BRIEF COMMUNICATION www.jasn.org Intravital and Kidney Slice Imaging of Podocyte Membrane Dynamics 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 im- ages 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 protru- sions (Figure 1D, Supplemental Movie 4). Simplification and retraction of larger processes in podocytes expressing CA-Rac1 were quantified by analyzing their perim- eter 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 in- dividual animals; P,0.001). The total size of the cells measured by the area in maxi- mum Z projections was unchanged (Figure 1F) (P=0.98), showing that CA-Rac1 pre- dominantly 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