complex-mediated glomerulonephritis in a cat

Case Summary A 5-year-old cat was examined for vomiting and anorexia of 2 days’ duration. Azotemia, hyperphosphatemia and hypoalbuminemia were the main biochemical findings. Serial analyses of the urine revealed isosthenuria, proteinuria and eventual glucosuria. Hyperechoic perirenal fat was detected surrounding the right kidney by ultrasonography. Histopathologic evaluation of ante-mortem ultrasound-guided needle biopsies of the right kidney was consistent with proliferative, necrotizing and crescentic glomerulonephritis with fibrin thrombi, proteinaceous and red blood cell casts, and moderate multifocal chronic-active interstitial nephritis. Owing to a lack of clinical improvement, the cat was eventually euthanized. Post-mortem renal biopsies were processed for light microscopy, transmission electron microscopy and immunofluorescence. This revealed severe focal proliferative and necrotizing glomerulonephritis with cellular crescent formation, podocyte injury and secondary segmental sclerosis. Ultrastructural analysis revealed scattered electron-dense deposits in the mesangium, and immunofluorescence demonstrated positive granular staining for λ light chains, consistent with immune complex-mediated glomerulonephritis. Severe diffuse acute tubular epithelial injury and numerous red blood cell casts were also seen. Relevance and novel information To our knowledge, this is the first report of naturally occurring proliferative, necrotizing and crescentic immune complex glomerulonephritis in a cat

Assessment of peritubular capillary rarefaction in kidneys of cats with chronic kidney disease

Abstract Background Hypoxia is a key driver of fibrosis and is associated with capillary rarefaction in humans. Objectives Characterize capillary rarefaction in cats with chronic kidney disease (CKD). Animals Archival kidney tissue from 58 cats with CKD, 20 unaffected cats. Methods Cross‐sectional study of paraffin‐embedded kidney tissue utilizing CD31 immunohistochemistry to highlight vascular structures. Consecutive high‐power fields from the cortex (10) and corticomedullary junction (5) were digitally photographed. An observer counted and colored the capillary area. Image analysis was used to determine the capillary number, average capillary size, and average percent capillary area in the cortex and corticomedullary junction. Histologic scoring was performed by a pathologist masked to clinical data. Results Percent capillary area (cortex) was significantly lower in CKD (median 3.2, range, 0.8‐5.6) compared to unaffected cats (4.4, 1.8‐7.0; P = <.001) and was negatively correlated with serum creatinine concentrations (r = −.36, P = .0013), glomerulosclerosis (r = −0.39, P = <.001), inflammation (r = −.30, P = .009), and fibrosis (r = −.30, P = .007). Capillary size (cortex) was significantly lower in CKD cats (2591 pixels, 1184‐7289) compared to unaffected cats (4523 pixels, 1801‐7618; P = <.001) and was negatively correlated with serum creatinine concentrations (r = −.40, P = <.001), glomerulosclerosis (r = −.44, P < .001), inflammation (r = −.42, P = <.001), and fibrosis (r = −.38, P = <.001). Conclusions and Clinical Importance Capillary rarefaction (decrease in capillary size and percent capillary area) is present in kidneys of cats with CKD and is positively correlated with renal dysfunction and histopathologic lesions

Mesangial cell process extension into the GBM of AS but not WT dogs.

<p>A-B: Dual immunofluorescence immunostaining of kidney from a WT dog and an AS dog, 63x1.4 n.a. oil with 3X zoom. Anti-laminin β2 and anti-integrin α8 antibodies were used to stain the GBM and mesangial cells, respectively. Staining reveals distinct delineation of mesangium absent from the GBM of the normal dog (A) but extension of mesangium within the GBM of the AS dog (B). C: Transmission electron microscopy of kidney tissue from an AS dog at milestone 2. Cytoplasmic extensions, also described as cellular interpositioning, are observed at the base of the capillary loops, consistent with invasion of mesangial cell processes (arrow) corresponding with extension of the mesangium (B).</p

Clinical parameters (average, range) at each milestone in AS (n = 8) vs WT (n = 4) dogs.

<p>(A) Serum creatinine (sCr); (B) Symmetric dimethylarginine (SDMA); (C) Urine protein: urine creatinine (UPC); (D) Iohexol clearance; *p<0.05.</p

Identification of laminin 211 in the GBM of AS but not WT dogs.

<p>Dual immunofluorescence immunostaining of kidney from a WT dog (A-C) and an AS dog at milestone 2 (D-F); 63x1.4 n.a. oil. Laminin 521 of the GBM was labeled with anti-laminin β2 (LAMB2), and laminin 211 produced by mesangial cells, was labeled with anti-laminin α2 (LAMA2), demonstrating co-localization of laminin 211 with the GBM of several capillary loops in the AS dog.</p

Immunofluorescence staining for fibronectin of kidney from WT and AS dogs at milestone 1.

<p>Staining for fibronectin reveals fibrosis in AS dogs (B) as early as milestone 1 on confocal microscopy when compared to WT littermates (A) at the same milestone, 10x0.3 n.a. dry.</p