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Mitogen-activated protein kinase phosphorylation in kidneys of beta(s) sickle cell mice
Militza Kiroycheva1, Fayyaz Ahmed1, Gillian M Anthony1
1Renal Division, Department of Medicine, Montefiore Medical Center, Bronx, New York.
Abstract:
Previous studies in beta(s) sickle cell mice demonstrated renal immunostaining for nitrotyrosine, which is putative evidence of peroxynitrite (ONOO(-)) formation. ONOO(-) is known to nitrate tyrosine residues of various enzymes, thereby interfering with phosphorylation and inactivating them. The present study examined the state of phosphorylation of mitogen-activated protein (MAP) kinase signal transduction enzymes, i.e., p38, c-Jun NH(2)-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK). Western blot performed with antibodies directed against specific phosphorylated threonine/tyrosine residues of these enzymes demonstrated reduced phosphorylation of renal p38 and a trend toward reduced phosphorylation of ERK. In contrast, phosphorylation of renal JNK was markedly increased compared with normal mice. The abundance of MAP kinase phosphatase-1 (MKP-1), a key upstream enzyme that modulates phosphorylation of MAP kinases, was not different in beta(s) versus normal mice. To determine whether nitration of tyrosine by ONOO(-) was responsible for reduced phosphorylation of p38 and ERK, mercaptoethylguanidine (MEG), a compound known to reduce inducible isoform of nitric oxide synthase activity and to scavenge ONOO(-), was administered to beta(s) mice for 5 d. MEG was found to restore phosphorylation of p38 and ERK toward normal levels. These observations provide evidence that ONOO(-) (or closely related reaction products of NO) contributes to dephosphorylation of p38 and ERK, and presumably reduces activity of these enzymes. The increased phosphorylation of JNK, which suggests activation of this signaling pathway by extracellular stress signals, may play a role in apoptosis in the kidneys of these mice. The changes in phosphorylation of MAP kinase pathways found in this study could have important consequences for regulation of nuclear transcription factors, and thus renal function and pathology in sickle cell kidneys.
Insights
Peroxynitrite formation in sickle cell kidneys reduces p38 and ERK phosphorylation, while increasing JNK phosphorylation. Mercaptoethylguanidine treatment restored normal p38 and ERK phosphorylation levels.
Area of Science:
- Nephrology
- Biochemistry
- Molecular Biology
Background:
- Sickle cell disease (SCD) is associated with kidney complications.
- Nitrotyrosine, a marker of peroxynitrite (ONOO(-)) formation, is found in SCD kidneys.
- ONOO(-) can inactivate enzymes by nitrating tyrosine residues, affecting phosphorylation.
Purpose of the Study:
- To investigate the phosphorylation status of mitogen-activated protein (MAP) kinases (p38, JNK, ERK) in beta(s) sickle cell mouse kidneys.
- To determine if ONOO(-) contributes to altered MAP kinase phosphorylation in SCD.
- To evaluate the therapeutic potential of ONOO(-) scavenging in SCD kidney pathology.
Main Methods:
- Western blot analysis of phosphorylated p38, JNK, and ERK in kidneys of beta(s) mice and normal controls.
- Assessment of MAP kinase phosphatase-1 (MKP-1) abundance.
- Administration of mercaptoethylguanidine (MEG), an ONOO(-) scavenger, to beta(s) mice.
Main Results:
- Reduced phosphorylation of renal p38 and a trend toward reduced ERK phosphorylation in beta(s) mice.
- Markedly increased phosphorylation of renal JNK in beta(s) mice compared to controls.
- MEG treatment restored p38 and ERK phosphorylation toward normal levels in beta(s) mice.
Conclusions:
- ONOO(-) contributes to dephosphorylation and presumed inactivation of p38 and ERK in sickle cell kidneys.
- Increased JNK phosphorylation suggests activation of stress signaling pathways, potentially contributing to kidney apoptosis.
- Altered MAP kinase signaling in SCD kidneys may impact nuclear transcription factors, renal function, and pathology.