A fast and simple clearing and swelling protocol for 3D in-situ imaging of the kidney across scales

Abstract

In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines. In recent years, many light-microscopy protocols have been published for visualization of nanoscale structures in the kidney. These protocols present researchers with new tools to evaluate both foot process anatomy and effacement, as well as protein distributions in foot processes, the slit diaphragm and in the glomerular basement membrane. However, these protocols either involve the application of different complicated super resolution microscopes or lengthy sample preparation protocols. Here, we present a fast and simple, five-hour long procedure for three-dimensional visualization of kidney morphology on all length scales. The protocol combines optical clearing and tissue expansion concepts to produce a mild swelling, sufficient for resolving nanoscale structures using a conventional confocal microscope. We show that the protocol can be applied to visualize a wide variety of pathologic features in both mouse and human kidneys. Thus, our fast and simple protocol can be beneficial for conventional microscopic evaluation of kidney tissue integrity both in research and possibly in future clinical routines. Numerous optical protocols for imaging nanoscale structures in the kidney have been presented in recent years.1Chozinski T.J. Mao C. Halpern A.R. et al.Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy.Sci Rep. 2018; 8: 10396Crossref PubMed Scopus (18) Google Scholar, 2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar, 3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar, 4Suleiman H. Zhang L. Roth R. et al.Nanoscale protein architecture of the kidney glomerular basement membrane.Elife. 2013; 2e01149PubMed Google Scholar, 5Unnersjö-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 (49) Google Scholar, 6Unnersjö-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 (26) Google Scholar, 7Zhao Y. Bucur O. Irshad H. et al.Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.Nat Biotechnol. 2017; 35: 757-764Crossref PubMed Scopus (86) Google Scholar, 8Bucur O. Fu F. Calderon M. et al.Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology.Nat Protoc. 2020; 15: 1649-1672Crossref PubMed Scopus (5) Google Scholar The power of these new techniques as tools for quantifying filtration barrier anatomy and pathology is exemplified in recent publications from our group and others.3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar59Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar,10Motrapu M. Świderska M.K. Mesas I. et al.Drug testing for residual progression of diabetic kidney disease in mice beyond therapy with metformin, ramipril, and empagliflozin.J Am Soc Nephrol. 2020; 31: 1729-1745Crossref PubMed Scopus (7) Google Scholar Although powerful, all these optical protocols require the use of advanced diffraction-unlimited microscopy or involve the introduction of complex polymer chemistry into the sample to clear and/or physically expand it. Many of the protocols are also time-consuming and require a significant amount of hands-on labor. This extra layer of complexity might be what is keeping these new methods from being routinely used by both researchers and clinical pathologists. Furthermore, many of the protocols lack access to deep 3-dimensional (3D) imaging of foot processes (FPs) beyond 5–15 μm in depth.1Chozinski T.J. Mao C. Halpern A.R. et al.Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy.Sci Rep. 2018; 8: 10396Crossref PubMed Scopus (18) Google Scholar, 2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar, 3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar, 4Suleiman H. Zhang L. Roth R. et al.Nanoscale protein architecture of the kidney glomerular basement membrane.Elife. 2013; 2e01149PubMed Google Scholar,7Zhao Y. Bucur O. Irshad H. et al.Nanoscale imaging of clinical specimens using pathology-optimized expansion microscopy.Nat Biotechnol. 2017; 35: 757-764Crossref PubMed Scopus (86) Google Scholar,8Bucur O. Fu F. Calderon M. et al.Nanoscale imaging of clinical specimens using conventional and rapid-expansion pathology.Nat Protoc. 2020; 15: 1649-1672Crossref PubMed Scopus (5) Google Scholar Therefore, we aimed to develop a drastically simplified and fast all-optical protocol that clears and spatially swells kidney tissue samples sufficiently to allow for in situ 3D high-resolution analysis of renal anatomy and pathology using a conventional diffraction-limited microscope available in almost all life science and pathology institutions. Previous protocol simplifications have shown that the introduction of acrylamide polymers into paraformaldehyde-fixed samples can be totally omitted while still preserving tissue integrity on sodium dodecyl sulfate (SDS) clearance.11Xu N. Tamadon A. Liu Y. et al.Fast free-of-acrylamide clearing tissue (FACT)—an optimized new protocol for rapid, high-resolution imaging of three-dimensional brain tissue.Sci Rep. 2017; 7: 9895Crossref PubMed Scopus (26) Google Scholar Another study has shown that tissue samples can be expanded by more than a factor of 2 using a simple polymer-free protocol based on immersion in different aqueous solutions.12Murakami T.C. Mano T. Saikawa S. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (115) Google Scholar Thus, we removed acrylamide infusion from our more tedious and complex published protocols5Unnersjö-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 (49) Google Scholar,6Unnersjö-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 (26) Google Scholar and changed the sample embedding medium to allow for a mild (1.3-fold linear) swelling of kidney tissue samples. By also optimizing imaging parameters, we achieved a sufficiently high effective resolution for 3D visualization of podocyte FPs and the glomerular basement membrane (GBM) using conventional confocal microscopy. The simple clearing/swelling protocol is depicted schematically in Figure 1a. Note that the SDS delipidation step was added not primarily for optical transparency, but rather to ensure sufficient swelling of samples.12Murakami T.C. Mano T. Saikawa S. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (115) Google Scholar Furthermore, when omitting delipidation, the staining quality is poor (Supplementary Figure S1A). We show that rapid delipidation at 70 °C for 1 hour produced good results without the sample degradation occurring at higher temperatures (Supplementary Figure S1B). As reported by Murakami et al.,12Murakami T.C. Mano T. Saikawa S. et al.A three-dimensional single-cell-resolution whole-brain atlas using CUBIC-X expansion microscopy and tissue clearing.Nat Neurosci. 2018; 21: 625-637Crossref PubMed Scopus (115) Google Scholar urea and urea-like molecules can effectively swell tissue samples, resulting in increased effective spatial resolution. We thus added 4 M urea in a mixture with 80% (wt/wt) fructose (dubbed FRUIT),13Hou B. Zhang D. Zhao S. et al.Scalable and DiI-compatible optical clearance of the mammalian brain.Front Neuroanat. 2015; 9: 19Crossref PubMed Scopus (109) Google Scholar which resulted in a linear swelling factor of ∼1.3 (Figure 1b). To shorten the labeling time, we used dye-conjugated primary antibodies. Initially, when using dye-conjugated nephrin antibodies in a standard staining buffer (phosphate-buffered saline with Tween 20), the signal and labeling efficiency were not high enough to outline individual FPs. However, by using an alternative staining buffer, HEPES-TSC,14Susaki E.A. Shimizu C. Kuno A. et al.Versatile whole-organ/body staining and imaging based on electrolyte-gel properties of biological tissues.Nat Commun. 2020; 11: 1982Crossref PubMed Scopus (36) Google Scholar staining quality was increased to a satisfactory degree (Supplementary Figure S1C). Compared with previously published protocols using conventional microscopy, our fast and simple 5-hour protocol outperformed them on several essential parameters (Figure 1c–f). The protocol presented by Pullman et al.2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar is slightly faster overall but has the drawbacks of requiring super-resolution structured illumination microscopy microscopy and using thin cryosections, precluding access to large imaging depths. Of note, our protocol is less complex than standard paraffin-embedding or cryosectioning when all evaluated parameters are considered. Because our protocol gives only a moderate swelling factor of 1.3, confocal imaging parameters had to be optimized to achieve sufficient resolving power for imaging FPs. Mouse kidney samples were treated according to the protocol and stained for nephrin using different fluorophores. The resolution of a confocal microscope depends on both the pinhole size and the wavelength of the excitation/emission light.15Wilson T. Resolution and optical sectioning in the confocal microscope.J Microsc. 2011; 244: 113-121Crossref PubMed Scopus (122) Google Scholar Thus, we used a smaller pinhole diameter of 0.3 Airy units (AU) together with a green-emitting dye, Alexa Fluor 488, and in this way achieved sufficient resolution to resolve FPs in a wild-type (WT) mouse kidney (Figure 2a). Thus, whenever mouse FPs are to be visualized, fluorophores with a maximum excitation wavelength of 488 nm are used. Under these optimized imaging conditions, we were able to visualize whole mouse glomeruli in 3D at an effective resolution of ∼115 nm, good enough to resolve individual FPs (Figure 2b–e). Moreover, we show that an antibody penetration depth of at least 70 μm is achieved with fast labeling (Supplementary Figure S2A–C). Using a glycerol objective with a correction collar, this provides access to larger Z-depths than any of the previously developed protocols, with the exception of clearing/stimulated emission depletion protocol.5Unnersjö-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 (49) Google Scholar Because the axial resolution of a confocal microscope is roughly 3 times worse than the lateral resolution,15Wilson T. Resolution and optical sectioning in the confocal microscope.J Microsc. 2011; 244: 113-121Crossref PubMed Scopus (122) Google Scholar capillaries should be oriented in the x-y plane for optimally resolving FPs. We show that with our protocol, we can find plenty (up to 25) areas for imaging in each glomerulus (Supplementary Figure S2D), allowing us to gather large amounts of data to also detect subtle variations in filtration barrier structures.9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar This demonstrates the importance of having access to the depth dimension when performing high-throughput quantitative analyses of glomerular health. Importantly, we also validated the protocol for resolving FPs in human patient material (Figure 2f and g), which is less challenging compared with mouse, owing to the larger dimensions of human FPs.9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar Moreover, using wheat germ agglutinin (WGA) lectin with affinity toward the glycocalyx of FPs, we were also able to resolve FPs in a cross-sectional manner in both mouse and human samples (Figure 2h and i). In addition, we costained for nephrin, with the expected localization in the slit membrane, showing that we can get data on protein localization in addition to morphologic information. As reported previously,1Chozinski T.J. Mao C. Halpern A.R. et al.Volumetric, nanoscale optical imaging of mouse and human kidney via expansion microscopy.Sci Rep. 2018; 8: 10396Crossref PubMed Scopus (18) Google Scholar, 2Pullman J.M. Nylk J. Campbell E.C. et al.Visualization of podocyte substructure with structured illumination microscopy (SIM): a new approach to nephrotic disease.Biomed Opt Express. 2016; 7: 302-311Crossref PubMed Scopus (13) Google Scholar, 3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar,5Unnersjö-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 (49) Google Scholar,6Unnersjö-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 (26) Google Scholar,9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar,10Motrapu M. Świderska M.K. Mesas I. et al.Drug testing for residual progression of diabetic kidney disease in mice beyond therapy with metformin, ramipril, and empagliflozin.J Am Soc Nephrol. 2020; 31: 1729-1745Crossref PubMed Scopus (7) Google Scholar en face FP imaging of the slit diaphragm can be readily used to visualize and quantify FP effacement. We validate that this approach also can be applied with our new protocol using a recently published mouse model for focal segmental glomerulosclerosis (FSGS)9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar (Figure 3). By staining for nephrin, the slit diaphragm pattern could be visualized in 3 dimensions in whole glomeruli of WT and mutated mice (Figure 3a and b). As reported previously,3Siegerist F. Ribback S. Dombrowski F. et al.Structured illumination microscopy and automatized image processing as a rapid diagnostic tool for podocyte effacement.Sci Rep. 2017; 7: 11473Crossref PubMed Scopus (26) Google Scholar59Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar the slit diaphragm length per area can be quantified semiautomatically, and this analysis provides a result consistent with what has been published previously for this mouse line9Butt L. Unnersjö-Jess D. Höhne M. et al.A molecular mechanism explaining albuminuria in kidney disease.Nat Metab. 2020; 2: 461-474Crossref PubMed Scopus (18) Google Scholar (Figure 3c). Furthermore, glomerular capillaries were imaged in cross-section showing both effacement of FPs as well as alterations to the GBM (Figure 3d–f); see Supplementary Methods and Figure S3 for details of GBM measurements. To further validate our protocol for possible clinical use, we applied it to human tissue from patients diagnosed with different types of kidney disease. Two patients with congenital nephrotic syndrome, either with a homozygous mutation in the NPHS1 for or in the and for the and We also 2 patients diagnosed with and disease. We could visualize and quantify of kidney as FP effacement, as well as GBM alterations (Figure could be both en face (Figure and from the (Figure Moreover, we the expected of nephrin in slit of the patient with an NPHS1 the of glomerular a of the slit Am Soc Nephrol. PubMed Google Scholar (Figure and As for mouse samples (Figure imaging data could be evaluated (Figure and and Supplementary Figure and Of note, mild GBM was in the diagnosed patients compared with The kidney in Figure 4 was from an all the samples were from or Because GBM with (Supplementary Figure a more significant be compared with We also validated that swelling was by our protocol (Supplementary Figure and also that a different GBM results (Supplementary Figure and Because not only filtration barrier but also to renal are of high to both researchers and we the of using our protocol to morphology We show that using we could detect and in protein in both human samples (Supplementary Figure and mouse samples (Supplementary Figure To further the possible use of the protocol in patient samples, we we could detect pathologic features of of the We were able to visualize pathologic features in the from the focal and in glomerular capillaries compared with present standard methods (Figure and quantitative with microscopy results using both methods (Figure and and Supplementary Figure see Supplementary Methods for a of FP We also applied the protocol to a from a patient diagnosed with (Figure and were able to the visualization of many diagnostic were in the filtration barrier (Figure and and a analysis resulted in and our protocol (Figure and the of the kidney was evaluated with both standard (Figure and (Figure We could visualize the alterations lack of in the from the as of (Figure and glomerular capillaries (Figure and (Figure using an antibody both on the large (Figure and and in (Figure with a pattern to that on standard (Figure and FP imaging mild effacement (Figure and The pathologic features that we could detect with our protocol are in Supplementary we present a protocol for high-throughput visualization of renal structures on all length that is both faster and than previously published protocols for FP analysis and glomerular filtration barrier imaging in the kidney. We show that the resolution we achieve is high enough to in the filtration barrier using simple confocal microscopy, a standard tool available in research and pathology The 3D of the protocol is when performing imaging of FPs a large imaging is allowing for and of of the sample for quantitative of our study is that our protocol not only and compared with the standard protocols used for standard analysis of kidney could also these into protocol, with in of and However, as the analysis is based on antibodies to a large the of labeling is high per which is also for the protocols presented in this In the of the protocol, we use dye-conjugated primary antibodies that sufficient staining quality for resolving FPs. 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