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Background: Marked podocyte depletion is an established key feature of glomerulosclerosis (FSGS). However, little is known about the consequences of mild podocyte loss. In addition, activation of parietal epithelial cells (PECs) has been proposed as a major effector in FSGS. This study investigates the consequences of graded podocyte depletion, the hypertrophic response of podocytes and associations with PEC activation and thereby glomerulosclerosis. Method(s): We induced selective podocyte depletion in PodCreiDTR mice by injection of diphtheria toxin (DT) at different doses. L-NAME induced hypertension was used as a second hit challenge after mild podocyte loss. The mammalian target of rapamycin (mTOR) signalling pathway was manipulated using mTOR inhibitors (RAD001 and INK128). Podocyte depletion and hypertrophy were examined by 3D analysis of whole glomeruli in optically-cleared kidney slices. Result(s): PodCreiDTR mice injected with a low dose of DT presented mild podocyte depletion, compensatory podocyte hypertrophy and reversible albuminuria without PEC activation or glomerulosclerosis, even following a second hit challenge (high blood pressure), suggesting a protective role of podocyte hypertrophy. Injection of a higher dose of DT in PodCreiDTR mice led to greater podocyte loss and hypertrophy. However, these mice showed PEC activation, glomerulosclerosis and persistent albuminuria, suggesting there is a limit for the protective role of podocyte hypertrophy. Pharmacological inhibition of mTOR during the induction of mild podocyte depletion led to persistent and exacerbated albuminuria, impairment of podocyte hypertrophy, PEC activation and glomerulosclerosis. Conclusion(s): Podocyte hypertrophy via mTOR signalling is required for the adaptive hypertrophic response of remaining podocytes after mild podocyte depletion. These results are relevant for the use of mTOR inhibitors in the context of FSGS and CKD.

Background: Podocyte depletion is a common feature of glomerulosclerosis (FSGS), but its role in crescentic nephritis remains unclear. This study combined genetic tagging of podocytes with three different optical clearing techniques to determine podocyte depletion in whole glomeruli from mice with crescentic nephritis. Method(s): Podocyte nuclei were labeled by eGFP-histone in of adult male Pod-rtTA/ H2B-eGFP mice by oral doxycycline, followed by a 7-day wash out period, and a single intra-peritoneal injection of nephrotoxic serum (NTS; 5mg/g). Experimental mice were killed 10 days after NTS injection, and compared to age-matched controls. Kidney slices were optically cleared with SCALE-A4, CLARITY and Ethyl Cinnamate (ECi). Highresolution serial optical images were obtained by confocal and two-photon microscopy. Result(s): Mean podocyte number per mouse showed very low variability within controls (1-5% variability, P>0.05) and within NTS-injected mice (1-9% variability, P>0.05) independent of the clearing technique. In NTS-injected mice, a similar degree of average podocyte depletion per mouse was identified with all clearing methods (60-63%, P<0.001). The technical (dis-)advantages of each clearing protocol were also analysed, including optimal penetration depth and resolution, compatibility with immunofluorescence, microscopy set-ups, and cost-efficiency. Importantly, total podocyte number per glomerulus showed great variability: controls (mean: 78,81; ranging from 49 to 128 podocytes per glomerulus) and in NTS-injected mice (mean: 29,99; ranging from 1 to 95 podocytes per glomerulus). Using the lowest value for podocyte number in controls as a cut-off reference, only 78% of analysed glomeruli (141 of 180) from NTS-injected mice had a certain degree of podocyte depletion. While deposition of the NTS within glomeruli occurred in a global and homogeneous fashion, podocyte loss was focal. Conclusion(s): This study has identified early focal podocyte depletion in mic

Background: Optical clearing and advanced light microscopy have revolutionised three-dimensional quantification in cell biology. Here, we present a Systematic Pipeline of Enhanced Clarification for Three-dimensional Rendering and Unbiased Morphometrics (SPECTRUM) as a unique tool for the comprehensive analysis of glomerular health and disease. Method(s): SPECTRUM is based on a combination of single cell identification (ie. lineage tracing), optical clearing, advanced light microscopy with single cell resolution, and 3D morphometrics. Podocytes and parietal epithelial cells (PECs) were genetically labelled with eGFP using inducible mouse systems (POD or PEC -rtTA/H2B-eGFP). Crescentic nephritis was used as a validation model. Several optical clearing protocols were optimized for kidney tissue, including aqueous, hydrogel, and solvent-based approaches. Image acquisition was based on whole structures (ie. glomeruli and lesions) using light sheet, confocal and two-photon microscopy. Subsequent tissue de-clarification with immunolabelling and classical histopathology allowed the combination of 3D and 2D analyses. Result(s): While IgG deposition showed a homogeneous distribution among all analysed glomeruli, only 80 showed signs of podocyte loss (podocyte loss per glomerulus of about 60). Only glomerular lesions showed significant increases in numbers of PECs (sign of PEC activation) in close association with reductions in glomerular capillary volume (sign of capillary injury). PEC activation was confirmed via immunofluorescence in recycled tissue samples (ie. de-novo CD44 expression). Based on 3D analysis of intraglomerular lesion location, we identified a progressive pattern from localised tubular lesions to segmental lesions and development of atubular glomeruli. Conclusion(s): SPECTRUM provides new roadmap for morphometrics in the kidney. In crescentic nephritis, SPECTRUM revealed that despite of uniform IgG deposition, there was focal lesion development and podocyte

Background: Marked podocyte depletion is an established key feature of glomerulosclerosis (FSGS). However, little is known about the consequences of mild podocyte loss. In addition, activation of parietal epithelial cells (PECs) has been proposed as a major effector in FSGS. This study investigates the consequences of graded podocyte depletion, the hypertrophic response of podocytes and associations with PEC activation and thereby glomerulosclerosis. Method(s): We induced selective podocyte depletion in PodCreiDTR mice by injection of diphtheria toxin (DT) at different doses. L-NAME induced hypertension was used as a second hit challenge after mild podocyte loss. The mammalian target of rapamycin (mTOR) signalling pathway was manipulated using mTOR inhibitors (RAD001 and INK128). Podocyte depletion and hypertrophy were examined by 3D analysis of whole glomeruli in optically-cleared kidney slices. Result(s): PodCreiDTR mice injected with a low dose of DT presented mild podocyte depletion, compensatory podocyte hypertrophy and reversible albuminuria without PEC activation or glomerulosclerosis, even following a second hit challenge (high blood pressure), suggesting a protective role of podocyte hypertrophy. Injection of a higher dose of DT in PodCreiDTR mice led to greater podocyte loss and hypertrophy. However, these mice showed PEC activation, glomerulosclerosis and persistent albuminuria, suggesting there is a limit for the protective role of podocyte hypertrophy. Pharmacological inhibition of mTOR during the induction of mild podocyte depletion led to persistent and exacerbated albuminuria, impairment of podocyte hypertrophy, PEC activation and glomerulosclerosis. Conclusion(s): Podocyte hypertrophy via mTOR signalling is required for the adaptive hypertrophic response of remaining podocytes after mild podocyte depletion. These results are relevant for the use of mTOR inhibitors in the context of FSGS and CKD.

Background: Podocyte depletion is a common feature of glomerulosclerosis (FSGS), but its role in crescentic nephritis remains unclear. This study combined genetic tagging of podocytes with three different optical clearing techniques to determine podocyte depletion in whole glomeruli from mice with crescentic nephritis. Method(s): Podocyte nuclei were labeled by eGFP-histone in of adult male Pod-rtTA/ H2B-eGFP mice by oral doxycycline, followed by a 7-day wash out period, and a single intra-peritoneal injection of nephrotoxic serum (NTS; 5mg/g). Experimental mice were killed 10 days after NTS injection, and compared to age-matched controls. Kidney slices were optically cleared with SCALE-A4, CLARITY and Ethyl Cinnamate (ECi). Highresolution serial optical images were obtained by confocal and two-photon microscopy. Result(s): Mean podocyte number per mouse showed very low variability within controls (1-5% variability, P>0.05) and within NTS-injected mice (1-9% variability, P>0.05) independent of the clearing technique. In NTS-injected mice, a similar degree of average podocyte depletion per mouse was identified with all clearing methods (60-63%, P<0.001). The technical (dis-)advantages of each clearing protocol were also analysed, including optimal penetration depth and resolution, compatibility with immunofluorescence, microscopy set-ups, and cost-efficiency. Importantly, total podocyte number per glomerulus showed great variability: controls (mean: 78,81; ranging from 49 to 128 podocytes per glomerulus) and in NTS-injected mice (mean: 29,99; ranging from 1 to 95 podocytes per glomerulus). Using the lowest value for podocyte number in controls as a cut-off reference, only 78% of analysed glomeruli (141 of 180) from NTS-injected mice had a certain degree of podocyte depletion. While deposition of the NTS within glomeruli occurred in a global and homogeneous fashion, podocyte loss was focal. Conclusion(s): This study has identified early focal podocyte depletion in mic

Background: Optical clearing and advanced light microscopy have revolutionised three-dimensional quantification in cell biology. Here, we present a Systematic Pipeline of Enhanced Clarification for Three-dimensional Rendering and Unbiased Morphometrics (SPECTRUM) as a unique tool for the comprehensive analysis of glomerular health and disease. Method(s): SPECTRUM is based on a combination of single cell identification (ie. lineage tracing), optical clearing, advanced light microscopy with single cell resolution, and 3D morphometrics. Podocytes and parietal epithelial cells (PECs) were genetically labelled with eGFP using inducible mouse systems (POD or PEC -rtTA/H2B-eGFP). Crescentic nephritis was used as a validation model. Several optical clearing protocols were optimized for kidney tissue, including aqueous, hydrogel, and solvent-based approaches. Image acquisition was based on whole structures (ie. glomeruli and lesions) using light sheet, confocal and two-photon microscopy. Subsequent tissue de-clarification with immunolabelling and classical histopathology allowed the combination of 3D and 2D analyses. Result(s): While IgG deposition showed a homogeneous distribution among all analysed glomeruli, only 80 showed signs of podocyte loss (podocyte loss per glomerulus of about 60). Only glomerular lesions showed significant increases in numbers of PECs (sign of PEC activation) in close association with reductions in glomerular capillary volume (sign of capillary injury). PEC activation was confirmed via immunofluorescence in recycled tissue samples (ie. de-novo CD44 expression). Based on 3D analysis of intraglomerular lesion location, we identified a progressive pattern from localised tubular lesions to segmental lesions and development of atubular glomeruli. Conclusion(s): SPECTRUM provides new roadmap for morphometrics in the kidney. In crescentic nephritis, SPECTRUM revealed that despite of uniform IgG deposition, there was focal lesion development and podocyte

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.Copyright © 2019 International Society of Nephrology

Beside the classical flat parietal epithelial cells (PECs), we investigated proximal tubular epithelial-like cells, a neglected subgroup of PECs. These cells, termed cuboidal PECs, make up the most proximal part of the proximal tubule and may also line parts of Bowman's capsule. Additionally, a third intermediate PEC subgroup was identified at the junction between the flat and cuboidal PEC subgroups at the tubular orifice. The transgenic mouse line PEC-rtTA labeled all three PEC subgroups. Here we show that the inducible Pax8-rtTA mouse line specifically labeled only cuboidal and intermediate PECs, but not flat PECs. In aging Pax8-rtTA mice, cell fate mapping showed no evidence for significant transdifferentiation from flat PECs to cuboidal or intermediate PECs or vice versa. In murine glomerular disease models of crescentic glomerulonephritis, and focal segmental glomerulosclerosis (FSGS), intermediate PECs became more numerous. These intermediate PECs preferentially expressed activation markers CD44 and Ki-67, suggesting that this subgroup of PECs was activated more easily than the classical flat PECs. In mice with FSGS, cuboidal and intermediate PECs formed sclerotic lesions. In patients with FSGS, cells forming the tip lesions expressed markers of intermediate PECs. These novel PEC subgroups form sclerotic lesions and were more prone to cellular activation compared to the classical flat PECs in disease. Thus, colonization of Bowman's capsule by cuboidal PECs may predispose to lesion formation and chronic kidney disease. We propose that tip lesions originate from this novel subgroup of PECs in patients with FSGS.Copyright © 2019 International Society of Nephrology

Recent developments in optical tissue clearing have been difficult to apply for the morphometric analysis of organs with high cellular content and small functional structures, such as the kidney. Here, we establish combinations of genetic and immuno-labelling for single cell identification, tissue clearing and subsequent de-clarification for histoimmunopathology and transmission electron microscopy. Using advanced light microscopy and computational analyses, we investigated a murine model of crescentic nephritis, an inflammatory kidney disease typified by immune-mediated damage to glomeruli leading to the formation of hypercellular lesions and the rapid loss of kidney function induced by nephrotoxic serum. Results show a graded susceptibility of the glomeruli, significant podocyte loss and capillary injury. These effects are associated with activation of parietal epithelial cells and formation of glomerular lesions that may evolve and obstruct the kidney tubule, thereby explaining the loss of kidney function. Thus, our work provides new high-throughput endpoints for the analysis of complex tissues with single-cell resolution.Copyright © 2019 International Society of Nephrology

Beside the classical flat parietal epithelial cells (PECs), we investigated proximal tubular epithelial-like cells, a neglected subgroup of PECs. These cells, termed cuboidal PECs, make up the most proximal part of the proximal tubule and may also line parts of Bowman's capsule. Additionally, a third intermediate PEC subgroup was identified at the junction between the flat and cuboidal PEC subgroups at the tubular orifice. The transgenic mouse line PEC-rtTA labeled all three PEC subgroups. Here we show that the inducible Pax8-rtTA mouse line specifically labeled only cuboidal and intermediate PECs, but not flat PECs. In aging Pax8-rtTA mice, cell fate mapping showed no evidence for significant transdifferentiation from flat PECs to cuboidal or intermediate PECs or vice versa. In murine glomerular disease models of crescentic glomerulonephritis, and focal segmental glomerulosclerosis (FSGS), intermediate PECs became more numerous. These intermediate PECs preferentially expressed activation markers CD44 and Ki-67, suggesting that this subgroup of PECs was activated more easily than the classical flat PECs. In mice with FSGS, cuboidal and intermediate PECs formed sclerotic lesions. In patients with FSGS, cells forming the tip lesions expressed markers of intermediate PECs. These novel PEC subgroups form sclerotic lesions and were more prone to cellular activation compared to the classical flat PECs in disease. Thus, colonization of Bowman's capsule by cuboidal PECs may predispose to lesion formation and chronic kidney disease. We propose that tip lesions originate from this novel subgroup of PECs in patients with FSGS.Copyright © 2019 International Society of Nephrology