Clearly imaging and quantifying the kidney in 3D

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 Clear Lipid-exchanged Acrylamide-hybridized Rigid Imaging/Immunostaining/In situ hybridization-compatible Tissue-hYdrogel [CLARITY]14Yang B. Treweek J.B. Kulkarni R.P. et al.Single-cell phenotyping within transparent intact tissue through whole-body clearing.Cell. 2014; 158: 945-958Abstract Full Text Full Text PDF PubMed Scopus (611) Google Scholar) was used for its ability to retain fluorescence of an endogenous tdTomato reporter. Analysis of tdTomato-labeling within the cleared tissue showed that cells within the early committing nephron can return to a progenitor state, where prior results suggested commitment was unidirectional.15Lawlor K.T. Zappia L. Lefevre J. et al.Nephron progenitor commitment is a stochastic process influenced by cell migration.Elife. 2019; 8e41156Crossref PubMed Scopus (29) Google Scholar Thus, tissue clearing has improved our understanding of dynamic developmental processes across scales and species. Advances in tissue clearing and quantitative analysis have enabled a new capacity for precision phenotyping. Statistically robust changes in branching and nephron number have been identified in mouse models with subtle phenotypes such as Ret+/− and Six2+/− mice (Figure 2), which were previously classified as "normal."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,16Combes A.N. Wilson S. Phipson B. et al.Haploinsufficiency for the Six2 gene increases nephron progenitor proliferation promoting branching and nephron number.Kidney Int. 2018; 93: 589-598Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar High-resolution confocal imaging of cleared Wnt11−/− kidneys identified a novel cellular phenotype where nephron progenitors appear disorganized due to an inability to form stable cellular attachments with the ureteric tip and prematurely differentiate.17O'Brien L.L. Combes A.N. Short K.M. et al.Wnt11 directs nephron progenitor polarity and motile behavior ultimately determining nephron endowment.Elife. 2018; 7e40392Crossref PubMed Scopus (28) Google Scholar These methods have also been used to quantify how new regulators of nephron progenitor cell state such as DNA methyltransferase 1, microRNAs Lin28 and Let7, and TSC1 influence kidney development and nephron number.18Volovelsky O. Nguyen T. Jarmas A.E. et al.Hamartin regulates cessation of mouse nephrogenesis independently of Mtor.Proc Natl Acad Sci U S A. 2018; 115: 5998-6003Crossref PubMed Scopus (19) Google Scholar, 19Wanner N. Vornweg J. Combes A. et al.DNA methyltransferase 1 controls nephron progenitor cell renewal and differentiation.J Am Soc Nephrol. 2019; 30: 63-78Crossref PubMed Scopus (36) Google Scholar, 20Yermalovich A.V. Osborne J.K. Sousa P. et al.Lin28 and Let-7 regulate the timing of cessation of murine nephrogenesis.Nat Commun. 2019; 10: 168Crossref PubMed Scopus (28) Google Scholar Precision phenotyping is likely to become an important tool in our effort to understand the additive effect of genetic and environmental factors that contribute to low nephron number and predisposition to chronic kidney disease. The data being produced from the cleared developing kidney are fueling a wave of image-based mathematical modeling that attempts to discern how the macroscopic form and function of a tissue arise from molecular interactions between progenitor cell populations.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,21Hannezo E. Scheele C. Moad M. et al.A unifying theory of branching morphogenesis.Cell. 2017; 171: 242-255e27Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22Menshykau D. Michos O. Lang C. et al.Image-based modeling of kidney branching morphogenesis reveals GDNF-RET based Turing-type mechanism and pattern-modulating WNT11 feedback.Nat Commun. 2019; 10: 239Crossref PubMed Scopus (40) Google Scholar, 23Zubkov V.S. Combes A.N. Short K.M. et al.A spatially-averaged mathematical model of kidney branching morphogenesis.J Theor Biol. 2015; 379: 24-37Crossref PubMed Scopus (16) Google Scholar Further application of whole organ imaging in cleared tissue will be critical to understand the molecular and cellular drivers of 3D tissue patterning and may hold the key to improving the structure and accuracy of stem cell models of the kidney. The intricate morphology of the adult kidney limits the use of 2D morphological tools as they may convey incomplete or misleading perceptions of 3D structure. Here, we discuss some of the important methodological advances in recent years that now allow robust 3D analysis of structures in the adult kidney, ranging from intact kidneys all the way to components of the glomerular filtration barrier (Figure 3).24Klingberg A. Hasenberg A. Ludwig-Portugall I. et al.Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy.J Am Soc Nephrol. 2017; 28: 452-459Crossref PubMed Scopus (178) Google Scholar, 25Østergaard M.V. Sembach F.E. Skytte J.L. et al.Automated image analyses of glomerular hypertrophy in a mouse model of diabetic nephropathy.Kidney360. 2020; 6: 469-479Crossref Google Scholar, 26Hasegawa S. Susaki E.A. Tanaka T. et al.Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.Kidney Int. 2019; 96: 129-138Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 27Puelles V.G. van der Wolde J.W. Schulze K.E. et al.Validation of a three-dimensional method for counting and sizing podocytes in whole glomeruli.J Am Soc Nephrol. 2016; 27: 3093-3104Crossref PubMed Scopus (40) Google Scholar, 28Puelles V.G. Fleck D. Ortz L. et al.Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.Kidney Int. 2019; 96: 505-516Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 29Unnersjo-Jess D. Butt L. Hohne M. et al.A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales.Kidney Int. 2021; 99: 1010-1020Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar The human kidney shows a high level of intra- and intersubject variability in nephron number and volume, which has been described in autopsy studies from subjects without overt kidney diseases1Bertram 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,30Puelles V.G. Hoy W.E. Hughson M.D. et al.Glomerular number and size variability and risk for kidney disease.Curr Opin Nephrol Hypertens. 2011; 20: 7-15Crossref PubMed Scopus (102) Google Scholar,31Puelles V.G. Zimanyi M.A. Samuel T. et al.Estimating individual glomerular volume in the human kidney: clinical perspectives.Nephrol Dial Transplant. 2012; 27: 1880-1888Crossref PubMed Scopus (30) Google Scholar and has been linked to developmental disturbances. For decades, the gold standard tool to quantify nephron number in experimental and human samples has been the disector/fractionator method, a design-based stereological method.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 While this approach is accurate and precise, it is also hands-on, time-consuming, and requires significant training. For this reason, alternative approaches have been proposed to devise efficient methods that can provide high-quality endpoints:32Puelles V.G. Bertram J.F. Counting glomeruli and podocytes: rationale and methodologies.Curr Opin Nephrol Hypertens. 2015; 24: 224-230PubMed Google Scholar for example, combinations of optical clearing methods, including hydrophobic (previously known as solvent-based), hydrophilic or hydrogel-based, with advanced light microscopy (e.g., confocal, multiphoton, or light sheet). To our knowledge, the first report that combined optical clearing and light sheet microscopy for the analysis of nephron number and size was published by Klingberg et al.24Klingberg A. Hasenberg A. Ludwig-Portugall I. et al.Fully automated evaluation of total glomerular number and capillary tuft size in nephritic kidneys using lightsheet microscopy.J Am Soc Nephrol. 2017; 28: 452-459Crossref PubMed Scopus (178) Google Scholar in a landmark paper that featured multiple important advances. First, this report introduced ethyl cinnamate, a nonhazardous solvent that could replace toxic chemicals such as benzyl alcohol–benzyl benzoate, which until then was regularly used in hydrophobic clearing protocols. Second, the investigators performed nephron counting in intact mouse kidneys in an aggressive and progressive model of immune-mediated kidney disease, identifying nephron loss during the disease course. In addition, significant steps toward computerized automation of image segmentation algorithms were introduced, providing a path to significant efficiency improvements. Recently, Østergaard et al.25Østergaard M.V. Sembach F.E. Skytte J.L. et al.Automated image analyses of glomerular hypertrophy in a mouse model of diabetic nephropathy.Kidney360. 2020; 6: 469-479Crossref Google Scholar presented a similar hydrophobic clearing method combined with light sheet microscopy and automated image segmentation that provides both total nephron number and single glomerular volume in a mouse model of diabetic nephropathy. Furthermore, this study showed elegant functional labeling of proximal tubuli via in vivo albumin injection to visualize the glomerular-tubular juncture and a simple protocol for correlative histopathology in the same optically cleared samples. This report shows the flexibility of these protocols, which are becoming multilayered pipelines for comprehensive tissue profiling. Following tracks of branching vasculature or nerve fibers in the adult kidney is almost impossible in 2D conventional histology as the size of the organ and the complexity of the structural distribution requires extensive and expert serial sectioning. Recently, Huang et al.33Huang J. Brenna C. Khan A.U.M. et al.A cationic near infrared fluorescent agent and ethyl-cinnamate tissue clearing protocol for vascular staining and imaging.Sci Rep. 2019; 9: 521Crossref PubMed Scopus (20) Google Scholar reported a cationic near infrared fluorescent dye (MHI148-PEI) to specifically label blood vessels, which can be combined with a modified and accelerated clearing protocol based on ethyl cinnamate to shorten tissue processing time. This approach opens new avenues for vascular research in the kidney, especially when high-resolution imaging in 3D is required. Similarly, Hasegawa et al.26Hasegawa S. Susaki E.A. Tanaka T. et al.Comprehensive three-dimensional analysis (CUBIC-kidney) visualizes abnormal renal sympathetic nerves after ischemia/reperfusion injury.Kidney Int. 2019; 96: 129-138Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar used Clear Unobstructed Brain/Body Imaging Cocktails and Computational analysis (CUBIC) together with a custom-built light sheet microscopy system to identify the distribution of sympathetic innervation in the intact mouse kidney. Sympathetic nerves were primarily distributed around arteries. After 10 days of ischemia or reperfusion injury, sympathetic innervation density was significantly decreased, correlating with reduced norepinephrine levels in kidney tissue. These changes partially persisted 28 days after the initial injury, suggesting that continuous sympathetic alterations may be found during the progression of chronic kidney disease. The renal tubular system is particularly difficult to analyze in 3D. The convoluted and complex morphology poses multiple challenges for conventional methods based on serial sectioning and standard light microscopy. However, optical clearing allows the analysis of intact tubuli and their surrounding microenvironment. Saritas et al.34Saritas T. Puelles V.G. Su X.T. et al.Optical clearing in the kidney reveals potassium-mediated tubule remodeling.Cell Rep. 2018; 25: 2668-2675e3Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar performed a combination of hydrogel-based and hydrophobic optical clearing followed by advanced light microscopy and semiautomatic 3D analysis to characterize distal tubular remodeling due to dietary potassium deprivation. The investigators quantified tubular hyperplasia and detected an increase in proliferating cells within the tubulointerstitium. This study utilized the versatility of both optical clearing and light microscopy to perform tubular analysis in intact kidneys using hydrogel-embedding and light sheet microscopy, as well as single-cell quantification in kidney slices using hydrophobic clearing and confocal microscopy, as each combination was better suited for the specific research question. Combinations of optical clearing and advanced light microscopy were used by Tahaei et al.35Tahaei E. Coleman R. Saritas T. et al.Distal convoluted tubule sexual dimorphism revealed by advanced 3D imaging.Am J Physiol Renal Physiol. 2020; 319: F754-F764Crossref PubMed Scopus (9) Google Scholar who characterized sexual dimorphism in the length of the distal convoluted tubule, which may determine its capacity to adapt to physiological stress, and Schuh et al.36Schuh C.D. Polesel M. Platonova E. et al.Combined structural and functional imaging of the kidney reveals major axial differences in proximal tubule endocytosis.J Am Soc Nephrol. 2018; 29: 2696-2712Crossref PubMed Scopus (34) Google Scholar who revealed axial differences in proximal tubule ligand uptake and endolysosomal function, highlighting that the S1 segment is highly specialized to reabsorb proteins via receptor-mediated endocytosis. In a recent study, Blanc et al.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 analyzed a model of cyst formation, showing that cysts developed only in specific nephron segments, which determined their shape and were located contiguous to normal nondilated tubules. Overall, we now have multiple examples of direct application of optical clearing for systematic analysis of the tubulointerstitial compartment. The glomerulus provides a unique example for the analysis of intact functional units within an organ. In particular, the study of podocyte depletion has directly benefited from these technological advances as the gold standard design-based methodologies require significant investment of time and resources.37Puelles V.G. Douglas-Denton R.N. Cullen-McEwen L. et al.Design-based stereological methods for estimating numbers of glomerular podocytes.Ann Anat. 2014; 196: 48-56Crossref PubMed Scopus (15) Google Scholar The first tool for 3D podocyte morphometric analysis of intact mouse glomeruli combined immunolabeling of kidney slices using a modified protocol of indirect immunofluorescence, hydrophobic optical clearing, and confocal microscopy, which facilitated the quantification of total podocyte number in whole individual glomeruli of known volume. We believe this approach is ideal for the study of processes that affect a specific subset of structures (e.g., podocyte loss leading to focal and segmental glomerulosclerosis).27Puelles V.G. van der Wolde J.W. Schulze K.E. et al.Validation of a three-dimensional method for counting and sizing podocytes in whole glomeruli.J Am Soc Nephrol. 2016; 27: 3093-3104Crossref PubMed Scopus (40) Google Scholar The same technique was later applied to characterize the role of podocyte hypertrophy as a compensatory response following podocyte loss, a mechanism that is mediated by the mammalian target of rapamycin signaling pathway.38Puelles V.G. van der Wolde J.W. Wanner N. et al.mTOR-mediated podocyte hypertrophy regulates glomerular integrity in mice and humans.JCI Insight. 2019; 4e99271Crossref PubMed Scopus (28) Google Scholar While these 2 studies focused on the analysis of glomerular podocytes, this pipeline based on conventional immunolabeling can be used to characterize morphological changes of any cell type at high spatial resolution. Another important tool that needed to be successfully integrated into the toolbox for 3D kidney morphometry is genetic lineage tracing. Most optical clearing methods tend to induce a certain degree of fluorescence quenching (which varies from mild to severe).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 We recently presented a protocol that combines lineage tracing, optical clearing, and multiphoton microscopy for the comprehensive analysis of podocyte loss and parietal epithelial cell activation.28Puelles V.G. Fleck D. Ortz L. et al.Novel 3D analysis using optical tissue clearing documents the evolution of murine rapidly progressive glomerulonephritis.Kidney Int. 2019; 96: 505-516Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar In this study, podocyte loss was identified as a feature of experimental crescentic nephritis using metabolic labeling of podocytes with enhanced green fluorescent protein. The evolution of cellular crescents was visualized and quantified based on the proliferation and migration of parietal epithelial cells, which were also marked by enhanced green fluorescent protein and tracked during the course of an experimental model of crescentic nephritis. Analysis of intact glomeruli allowed the identification of focal podocyte loss and lesion formation, which progressed to tubular occlusions by parietal epithelial cells, leading to formation of atubular glomeruli. This study paves the way for future studies of complex immune-epithelial interactions within intact kidneys. An important diagnostic tool for routine assessment of glomerular diseases is the ultrastructural analysis of renal biopsies by electron microscopy. An alternative to electron microscopy can be found in the field of expansion microscopy, which by definition aims to increase optical resolution by increasing tissue dimensions. Over the years, Unnersjo-Jess et al.39Unnersjo-Jess D. Scott L. Blom H. Brismar H. Super-resolution stimulated emission depletion imaging of slit diaphragm proteins in optically cleared kidney tissue.Kidney Int. 2016; 89: 243-247Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,40Unnersjo-Jess D. Scott L. Sevilla S.Z. et al.Confocal super-resolution imaging of the glomerular filtration barrier enabled by tissue expansion.Kidney Int. 2018; 93: 1008-1013Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar have provided multiple protocols for the visualization of nanoscale structures in the kidney. Recently, these investigators reported a fast and simple procedure for 3D visualization of kidney morphology, combining optical clearing and tissue expansion concepts to resolve nanoscale structures using conventional confocal microscopy.29Unnersjo-Jess D. Butt L. Hohne M. et al.A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales.Kidney Int. 2021; 99: 1010-1020Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar This protocol was applied to visualize and quantify multiple pathologic features in mouse and human kidney biopsies including foot process effacement, alterations of the glomerular basement membrane, slit diaphragm length, IgG deposits, and classical pathology features (e.g., glomerulosclerosis, tubulointerstitial fibrosis, and tubular atrophy). In summary, this method allows multiscale visualization and quantification of renal pathology using a simple method and accessible image analysis tools. The scalability and implementation of this approach in clinical practice will likely be determined by the need of advanced light microscopy equipment and the development of automated image analysis tools. The current spectrum of tissue clearing methods includes simple techniques that can be implemented in most laboratories to improve 3D imaging on commonly available microscopes. As the opportunities afforded by these new approaches become apparent, we anticipate a wider uptake of specialized microscopy systems to effectively image cleared tissue at high resolution and the development of tailored research and clinical workflows to address specific questions. The field of neuroscience has served as a testing ground for cleared tissue imaging methods and applications. Automated image analysis, novel approaches to studying cellular connectivity, and tissue-wide gene and protein expression atlas projects have been developed in this context. It is likely that similar approaches will yield new insights into the cellular anatomy and interdependency of cell populations in the developing and adult kidney.