Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli

Puelles, Victor G.; Wolde, James W. van der; Schulze, Keith E.; Short, Kieran M.; Wong, Milagros; Bensley, Jonathan G.; Cullen‐McEwen, Luise A.; Caruana, Georgina; Hokke, Stacey; Li, Jinhua; Firth, Sonja; Harper, Ian S.; Nikolic-Paterson, David J.; Bertram, John F.

doi:10.1681/asn.2015121340

Cited by 60 publications

(78 citation statements)

References 48 publications

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“…The extent of albuminuria, as assessed by the albumin-to-creatinine ratio of spot-collected urine samples over 12 weeks duration and by 24-hour urine albumin excretion at 12 weeks post-DM, was markedly attenuated in diabetic Lrg1 2/2 mice compared with diabetic Lrg1 +/+ mice (Figure 2, F and G). DM-induced podocyte foot process effacement and loss (as ascertained by podocyte marker p57 48,49 ) were also reduced in diabetic Lrg1 2/2 mice (Figure 3, A-D). Together, these data indicate that the genetic deletion of LRG1 results in marked attenuation of diabetic glomerulopathy.…”

Section: Genetic Deletion Of Lrg1 Attenuates Diabetes-induced Albuminmentioning

confidence: 89%

“…Diabetes was similarly induced with low-dose STZ injections 3 weeks after UNx in Lrg1 +/+ ;Flk1-H2B-EYFP (Lrg1 +/+ ; EYFP) and in Lrg1 2 /2 ;Flk1-H2B-EYFP (Lrg1 2 /2 ;EYFP) mice. Because the above directed in vivo angiogenesis assay results showed differential angiogenic potential when examined between 7-9 weeks post-STZ injection, Lrg1 +/+ ; EYFP and Lrg1 2/2 ;EYFP mice were sacrificed at 8 weeks post-DM, and the total number of EYFP+ cells per glomeruli were quantified in 500 mm optically cleared kidney sections 49 by two-photon microscopy. Indeed, the number of EYFP+ cells in the diabetic Lrg1 +/+ ;EYFP mouse glomeruli was found to be increased at 8 weeks post-DM, but this increase was mitigated in the diabetic Lrg1 +/+ ;EYFP mouse glomeruli ( Figure 4D, Supplemental Files 1-4).…”

Section: Lrg1 Deletion Attenuates Diabetes-induced Angiogenesismentioning

confidence: 99%

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LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-β–Induced Angiogenesis

Hong

Lü

et al. 2019

JASN

110

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Background Glomerular endothelial dysfunction and neoangiogenesis have long been implicated in the pathogenesis of diabetic kidney disease (DKD). However, the specific molecular pathways contributing to these processes in the early stages of DKD are not well understood. Our recent transcriptomic profiling of glomerular endothelial cells identified a number of proangiogenic genes that were upregulated in diabetic mice, including leucine-rich a-2-glycoprotein 1 (LRG1). LRG1 was previously shown to promote neovascularization in mouse models of ocular disease by potentiating endothelial TGF-b/activin receptorlike kinase 1 (ALK1) signaling. However, LRG1's role in the kidney, particularly in the setting of DKD, has been unclear.Methods We analyzed expression of LRG1 mRNA in glomeruli of diabetic kidneys and assessed its localization by RNA in situ hybridization. We examined the effects of genetic ablation of Lrg1 on DKD progression in unilaterally nephrectomized, streptozotocin-induced diabetic mice at 12 and 20 weeks after diabetes induction. We also assessed whether plasma LRG1 was associated with renal outcome in patients with type 2 diabetes.Results LRG1 localized predominantly to glomerular endothelial cells, and its expression was elevated in the diabetic kidneys. LRG1 ablation markedly attenuated diabetes-induced glomerular angiogenesis, podocyte loss, and the development of diabetic glomerulopathy. These improvements were associated with reduced ALK1-Smad1/5/8 activation in glomeruli of diabetic mice. Moreover, increased plasma LRG1 was associated with worse renal outcome in patients with type 2 diabetes.Conclusions These findings identify LRG1 as a potential novel pathogenic mediator of diabetic glomerular neoangiogenesis and a risk factor in DKD progression.Diabetic kidney disease (DKD) is a frequent complication of diabetes mellitus (DM) and the most common cause of ESRD in the United States. 1 Intensive glycemic control, BP control, and blockade of the renin-angiotensin-aldosterone system are the gold standard for current treatment of patients with DKD. 2-4 However, these established regimens provide only partial therapeutic effects, 5 indicating that pathogenic mechanisms driving DKD progression may not be targeted by current treatments. In addition, several recent clinical trials in DKD treatments were unsuccessful, 5-8 highlighting an unmet need for better understanding of mechanisms mediating the early stages of DKD for the design of novel preventive therapeutic strategies.Lrg1 expression that colocalize with CD31 outside the tubules. Scale bars, 20 mm. (D) Semiquantitative scoring of Lrg1 mRNA colocalized with Pecam mRNA (n=3 mice per group, 20 gloms scored per mouse). (E) In situ hybridization of LRG1 mRNA (red) combined with CD31 immunofluorescence (green) on human kidney sections with nuclear counterstain. Glomeruli are outlined with dotted lines on upper panels, and magnified view of the box is shown in lower panels. Scale bar, 50 mm. (F) Semiquantitative scoring of LRG1 mRNA colocalized with CD31...

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Section: Genetic Deletion Of Lrg1 Attenuates Diabetes-induced Albuminmentioning

confidence: 89%

Section: Lrg1 Deletion Attenuates Diabetes-induced Angiogenesismentioning

confidence: 99%

LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-β–Induced Angiogenesis

Hong

Lü

et al. 2019

JASN

110

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“…The application of optical clearing to kidney morphometrics has been limited, despite recent evidence showing that conventional 2-dimensional (2D) analyses may not be accurate or precise enough to detect existing differences and may introduce methodological bias. 13 Implementation of 3D morphometrics has been difficult for parenchymal organs, in particular of the kidney. This study introduces a new combination of diverse technologies allowing single-cell identification, kidney-optimized reversible optical clearing for subsequent application of standard histology, immunofluorescence and transmission electron microscopy in the same sample, flexible advanced light microscopy based on adaptable and solvent-resistant imaging chambers that allowed high-throughput imaging of whole slices and intact kidney structures with single-cell resolution, and quantitative volumetric computational analytics.…”

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confidence: 99%

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis

Puelles

Fleck

Ortz

et al. 2019

Kidney International

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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 highthroughput endpoints for the analysis of complex tissues with single-cell resolution.

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“…24 The number of podocytes per mouse kidney is smaller (about 75 per glomerulus). 25 This would give 20220 glomeruli per mouse kidney X 75 podocytes per glomerulus = 1.5 X 10 6 podocytes per mouse kidney. This comes out to 3.0% of total epithelial cells in mouse kidney (Table 3).…”

Section: How Much Do Various Kidney Tubule Cell Types Contribute To Tmentioning

confidence: 99%

Cell-Type Selective Markers Represented in Whole-Kidney RNA-Seq Data

Clark

Chen

Chou

et al. 2018

Preprint

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Bulk-tissue RNA-Seq is seeing increasing use in the study of physiological and pathophysiological processes in the kidney. However, the presence of multiple cell types in kidney complicates the data interpretation. Here we address the question, “What cell types are represented in whole-kidney RNA-Seq data?” to identify circumstances in which bulk-kidney RNA-Seq can be successfully interpreted. We carried out RNA-Seq in mouse whole kidneys and microdissected renal tubule segments. To aid in the interpretation of the data, we compiled a database of cell-type selective protein markers for 43 cell types believed to be present in kidney tissue. The whole-kidney RNA-Seq analysis identified transcripts corresponding to 17742 genes, distributed over 5 orders of magnitude of expression level. Markers for all 43 curated cell types were detectable. Analysis of the cellular makeup of mouse and rat kidney, calculated from published literature, suggests that proximal tubule cells account for more than half of the mRNA in a kidney. Comparison of RNA-Seq data from microdissected proximal tubules with whole-kidney data supports this view. RNA-Seq data for cell-type selective markers in bulk-kidney samples provide a valid means to identify changes in minority-cell abundances in kidney tissue. Because proximal tubules make up a substantial fraction of whole-kidney samples, changes in proximal tubule gene expression can be assessed presumptively by bulk-kidney RNA-Seq, although results could potentially be obscured by the presence of mRNA from other cell types. The dominance of proximal tubule cells in whole-kidney samples also has implications for the interpretation of single-cell RNA-Seq data.

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Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli

Cited by 60 publications

References 48 publications

LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-β–Induced Angiogenesis

LRG1 Promotes Diabetic Kidney Disease Progression by Enhancing TGF-β–Induced Angiogenesis

Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis

Cell-Type Selective Markers Represented in Whole-Kidney RNA-Seq Data

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