Fig 2: Immunohistochemistry (IHC) of DACH1 in formalin-fixed paraffin-embedded human adrenal sections. A, IHC of DACH1 in adjacent adrenal glands. DACH1 is highly expressed in zona glomerulosa (ZG) cell’s nuclei. Pictures are representative of 13 adrenals from 10 patients with aldosterone-producing adenomas (APA) and 3 with pheochromocytomas. B, IHC of DACH1 in APAs. ZG-like APAs appeared to have similar staining of DACH1 to their adjacent zona glomerulosa. Left, Scanned images. Right, Zoomed insets of left panel. C, Colocalization of DACH1 cytoplasmic staining with PCP4 in cell clusters. D, Hematoxylin and eosin (H&E) stain and IHC of DACH1, CYP11B1 (as a zona fasciculata [ZF] marker), and ZG markers, CYP11B2, PCP4, and KCNJ5, in serial adrenal sections from a patient with Conn’s syndrome.

Fig 3: Diabetic nephropathy in human patients caused a severe down-regulation of DACH1 and synaptopodin. A, The expression of DACH1 (red) and synaptopodin (green) was reduced in glomeruli of patients suffering from diabetic nephropathy (DN) as compared to control tissue (Ctrl) imaged by laser scanning microscopy (each n = 3). Scale bar represents 50 µm. B, Expression of DACH1 and synaptopodin in human kidneys imaged by super resolution microscopy (structured illumination microscopy—SIM). The expression of DACH1 and synaptopodin was reduced in DN patients in contrast to the controls. C, Immunohistochemistry of human kidneys showed the reduced DACH1 expression in DN compared to controls. Scale bar represents 20 µm

Fig 4: Segmentation U-Net for molecular morphometrics in kidney samples.(A) Biopsies from patients with immune-mediated kidney diseases, which are diagnosed, treated, and monitored based on clinical, pathological, and integrative data, are used to perform molecular labeling of kidney podocytes, based on indirect immunofluorescence (24). (B) Glomerular area and podocyte nuclei are virtually dissected from high-resolution confocal images with a segmentation U-Net for 2 simultaneous outputs that was trained using a balanced 2-layer binary cross-entropy loss. (C) 3D podocyte morphometrics (podometrics) were generated by model-based stereology, which extrapolates 3D from 2D data; in this case, glomerular and podocyte areas and podocyte spatial location were used to estimate 3D glomerular dimensions, as well as numbers, sizes, and distributions of podocytes. ANCA-GN, antineutrophil cytoplasmic antibody–associated glomerulonephritis; eGFR, estimated glomerular filtration rate; DACH1, Dachshund Family Transcription Factor 1; WT1, Wilms’ Tumor 1; BCE, binary cross-entropy; Conv, convolution; ReLU, rectified linear unit; Max Pool, max pooling.

Fig 5: Sirolimus treatment of experimental FSGS did not change albuminuria, podocyte injury and podocyte number. (A) Sirolimus treated mice did not show a lower albumin/creatinine ratio compared to control mice at day 4 and (B) day 7. Sirolimus treated mice n = 12, phosal treated mice n = 8. Mean with SD is shown. ns P = 0.05. (C, D, E) Representative images of glomeruli stained against synaptopodin (C, D, red) and desmin (C, E, green). (C) A healthy glomerulus (a) only shows desmin expression in the mesangial cells. In diseased glomeruli (b) desmin staining can also be detected in podocytes (co-localizing with synaptopodin expression (C, yellow, arrow)). (F) Glomerular desmin expression was similar in the sirolimus and control group. Sirolimus treated mice n = 4, phosal treated mice n = 4. Mean with SD is shown. ns P = 0.05. (G, H, I) Representative images of glomeruli stained against synaptopodin (G, H, red) and DACH1 (G, I, yellow). (G) A line was drawn surrounding the tuft area to measure it. The whole tuft area was selected also in case no or less synaptopodin signal was present (example glomerulus a). (J) Phosal and sirolimus treated mice have a similar number of DACH1 positive podocytes per mm2 of the tuft area. Sirolimus treated mice n = 6, phosal treated mice n = 6. Mean with SEM is shown. ns P = 0.05.