Date of Award

4-1-2008

Degree Type

Restricted Access Dissertation

Degree Name

Doctor of Philosophy

Field of Study

Integrative Physiology

Department

Graduate School of Biomedical Sciences

First Advisor

Rong Ma

Second Advisor

Joan Carroll

Third Advisor

Glenn Dillon

Abstract

Sours-Brothers, Sherry. Store-operated Calcium Entry in Glomerular Mesangial Cells. Doctor of Philosophy (Integrative Physiology, Molecular Cardiovascular Science Track), April, 2008, 181 pp; 1 table, 25 figures, bibliography, 241 titles. Mesangial cells (MCs) are found within the glomerulus, where they contribute to the regulation of glomerular filtration (GFR). Their contractile function is similar to that of vascular smooth muscle cells, regulated by a number of different Ca2+ release and entry mechanisms in response to vasoactive substances. Among these are store-operated channels (SOC), which have been identified in MC, but whose molecular components are unknown. Deficiency of store-operated Ca2+ entry (SOCE) has also been associated with loss of MC contractile function found during early diabetic renal hyperfiltration. For these reasons, it is imperative to clarify the mechanisms underlying SOCE in MCs. Members of the canonical transient receptor potential (TRCP) family of proteins have been identified as candidates for SOC function in a number of cell types. The distribution of TRPC subtypes, and their combination to form heterotetrameric channels is cell-type specific, possibly allowing for variation SOCE mechanisms in different cells. Recently, the endoplasmic reticulum (ER) resident protein stromal interaction molecule 1 (STIM1) has been identified as a regulator of SOCs, including TRPCs. With this in mind, the following studies were carried out to identify the distribution and function of TRPC proteins in MCs, including their role in the mediation of MC contractile function and potential regulation by STIm1. In the first study, TRPC1, -3, -6, and -7 were identified in cultured human MMCs as well as rat and human kidney sections. TRPC1 was found to associate with TRPC4 and TRPC6 by co-immunoprecipitation and colocalization by immunocytochemistry. Overexpression of TRPC1 by transient transfection increased, while knockdown of TRPC1 expression by RNAi decreased thapsigargin-mediated SOCE. These results indicate a role for TRPC1 in SOCE in MCs. In the second study, the contribution of TRPC1-mediated SOCE to Ang II-stimulated MC contractile function was examined. Ang II-mediated SOCE was attenuated by TRPC1-RNAi or by treatment with a TRPC1 antibody known to block channel activity. TRPC1-RNAi and antibody blockade also inhibited Ang II-stimulated single channel activity as measured by cell-attached patch clamp, while TRPC1-RNAi attenuated Ang II-mediated MC contraction. This effect was also examined in vivo in rats. Infusion of TRPC1 antibody blocked Ang II-induced decline in GFR. In the final study, the formation of SOC by TRPC heteromultimerization was assessed. Both TRPC1 and TRPC4 were found to contribute to TG-stimulated SOCE and single-channel activity in cultured MCs. The interaction between these two subtypes increased upon store-depletion with TG, while translocation of TRPC1 but not TRPC4 to the plasma membrane was induced by TG. STIM1 was also found to contribute to regulation of SOC, but co-immunoprecipitation demonstrated an interaction with TRPC1 but not TRPC4. These data suggest that SOC activity is mediated by interaction between TRPC1 and TRPC4, and translocation of TRPC1 to the plasma membrane may be responsible for increasing channel activity upon store depletion. STIM1 may play a regulatory role by activity channel complexes via TRPC4. Taken together these studies indicate an important role for TRPC function in MCs. Not only do these studies further understanding of SOC function in MCs specifically, they also contribute to the delineation of TRPC channel activity, complex formation, and regulation by STIM1. Futures studies are needed to further examine TRPC activation mechanisms and their potential role in other physiological and pathophysiological MC functions.

Comments

Sours-Brothers, Sherry. Store-operated Calcium Entry in Glomerular Mesangial Cells. Doctor of Philosophy (Integrative Physiology, Molecular Cardiovascular Science Track), April, 2008, 181 pp; 1 table, 25 figures, bibliography, 241 titles. W 4 S724S 2008

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