Increased Apoptosis Susceptibility in Mesangial Cells from Spontaneously Hypertensive Rats

Abstract

We have examined the susceptibility to apoptosis in mesangial cells from spontaneously hypertensive rats (SHR) or from normotensive rats (WKY) and the possible involvement of nitric oxide in this process. Mesangial cells monolayers from either SHR or normal rats were incubated for 12 h in medium with or without fetal calf serum (FCS) and with or without thapsigargin (Tg, 10−6 M). A series of cultures from rats of both groups was treated with NG-nitro-l-arginine methyl ester (l-NAME, 10−4 M). We assessed apoptosis by propidium iodide staining, by TUNEL nitrite production (Griess reaction), by inducible nitric oxide synthase (iNOS) and Bcl-2 and Bax by Western blot. Incubated with a FCS-free medium, cells from SHR showed a significantly higher apoptotic rate (10.7 ± 2.0) than with 10% FCS (10% FCS, 4.7 ± 0.3), while WKY cells did not show this increment (10% FCS, 4.7 ± 0.3; 0% FCS, 5.9 ± 0.3). Apoptosis in cells from WKY increased when incubated with thapsigargin in FCS-free medium (0% FCS+ Tg, 17.7 ± 2.9%) and increased even more in SHR cells (0% FCS+ Tg, 19.7 ± 2.9%). Treatment with l-NAME decreased thapsigargin-induced apoptosis in both SHR (8.2 ± 2.4%) and WKY cells (9.3 ± 2.4%). An increase in nitrite production and iNOS expression was detected in groups in which the apoptosis rate was elevated. A high rate of apoptosis was also associated with a decrease in the Bcl-2/Bax ratio. Our results indicate that in SHR cells, short-term serum deprivation and the increase in intracellular free calcium concentration with thapsigargin are able to enhance the apoptosis rate in primary cultures and that the expression of iNOS, and hence NO production, is involved in this effect.