Bruce L. Riser Stephanie Ladson-Wofford Abdulkarim Sharba Pedro Cortes Katie Drake Christopher J. Guerin Jerry Yee Mary E. Choi Patricia R. Segarini Robert G. Narins
Background. Transforming growth factor-β (TGF-β) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured me..
Background. Transforming growth factor-β (TGF-β) is a causal factor in experimental glomerulosclerosis, and it mediates the increased extracellular matrix (ECM) accumulation that occurs in cultured mesangial cells (MCs) exposed to high glucose concentrations and cyclic mechanical strain. This change is associated with increased levels of TGF-β, but may also involve alterations in receptor expression and binding. Methods. Rat MCs cultured in media containing either 8 or 35 mM glucose were seeded into culture plates with elastin-coated flexible bottoms. Thereafter, they were subjected to cyclic stretch or static conditions and then examined for 125I-TGF-β1 binding and expression of TGF-β receptors at the gene and protein levels. Results. Kinetic studies showed that MCs bound TGF-β1 in a time- and concentration-dependent manner, expressing 6800 high-affinity receptors per cell, with an apparent dissociation constant (K(d)) of 15.4 pM, while cross- linking analysis identified three TGF-β receptors (βR) corresponding to βRI, βRII, and βRIII of 54, 73, and 200 kDa, respectively. Immunocytochemical studies of βRI and βRII protein revealed MC expression in a homogeneous, punctate distribution, whereas Northern analysis demonstrated the presence of the corresponding mRNAs. Exposure to cyclic stretching significantly increased (10%) the overall number of TGF-β receptors, whereas ligands associated with βRs I, II, and III also increased (25 to 50%). The finding of increased (30 to 40%) βRI and βRII transcript levels and immunoreactive protein (163 and 59%, respectively) in the absence of significant changes in the apparent K(d) indicated that stretch-induced binding was the result of increased receptor synthesis and expression and not due to a change in binding affinity. In a similar, but more dramatic fashion, exposure to high glucose also elevated (50%) the receptor number, as well as the amount of ligands associated with βRs I, II, and III (100 to 250%). This same treatment also increased the levels of βRI and βRII mRNA (30 to 40%) and the immunoreactive protein (82 and 82%, respectively), without significantly altering the binding affinity of the receptor. A concerted or synergistic effect of both stimuli was not evidenced. Conclusion. These results suggest that the modulation of TGF-β receptors may be an additional control point in mediating the glucose- and mechanical force-induced increase in ECM deposition by MCs.