Evaluation of cyclophilin B as a candidate essential hypertension gene
Kainer, Daniel Bryan
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Cyclophilins comprise a family of peptidyl-prolyl isomerases originally thought to contribute to protein folding, but are now thought to mediate multiple intra- and extracellular functions. These proteins, when liganded with cyclosporine A, bind and inhibit calcineurin (CaN), a calcium-calmodulin dependent protein phosphatase known to regulate proximal tubule sodium, potassium ATPase (NKA) activity. NKA, the protein responsible for the maintenance of the electrochemical gradient driving renal ion transport is altered in hypertension in both the spontaneously hypertensive rat (SHR) and human patients. Therefore, we viewed cyclophilin as a prime hypertension candidate gene that may exert its effects upstream of NKA. Cyclophilin B is an ER-resident cyclophilin isoform thought to influence [Ca++]i., an important parameter in both the etiology of hypertension and CaN regulation This observation, coupled with the observation that CypB expression levels are increased by various sodium-retaining conditions, led us to investigate this gene within the context of hypertension. No CypB sequence differences between samples derived from hypertensive and normotensive (WKY) animals were observed. However, quantitative alterations in genes contributing to hypertension, which itself is defined quantitatively, may play a crucial role in the underlying etiology. Indeed, SHR displayed enhanced expression in whole kidney and microdissected proximal tubule, with the latter alteration becoming observable even in prehypertensive animals. Moreover, this difference was maintained in immortalized cells derived from SHR and WKY proximal tubules, suggesting that this alteration in expression is not due to a compensatory mechanism correcting for elevated blood pressure. We next evaluated the influence of angiotensin II (All), an NKA-activating hormone and two antagonistic first messengers, dopamine (DA) and atrial natriuretic peptide (ANP) on CypB expression. All and DA increased CypB expression, while ANP had no effect. No strain-specific differences were observed. We examined CypB subcellular localization and determined that CypB is largely restricted to the ER and cytosol with possible nuclear localization. Yeast two-hybrid analysis revealed interactions between CypB and hnRNP A2, subunit I of cytochrome oxidase, aminoacylase 1, very long chain acyl Co A synthetase, ribosomal protein LI 9, and eukaryotic initiation factor-2. These newly-characterized interactions add to an already extensive list and likely underscore a rich diversity of intracellular CypB functions. The current studies also provide evidence for a link between CypB and SHR hypertension. However, mechanisms underlying such a link are still unclear and require further investigation.