Transmission electron microscopy (TEM) has become an essential technique for observing subcellular ultrastructure, and is widely used in both clinical diagnosis and biomedical research. However, analysis of TEM data remains extremely labor-intensive and often inconsistent across operators due to the lack of dedicated computational methods. Here, we present TEAMKidney, a deep learning framework for accurate and scalable measurement of ultrastructures in TEM images across species, magnifications, and instrument platforms. We collected 12,991 TEM images from patients with multiple kidney diseases and from different animal models. By combining a self-training-based semantic segmentation stage with a TEM-tailored panoptic segmentation model, we address two major challenges in TEM data analysis: the lack of accurately labeled training data and the difficulty of achieving high segmentation accuracy for complex ultrastructure. Application of TEAMKidney to both human and animal images successfully reveals disease-associated changes in two critical glomerular ultrastructures: the glomerular basement membrane and podocyte foot processes. In addition to significantly outperforming existing tools, TEAMKidney shows close agreement with pathological expert measurements used in clinical assessment protocols. By reducing dependence on manual tracing while preserving expert-level accuracy, TEAMKidney demonstrates that deep learning can substantially reduce the burden of image analysis in both clinical pathology and biomedical research settings.

Mowat-Wilson Syndrome (MWS) is an autosomal dominant genetic disorder caused by heterozygous mutations or deletions in the Zinc finger E-box-binding homeobox 2 (ZEB2) gene. Congenital anomalies of the kidney and urinary tract (CAKUT), including hydroureter and hydronephrosis, have been reported in patients with MWS. However, the role of the ZEB2 gene in urinary tract development and the cellular and molecular mechanisms underlying the CAKUT phenotypes in MWS remain unknown. In this study, we examined ZEB2 expression in the developing mouse ureter and generated Zeb2 ureteral mesenchyme-specific conditional knockout mice (Zeb2 cKO) by crossing Zeb2 floxed mice with Tbx18Cre+ mice. The urinary tract of Zeb2 cKO mice and their wild-type littermates was analyzed for morphological and histological changes. Our results show that ZEB2 is expressed in TBX18+ ureteral mesenchymal cells during mouse ureter development. Deleting Zeb2 in these cells caused hydroureter and hydronephrosis, indicating obstructive uropathy. Cellular and molecular marker analysis revealed that the TAGLN+ACTA2+ ureteral smooth muscle cell (SMC) layer was absent in Zeb2 cKO mice. In contrast, the tunica adventitia cell layer was significantly expanded compared to controls. At the molecular level, Zeb2 cKO mice had significantly decreased TBX18 expression but increased SOX9 expression in the developing ureter compared to wild-type controls. Our findings demonstrate that ZEB2 is crucial for normal ureteral SMC differentiation during ureter development. Additionally, our study suggests that MWS patients may have abnormal ureteral SMC development, which contributes to the abnormalities of the urinary tract.

Introduction: Pathogenic (P) variants in COL4A3/A4/A5 genes are known to cause thin glomerular basement membrane (GBM) or Alport-related kidney disease; however, the exact diagnostic yield of genetic testing remains unknown. Methods: In this retrospective genotype-phenotype correlation study, we screened the patient populations of 2 major US medical centers for individuals who underwent kidney biopsy, and who had documented genetic testing results on a large 385-kidney disease gene panel. We correlated GBM thickness, estimated glomerular filtration rate, proteinuria, and hematuria with genotyping results. Results: We identified 115 patients with coexisting histopathology and genetic testing data, of which 49 had ultrastructural abnormalities of the GBM. Among those 49 cases, 9 had a heterozygous pathogenic or likely pathogenic (P/LP) variant in one of the COL4A genes, and 9 additional patients had COL4A variants of uncertain significance (VUS). Thirty-one patients with thin GBM were COL4A3/A4/A5 wildtype. One patient with a P variant in COL4A4 had no GBM abnormalities. Three COL4A VUS were upgraded to P/LP through experimental testing. Presence of P/LP variants in COL4A genes correlated with GBM thickness, but not with other clinical parameters. Among 31 thin GBM cases with no COL4A variant, we found variants in steroid-resistant nephrotic syndrome-, congenital anomalies of the kidneys and urinary tract (CAKUT)-, and autosomal dominant tubulointerstitial kidney disease (ADTKD) genes characterized as P/ LP or as "high-risk VUS." Immune-mediated glomerular injury was as frequent in biopsy specimens with thin GBM as with normal GBM. Conclusion: In our study, almost two-thirds of patients with thin GBM have no variant in COL4A3/A4/A5 genes. Our data suggest that genetic testing may not obviate the need for kidney biopsy.

OBJECTIVE:Amyloidosis is a group of disorders characterized by aggregation of abnormal amyloid protein in various tissues, often leading to organ dysfunction and failure. We optimized the immunogold electron microscopy (IEM) technique to enable efficient amyloid typing in 4% paraformaldehyde-fixed (PFA) and formalin-fixed paraffin-embedded (FFPE) tissues. METHODS:The optimized IEM technique was applied to 151 Congo-red positive specimens from various tissues representing different amyloidosis types; 117 were fixed in 4% PFA and 34 were FFPE samples. Specimens were embedded in Lowicryl/K4M and stained with modified Richardson's blue solution to differentiate amyloid (lavender-plum coloration stained) from non-amyloid (blue stained) areas under light microscopy. Antibodies against kappa and lambda light chains, transthyretin and amyloid A were used for amyloid typing by IEM. RESULTS:The optimized IEM technique enabled precise localization of amyloid deposits and rapid identification of target area under light microscopy for thin-section placement on nickel grids for immunogold staining. Of 151 specimens, 147 (97.4%) were classified as kappa or lambda light chains, transthyretin or amyloid A. Lambda light chain predominated in fat pad aspirates (56.8%), while transthyretin was most common in heart tissues (71.7%). CONCLUSION:This optimized IEM technique enhances the accuracy and efficiency of amyloid typing, especially in samples with trace amyloid deposit. It offers significant advantages over traditional epoxy embedding with toluidine blue staining, supporting timely clinical diagnosis and therapeutic decision-making.

Slit Guidance Ligand 2 (SLIT2) binds Roundabout (ROBO) guidance receptors to direct axon pathfinding and neuron migration during nervous system development. SLIT2 expression has previously been linked to dementia risk. To study the association between SLIT2 expression in human vitreous humor and plasma samples and neurocognitive test scores in a cross-sectional cohort study utilizing a novel, highly-sensitive Meso Scale Discovery (MSD) assay for SLIT2 detection. Seventy-nine individuals with a mean age of 55.79 ± 12.03 years underwent eye surgery with collection of vitreous humor, blood (plasma) collection, and neurocognitive assessment. Vitreous humor and plasma samples were analyzed by SLIT2 MSD electrochemiluminescence immunoassay. Associations between SLIT2 levels in vitreous humor and plasma were analyzed using GraphPad Prism. We found up to a 7-fold higher level of SLIT2 in human vitreous humor compared to plasma. Lower vitreous SLIT2 levels were associated with a lower Montreal Cognitive Assessment (MoCA) score and Immediate Recall Verbatim (IRV) z-score, and higher plasma SLIT2 was associated with a lower MoCA score. In multivariate analysis using single and multiple predictor models, the same significant associations were found when adjusted for age, sex, race, diabetic status, diabetic retinopathy status, glaucoma status, and Apolipoprotein E ( APOE ) genotype. SLIT2 protein levels are significantly associated with MoCA score and IRV z-score in middle-aged individuals. The relationship remained significant when adjusted for demographics, co-morbidity, and APOE genotype, suggesting SLIT2 may be a sensitive biomarker for detection of mild cognitive impairment and early dementia, and warrants further studies.

Transmission electron microscopy (TEM) images can visualize kidney glomerular filtration barrier ultrastructure, including the glomerular basement membrane (GBM) and podocyte foot processes (PFP). Podocytopathy is associated with glomerular filtration barrier morphological changes observed experimentally and clinically by measuring GBM or PFP width. However, these measurements are currently performed manually. This limits research on podocytopathy disease mechanisms and therapeutics due to labor intensiveness and inter-operator variability. We developed a deep learning-based digital pathology computational method to measure GBM and PFP width in TEM images from the kidneys of Integrin-Linked Kinase (ILK) podocyte-specific conditional knockout (cKO) mouse, an animal model of podocytopathy, compared to wild-type (WT) control mouse. We obtained TEM images from WT and ILK cKO littermate mice at 4 weeks old. Our automated method was composed of two stages: a U-Net model for GBM segmentation, followed by an image processing algorithm for GBM and PFP width measurement. We evaluated its performance with a 4-fold cross-validation study on WT and ILK cKO mouse kidney pairs. Mean [95% confidence interval (CI)] GBM segmentation accuracy, calculated as Jaccard index, was 0.73 (0.70-0.76) for WT and 0.85 (0.83-0.87) for ILK cKO TEM images. Automated and manual GBM width measurements were similar for both WT (P = .49) and ILK cKO (P = .06) specimens. While automated and manual PFP width measurements were similar for WT (P = .89), they differed for ILK cKO (P < .05) specimens. WT and ILK cKO specimens were morphologically distinguishable by manual GBM (P < .05) and PFP (P < .05) width measurements. This phenotypic difference was reflected in the automated GBM (P < .05) more than PFP (P = .06) widths. Our deep learning-based digital pathology tool automated measurements in a mouse model of podocytopathy. This proposed method provides high-throughput, objective morphological analysis and could facilitate podocytopathy research.

Heterozygous mutations or deletions in the ZEB2 gene cause Mowat-Wilson syndrome (MWS, OMIM #235730), a congenital disorder with multiple defects, including renal anomalies. Some MWS patients reported having hypotension. ZEB2 has been reported as a super-enhancer for the renin (REN) gene, and renin-null cells have upregulated ZEB2 expression. However, the role of ZEB2 in renin cell biology and the cellular and molecular mechanisms underlying the hypotension phenotype in MWS remain unclear. Renin precursor cells derive from FOXD1-positive kidney stromal progenitors. ZEB2 is highly expressed in FOXD1-positive kidney stromal progenitors and regulates their differentiation. We hypothesize that ZEB2 is required for the differentiation of FOXD1-positive kidney stromal progenitors into renin + cells. We deleted the Zeb2 gene in the kidney stroma using Foxd1Cre and analyzed the Zeb2 flox/flox ; Foxd1Cre + conditional knockout mice ( Zeb2 cKO) and their wild-type (WT) littermate controls for the renin cell number and arterial blood pressure. Single-cell RNA sequencing (scRNA-seq) was performed using the 10x Genomics Chromium platform on kidney tissues from 3-week-old Zeb2 cKO and WT mice. The data were analyzed using our in-house computational pipeline. Immunostaining was performed to validate scRNA-seq findings. We found that 4-week-old Zeb2 cKO mice had reduced renin-expressing cells and lower arterial blood pressure compared to WT controls. Our clustering analysis identified nine major cell types in both groups. The scRNA-seq and gene expression analysis revealed a significant increase in the proportion of podocytes and parietal epithelial cells (PECs) and ectopic expression of podocyte and PEC markers (WT1, TLE4, and CLDN1) within the vascular compartment (also expressing renin) of Zeb2 cKO kidneys. Trajectory and functional enrichment analyses identified significant alterations in angiogenesis and chromatin remodeling pathways in Zeb2 cKO mouse kidneys. In summary, ZEB2 is required for the differentiation of FOXD1-positive stromal progenitors in the kidney. In the absence of ZEB2, FOXD1-positive stromal progenitors adopt a podocyte—and PEC-like cell fate instead of renin-expressing cells, leading to a hypotension phenotype. Our results suggest that ZEB2 is a critical determinant of renin + cell lineage development.

Mowat-Wilson Syndrome (MWS) is a multiple congenital anomaly syndrome caused by mutations in the ZEB2, which plays a critical role in cell fate determination and differentiation during development. Congenital anomalies of the kidney and urinary tract (CAKUT) have been reported in MWS patients. However, the role of ZEB2 in urinary tract development and the cellular and molecular mechanism underlining the CAKUT phenotypes in MWS remains unknown. We performed ZEB2 protein expression analysis in the developing mouse ureter. We generated Zeb2 ureteral mesenchyme-specific conditional knockout mice by crossing Zeb2 floxed mice with Tbx18Cre mice (Zeb2 cKO) and analyzed the urinary tract phenotypes in Zeb2 cKO mice and wild-type littermate controls by gross and histological examination. Ureteral cellular and molecular phenotypes were studied using TAGLN, ACTA2, FOXD1, POSTN, CDH1, TBX18, and SOX9 ureteral cell-specific markers. We found that ZEB2 is expressed in TBX18+ ureteral mesenchymal cells during mouse ureter development. Deletion of Zeb2 in developing ureteral mesenchymal cells causes hydroureter and hydronephrosis phenotypes, leading to obstructive uropathy, kidney failure, and early mortality. Cellular and molecular marker analyses showed that the TAGLN+ACTA2+ ureteral smooth muscle cells (SMCs) layer is not formed in Zeb2 cKO mice at E15.5, but the FOXD1+ and POSTN+ tunica adventitia cells layer is significantly expanded compared to wild-type controls. CDH1+ urothelium cells are reduced considerably in the Zeb2 cKO ureters at E15.5. Mechanistically, we found that Zeb2 cKO mice have significantly decreased TBX18 expression but an increased SOX9 expression in the developing ureter at E14.5 and E15.5 compared to wild-type littermate controls. Our results show that ZEB2 is essential for ureter development by maintaining ureteral mesenchymal cell differentiation into normal ureteral SMCs. Our study also shed new light on the pathological mechanism underlying the developmental abnormalities of the urinary tract phenotypes in MWS patients.

Kumar, Sudhir; Fan, Xueping; Zaltz, Emily; Song, Paul; Jiang, Yuqiao; Lu, Weining Author Information