The role of TGF-β1 activation in the hypertension-associated vascular pathology of Dahl Salt Sensitive (SS) rats

We have shown that hypertension-induced afferent arteriolar remodeling and autoregulatory dysfunction in SS rats produces kidney damage; matrix metalloproteinase-9 (MMP-9) knock-out (KO) significantly improves the kidney arteriolar pathophysiology and alleviates kidney damage in SS rats. In this study, we hypothesized that MMP-9 activates TGF-β1, which increases arterial expression of Chemokine (C-C motif) Ligand 2 (CCL2) by smooth muscle cells (SMCs) during cyclic strain. Primary cultures of SS or SS Mmp9-/- aortic smooth muscle cells (ASMC) or kidney microvascular smooth muscle cells (KSMC) in early (<6) passages were grown in standard fashion in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. Cultured cells were identified as smooth muscle cells by their characteristic morphology and positive immunostaining for α-smooth muscle actin. The first study used ASMCs of SS and SS Mmp9-/- grown on 6-well BioFlex plates. Cyclic strain was generated using the FlexCell 6000 and a standardized regimen (8 h, 15% elongation @ 1 Hz). When compared with expression by ASMCs maintained under static condition, cyclic strain increased medium levels of active TGF-β1 (static condition vs cyclic strain: 0.286±0.124 vs 1.66±0.42 pg/mL per g cell protein, n=7; p<0.05). Importantly, Mmp9 KO inhibited the increase in medium active TGF-β1 (control vs mmp9 KO: 1.66±0.142 vs 0.481±0.070 pg/mL per g cell protein, n =7; p<0.05). Loss of Mmp9 inhibited cyclic strain-induced increases in medium levels of CCL2 (control vs mmp9 KO: 141.2±5.8 vs 45.1±1.7 pg/mL per g cell protein, n=8; p<0.05). In a second study, KSMCs under static conditions were treated with recombinant active TGF-β1 (1 ng/mL and 10 ng/mL) for 6 h. TGF-β1 increased Ccl2 mRNA expression by 2.18 and 4.37-fold in KSMC incubated in medium containing 1 ng/mL and 10 ng/mL of TGF-β1, respectively. Pre-treatment of KSMC with an ALK4/5 inhibitor, 10 µmol/mL, reduced cyclic strain-induced release of CCL2 (vehicle vs ALK4/5 inhibitor: 60.8±2.1 vs 32.0±1.0 pg/mL per g cell protein; n=8, p<0.05) into the medium. Our studies show that MMP-9 plays an important role in the activation of vascular TGF-β1 in SS rats during cyclic strain. Activation of the TGF-β1 pathway also increased cyclic strain-induced CCL-2 expression, which may integrally contribute to kidney microvascular remodeling and dysfunction in this model of hypertension-associated nephropathy. a Merit Award (1 I01 BX005640) from U.S. Dept. of Veterans Affairs, a NIH George M. O'Brien Kidney and Urological Research Centers Program (P30 DK079337) This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.