Jennifer L. Gooch - US grants

DESCRIPTION (provided by applicant): Calcineurin is a calcium-dependent phosphatase that has emerged as an important signaling molecule in the kidney. Calcineurin is the target of drugs such as cyclosporin A (CsA), whose therapeutic use is limited by associated nephrotoxicity. Currently, calcineurin is known to function in signal transduction of key molecules including Agll, IGF-I and TGFbeta. However, action of calcineurin is cell-specific and there are at least two distinct models of calcineurin action in the kidney. First, we have shown that calcineurin is required for TGFbeta-mediated ECM accumulation in cultured mesangial cells (MCs) and inhibition of calcineurin protects glomeruli from ECM accumulation associated with type I diabetes in vivo. Conversely, CsA induces ECM accumulation in tubule epithelial cells (TECs) in vitro and in the tubulointerstitium in vivo. Furthermore, there is no significant protection from diabetes-induced ECM accumulation with calcineurin inhibition in the cortical tubulointerstitium. Understanding mechanisms of calcineurin signaling specificity in the kidney is key to targeting inhibition of calcineurin to prevent ECM accumulation in glomeruli and avoid CsA-mediated nephrotoxicity in the tubulointerstitium. Signaling mechanisms of calcineurin in renal cells are poorly understood and few targets of calcineurin phosphatase action have been described in the kidney. Our work has identified candidate pathways downstream of calcineurin that may be critical to cell-specific regulation of ECM proteins. Moreover, we have discovered that calcineurin A isoforms are differentially regulated in the diabetic kidney, suggesting that this may be a further level of cell-specific signaling in the kidney. Therefore, our hypothesis is the following: Cell-specific action of calcineurin in the kidney is the result of signaling specificity downstream of calcineurin, dephosphorylation of cell-specific targets, and/or action of different calcineurin isoforms. First, we will delineate downstream signaling pathways that may be involved in regulation of ECM accumulation in MCs and TECs. Next, we will identify cell-specific targets of calcineurin dephosphorylation. Finally, we will evaluate the specific contribution of calcineurin A alpha isoform and calcineurin A beta isoform to regulation of ECM accumulation in MCs and TECs. The goal of these experiments is to distinguish mechanisms of calcineurin-mediated regulation of ECM in glomeruli from calcineurin action that contributes to CsA-nephrotoxicity.

[unreadable] DESCRIPTION (provided by applicant): L-Calcineurin is an important regulatory enzyme that functions in many cellular processes including T cell signal transduction. Drug such as cyclosporin and FK506 that inhibit calcineurin (CIs) are clinically useful to suppress the immune system following organ transplantation. Unfortunately, inhibition of calcineurin also affects tissues other than the immune system and long-term use of CIs often produces therapeutically-limiting side-effects including nephrotoxicity and cardiovascular disease. Work in our laboratory is focused on specificity of calcineurin action in the kidney. Interestingly, when we examined mice that lack the alpha isoform of the catalytic subunit of calcineurin and mice that lack the beta isoform, we find very different effects. Namely, loss off alpha severely impairs development of the kidney, alters cell cycle regulation, and results in increased matrix production - features consistent with Cl nephrotoxicity while mice lacking the beta isoform lack signs of significant renal damage. Moreover, we find that alpha-/- mice develop hyperlipidemia, demonstrating a direct connection between calcineurin action and development of cardiovascular disease. Finally, mice lacking the beta isoform have been previously described to have an "immune-suppressed"-like phenotype while T cell function in alpha null animals can still be suppressed with cyclosporin. These mouse models clearly demonstrate that calcineurin isoforms have distinct functions that are particularly relevant in the transplant setting. Inhibition of the alpha isoform produces nephrotoxicity and hyperlipidemia while inhibition of the beta isoform results in suppression of T cell signaling. Therefore, we hypothesize that selective inhibition of the beta isoform will result in immune suppression with fewer toxic side-effects. To test this hypothesis, it will be necessary to generate a mouse model that will allow us to carefully compare the effects of selective loss of calcineurin isoforms. Since alpha-/- mice live only a few weeks, it is not currently possible to perform such studies. However, recent advances in gene manipulations provide innovative tools to generate inducible, isoform-specific knockout mice. We will create these mice and characterize the effect of inducible loss of each isoform in immune suppression and nephrotoxic / cardio- vascular side- effects. Using a well-characterized skin transplant model, we will then test the hypothesis that selective inhibition of calcineurin isoforms results in immune suppression with fewer side-effects. [unreadable] [unreadable] [unreadable]