ASN's Mission

To create a world without kidney diseases, the ASN Alliance for Kidney Health elevates care by educating and informing, driving breakthroughs and innovation, and advocating for policies that create transformative changes in kidney medicine throughout the world.

learn more

Contact ASN

1401 H St, NW, Ste 900, Washington, DC 20005

email@asn-online.org

202-640-4660

The Latest on X

Kidney Week

ASN / Education & Meetings / Kidney Week /

Please note that you are viewing an archived section from 2021 and some content may be unavailable. To unlock all content for 2021, please visit the archives.

Abstract: PO0516

Enhancement of In Vitro hPTH1-84 Bioactivity by hPTH38-84 and hPTH45-84

Session Information

Category: Bone and Mineral Metabolism

  • 401 Bone and Mineral Metabolism: Basic

Authors

  • Kritmetapak, Kittrawee, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Losbanos, Louis A., Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Hines, Jolaine M., Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Singh, Ravinder, Mayo Clinic Minnesota, Rochester, Minnesota, United States
  • Kumar, Rajiv, Mayo Clinic Minnesota, Rochester, Minnesota, United States
Background

Recent research using high resolution mass spectrometry demonstrates that serum concentrations of full-length parathyroid hormone (hPTH1-84) and its fragments (hPTH28-84, 34-77, 34-84, 37-77, 37-84, 38-77, 38-84, and 45-84) are increased significantly in CKD patients with an eGFR of <17-23 mL/min/1.73m2 (Kritemetapak et al. Clin Chem. 2021 Mar 6:hvab013. doi: 10.1093/clinchem/hvab013. Online ahead of print.PMID: 33693557). Information about the bioactivity of these newly discovered hPTH fragments is lacking.

Methods

Recombinant hPTH1-84 was synthesized in Escherichia coli and purified by immobilized metal-ion affinity chromatography. hPTH28-84, hPTH38-84, and hPTH45-84 were synthesized by solid phase peptide synthesis. The identity of hPTH1-84 and hPTH fragments was confirmed by mass spectrometry. To determine whether different-sized hPTH fragments modulate the bioactivity of hPTH1-84, we studied their effects on the generation of the cellular second messenger, cAMP, which mediates the intracellular signaling of hPTH1-84, in murine preosteoblasts (MC3T3-E1) in vitro. Forskolin, an adenylyl cyclase activator, was used as a positive control for cAMP production. All experiments were performed in triplicate.

Results

In MC3T3-E1 cells, cAMP increased to 28.5+5.5, 64.4+9.9, 91.5+11.6, 114.6+15.5, and 109.1+6.5 pmol/mL 30 minutes after treatment with 1, 3, 10, 50, and 100 nM,hPTH1-84 . The same concentrations of hPTH28-84, hPTH38-84, and hPTH45-84 had no effects. When hPTH1-84 was added to cells concurrently with 100 nM hPTH38-84 or hPTH45-84 in 1:100, 3:100, 1:10, 1:2, and 1:1 molar ratios, cAMP responses to hPTH1-84 increased by 65.3% (95% CI, 63.2% to 67.4%; P<0.01) in the presence of hPTH38-84 and increased by 77.0% (95% CI, 65.2% to 88.8%; P<0.01) in the presence of hPTH45-84. hPTH28-84, added concurrently with PTH1-84, did not enhance cAMP generation in MC3T3 cells.

Conclusion

The small hPTH fragments, hPTH38-84 and 45-84, but not hPTH28-84, enhance the hPTH-induced generation of cAMP in MC3T3-E1, suggesting a novel biological role and a novel mechanism of action for these PTH fragments in osteoblast-like cells. It is plausible that in vivo these fragments may enhance the activity of full-length PTH by novel mechanisms. These findings may partly explain the discrepancy between PTH levels and bone histology in patients with CKD.

Funding

  • Other NIH Support