Microperfusion of dissected tubules is an established technique that can provide information about cellular function within isolated nephron segments in the absence of confounding feedback variables. This information cannot be obtained using other approaches, including intravital microscopy. Recent advances in transduction approaches to express heterologous constructs as well as to efficiently knock down endogenous proteins in kidney and bladder have expanded the opportunity to use live cell approaches, including FRET, to investigate physiologic regulation of membrane trafficking and signaling pathways in real time in isolated tubules. This request is for funds to purchase a perfusion imaging system to enable high speed, high resolution imaging of microperfused kidney tubules and blood vessels as well as other tissues and cells. The system consists of a single photon Nikon A1R confocal microscope with a resonant scanner, spectral detector and FRET module, dual micromanipulators, and an environmental chamber. The system will be housed within the P30-funded George M. O'Brien Pittsburgh Center for Kidney Research at the University of Pittsburgh School of Medicine. The mandate of this center is to facilitate multidisciplinary research, trainin and information transfer related to kidney physiology, cell biology, pharmacology, and pathophysiology. The Center includes cores for Kidney Imaging, Cellular Physiology, Single Nephron and Metabolomics, and Model Organisms, and serves a large group of NIH-funded investigators at the University of Pittsburgh and at other institutions. Our center lacks a dedicated microscope for live imaging of perfused tubules, and the availability of the proposed system will significantly enhance the services we are able to provide to center users. Relevance- The proposed system will provide a crucial tool for the research of numerous biomedical investigators studying the cellular basis of disease. In particular, studies of isolated tubules and vessels conducted with this system are uniquely powerful for elucidating signal transduction processes associated with normal cell function and disease processes.
The proposed system will provide NIH-funded investigators the opportunity to study cellular functions in individually microdissected kidney tubules and other vessels in real time. We are requesting funds to purchase a live cell confocal microscope with the necessary requirements to enable us to perform these techniques. The microscope will be housed within a core facility of the Pittsburgh Center for Kidney Research and will significantly enhance the research capabilities of our large and expanding user base.
| Long, Kimberly R; Shipman, Katherine E; Rbaibi, Youssef et al. (2017) Proximal tubule apical endocytosis is modulated by fluid shear stress via an mTOR-dependent pathway. Mol Biol Cell 28:2508-2517 |
