Escherichia coli lipopolysaccharide impairs the calcium signaling pathway in mesangial cells: role of angiotensin II receptors.
Sepsis causes impaired vascular reactivity, hypotension and acute renal failure. The ability of the Escherichia coli endotoxin (lipopolysaccharide [LPS]) to impair agonist-induced contractility in mesangial cells, which contributes to LPS-induced renal dysfunction, was evaluated. Agonist-induced intracellular calcium ([Ca(2+)]i) mobilization was analyzed using angiotensin II (AngII). The effect of LPS on the levels of the renin-angiotensin system (RAS) components and the roles of vasodilatation-inducing molecules including AT2 receptor (AT2R) and nitric oxide (NO) in the cell reactivity were also evaluated. Confluent human mesangial cells (HMCs) were stimulated with LPS (0111-B4, 100 microg/mL). AngII-induced [Ca(2+)]i mobilization was measured by fluorometric analysis using Fura-2AM in the absence and presence of an AT2R antagonist (PD123319). The mRNA and protein levels for angiotensinogen, renin, angiotensin-converting enzyme, AT1R and AT2R were analyzed by realtime reverse transcriptase-polymerase chain reaction and Western blot, respectively. NO production was measured by the chemiluminescence method in the culture media after 24, 48 and 72 h of LPS incubation. After 24 h, LPS-stimulated HMCs displayed lower basal [Ca(2+)]i and an impaired response to AngII-induced rise in [Ca(2+)]i. LPS significantly increased AT2R levels, but did not cause significant alterations of RAS components. PD123319 restored both basal and AngII-induced [Ca(2+)]i peak, suggesting an involvement of AT2R in these responses. The expected increase in NO production was significant only after 72 h of LPS incubation and it was unaffected by PD123319. Results showed that LPS reduced the reactivity of HMCs to AngII and suggest that the vasodilatation induced by AT2R is a potential mediator of this response through a pathway independent of NO.
Department of Medicine, Renal Division, Federal University of São Paulo, São Paulo, Brazil.
Edgar
E
Maquigussa
Carine P
CP
Arnoni
Priscila C
PC
Cristovam
Andrea S
AS
de Oliveira
Elisa M S
EM
Higa
Mirian A
MA
Boim
D000804:Angiotensin II / Q000378:metabolism; D000808:Angiotensinogen / Q000096:biosynthesis; D015153:Blotting, Western; D020013:Calcium Signaling / Q000187:drug effects*; D004926:Escherichia coli / Q000472:pathogenicity*; D020869:Gene Expression Profiling; D006801:Humans; D008070:Lipopolysaccharides / Q000633:toxicity*; D050527:Mesangial Cells / Q000187:drug effects*; D009569:Nitric Oxide / Q000378:metabolism; D007703:Peptidyl-Dipeptidase A / Q000096:biosynthesis; D011945:Receptors, Angiotensin / Q000096:biosynthesis / Q000187:drug effects*; D012083:Renin / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction
D008070:Lipopolysaccharides; D011945:Receptors, Angiotensin; D000808:Angiotensinogen; D000804:Angiotensin II; D009569:Nitric Oxide; D007703:Peptidyl-Dipeptidase A; D012083:Renin