Clearly imaging and quantifying the kidney in 3D

Abstract

For decades, measurements of kidney microanatomy using 2-dimensional sections has provided us with a detailed knowledge of kidney morphology under physiological and pathological conditions. However, the rapid development of tissue clearing methods in recent years, in combination with the development of novel 3-dimensional imaging modalities have provided new insights into kidney structure and function. This review article describes a range of novel insights into kidney development and disease obtained recently using these new methodological approaches. For example, in the developing kidney these approaches have provided new understandings of ureteric branching morphogenesis, nephron progenitor cell proliferation and commitment, interactions between ureteric tip cells and nephron progenitor cells, and the establishment of nephron segmentation. In whole adult mouse kidneys, tissue clearing combined with light sheet microscopy can image and quantify the total number of glomeruli, a major breakthrough in the field. Similar approaches have provided new insights into the structure of the renal vasculature and innervation, tubulointerstitial remodeling, podocyte loss and hypertrophy, cyst formation, the evolution of cellular crescents, and the structure of the glomerular filtration barrier. Many more advances in the understanding of kidney biology and pathology can be expected as additional clearing and imaging techniques are developed and adopted by more investigators. For decades, measurements of kidney microanatomy using 2-dimensional sections has provided us with a detailed knowledge of kidney morphology under physiological and pathological conditions. However, the rapid development of tissue clearing methods in recent years, in combination with the development of novel 3-dimensional imaging modalities have provided new insights into kidney structure and function. This review article describes a range of novel insights into kidney development and disease obtained recently using these new methodological approaches. For example, in the developing kidney these approaches have provided new understandings of ureteric branching morphogenesis, nephron progenitor cell proliferation and commitment, interactions between ureteric tip cells and nephron progenitor cells, and the establishment of nephron segmentation. In whole adult mouse kidneys, tissue clearing combined with light sheet microscopy can image and quantify the total number of glomeruli, a major breakthrough in the field. Similar approaches have provided new insights into the structure of the renal vasculature and innervation, tubulointerstitial remodeling, podocyte loss and hypertrophy, cyst formation, the evolution of cellular crescents, and the structure of the glomerular filtration barrier. Many more advances in the understanding of kidney biology and pathology can be expected as additional clearing and imaging techniques are developed and adopted by more investigators. Editor’s NoteIn this review, which is part of the "Visualizing Techniques" series, new methods are discussed that very much facilitate the imaging and 3-dimensional reconstruction of complex structures such as glomeruli, tubules, particular tubular segments, or the kidney vasculature. What took weeks to months in the past can now be done rapidly thanks to the discovery of tissue clearing and smart new combinations of techniques. In this review, which is part of the "Visualizing Techniques" series, new methods are discussed that very much facilitate the imaging and 3-dimensional reconstruction of complex structures such as glomeruli, tubules, particular tubular segments, or the kidney vasculature. What took weeks to months in the past can now be done rapidly thanks to the discovery of tissue clearing and smart new combinations of techniques. Total nephron number in each normal human kidney ranges from approximately 200,000 to more than 2 million,1Bertram J.F. Douglas-Denton R.N. Diouf B. et al.Human nephron number: implications for health and disease.Pediatr Nephrol. 2011; 26: 1529-1533Crossref PubMed Scopus (300) Google Scholar averaging approximately 1 million. These nephrons as well as the other components of the kidney including collecting ducts, blood vessels, interstitia, and nerve fibers are arranged with exquisite microanatomical precision.2Blanc T. Goudin N. Zaidan M. et al.Three-dimensional architecture of nephrons in the normal and cystic kidney.Kidney Int. 2021; 99: 632-645Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar This precise patterning underpins normal kidney function and health, which can be severely disturbed when tissue architecture is altered due to glomerular or tubular hypertrophy or shrinkage, nephron loss, congenital abnormalities, vessel rarefaction, and interstitial fibrosis. A range of imaging approaches have contributed to our comprehensive understanding of kidney microanatomy during development and in the healthy and diseased adult kidney. For the most part, this understanding has come from analysis of 2-dimensional (2D) sections whether they be light or electron microscopic physical sections or confocal optical sections. Quantitation of this microanatomy has mostly depended on stereological analyses of sections that have provided estimates of numbers, lengths, surface areas, and volumes of glomeruli, tubules, vessels, interstitia, and their cellular components.3Bertram J.F. Analyzing renal glomeruli with the new stereology.Int Rev Cytol. 1995; 161: 111-172Crossref PubMed Scopus (143) Google Scholar,4Nyengaard J.R. Stereologic methods and their application in kidney research.J Am Soc Nephrol. 1999; 10: 1100-1123Crossref PubMed Google Scholar Until recently, 3D visualization of kidney microstructure was largely limited to confocal microscopy, which enables optical sectioning and 3D reconstruction up to a depth of 50 to 80 μm. Unfortunately, the refractive properties of protein and lipid components within a tissue scatter light and dramatically reduce image quality as depth increases. In recent years, a range of clearing methods have been developed to remove lipid and pigment content of a tissue and match the refractive properties of tissue and mounting media to render the tissue transparent.5Puelles V.G. Moeller M.J. Bertram J.F. We can see clearly now: optical clearing and kidney morphometrics.Curr Opin Nephrol Hypertens. 2017; 26: 179-186Crossref PubMed Scopus (11) Google Scholar Clearing methods range in complexity from incubating tissue in various solutions over a period of hours to extended procedures involving custom electrochemical equipment. We direct readers to recent reviews5Puelles V.G. Moeller M.J. Bertram J.F. We can see clearly now: optical clearing and kidney morphometrics.Curr Opin Nephrol Hypertens. 2017; 26: 179-186Crossref PubMed Scopus (11) Google Scholar,6Ueda H.R. Erturk A. Chung K. et al.Tissue clearing and its applications in neuroscience.Nat Rev Neurosci. 2020; 21: 61-79Crossref PubMed Scopus (184) Google Scholar for a detailed overview of current methods, and the papers cited herein for specific methods and use cases. Subcellular structures and entire populations of cells can be resolved within cleared whole organs or thick sections. Accurately imaging features within cleared tissue requires specialized microscopes and image processing to capture and reconstruct volumetric data from samples that can range from hundreds of microns to centimeters in size. Point scanning and spinning disk confocal microscopes have been effectively used to image features within cleared kidney tissue but suffer from reduced resolution in the Z axis and a progressive reduction in fluorescence intensity at depth due to a single axis of illumination and imaging. Light sheet microscopy techniques are particularly suited to imaging cleared tissues as they enable fast acquisition of large 3D volumes and can include multiangle illumination and imaging. Optical projection tomography is another approach that incorporates multiangle imaging and digital reconstruction to produce data in which the X, Y, and Z axes of each voxel are the same resolution. Regardless of the specific approach, tissue clearing and whole mount imaging provide a new opportunity to explore uncharted cellular microenvironments and higher-order tissue structure. In this review, we highlight some new insights into kidney development, adult kidney structure, and pathology that have resulted from tissue clearing followed by 3D quantitative analysis. Decades of gene expression profiling and knockout studies have led to a detailed understanding of the cell types, gene regulatory networks, and signaling pathways that control mammalian kidney development. This knowledge informed diagnosis of congenital disease and enabled production of kidney cell types from pluripotent stem cells.7Little M.H. Combes A.N. Kidney organoids: accurate models or fortunate accidents.Genes Dev. 2019; 33: 1319-1345Crossref PubMed Scopus (57) Google Scholar However, the size, opacity, and complexity of the developing kidney presented a significant barrier to precisely measuring the morphogenesis of this organ and the 3D distribution of cells and proteins within it. The application of tissue clearing and multiscale imaging in the kidney has enabled visualization and quantitative analysis of ureteric branching morphogenesis, progenitor cell populations, and nephron endowment within intact organs (Figure 1).8Short K. Hodson M. Smyth I. Spatial mapping and quantification of developmental branching morphogenesis.Development. 2013; 140: 471-478Crossref PubMed Scopus (66) Google Scholar These approaches underpin a new capacity for precision phenotyping and open avenues to analyze the cellular and molecular drivers of kidney development and congenital disease. Establishment of the ureteric tree is a defining feature of kidney development and has been extensively studied using mouse genetics and flattened kidney explant cultures. However, understanding how individual genes contribute to the growth, form, and patterning of this branched epithelial duct structure in vivo required the capacity to image and analyze this complex network across a wide range of sample sizes. Short et al.8Short K. Hodson M. Smyth I. Spatial mapping and quantification of developmental branching morphogenesis.Development. 2013; 140: 471-478Crossref PubMed Scopus (66) Google Scholar addressed these issues using whole mount immunofluorescence, solvent-based clearing (benzyl alcohol–benzyl benzoate) and imaging whole organs with optical projection tomography. Employing this approach to capture 3D snapshots from early to mid-kidney development, the team developed custom analysis software to derive unprecedented detail about the ureteric tree including tip number, branch angles and length, branch volumes, and the number of generations of branching from intact ureteric trees (Figure 1c and d).8Short K. Hodson M. Smyth I. Spatial mapping and quantification of developmental branching morphogenesis.Development. 2013; 140: 471-478Crossref PubMed Scopus (66) Google Scholar Initially used to characterize and identify novel aspects of renal branching,9Combes A.N. Short K.M. Lefevre J. et al.An integrated pipeline for the multidimensional analysis of branching morphogenesis.Nat Protoc. 2014; 9: 2859-2879Crossref PubMed Scopus (28) Google Scholar,10Short K.M. Combes A.N. Lefevre J. et al.Global quantification of tissue dynamics in the developing mouse kidney.Dev Cell. 2014; 29: 188-202Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar ongoing efforts seek to define the principles and key regulators that govern branch patterning in vivo.11Lefevre J.G. Short K.M. Lamberton T.O. et al.Branching morphogenesis in the developing kidney is governed by rules that pattern the ureteric tree.Development. 2017; 144: 4377-4385PubMed Google Scholar Molecular interactions between ureteric tip cells and nephron progenitor cells play a major role in establishing the complement of nephrons that facilitate kidney function in adult life. Uninduced nephron progenitors produce factors that promote kidney growth through ureteric branching while spatially restricted cues within the ureteric tip both maintain nephron progenitor state and induce a subset of these progenitors to differentiate into an early nephron. These interactions drive a cycle of branching and nephron induction that ceases around birth, when remaining nephron progenitors differentiate. Prior to tissue clearing methods, our understanding of the dynamics of kidney morphogenesis and the relative abundance of progenitor cell populations across time was severely limited. This changed with the development of multiscale imaging approaches utilizing extended immunofluorescence protocols, tissue clearing, and imaging approaches to quantify kidney development at the cellular (confocal) and whole organ level (optical projection tomography, light sheet).9Combes A.N. Short K.M. Lefevre J. et al.An integrated pipeline for the multidimensional analysis of branching morphogenesis.Nat Protoc. 2014; 9: 2859-2879Crossref PubMed Scopus (28) Google Scholar,10Short K.M. Combes A.N. Lefevre J. et al.Global quantification of tissue dynamics in the developing mouse kidney.Dev Cell. 2014; 29: 188-202Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar It became apparent that the rates of kidney growth vary dramatically with initially rapid branching and proliferation rates declining in phases that correlate with reductions in the number of progenitor cells within the nephrogenic niche.10Short K.M. Combes A.N. Lefevre J. et al.Global quantification of tissue dynamics in the developing mouse kidney.Dev Cell. 2014; 29: 188-202Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar Three-dimensional confocal imaging of cleared whole human fetal kidneys (week 11) or samples from the kidney cortex (weeks 11–23) affirm that this decrease in niche size is conserved across species.12Lindstrom N.O. McMahon J.A. Guo J. et al.Conserved and divergent features of human and mouse kidney organogenesis.J Am Soc Nephrol. 2018; 29: 785-805Crossref PubMed Scopus (109) Google Scholar Further comparative analysis of the human and mouse nephrogenic niche in cleared tissue led to a novel time-based recruitment model of nephron formation, wherein progenitor cells are progressively added to an early nephron and adopt a segment identity according to the order in which they arrive.13Lindstrom N.O. De Sena Brandine G. Tran T. et al.Progressive recruitment of mesenchymal progenitors reveals a time-dependent process of cell fate acquisition in mouse and human nephrogenesis.Dev Cell. 2018; 45: 651-660e4Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar In another study focused on nephron progenitor commitment, a hydrogel-based tissue clearing method (passive B. et phenotyping within intact tissue through 2014; Full Text Full Text PDF PubMed Scopus Google was used for its to fluorescence of an of within the cleared tissue that cells within the early nephron can to a progenitor was Lefevre J. et progenitor is a process by cell 2019; PubMed Scopus Google Scholar tissue clearing has our understanding of developmental across and in tissue clearing and quantitative analysis have enabled a new capacity for precision in branching and nephron number have been in mouse models with such as and (Figure which as K.M. Combes A.N. Lefevre J. et al.Global quantification of tissue dynamics in the developing mouse kidney.Dev Cell. 2014; 29: 188-202Abstract Full Text Full Text PDF PubMed Scopus (175) Google A.N. B. et for the gene nephron progenitor proliferation branching and nephron Int. 2018; Full Text Full Text PDF PubMed Scopus Google Scholar confocal imaging of cleared kidneys a novel cellular nephron progenitors due to an to cellular with the ureteric tip and Combes A.N. Short K.M. et nephron progenitor and nephron 2018; PubMed Scopus (28) Google Scholar These methods have been used to quantify how new regulators of nephron progenitor cell state such as and and kidney development and nephron T. et of mouse of A. 2018; PubMed Scopus Google N. J. Combes A. et 1 nephron progenitor cell and Am Soc Nephrol. 2019; PubMed Scopus Google et and the of of 2019; 10: PubMed Scopus (28) Google Scholar phenotyping is to an in our to the of and factors that contribute to nephron number and to kidney disease. The data from the cleared developing kidney are a of that to how the and function of a tissue from molecular interactions between progenitor cell J.G. Short K.M. Lamberton T.O. et al.Branching morphogenesis in the developing kidney is governed by rules that pattern the ureteric tree.Development. 2017; 144: 4377-4385PubMed Google M. et of branching 2017; Full Text Full Text PDF PubMed Scopus Google et of kidney branching morphogenesis reveals and 2019; 10: PubMed Scopus Google Combes A.N. Short K.M. et model of kidney branching PubMed Scopus Google Scholar Further application of whole organ imaging in cleared tissue be to the molecular and cellular drivers of 3D tissue patterning and the key to the structure and of stem cell models of the kidney. 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Diouf B. et al.Human nephron number: implications for health and disease.Pediatr Nephrol. 2011; 26: 1529-1533Crossref PubMed Scopus (300) Google V.G. et number and size and for kidney Opin Nephrol Hypertens. 2011; PubMed Scopus Google V.G. T. et individual glomerular in the human PubMed Scopus Google Scholar and has been to developmental For decades, the to quantify nephron number in and human samples has been the a stereological J.F. Analyzing renal glomeruli with the new stereology.Int Rev Cytol. 1995; 161: 111-172Crossref PubMed Scopus (143) Google Scholar,4Nyengaard J.R. Stereologic methods and their application in kidney research.J Am Soc Nephrol. 1999; 10: 1100-1123Crossref PubMed Google Scholar this approach is accurate and is and requires significant For this approaches have been to methods that can provide V.G. Bertram J.F. glomeruli and and Opin Nephrol Hypertens. 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