Expression and Signaling of Parathyroid Hormone-Related Protein in Cultured Podocytes

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Abstract
Podocyte function appears to be regulated by vasoactive factors. In vivo podocytes express parathyroid hormone-related protein (PTHrP), the N-terminal fragment of which has vasoactive properties. Since the signaling pathway(s) of PTHrP(1–36) are unknown in podocytes, differentiated cells of a conditionally immortalized mouse podocyte cell line were studied. Gene expression of PTHrP and the PTH/PTHrP receptor was investigated by RT-PCR; protein distribution of PTHrP was examined by immunofluorescence. Accumulation of cAMP was determined by an enzyme immunoassay; [Ca2+]i was measured by fura-2 ratio imaging. PTHrP and PTH/PTHrP receptor mRNA was detected in differentiated podocytes. Immunoreactive PTHrP exhibited a granular distribution in the cytoplasm of differentiated podocytes. With regard to the signaling pathway(s) of PTHrP(1–36), a concentration-dependent increase of cAMP levels with an EC50 value of 4 ± 2 nM was found. PTHrP(1–36) (1 µM) increased cAMP levels 5.5 ± 1.1-fold above baseline as compared with a 25.4 ± 4.2-fold increase in response to forskolin (10 µM). The PTH/PTHrP receptor antagonist PTHrP(7–34) significantly diminished the PTHrP(1–36)-induced cAMP increase. While superfusion of podocytes with bradykinin (100 nM) increased [Ca2+]i, PTHrP(1–36) (100 nM) was without effect on [Ca2+]i. However, PTHrP(1–36) attenuated the bradykinin-induced increase in [Ca2+]i. Our results suggest that PTHrP is an autocrine hormone in podocytes, which selectively activates the cAMP pathway through the PTH/PTHrP receptor.