Date

8-5-2015

Document Type

Dissertation

Degree Name

Ph.D.

Department

Department of Physiology & Pharmacology

Institution

Oregon Health & Science University

Abstract

Tacrolimus (FK-506) is an immunosuppressive drug often used to prevent graft rejection in organ transplant patients. It inhibits the phosphatase calcineurin, when bound to an endogenous protein FKBP12. In T-cells this leads to immunosuppression. While effective, tacrolimus also causes side effects including hypertension, diurnal dipping disruption, hyperkalemia, hypomagnesemia and hypercalciuria. In an effort to develop safer immunosuppressive drugs and more effectively treat patients, unraveling these pathological mechanisms has become a priority.

Tacrolimus leads to hypertension by pleiotropic effects in the vasculature, central nervous system, renin-angiotensin/aldosterone axis and the kidney. We recently found, however, that the renal sodium chloride cotransporter (NCC) is necessary for the development of tacrolimus-induced hypertension. Furthermore, tacrolimus increases NCC phosphorylation, a mark of activity. The essential nature of NCC in this multi-faceted pathology, suggests that understanding, and avoiding, NCC activation may be the key to developing safer immunosuppressive drugs. It is currently unclear, however, whether tacrolimus is activating NCC directly, or by affecting extra-or intra-renal targets upstream of NCC activation. It is also unknown whether calcineurin or FKBP12 inhibition is involved.

I hypothesize that calcineurin is responsible for dephosphorylating NCC. Thus, its inhibition in the DCT leads to an increase in NCC phosphorylation, which is essential for the development of tacrolimus-induced hypertension and electrolyte disorders. To test this I generated a mouse model in which FKBP12, the necessary binding partner of tacrolimus, can be inducibly deleted along the nephron (KS-FKBP12-/-). I tested in vivo 1) whether FKBP12 disruption along the nephron contributes to NCC phosphorylation, hypertension or electrolyte disorders and 2) whether protecting calcineurin from inhibition along the nephron is sufficient to ameliorate these side effects.

I found that KS-FKBP12-/- mice were phenotypically normal at baseline, suggesting that FKBP12 disruption along the nephron does not underlie tacrolimus-induced hypertension or electrolyte disorders. This also implies that immunophilin-sparing calcineurin inhibitors will not alleviate the renal-component of these side effects.

Conversely, KS-FKBP12-/- mice treated with tacrolimus had lower blood pressures and more effectively recovered their diurnal dipping patterns than tacrolimus-treated control mice. They also had lower levels of phosphorylated NCC (pNCC), maintained a normal pNCC: plasma [K+] relationship (suggesting protection from K+ dysregulation), and were protected from hypomagnesemia and hypercalciuria. Additionally, in vitro I found that tacrolimus inhibits NCC dephosphorylation. Taken together, this suggests that calcineurin plays a role in NCC dephosphorylation and that its inhibition in DCT cells leads to an increase in NCC activity, hypertension and hyperkalemia. An FKBP12-dependent event along the nephron, such as calcineurin inhibition, also causes hypomagnesemia, likely by reducing TRPM6 mRNA abundance, and hypercalciuria by reducing calcium transport across the DCT.

Collectively this supports the idea that tacrolimus-induced dysregulation of the DCT contributes to a host of pathologies including an increase in NCC activity. Calcineurin inhibitors which can’t access the nephron may be substantially safer therapeutics. Until then, thiazide diuretics, which directly inhibit NCC activity, will likely be particularly effective at treating hypertensive or hyperkalemic tacrolimus patients.

Identifier

doi:10.6083/M4C24V8C

School

School of Medicine

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