Abstract: PUB282
pH Stability Is Essential to Chronic Cellular Models of Cardiac Fibrosis in CKD
Session Information
Category: Hypertension and CVD
- 1601 Hypertension and CVD: Basic
Authors
- Jain, Krishan G, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Halim, Arvin, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Campos, Monique Opuszcka, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Narayanan, Gayatri, Indiana University School of Medicine, Indianapolis, Indiana, United States
- Lu, Tzongshi, Harvard Medical School, Boston, Massachusetts, United States
- Lim, Kenneth, Indiana University School of Medicine, Indianapolis, Indiana, United States
Background
Fibrogenesis of the heart wall is a major complication in CKD and is driven by multiple components of uremia, including mineral stressors and pro-inflammatory cytokines. Long-term in vitro cardiac fibrosis models are pivotal to studying this disease process. However, exposure of myocardial fibroblasts to CKD-associated stressors are limited by short cellular viability due to pH instability. Herein, we assessed the role of standard buffer systems, sodium bicarbonate (NaHCO3) and HEPES in primary cardiac fibrosis models.
Methods
Fibrosis models were developed using human adult ventricular fibroblasts exposed to 1) high mineral stressors (2mM Ca2+ + 3.8mM Pi, “Ca/Pi”), 2) TGF-β (5, 10, 15 ng/mL), or 3) neither (control) for 10 days. Fibroblasts were cultured in high glucose DMEM media (2.5% FBS) buffered with 44mM NaHCO3 alone or in combination with 20mM HEPES. Cell morphology and attachment were qualitatively assessed every other day for 10 days. Fibrosis was assessed by collagen type 1 (COL1A1) protein expression. Cell culture media was assessed for pH at various time intervals.
Results
Ca/Pi-exposed cells in NaHCO3 only buffered medium had reduced cell adhesion to 45% by day 5 and increased cell death that made COL1A1 expression difficult to assess. Cells exposed to Ca/Pi and cultured in NaHCO3 + HEPES maintained >90% cell adherence for up to 10 days with increased COL1A1 expression ~5-fold higher than controls (P=0.0001). TGF-β induced fibrosis models had no reduction in cell attachment and viability regardless of the type of buffered medium. TGF-β at 5, 10, and 15ng/mL induced ~20-, ~23-, and ~20-fold increase in COL1A1 compared to controls. NaHCO3 + HEPES buffered media maintained a near physiological pH (pH 7.36) compared to NaHCO3-only buffered media (pH 7.6) up to 48hrs in Ca/Pi-exposed cells (P<0.001). Ca/Pi exposure for 72 hours increased media pH to 7.6 in NaHCO3 + HEPES buffered media and up to 7.86 in NaHCO3-only buffered media.
Conclusion
TGF-β is more potent inducer of fibrosis compared to Ca/Pi. HEPES provides a more stable pH than NaHCO3 alone for long-term cell viability in in vitro fibrosis models. These findings highlight the differential role of buffering agents and CKD-associated stressors (Ca/Pi and TGF-β) as critical determinants of in vitro fibrosis models
Funding
- Private Foundation Support