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

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.

[1]  Etsuo A. Susaki,et al.  CUBIC pathology: three-dimensional imaging for pathological diagnosis , 2017, Scientific Reports.

[2]  P. Tipping,et al.  T cells in crescentic glomerulonephritis. , 2006, Journal of the American Society of Nephrology : JASN.

[3]  S. Akira,et al.  Genetic and pharmacological inhibition of microRNA-92a maintains podocyte cell cycle quiescence and limits crescentic glomerulonephritis , 2017, Nature Communications.

[4]  Philip Webster,et al.  Glomerular disease: Crescentic glomerulonephritis: beyond the immune system , 2017, Nature Reviews Nephrology.

[5]  Neil R. Powe,et al.  Global kidney health 2017 and beyond: a roadmap for closing gaps in care, research, and policy , 2017, The Lancet.

[6]  B. Smeets,et al.  Origin of regenerating tubular cells after acute kidney injury , 2014, Proceedings of the National Academy of Sciences.

[7]  Heino Velazquez,et al.  Three-Dimensional Morphology by Multiphoton Microscopy with Clearing in a Model of Cisplatin-Induced CKD. , 2016, Journal of the American Society of Nephrology : JASN.

[8]  Anna Medyukhina,et al.  Fully Automated Evaluation of Total Glomerular Number and Capillary Tuft Size in Nephritic Kidneys Using Lightsheet Microscopy. , 2017, Journal of the American Society of Nephrology : JASN.

[9]  I. Pastan,et al.  Tubulointerstitial fibrosis can sensitize the kidney to subsequent glomerular injury. , 2017, Kidney international.

[10]  Dimitri Perrin,et al.  Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging , 2015, Nature Protocols.

[11]  R. Wiggins,et al.  Urinary podocyte and TGF-&bgr;1 mRNA as markers for disease activity and progression in anti-glomerular basement membrane nephritis , 2017, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[12]  G. Remuzzi,et al.  Podocyte–actin dynamics in health and disease , 2016, Nature Reviews Nephrology.

[13]  Victor G Puelles,et al.  Glomerular number and size variability and risk for kidney disease , 2011, Current opinion in nephrology and hypertension.

[14]  N. Plesnila,et al.  Shrinkage-mediated imaging of entire organs and organisms using uDISCO , 2016, Nature Methods.

[15]  Atsushi Miyawaki,et al.  Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain , 2011, Nature Neuroscience.

[16]  N. Gretz,et al.  Pathways to recovery and loss of nephrons in anti-Thy-1 nephritis. , 2003, Journal of the American Society of Nephrology : JASN.

[17]  Victor G. Puelles,et al.  New insights on glomerular hyperfiltration: a Japanese autopsy study. , 2017, JCI insight.

[18]  W. Kriz,et al.  Pathways to nephron loss starting from glomerular diseases-insights from animal models. , 2005, Kidney international.

[19]  Haruo Kasai,et al.  Chemical Landscape for Tissue Clearing Based on Hydrophilic Reagents. , 2018, Cell reports.

[20]  Aurelien Dugourd,et al.  Gli1+ Mesenchymal Stromal Cells Are a Key Driver of Bone Marrow Fibrosis and an Important Cellular Therapeutic Target. , 2017, Cell stem cell.

[21]  T. Wynn,et al.  Macrophages in Tissue Repair, Regeneration, and Fibrosis. , 2016, Immunity.

[22]  B. Smeets,et al.  Tracing the origin of glomerular extracapillary lesions from parietal epithelial cells. , 2009, Journal of the American Society of Nephrology : JASN.

[23]  Tobias B Huber,et al.  New Insights into Podocyte Biology in Glomerular Health and Disease. , 2017, Journal of the American Society of Nephrology : JASN.

[24]  B. Smeets,et al.  Investigations of Glucocorticoid Action in GN. , 2017, Journal of the American Society of Nephrology : JASN.

[25]  Jeff W. Lichtman,et al.  Clarifying Tissue Clearing , 2015, Cell.

[26]  Rajan P Kulkarni,et al.  Single-Cell Phenotyping within Transparent Intact Tissue through Whole-Body Clearing , 2014, Cell.

[27]  Victor G. Puelles,et al.  We can see clearly now: optical clearing and kidney morphometrics , 2017, Current opinion in nephrology and hypertension.

[28]  R. Fryer,et al.  Quantification and Comparison of Anti-Fibrotic Therapies by Polarized SRM and SHG-Based Morphometry in Rat UUO Model , 2016, PloS one.

[29]  B. Humphreys,et al.  Lineage-tracing methods and the kidney , 2013, Kidney international.

[30]  B. Najafian,et al.  Atubular glomeruli and glomerulotubular junction abnormalities in diabetic nephropathy. , 2003, Journal of the American Society of Nephrology : JASN.

[31]  Victor G. Puelles,et al.  Validation of a Three-Dimensional Method for Counting and Sizing Podocytes in Whole Glomeruli. , 2016, Journal of the American Society of Nephrology : JASN.

[32]  C. Heuser,et al.  Exclusive CX3CR1 dependence of kidney DCs impacts glomerulonephritis progression. , 2013, The Journal of clinical investigation.

[33]  C. Daniel,et al.  Cardiovascular and Renal Effects of High Salt Diet in GDNF+/- Mice with Low Nephron Number , 2013, Kidney and Blood Pressure Research.

[34]  Markus M. Rinschen,et al.  A Multi-layered Quantitative In Vivo Expression Atlas of the Podocyte Unravels Kidney Disease Candidate Genes , 2018, Cell reports.

[35]  J. Hodgin,et al.  Podocyte Depletion in Thin GBM and Alport Syndrome , 2016, PloS one.

[36]  J. Hodgin,et al.  Quantitative podocyte parameters predict human native kidney and allograft half-lives. , 2016, JCI insight.

[37]  M. Jeansson,et al.  New insights into the pathogenesis of cellular crescents , 2011, Current opinion in nephrology and hypertension.

[38]  Gregory B. Lim.  Heart failure: Macrophages promote cardiac fibrosis and diastolic dysfunction , 2018, Nature Reviews Cardiology.

[39]  Hiroshi Sekiya,et al.  A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing , 2018, Nature Neuroscience.

[40]  Aaron S. Andalman,et al.  Structural and molecular interrogation of intact biological systems , 2013, Nature.

[41]  Hiroki R Ueda,et al.  Whole-body and Whole-Organ Clearing and Imaging Techniques with Single-Cell Resolution: Toward Organism-Level Systems Biology in Mammals. , 2016, Cell chemical biology.

[42]  Kohei Miyazono,et al.  Whole-Body Profiling of Cancer Metastasis with Single-Cell Resolution. , 2017, Cell reports.

[43]  Quan Yuan,et al.  Tissue clearing of both hard and soft tissue organs with the PEGASOS method , 2018, Cell Research.

[44]  Sergey Plotnikov,et al.  Second harmonic generation microscopy for quantitative analysis of collagen fibrillar structure , 2012, Nature Protocols.

[45]  E. Susaki,et al.  Whole-Brain Imaging with Single-Cell Resolution Using Chemical Cocktails and Computational Analysis , 2014, Cell.

[46]  T. Maher,et al.  Pulmonary Macrophages: A New Therapeutic Pathway in Fibrosing Lung Disease? , 2016, Trends in molecular medicine.

[47]  R. Kramann,et al.  Gli1+ Pericyte Loss Induces Capillary Rarefaction and Proximal Tubular Injury. , 2017, Journal of the American Society of Nephrology : JASN.

[48]  N. Marcussen,et al.  Ultrastructural quantitation of atubular and hypertrophic glomeruli in rats with lithium-induced chronic nephropathy , 2005, Virchows Archiv A.