Cells treated with Ang II or NIC alone had lower relative densitometry values. However, Table 1 indicates that Ang II combined with NIC induced an approximate 12-fold increase in FN expression. Especially when considering lower concentrations (10-8 M or less), there is a significant increase (p=0.01773) in samples treated with both nicotine and angiotensin II compared to samples treated only with nicotine or angiotensin II. These multiple-fold increases of the combination samples compared to samples with just Ang II or NIC suggest a synergistic effect of Ang II and NIC on FN expression. The NIC at 10-8 M sample and Ang II at 10-10 M sample have relative densitometry values of approximately 0.7 relative densitometry units (RDUs). The cell sample treated with both NIC at 10-8 M and Ang II at 10-10 M shows a significant increase in relative densitometry with a value of 5.83 RDUs suggesting more than an additive effect. At concentrations that by themselves do not considerably increase FN production, Ang II and NIC combined result in significant increases in FN production.
Table 1. Densitometry analysis of western blots using rat mesangial cell samples treated with NIC and/or Ang II (ranging from 0 to 10-7 M) measured in relative densitometry units. (N=4 per treatment group)
0 10-7 10-8 10-9 10-10
0
10-7
10-8
10-9
10-10
1.00±
0.65±
0.81 ±
1.46 ±
1.14 ±
0.00
0.19
0.31
0.23
0.54
1.27 ±
1.12 ±
4.62±
3.80±
31.82±
0.098
0.26
1.72
1.85
15.8
1.11±
1.31±
0.77±
34.59±
20.02±
0.24
0.33
0.25
16.41
9.88
0.72 ±
3.99±
9.52±
4.63±
6.28±
0.18
2.14
1.99*#
1.12
4.53
0.81 ±
0.68±
5.83±
16.07±
42.45±
0.14
0.22
1.11*#
14.2
21.0
*
*p<0.05 vs. Nicotine alone #p<0.05 vs. Angiotensin II alone N=4
Summary These results suggest a synergistic effect between Ang II and NIC on mesangial cell fibronectin production and proliferation. Rat mesangial cells treated with both NIC and Ang II showed a significant increase in proliferation and fibronectin expression compared to cells treated with NIC or Ang II alone. In many cases, the effects were multiple-fold increases of FN expression 56 • inquiro | no. 6 | 2012
Conclusion These studies show that a synergistic relationship between Ang II and NIC exists. The combined effects of the two compounds increase mesangial cell FN production. This relationship may explain some of the deleterious effects of tobacco smoking in renal diseases associated with RAS activation. References 1. Cheng TH, et al. J Am College Cardiology. 2003;42:18451854. 2. Jaimes EA, Tian RX, Leopoldo R. Am J Physiology. 2007;292:H76-82. 3. Johnson RJ, Alpers CE, Yoshimura A, et al. Hypertension. 1992;19:464-474. 4. Kagami S, Border WA, Miller DA, et al. J. Clin. Invest. 1994;93:2431-2437.
NIC (M) ANG II (M)
that exceeded the additive expression of both compounds independently, suggesting a synergistic effect. These effects were more evident at concentrations of Ang II or NIC that by themselves did not have significant effects on mesangial cell proliferation or FN production but induced robust proliferation and FN production when the cells were stimulated with both agents. At higher concentrations, their combined effects were of lower magnitude, which suggests the presence of feedback mechanisms that modulate these responses.
5. Life Technologies, Click it EdU Microplate Assay. http:// products.invitrogen.com 6. Myers BD, Boothroyd D, Olshen RA. J Am Soc Nephrol. 1998;9:S66-70. 7. Orth SR, Hallan SI. Clin. J. Am Soc Nephrol. 2008;3:226236. Acknowledgments • Funding support from NIH and FAMRI to E.A.J. • Travel grants provided by Department of Biology and American Heart Association to C.Y.R. • Special thanks to Madison Duckworth, student researcher • All experiments performed in the lab of E.A.J.