The Application of Digital Pathology to Improve Accuracy in Glomerular Enumeration in Renal Biopsies.

BACKGROUND: In renal biopsy reporting, quantitative measurements, such as glomerular number and percentage of globally sclerotic glomeruli, is central to diagnostic accuracy and prognosis. The aim of this study is to determine the number of glomeruli and percent globally sclerotic in renal biopsies by means of registration of serial tissue sections and manual enumeration, compared to the numbers in pathology reports from routine light microscopic assessment. DESIGN: We reviewed 277 biopsies from the Nephrotic Syndrome Study Network (NEPTUNE) digital pathology repository, enumerating 9,379 glomeruli by means of whole slide imaging. Glomerular number and the percentage of globally sclerotic glomeruli are values routinely recorded in the official renal biopsy pathology report from the 25 participating centers. Two general trends in reporting were noted: total number per biopsy or average number per level/section. Both of these approaches were assessed for their accuracy in comparison to the analogous numbers of annotated glomeruli on WSI. RESULTS: The number of glomeruli annotated was consistently higher than those reported (p CONCLUSIONS: Although glass slides were not available for direct comparison to whole slide image annotation, this study indicates that routine manual light microscopy assessment of number of glomeruli is inaccurate, and the magnitude of this error is proportional to the total number of glomeruli

The Application of Digital Pathology to Improve Accuracy in Glomerular Enumeration in Renal Biopsies

In renal biopsy reporting, quantitative measurements, such as glomerular number and percentage of globally sclerotic glomeruli, is central to diagnostic accuracy and prognosis. The aim of this study is to determine the number of glomeruli and percent globally sclerotic in renal biopsies by means of registration of serial tissue sections and manual enumeration, compared to the numbers in pathology reports from routine light microscopic assessment

Reproducibility of the NEPTUNE descriptor-based scoring system on whole-slide images and histologic and ultrastructural digital images

The multicenter Nephrotic Syndrome Study Network (NEPTUNE) digital pathology scoring system employs a novel and comprehensive methodology to document pathologic features from whole-slide images, immunofluorescence and ultrastructural digital images. To estimate inter- and intra-reader concordance of this descriptor-based approach, data from 12 pathologists (eight NEPTUNE and four non-NEPTUNE) with experience from training to 30 years were collected. A descriptor reference manual was generated and a webinar-based protocol for consensus/cross-training implemented. Intra-reader concordance for 51 glomerular descriptors was evaluated on jpeg images by seven NEPTUNE pathologists scoring 131 glomeruli three times (Tests I, II, and III), each test following a consensus webinar review. Inter-reader concordance of glomerular descriptors was evaluated in 315 glomeruli by all pathologists; interstitial fibrosis and tubular atrophy (244 cases, whole-slide images) and four ultrastructural podocyte descriptors (178 cases, jpeg images) were evaluated once by six and five pathologists, respectively. Cohen’s kappa for inter-reader concordance for 48/51 glomerular descriptors with sufficient observations was moderate (0.40<kappa ≤0.60) for 17 and good (0.60<kappa ≤0.80) for 8, for 52% with moderate or better kappas. Clustering of glomerular descriptors based on similar pathologic features improved concordance. Concordance was independent of years of experience, and increased with webinar cross-training. Excellent concordance was achieved for interstitial fibrosis and tubular atrophy. Moderate-to-excellent concordance was achieved for all ultrastructural podocyte descriptors, with good-to-excellent concordance for descriptors commonly used in clinical practice, foot process effacement, and microvillous transformation. NEPTUNE digital pathology scoring system enables novel morphologic profiling of renal structures. For all histologic and ultrastructural descriptors tested with sufficient observations, moderate-to-excellent concordance was seen for 31/54 (57%). Descriptors not sufficiently represented will require further testing. This study proffers the NEPTUNE digital pathology scoring system as a model for standardization of renal biopsy interpretation extendable outside the NEPTUNE consortium, enabling international collaborations

cGMP-Dependent Protein Kinase 1 Polymorphisms Underlie Renal Sodium Handling Impairment

Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na(+) reabsorption via a direct action on basolateral Na-K ATPase and luminal Na-H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n=574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na(+) loading, and the slope of the pressure-natriuresis relationship between blood pressure and Na(+) excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure-natriuresis curve (0.017±0.004 μEq/mm Hg per minute) compared with the ACC (0.0013±0.003 μEq/mm Hg per minute; P=0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r=0.83; P<0.001) or α1 Na-K ATPase (r=0.557; P<0.01) and between α1 Na-K ATPase and Na-H exchanger type 3 (r=0.584; P<0.01) or Src (r=0.691; P<0.001) was observed in patients carrying PRKG1 risk GAT (n=23) but not ACC (n=14) variants. A functional signaling complex among PRKG1, α1 Na-K ATPase, and Src was shown by immunoprecipitation from human renal caveolae. These findings indicate that PRKG1 risk alleles associate with salt-sensitivity related to a loss of the inhibitory control of renal Na(+) reabsorption, suggestive of a blunt pressure-natriuresis response.status: publishe

cGMP-Dependent Protein Kinase 1 Polymorphisms Underlie Renal Sodium Handling Impairment

Defective pressure-natriuresis related to abnormalities in the natriuretic response has been associated with hypertension development. A major signaling pathway mediating pressure natriuresis involves the cGMP-dependent protein kinase 1 (PRKG1) that, once activated by Src kinase, inhibits renal Na(+) reabsorption via a direct action on basolateral Na-K ATPase and luminal Na-H exchanger type 3, as shown in renal tubuli of animals. Because a clear implication of PRKG1 in humans is still lacking, here we addressed whether PRKG1 polymorphisms affect pressure-natriuresis in patients. Naive hypertensive patients (n = 574), genotyped for PRKG1 rs1904694, rs7897633, and rs7905063 single nucleotide polymorphisms (SNPs), underwent an acute Na(+) loading, and the slope of the pressure-natriuresis relationship between blood pressure and Na(+) excretion was calculated. The underlying molecular mechanism was investigated by immunoblotting protein quantifications in human kidneys. The results demonstrate that the PRKG1 risk haplotype GAT (rs1904694, rs7897633, rs7905063, respectively) associates with a rightward shift of the pressure-natriuresis curve (0.017 ± 0.004 μEq/mm Hg per minute) compared with the ACC (0.0013 ± 0.003 μEq/mm Hg per minute; P = 0.001). In human kidneys, a positive correlation of protein expression levels between PRKG1 and Src (r = 0.83;

Digital annotation of whole slide images improves accuracy of glomerular enumeration.

<p>(A,B) Scatter plot of annotated glomerular number versus reported glomerular number by diagnosis in cases reporting (A) total number of glomeruli or (B) average number of glomeruli per level. Dotted diagonal line shows where annotated = reported, and highlights the increase in number of glomeruli found by annotation. (A: n = 139; B: n = 138). (C,D) Box plot of annotated glomerular number and reported total glomerular number stratified by diagnosis in cases reporting (C) total number of glomeruli (n = 139) or (D) average number of glomeruli (n = 139).</p

Number of annotated versus reported glomeruli stratified by diagnosis.

<p>Number of annotated versus reported glomeruli stratified by diagnosis.</p

Digital review and annotation of whole slide images in the NEPTUNE digital pathology repository.

<p>A) Example of multiple levels visualized at the same time. The software allows overlapping and orientation of the sections to facilitate multilevel reconstruction and representation of the biopsy levels. B) Digital annotation of whole slide images. Two levels are shown in alignment. Glomeruli in the left panel are annotated in red. In a subsequent level (right panel), two of the red-annotated glomeruli are still present (3 and 4), while one has disappeared (6). Three additional glomeruli, including one adjacent to glomerulus 3 and 6 have appeared in the deeper level (annotated in blue).</p

Difference in glomerular count of number of annotated versus reported glomeruli stratified by number of glomeruli.

<p>Difference in glomerular count of number of annotated versus reported glomeruli stratified by number of glomeruli.</p