Podocytes are critical in the maintenance of a healthy glomerular filter, however they have been difficult to study in the intact kidney due to technical limitations. Here we report the development of serial multiphoton microscopy (MPM) of the same glomeruli over several days to visualize the motility of podocytes and parietal epithelial cells (PEC) in vivo. In Podocin-GFP mice podocytes formed sporadic multi-cellular clusters after unilateral ureteral ligation (UUO) and migrated into the parietal Bowman’s capsule. The tracking of single cells in Podocin-confetti mice featuring cell-specific expression of CFP, GFP, YFP, or RFP revealed the simultaneous migration of multiple podocytes. In PEPCK-GFP mice serial MPM found PEC-to-podocyte migration and nanotubule connections. Our data support the highly dynamic rather than static nature of the glomerular environment and cellular composition. Future application of this new approach promises to advance our understanding of the mechanisms of glomerular injury and regeneration.

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Transplantation is invariably associated with ischemia-reperfusion injury (IRI), inflammation and rejection. Resultant cell death has morphological features of necrosis but programmed cell death has been synonymous with apoptosis until pathways of regulated necrosis (RN) have been described. The best-studied RN pathway, necroptosis, is triggered by perturbation of caspase-8-mediated apoptosis and depends on receptor-interacting protein kinases 1 and 3 (RIPK1/ RIPK3) as well as mixed linage kinase domain like to form the necroptosome. The release of cytosolic content and cell death-associated molecular patterns (CDAMPs) can trigger innate and promote adaptive immune responses. Thus, the form of cell death can substantially influence alloimmunity and graft survival. Necroptosis is a key element of IRI, and RIPK1 interference by RN-specific inhibitors such as necrostatin-1 protects from IRI in kidney, heart and brain. Necroptosis may be a general mechanism in response to other forms of inflammatory organ injury, and will likely emerge as a promising target in solid organ transplantation. As second-generation RIPK1 and RIPK3 inhibitors become available, clinical trials for the prevention of delayed graft function and attenuation of allograft rejection-mediated injury will emerge. These efforts will accelerate upon further identification of critical necroptosis-triggering receptor(s).

signaling is a robust and key pathogenic mechanism in podocyte injury. This in vivo imaging approach will allow future detailed investigation of the molecular and cellular mechanisms of glomerular disease in the intact living kidney.

Peti-Peterdi J, Burford JL, Hackl MJ. The first decade of using multiphoton microscopy for high-power kidney imaging. Am J Physiol Renal Physiol 302: F227-F233, 2012. First published October 26, 2011 doi:10.1152/ajprenal.00561.2011In this review, we highlight the major scientific breakthroughs in kidney research achieved using multiphoton microscopy (MPM) and summarize the milestones in the technological development of kidney MPM during the past 10 years. Since more and more renal laboratories invest in MPM worldwide, we discuss future directions and provide practical, useful tips and examples for the application of this still-emerging optical sectioning technology. Advantages of using MPM in various kidney preparations that range from freshly dissected individual glomeruli or the whole kidney in vitro to MPM of the intact mouse and rat kidney in vivo are reviewed. Potential combinations of MPM with micromanipulation techniques including microperfusion and micropuncture are also included. However, we emphasize the most advanced and complex, quantitative in vivo imaging applications as the ultimate use of MPM since the true mandate of this technology is to look inside intact organs in live animals and humans. intravital imaging; juxtaglomerular apparatus; podocyte; two-photon microscopy IT HAS BEEN MORE THAN 10 YEARS since two-or three-photon excitation, collectively called multiphoton microscopy (MPM), was first applied to the study of the living kidney tissue by the Peti-Peterdi-Bell (37, 38) and Dunn-Molitoris groups (6, 42) that pioneered several applications of this powerful new imaging technology. Over the past decade, MPM has provided stunning images and real-time movies of the structure and function of the intact kidney in unparalleled spatial and temporal resolution. Because of the ability of this noninvasive imaging approach to directly visualize dynamic intrarenal processes in vivo and in near real-time without causing tissue damage, MPM has revolutionized renal physiology research and served as the perfect complement to more traditional biological and histological techniques. MPM also opened the door to studying otherwise inaccessible cell types and complex tissue structures like the glomerular podocyte (30, 39) and the juxtaglomerular apparatus (JGA) (37) in their intact environment. Applications of MPM helped to change several paradigms in renal (patho)physiology. Imaging data have been incorporated into a number of textbooks on kidney function (3,8,41), and MPM videos (30,37,39,40) have been used as visual aid and teaching material in graduate and medical student classrooms worldwide.MPM offers a state-of-the-art imaging technique superior for deep optical sectioning of living tissues. The higher resolution and minimal phototoxicity of this method permit longer time periods of continuous tissue scanning with uses in near realtime imaging of intact organs in vivo. MPM has applications far beyond the generation of superior images: dynamic intracellular processes as well as more complex physiological ...

Mutations in proteins localized to cilia and basal bodies have been implicated in a growing number of human diseases. Access of these proteins to the ciliary compartment requires targeting to the base of the cilia. However, the mechanisms involved in transport of cilia proteins to this transitional zone are elusive. Here we show that nephrocystin, a ciliary protein mutated in the most prevalent form of cystic kidney disease in childhood, is expressed in respiratory epithelial cells and accumulates at the base of cilia, overlapping with markers of the basal body area and the transition zone. Nephrocystin interacts with the phosphofurin acidic cluster sorting protein (PACS)-1. Casein kinase 2 (CK2)-mediated phosphorylation of three critical serine residues within a cluster of acidic amino acids in nephrocystin mediates PACS-1 binding, and is essential for colocalization of nephrocystin with PACS-1 at the base of cilia. Inhibition of CK2 activity abrogates this interaction and results in the loss of correct nephrocystin targeting. These data suggest that CK2-dependent transport processes represent a novel pathway of targeting proteins to the cilia.

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