Intravital and Kidney Slice Imaging of Podocyte Membrane Dynamics

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Sebastian Brähler,* Haiyang Yu,* Hani Suleiman,* Gokul M. Krishnan,* Brian T. Saunders,* † ‡ Jeffrey B. Kopp, Jeffrey H. Miner, Bernd H. Zinselmeyer,* and Andrey S. Shaw*

*Department of Pathology and Immunology and ‡Division of Nephrology, Washington University School of Medicine, St. Louis, Missouri; and †Kidney Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland

ABSTRACT In glomerular disease, podocyte injury results in a dramatic change in cell morphology minutes.10 Later, the same group, imaging known as foot process effacement. Remodeling of the actin cytoskeleton through the in time intervals of 24 hours, showed that activity of small GTPases was identified as a key mechanism in effacement, with podocytes populate Bowman’scapsulein increased membrane activity and motility in vitro. However, whether podocytes are the unilateral ureteral obstruction model stationary or actively moving cells in vivo remains debated. Using intravital and kidney of AKI, suggesting that podocytes move slice two–photon imaging of the three-dimensional structure of mouse podocytes, we after injury.8 Thus, the dynamics of podo- found that uninjured podocytes remained nonmotile and maintained a canopy-shaped cytes in their native state and how they re- structure over time. On expression of constitutively active Rac1, however, podocytes act after acute injury are still unanswered changed shape by retracting processes and clearly exhibited domains of increased questions. Here, we aimed to provide an membrane activity. Constitutive activation of Rac1 also led to podocyte detachment analysis of three–dimensional dynamic from the glomerular basement membrane, and we detected detached podocytes changes of the podocyte structure in health crawling on the surface of the tubular epithelium and occasionally, in contact with and disease in mice. peritubular capillaries. Podocyte membrane activity also increased in the inflammatory To analyze podocyte motility in vivo, environment of immune complex–mediated GN. Our results provide evidence that we used two-photon microscopy to image podocytes transition from a static to a dynamic state in vivo, shedding new light on kidneys in Confetti mice, in which podo- mechanisms in foot process effacement. cytes are randomly labeled with one of fl 11 J Am Soc Nephrol 27: 3285–3290, 2016. doi: 10.1681/ASN.2015121303 four possible uorophores (Confetti/ Podo:Cre). Glomerular and interstitial capillaries were labeled with a single

The kidney filtration barrier consists of were identified as the underlying mecha- in vitro 4–6 endothelial cells, the glomerular basement nisms . The relevance of these Received December 4, 2015. Accepted February membrane, and a highly specialized epi- findings on the stationary or dynamic na- 18, 2016. thelial cell, the podocyte, linked by the slit ture of podocytes in vivo is still unresolved. S.B. and H.Y. contributed equally to this work. diaphragm. When glomeruli are injured, Multiphoton microscopy has allowed podocytes undergo a dramatic remodel- groundbreaking insights into questions Published online ahead of print. Publication date available at www.jasn.org. ing of their actin cytoskeleton, resulting about cell mobility in many areas of bi- in a morphologic change known as foot ology,7,8 potentially allowing podocyte Present addresses: Dr. Haiyang Yu, Ludwig Institute for Cancer Research, La Jolla, California, and Dr. process effacement. The importance of membrane dynamics to be visualized. El- Andrey S. Shaw, Genentech, South San Francisco, the actin cytoskeleton is emphasized by egant imaging of zebrafish larvae showed California. the large number of actin-associated genes that podocytes are motile during the for- Correspondence: Dr. Bernd H. Zinselmeyer, De- that are mutated in hereditary forms of mation of the pronephros but stationary partment of Pathology and Immunology, Washington FSGS.1–3 Dysregulation of the small at later developmental stages over the University School of Medicine, 660 S. Euclid Avenue, GTPases, RhoA, Rac1, and Cdc42, critical course of several hours.9 Others, however, Campus Box 8118, St. Louis, MO 63110, or Dr. Andrey fl S. Shaw, Genentech, One DNA Way, Mail Stop 93b, regulators of the actin cytoskeleton, also using uorescent dextrans in the blood to South San Francisco, CA 94080. Email: bzinselmeyer@ leads to foot process effacement and pro- generate a negative image of the podocyte, path.wustl.edu or [email protected] teinuria, and remodeling of podocyte suggested that rat podocytes are motile, Copyright © 2016 by the American Society of structure, membrane activity, and motility changing position on the scale of Nephrology

J Am Soc Nephrol 27: 3285–3290, 2016 ISSN : 1046-6673/2711-3285 3285 BRIEF COMMUNICATION www.jasn.org intravenous injection of fluorescently imaging of podocytes is limited to mice several hours.12,13 To minimize cutting labeled tomato lectin, and kidneys were of 3–5 weeks of age, because glomeruli artifacts, only glomeruli with an intact exteriorized. Three-dimensional recon- move away from the capsule as mice age. Bowman’s capsule were imaged. The val- structions were made from successive im- This also limits the usefulness of this ap- idity of this approach was first confirmed ages acquired in the Z plane (Z stack), proach in disease models where it is nec- in Confetti mice. We observed the same which allowed us to visualize the complex essary to image older animals. Induction elaborate podocyte morphology as seen in three–dimensional structure of major po- of CA-Rac1 at 2–3 weeks of age resulted the intravital images obtained from intact docyte processes under healthy conditions in a considerable change in cell morphol- kidneys (Figure 1C, Supplemental Movie in vivo. The resulting images are remark- ogy with retracted or lost processes in vivo 3), and this structure was maintained for able in how strongly they resemble the (Figure 1B, Supplemental Movie 2). at least 3 hours. An analysis of cell viability canopy morphology of podocytes visual- To determine whether the same using propidium iodide (PI) and Hoechst ized by scanning electron microscopy changes would occur in older animals, 33342 costaining showed a marginal in- (Figure 1A, Supplemental Movie 1). we used a vibratome to cut 1-mm-thick crease of dead cells (PI positive) but only Previously, we showed that inducible sections from freshly isolated kidneys that after 90 minutes of incubation (13.266 expression of a constitutively active form were placed in a pressurized organ bath 0.53% at 0 minutes versus 19.1461.50% of Rac1 (CA-Rac1) specifically in podo- that provides a nutrient environment to dead cells at 90 minutes) (Supplemental cytes (CA-Rac1/NEFTA) results in acute prolong cell viability. This method is used Figure 1, B and C), comparable with a pre- foot process effacement and proteinuria routinely for live two–photon imaging of vious study.14 Because imaging depth was (Supplemental Figure 1A).5 Intravital tissue, because viability is preserved over shallower, the structural detail using vibratome slices was superior to that of images obtained from the intravital prepara- tions. Both imaging techniques, however, only allowed for analysis of primary and larger secondary processes. Slice imaging of glomeruli from the CA-Rac1/NEFTA mice revealed retracted primary processes and lamellipodia–like membrane protrusions (Figure 1D, Supplemental Movie 4). Simplification and retraction of larger processes in podocytes expressing CA-Rac1 were quantified by analyzing their perimeter in maximum Z projections (maximum intensity projections). The cell perimeter was significantly reduced in comparison with control Confetti–labeled podocytes (Figure 1E, Supplemental Figure 1D) (52.3364.0 mm; n=36 cells versus 101.86 5.7 mm; n=37 cells from three or more individual animals; P,0.001). The total size of the cells measured by the area in maximum Z projections was unchanged (Figure 1F) (P=0.98), showing that CA-Rac1 predominantly reduces the complexity of larger processes but not the cell size. Figure 1. CA-Rac1 expression leads to morphologic changes in podocyte structure in vivo. Toquantify podocyte movement invivo, Three-dimensional reconstructions of Z stacks from intravitally imaged glomeruli in (A) we acquired Z stacks in 90-second time Confetti/Podo:Cre mice and (B) CA-Rac1/NEFTA mice after 4 days of doxycycline treat- intervals over $30 minutes. The processes – ment. (C and D) Three-dimensional reconstructions of podocytes in vibratome cut kidney of the Confetti-labeled podocytes were re- slices in an organ bath from (C) Confetti/Podo:Cre mice and (D) CA-Rac1/NEFTA mice after markably stable, exhibiting only minor 4 days of doxycycline treatment. In (A–D) glomerular capillaries were highlighted via a passive movements caused by the heart single injection of DyLight 594-Labeled Tomato Lectin. Podocytes are visible in yellow (YFP), blue (CFP), red (RFP), green (GFP) in Confetti/Podo:Cre mice or green (GFP) in CA- beat (Figure 2A, Supplemental Movie 5). Rac1/NEFTA mice. Quantification of (E) podocyte perimeter and (F) area in flattened In contrast, we could easily detect active Z stacks from Confetti/Podo:Cre and CA-Rac1/NEFTA mice (n=36 podocytes versus n=37 membrane protrusions in CA-Rac1– podocytes, respectively, from at least three individual animals imaged intravitally or in expressing podocytes (Figure 2B, Supple- vibratome-cut kidney slices). ***P#0.001. mental Movie 6). To quantify membrane

3286 Journal of the American Society of Nephrology J Am Soc Nephrol 27: 3285–3290, 2016 www.jasn.org BRIEF COMMUNICATION activity, we developed a computational membrane dynamics in the CA-Rac1 in vivo clearly resembled protruding and method. Maximum intensity projections podocytes. retracting lamellipodia (Figure 2D, Sup- of each Z stack were generated by overlay- We also imaged podocytes at a higher plemental Movie 8). ing all of the Z-stack images for each time time resolution of 30-second intervals in Previously, we showed that the induc- point, and then, each stack was compared kidney slices using a resonant scanner. tion of overactive Rac1 results in podocyte with each other. Movement of cells or Using this technique, the stability of shedding detected by Western blot in the membranes resulted in changes in pixel podocyte processes under healthy, non- urine.5 After doxycycline induction, here, intensity over time, which was absent in proteinuric conditions was again evident we could visualize podocyte shedding sessile cells. Our method plotted these inConfetti mice (Figure 2C, Supplemental from glomeruli and individual podocytes changes in pixel intensity over 15 minutes Movie 7), whereas CA-Rac1 induction passing through kidney tubules in and identified areas of change on a color– caused localized and rapid changes in po- live CA-Rac1/NEFTA mice (Figure 3A, coded heat map. This approach clearly docyte membrane shape. At this resolu- Supplemental Movie 9). Podocytes were documented domains of increased tion, the changes in membrane dynamics also observed as attached to the tubular epithelium and crawling on the epithelial surface (Figure 3B, Supplemental Movie 9). Occasionally, detached podocytes could be observed to integrate into the tubular epithelium and make contact with interstitial capillaries, a phenomenon that was never observed in healthy Con- fetti mice (Figure 3C, Supplemental Movie 9). To confirm that higher membrane activity is not unique to the podocytes expressing CA-Rac1, we imaged podocytes in Confetti mice after administra- tion of nephrotoxic serum (NTS), an established model of immune–mediated podocyte injury (Supplemental Figure 2). Although podocytes did not show the same degree of morphologic changes that was seen in CA-Rac1-expressing podocytes, nephrotoxic serum clearly induced increased membrane activity (Fig- ure 4, A and B, Supplemental Movie 10). The significance of these findings was established by choosing representative heat maps (25325 mm) from three or more individual mice per group using the method described above and plotting y Figure 2. Podocytes are stable cells in vivo, and Rac1 overactivity increases podocyte the intensity change of the pixels on the membrane dynamics. Shown in each row are time course images representing three- axis and the percentage of pixels with a dimensional reconstructions of Z stacks acquired at 0, 3, and 6 minutes; total imaging specific intensity change on the x axis. periods were up to 49.5 minutes. (A) Intravitally imaged glomeruli in Confetti/Podo:Cre The results showed significantly higher mice show stable podocyte processes. Membrane movement was visualized by using a changes in pixel intensity in CA-Rac1/ heat map (column 4) depicting the pixel intensity change in a flattened Z stack over NEFTA podocytes in comparison with 15 minutes. Because the two cells depicted expressed different fluorophores, the analysis control mice (84.5 versus 47 arbitrary for each channel was done separately, and the pictures were combined (dashed line). units; P,0.001) (Figure 4C). Confetti (B) Under the same conditions, CA-Rac1-expressing podocytes showed increased mem- mice treated with nephrotoxic serum injec- brane ruffling, indicated by a higher intensity in the heat map along the cell borders. (C and tion also showed a significant difference D) Imaging of kidney slices in an incubation chamber allowed for more detailed visualization P, of the stable three–dimensional structure of major processes in (C) Confetti/Podo:Cre (58 arbitrary units; 0.01) in comparison podocytes, whereas (D) CA-Rac1/NEFTA podocytes showed rapid rearrangement of short, with the controls. Visible membrane dy- lamellipodia–like protrusions. Please note that changes in the focal plane caused by namics were detected in 16.8% of NTS– heartbeat or tissue drift can create small artifactual movements that are detected as a treated Confetti/Podo:Cre podocytes and background pixel intensity change as seen in the heat maps in A and C. 42.3% of Dox–induced CA-Rac1/NEFTA

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kidney was exteriorized. Animals were kept in a 37°C chamber and closely monitored. Ketamine/xylazine (50% of the initial dose) injections were repeated every 30 minutes. For kidney slice imaging, freshly isolated kidneys were cut into 1-mm-thick sections using a vibratome (Leica Microsystems,

Buffalo Grove, IL) in CO2-independent medium and then, immediately imaged in an incubation chamber (37°C RPMI medium; 95% O and 5% CO ;Gibco,GrandIsland,NY). Figure 3. Podocytes expressing CA-Rac1 are shed and can be detected within tubules. 2 2 Only glomeruli with an intact Bowman’scap- (A) Intravital imaging of a glomerulus in a CA-Rac1/NEFTA mouse after 4 days of doxycycline treatment reveals an GFP-positive podocyte (green) within a tubule (arrow). (B) Intravital sule that were surrounded by at least one layer imaging of a CA-Rac1–expressing podocyte actively migrating within a tubule and ex- of tubular epithelium were imaged to mini- tending lamellipodia (arrows). (C) Intravital three–dimensional reconstruction of a podocyte mize the possibility of cutting artifacts. that has integrated into the tubular epithelium. Whereas much of the cell body remained in the tubular lumen (T), the podocyte (P; green) contacted the DyLight 594-Labeled Tomato Two-Photon Microscopy Lectin intertubular blood vessels (V; red), which is highlighted by arrows. Images were collected using a customized Leica SP8 Two-Photon Microscope (Leica Microsys- podocytes. Importantly, no significant dif- epithelium and an occasional podocyte tems) equipped with a 253 and 0.95 numerical ferences were detected between podocytes that appeared to have integrated into the aperture water–immersion objective and a Mai expressing CA-Rac1 or after nephrotoxic tubular epithelium. The physiologic signif- Tai HP DeepSee Laser (Spectra-Physics) tuned injury. These results suggest that an in- icance of this observation is not clear. The to 895 nm. Fluorescence emission was guided crease in podocyte membrane dynamics combination of two-photon microscopy directly to supersensitive external hybrid photo- is a general feature of podocyte injury. with the new technical and analytic meth- detectors (Leica/Hammamatsu). For signal sep- Here,weshow,usingthree–dimensional, ods described here should provide new aration, we used the following dichroic beam two–photon imaging, that podocytes are insights into the molecular basis of splitters without bandpass filters (Semrock): stationary cells that maintain the structure proteinuric kidney diseases. 484-nm edge BrightLine (FF484-FDi01), 526- of their processes over time. Differences be- nm edge BrightLine (FF526-Di01), and 562- tween our work and previous studies could nm edge BrightLine (FF562-Di03). The mirrors be because of the use of rats versus mice or CONCISE METHODS were arranged as follows: fluorescence light was the use of single–plane dye exclusion10 ver- first split by the 526-nm filter; light with a wave- sus the direct imaging of fluorescently la- Animal Studies length .526 nm was then separated by the beled podocytes. Rac1 activation changed Animal studies were approved by the Washing- 562-nm beam splitter, whereas light with a podocyte morphology from a stable, ex- ton University Animal Studies Committee. CA- wavelength ,526 nm was further separated tended structure to a dynamic state with Rac1/Nphs1-rtTA (CA-Rac1/NEFTA) mice by the 484-nm beam splitter. These settings blunted motile processes. This suggests were generated as described previously.5 Expres- generated four channels: approximately that increased membrane dynamics may sion of CA-Rac1 was induced by oral adminis- 390–484 nm (color coded as blue), approxi- be a general feature of proteinuric kidney tration of doxycycline over 4 days. Proteinuria mately 484–526 nm (color coded as green), disease and that foot process effacement re- was verifiedbyCoomassiebluestaining approximately 526–562 nm (color coded as flects this change in membrane dynamics. of urine samples in an SDS-PAGE gel. yellow), and approximately 562–680 nm It is now clear that loss of podocytes is a Confetti/Podo:Cre mice were created by mat- (color coded as red). On the basis of the per- constitutive process in the normal kidney ing the R26R.Confetti mice16 with Nphs2:Cre centage of emission in these channels, we that is accelerated after podocyte injury.15 mice.17 Nephrotoxic nephritis was induced were able to visualize CFP, YFP, RFP, GFP, In the CA-Rac1 model, we could detect de- by a single body weight–adapted intravenous DyLight 594, and the second harmonic tached podocytes passing through the tu- injection of the nephrotoxic serum. Nephro- signal generated predominantly by collagen bules, suggesting that changes in podocyte toxic serum was produced as described previ- fibers. morphology result in podocyte detach- ously.18 Animals were imaged at day 3 of ment. Although not as significant after nephrotoxic nephritis. Tissue Viability nephrotoxic nephritis, podocyte detach- For intravital imaging, animals were an- To analyze tissue viability in kidney sections, ment was still detectable. Peti-Peterdi and esthetized (ketamine at 0.1 mg/g body wt and we used PI (Thermo Fisher Scientific, Vernon coworkers8 reported earlier that damaged xylazine at 0.02 mg/g body wt intraperitone- Hills, IL) and Hoechst 33342 (Thermo Fisher podocytes can extend into the proximal ally) and injected once intravenously with Scientific) costaining to analyze cells with a tubulus lumen. Remarkably, we de- DyLight 594-Coupled Tomato Lectin (Vector compromised cell membrane (PI positive) in tected podocytes crawling on the tubular Laboratories, Burlingame, CA), and the left comparison with all cells (Hoechst positive).

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nuclei. Results were compared using a one- way ANOVA with a Tukey post-test.

Measurement of Cell Perimeter and Volume in Maximum Intensity Projections To statistically evaluate our observation that CA-Rac1 podocytes lose their elaborate net- work of primary processes, we created a maximum intensity projection of our Z stack using the Imaris software (Bitplane). The resulting pictures were exported to ImageJ (the National Institutes of Health, Bethesda, MD), and the perimeter was manually drawn around a ﬂuorescently labeled podocyte. We then analyzed the resulting perimeter and the enclosed area and compared the values using a t test.

Generation of Heat Maps and Quantification of Movement Maximum intensity projections of each Z stack from representative glomerular areas (25325 mm from at least three animals per group) were created. Images were calibrated to 10 pixels per micrometer, and the average intensity for each pixel over time was calculated. The difference from the first frame was calculated, and the collective results were depicted as a color-coded map. The average values 6SEM (from 12 repre- Figure 4. Increased membrane dynamics are a feature of injured podocytes in an in- sentative areas from three individual experi- flammatory environment. (A) Time course of three-dimensional reconstructions of Z stacks ments) were plotted in a histogram, and a – in a vibratome cut kidney slice from a healthy Confetti/Podo:Cre mouse. (B) Time course of sigmoidal dose-response curve was fitted to Z – three-dimensional reconstructions of stacks in a vibratome cut kidney slice from a the data using the standard four–parameter Confetti/Podo:Cre littermate at day 3 after injection of nephrotoxic serum. Note the logistic equation. Significance was determined membrane movement in the RFP-expressing cell (orange; circle). (C) Quantification of pixel by calculating the half-maximum percentage intensity changes over 15 minutes in vibratome–cut kidney slices. Pixel intensity changes in fi representative glomerular areas of 25325 mm (12 representative areas from at least three in a histogram for each quanti ed area and x individual animals per group) were quantified. Shown are the average maximum intensity then, determining the values on the axis changes on the y axis and the percentage of pixels on the x axis. A sigmoidal curve was (pixel intensity) and the y axis (percentage of fitted to the data, and half-maximum percentages were determined. For analysis of sta- pixels) at half-maximum percentage. The de- tistical significance, values on the x and y axes at the half-maximum from each area were termined values for the three groups were an- compared separately between the three groups. CA-Rac1/NEFTA and nephrotoxic alyzed for significance using a one-way serum–injected Confetti/Podo:Cre mice showed significantly increased movement versus ANOVA with a Tukey post hoc test (x and y P, P, controls. ** 0.01; *** 0.001. values were analyzed separately).

To label capillaries, we injected 80 ml DyLight washed twice brieﬂyinPBSafterstaining 488-Coupled Tomato Lectin (Vector Labora- and then, imaged immediately on an Olym- ACKNOWLEDGMENTS tories) intravenously before harvesting tis- pus IX80 Confocal Microscope (Olympus, sue. Kidney slices were immersed in the Tokyo, Japan). Three independent experi- We thank Christine Stander and Lacy LaFata staining solution (5 mg/ml Hoechst 33342 ments were performed. For quantiﬁcation, for excellent technical support. and 1 mg/ml PI in PBS for 15 minutes at we chose at least 11 representative images This work was supported by the Deutsche room temperature) immediately after vibra- (approximately 3003300 mm each), counted Forschungsgemeinschaft scholarship BR4917/1- tome cutting and after 45- and 90-minute PI- and Hoechst 33342–positive nuclei, and 1 (to S.B.), the National Institutes of Health grants incubations in the organ bath. Slices were calculated the percentage of PI-positive R01DK078314 (to J.H.M.) and R01DK058366

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(to A.S.S.), and the Howard Hughes Medical 6. Blattner SM, Hodgin JB, Nishio M, Wylie SA, cell motility paralysis. JExpMed210: 757– fi Institute (A.S.S.). Saha J, Soo AA, Vining C, Randolph A, 774, 2013 Herbach N, Wanke R, Atkins KB, Gyung Kang 13. Zinselmeyer BH, Dempster J, Wokosin DL, H, Henger A, Brakebusch C, Holzman LB, Cannon JJ, Pless R, Parker I, Miller MJ: Kretzler M: Divergent functions of the Rho Chapter 16. Two-photon microscopy and DISCLOSURES GTPasesRac1andCdc42inpodocyteinjury. multidimensional analysis of cell dynamics. Kidney Int – Methods Enzymol – A.S.S. is an employee of Genentech (South San 84: 920 930, 2013 461: 349 378, 2009 7. Burford JL, Villanueva K, Lam L, Riquier- 14. Crawford C, Kennedy-Lydon T, Sprott C, Francisco, CA). Brison A, Hackl MJ, Pippin J, Shankland SJ, Desai T, Sawbridge L, Munday J, Unwin RJ, Peti-Peterdi J: Intravital imaging of podocyte Wildman SSP, Peppiatt-Wildman CM: An calcium in glomerular injury and disease. J intact kidney slice model to investigate vasa REFERENCES Clin Invest 124: 2050–2058, 2014 recta properties and function in situ. Neph- 8. Hackl MJ, Burford JL, Villanueva K, Lam L, ron, Physiol 120: 17–31, 2012 1. Kaplan JM, Kim SH, North KN, Rennke H, Correia Suszták K, Schermer B, Benzing T, Peti- 15. Vogelmann SU, Nelson WJ, Myers BD, LA, Tong HQ, Mathis BJ, Rodríguez-Pérez JC, Peterdi J: Tracking the fate of glomerular Lemley KV: Urinary excretion of viable Allen PG, Beggs AH, Pollak MR: Mutations in epithelial cells in vivo using serial multipho- podocytes in health and renal disease. Am ACTN4, encoding alpha-actinin-4, cause familial ton imaging in new mouse models with J Physiol Renal Physiol 285: F40–F48, focal segmental glomerulosclerosis. Nat Genet fluorescent lineage tags. Nat Med 19: 1661– 2003 24: 251–256, 2000 1666, 2013 16. Livet J, Weissman TA, Kang H, Draft RW, Lu J, 2. Brown EJ, Schlöndorff JS, Becker DJ, 9. Endlich N, Simon O, Göpferich A, Wegner H, Bennis RA, Sanes JR, Lichtman JW: Trans- Tsukaguchi H, Tonna SJ, Uscinski AL, Higgs Moeller MJ, Rumpel E, Kotb AM, Endlich K: genic strategies for combinatorial expression HN, Henderson JM, Pollak MR: Mutations in Two-photon microscopy reveals stationary of fluorescent proteins in the nervous system. theformingeneINF2causefocalsegmental podocytes in living zebrafish larvae. JAmSoc Nature 450: 56–62, 2007 glomerulosclerosis. Nat Genet 42: 72–76, 2010 Nephrol 25: 681–686, 2014 17. Moeller MJ, Sanden SK, Soofi A, Wiggins RC, 3. Kim JM, Wu H, Green G, Winkler CA, Kopp JB, 10. Peti-Peterdi J, Sipos A: A high-powered view Holzman LB: Podocyte-specific expression of Miner JH, Unanue ER, Shaw AS: CD2-associated of the filtration barrier. JAmSocNephrol21: cre recombinase in transgenic mice. Genesis protein haploinsufficiency is linked to glomeru- 1835–1841, 2010 35: 39–42, 2003 lar disease susceptibility. Science 300: 1298– 11. Snippert HJ, van der Flier LG, Sato T, van Es 18. YoY,BraunMC,BarisoniL,MobarakiH,LuH, 1300, 2003 JH, van den Born M, Kroon-Veenboer C, Shrivastav S, Owens J, Kopp JB: Anti-mouse 4. Zhu L, Jiang R, Aoudjit L, Jones N, Takano T: Barker N, Klein AM, van Rheenen J, Simons mesangial cell serum induces acute glomer- Activation of RhoA in podocytes induces fo- BD, Clevers H: Intestinal crypt homeostasis ulonephropathy in mice. Nephron Exp Nephrol cal segmental glomerulosclerosis. JAmSoc results from neutral competition between 93: e92–e106, 2003 Nephrol 22: 1621–1630, 2011 symmetrically dividing Lgr5 stem cells. Cell 5. Yu H, Suleiman H, Kim AHJ, Miner JH, Dani 143: 134–144, 2010 A, Shaw AS, Akilesh S: Rac1 activation in 12. Zinselmeyer BH, Heydari S, Sacristán C, podocytes induces rapid foot process ef- Nayak D, Cammer M, Herz J, Cheng X, Davis This article contains supplemental material online facement and proteinuria. MolCellBiol33: SJ, Dustin ML, McGavern DB: PD-1 promotes at http://jasn.asnjournals.org/lookup/suppl/doi:10. 4755–4764, 2013 immune exhaustion by inducing antiviral T 1681/ASN.2015121303/-/DCSupplemental.

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Supplementary Figure 1: (A) Coomassie blue staining of urine samples assayed by SDS-PAGE shows proteinuria in CA-Rac1/NEFTA animals after 4 days of doxycycline treatment, whereas untreated mice show no proteinuria. (B) Cell viability staining: Kidney slices were incubated in the organ bath for the indicated times and then stained with Propidium iodide (magenta; to stain the nuclei of cells with a compromised cell membrane) and Hoechst 33342 (blue; to stain all nuclei). DyLight 488-coupled tomato lectin (green) was injected i.v. before the experiment to mark blood vessels. (C) Quantification of cell viability: Representative images (300 x 300 µm each) were taken from three individual experiments, and the percentage of PI-positive cells was calculated. Each dot in the scatter-dot plot represents one quantified image; also depicted are mean and SEM. (D) Images of maximum intensity projections (MIP) of a CA-Rac1/NEFTA podocyte after 4 days of doxycycline treatment (right) and a Confetti/Podo:Cre podocyte (left) illustrate how perimeter and area were determined.

Supplementary Figure 2: Coomassie blue staining shows albuminuria in Confetti/Podo:Cre mice 3 days after a single injection of nephrotoxic antibody.

Supplementary Movies: Movie S1: Intravital Z-stack fly-through followed by a 3-D reconstruction of a glomerulus in a Confetti/Podo:Cre mouse. DyLight 594-labeled tomato lectin was injected once before the imaging i.v. to label capillaries red. A blood cell labeled with tomato lectin is seen in the glomerular capillaries. Two labeled podocytes (cytoplasmic YFP, yellow, and membrane-tagged CFP, blue) are visible. Note the three-dimensional structural details that are imparted by reconstruction of a complete Z-stack, showing the elaborate structure of the podocytes attached to the capillaries.

Movie S2: Intravital Z-stack fly-through followed by a 3-D reconstruction of a glomerulus in a CA- Rac1/NEFTA mouse. DyLight 594-labeled tomato lectin was injected i.v. before the imaging to label capillaries red. eGFP.CA-Rac1-expressing podocytes are depicted in green. Note the absence of the elaborate podocyte processes in comparison with the Confetti/Podo:Cre mouse (Movie S1).

Movie S3: Z-stack fly-through of a vibratome-cut Confetti/Podo:Cre mouse kidney slice in an organ bath, followed by 3-D reconstruction of a glomerulus. DyLight 594-labeled tomato lectin was injected i.v. before the imaging to label capillaries red. Podocytes are labeled in orange (cytoplasmic tomato), light-blue (membrane-tagged CFP), yellow (cytoplasmic YFP), or green (nuclear-tagged GFP). Collagen fibers in Bowman’s capsule are depicted in blue. The 3-D reconstruction shows higher detail in comparison to the intravital imaging. The glomerulus can be completely imaged in the Z-axis without losing quality in the deepest images.

Movie S4: Z-stack fly-through of a vibratome-cut CA-Rac1/NEFTA mouse kidney slice in an organ bath, followed by a 3-D reconstruction of a glomerulus. DyLight 594-labeled tomato lectin was injected i.v. before the imaging to label capillaries red. Podocytes expressing eGFP.CA-Rac1 are depicted in green. Collagen fibers in Bowman’s capsule and the interstitium are depicted in blue (second harmonic signal). CA-Rac1 overexpression dramatically altered podocyte cell shape, with blunted, lamellipodia-like processes.

Movie S5: Intravital time-lapse (interval = 90s) 3-D reconstruction of a glomerulus in a Confetti/Podo:Cre mouse over 36 minutes. Podocytes maintained their structure, with very stable major processes. Capillaries were visualized via a single injection of DyLight 594-coupled tomato lectin; blood cells that have taken up the lectin were observed moving inside the capillaries, indicating intact perfusion.

Movie S6: Intravital time-lapse (interval = 90s) 3-D reconstruction of a glomerulus in a CA- Rac1/NEFTA mouse over 49.5 minutes. Podocytes have lost their typical architecture and show rapid movements and extensions at the periphery.

Movie S7: Time-lapse (interval = 30s) 3-D reconstruction of a vibratome-cut kidney slice from a healthy Confetti/Podo:Cre mouse over 25 minutes. Using this technique podocyte structure can be imaged in greater detail and in the whole glomerulus without loss of quality at deeper levels over time. To minimize cutting artifacts, only glomeruli with an intact Bowman’s capsule were imaged. Podocytes maintained their elaborate 3-D structure in these organ slices over time. Please note that changes in the focal plane due to heartbeat or tissue drift can create small artifactual movements.

Movie S8: Time-lapse (interval = 30s) 3-D reconstruction of a vibratome-cut kidney slice from a CA- Rac1/NEFTA mouse over 16 minutes revealed multiple areas of rapid membrane changes. These could be observed by intravital imaging (Movie S6), but only the kidney- slice imaging showed the lamellipodia-like structure of the rapidly moving membrane domains.

Movie S9: A three part video showing the fate of displaced podocytes in a living mouse. (A) Video of a glomerulus from a CA-Rac1/NEFTA mouse showing a shed podocyte (arrow) within a tubule. (B) Intravital imaging in a CA-Rac1/NEFTA mouse shows podocytes actively migrating within a tubule by extending lamellipodia-like protrusions (arrowhead). (C) Intravital 3-D reconstruction of a podocyte that is integrated in the tubular epithelium. Note that the podocyte appears to send out processes that reach the intertubular capillaries, labeled with DyLight 594-coupled tomato lectin.

Movie S10: Time-lapse (interval = 30s) 3-D reconstruction of a vibratome-cut kidney slice from a Confetti/Podo:Cre mouse 3 days after injection of nephrotoxic antibody over 29 minutes . A tomato expressing podocyte (orange) shows rapid rearrangements of its cell membrane over time (circle).