Kidney fibrosis is the hallmark of chronic kidney disease progression, however, currently no antifibrotic therapies exist. This is largely because the origin, functional heterogeneity and regulation of scar-forming cells during human kidney fibrosis remains poorly understood. Here, using single cell RNA-seq, we profiled the transcriptomes of proximal tubule and non-proximal tubule cells in healthy and fibrotic human kidneys to map the entire human kidney in an unbiased approach. This enabled mapping of all matrix-producing cells at high resolution, revealing distinct subpopulations of pericytes and fibroblasts as the major cellular sources of scar forming myofibroblasts during human kidney fibrosis. We used genetic fate-tracing, time-course single cell RNA-seq and ATAC-seq experiments in mice, and spatial transcriptomics in human kidney fibrosis to functionally interrogate these findings, shedding new light on the origin, heterogeneity and differentiation of human kidney myofibroblasts and their fibroblast and pericyte precursors at unprecedented resolution. Finally, we used this strategy to facilitate target discovery, identifying Nkd2 as a myofibroblast-specific target in human kidney fibrosis.

Morphologic examination of tissue biopsies is essential for histopathological diagnosis. However, accurate and scalable cellular quantification in human samples remains challenging. Here, we present a deep learning–based approach for antigen-specific cellular morphometrics in human kidney biopsies, which combines indirect immunofluorescence imaging with U-Net–based architectures for image-to-image translation and dual segmentation tasks, achieving human-level accuracy. In the kidney, podocyte loss represents a hallmark of glomerular injury and can be estimated in diagnostic biopsies. Thus, we profiled over 27,000 podocytes from 110 human samples, including patients with antineutrophil cytoplasmic antibody–associated glomerulonephritis (ANCA-GN), an immune-mediated disease with aggressive glomerular damage and irreversible loss of kidney function. We identified previously unknown morphometric signatures of podocyte depletion in patients with ANCA-GN, which allowed patient classification and, in combination with routine clinical tools, showed potential for risk stratification. Our approach enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.

Background: Dual inhibition of the renin-angiotensin system (RAS) plus sodium-glucose transporter (SGLT)-2 or the mineralocorticoid receptor (MR) demonstrated additive renoprotective effects in large clinical trials. We hypothesized that triple therapy with RAS/SGLT2/MR inhibitors would be superior to dual RAS/SGLT2 blockade in attenuating CKD progression. Methods: We performed a preclinical randomized controlled trial (PCTE0000266) in Col4a3-deficient mice with established Alport nephropathy. Treatment was initiated late (6 weeks of age) in mice with elevated serum creatinine and albuminuria and with glomerulosclerosis, interstitial fibrosis, and tubular atrophy. We block-randomized 40 male and 40 female mice to either nil (vehicle) or late onset food admixes of ramipril monotherapy (10 mg/kg), ramipril plus empagliflozin (30 mg/kg), or ramipril plus empagliflozin plus finerenone (10 mg/kg). Primary endpoint was mean survival. Results: Mean survival was 63.7 ± 10.0 days (vehicle), 77.3 ± 5.3 days (ramipril), 80.3 ± 11.0 days (dual), and 103.1 ± 20.3 days (triple). Sex did not affect outcome. Histopathology, pathomics, and RNA sequencing revealed that finerenone mainly suppressed the residual interstitial inflammation and fibrosis despite dual RAS/SGLT2 inhibition. Conclusion: Experiments in mice suggest that triple RAS/SGLT2/MR blockade may substantially improve renal outcomes in Alport syndrome and possibly other progressive chronic kidney diseases due to synergistic effects on the glomerular and tubulointerstitial compartments.

Morphologic examination of tissue biopsies is essential for histopathological diagnosis. However, accurate and scalable cellular quantification in human samples remains challenging. Here, we present a deep learning-based approach for antigen-specific cellular morphometrics in human kidney biopsies, which combines indirect immunofluorescence imaging with U-Net-based architectures for image-to-image translation and dual segmentation tasks, achieving human-level accuracy. In the kidney, podocyte loss represents a hallmark of glomerular injury and can be estimated in diagnostic biopsies. Thus, we profiled over 27,000 podocytes from 110 human samples, including patients with anti-neutrophil cytoplasmic antibody-associated glomerulonephritis (ANCA-GN), an immune-mediated disease with aggressive glomerular damage and irreversible loss of kidney function. Previously unknown morphometric signatures of podocyte depletion were identified in patients with ANCA-GN, which allowed patient classification and showed potential for risk stratification in combination with routine clinical tools. Together, our approach enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.SummaryDeep learning enables robust and scalable molecular morphometric analysis of human tissues, yielding deeper biological insights into the human kidney pathophysiology.

No abstract

Background and Aims Kidney function, as well as its morphology, changes markedly with age and disorders such as diabetes. This process is associated with structural and functional alterations in cortical and juxtamedullary glomeruli. Currently, data on differences in cortical and juxtamedullary glomeruli associated with sex, age, genetic factors, and diabetes are limited. In this study, we investigated the abundance and morphometry of podocytes and glomeruli in mice of different ages and sex’, and suffering from diabetes or not using a deep-learning based analysis of immuno-stained kidney sections. Methods Male and female non-diabetic C57BL/6J mice and diabetes type II (db/db) mice were sacrificed at different time points: 4, 10, 20, 30, 34, 40 weeks and 6, 24 weeks, respectively. Subsequently, kidneys were extracted, embedded in paraffin, cut into sections, stained, and imaged for histological analysis. We used immunhistochemistry staining with Wilms Tumor 1 (WT1) antibody to specifically stain podocyte nuclei. Both manual and deep-learning based image segmentation were performed to analyze abundance and morphometry of podocytes and glomeruli. In total, 4134 glomerular structures were detected, and morphometry of glomeruli and podocytes was extracted and analyzed by an automated algorithm. Results Our study aimed to investigate aging- and diabetes-related differences in cortical and juxtamedullary glomeruli with respect to podocyte abundance and loss. Using a customized deep learning algorithm, podocyte nuclei could be quantified with comparable quality as a time-tedious manual analysis, which takes approximately 1 minute per glomerulus. Extracted morphometric features showed that juxtamedullary glomeruli had a larger cross-sectional area than cortical glomeruli (Figure 1a). For both cortical and juxtamedullary glomeruli the cross-sectional area slightly increased on average with aging (Figure 1b). As expected, podocyte endowment in cortical glomeruli was lower than in juxtamedullary glomeruli with podocyte density being higher (Figure 2a, 2b). Over life-time the number of podocytes and podocyte density per glomerulus slightly decreased both in cortical and juxtamedullary glomeruli (Figure 2b, 3b). Interestingly, female cortical glomeruli were on average smaller and had a higher podocyte density compared to males (Figure 1c, 3c). However, podocyte numbers did not differ between male and female (Figure 2c). Finally, we found that 24 weeks old db/db mice presented with glomerular hypertrophy in contrast to non-diabetic C57BL/6J mice of the same age (Figure 1d). Db/db mice lost podocytes from 6 weeks to 24 weeks of age with a decreased podocyte density. Surprisingly, podocyte loss occurred to a lower extent in non-diabetic mice (Figure 2d, 3d). Conclusion During aging and early diabetic disease both podocyte loss and glomerular hypertrophy occur. Similar changes occurred in juxtamedullary and cortical glomeruli in both sex’. Hyperfiltration might explain the pronounced extent in glomerular area in diabetic mice and should represent an increase in filtration surface to handle diabetes-related hyperfiltration. Less podocyte loss in diabetic mice at 24 weeks age compared to C57BL/6J mice might relate to the different mouse model and still moderate podocyte stress during the early stage of diabetic kidney disease.

Background and Aims Inhibitors of the renin-angiotensin system (RAS), sodium-glucose transporter (SGLT)-2, and the mineralocorticoid receptor (MR) have all demonstrated renoprotective effects in large clinical trials of diabetes-related CKD. Furthermore, dual RAS/SGLT2 blockade showed additive renoprotective effects also in non-diabetic CKD. We hypothesized that triple RAS/SGLT2/MR blockade would be even superior to dual RAS/SGLT2 blockade in non-diabetic CKD. Method We performed a “no touch” preclinical randomized controlled trial in Col4a3-deficient mice with spontaneous and progressive CKD (registry ID: PCTE0000266). Treatments were administered as food admix from 6-14 weeks of age at the following estimated doses: 10 mg/kg ramipril, 30 mg/kg empagliflozin, 10 mg/kg finerenone. The prespecified primary endpoint was total lifespan up to uremic death. Ancillary studies addressed baseline histology, and mechanistic studies on a subset of mice after 2.5 weeks of treatment. Results At the time of randomization, Col4a3-/- mice had albuminuria, elevated serum creatinine, glomerulosclerosis, tubular atrophy, and interstitial fibrosis. Total lifespan was 63.7 ± 9.99 days (vehicle), 77.25 ± 5.34 days (ramipril), 80.3 ± 10.98 days (ramipril+empagliflozin), and 103.05 ± 20.28 days (triple therapy), respectively. Artificial intelligence-based histopathology and RNA sequencing analysis documented a potent anti-sclerotic, -inflammation and -fibrotic effect of the triple combination. Conclusion Adding finereone to dual RAS/SGLT2 blockade significantly prolongs uremia-free lifespan even when started at an advanced stage of Alport nephropathy. Triple RAS/SGLT2/MR blockade could be a potent treatment strategy to prolong uremia-free lifespan in patients with CKD related to Alport syndrome and possibly other progressive kidney disorders.