Abstract
Maternal hyperglycemia can inhibit morphogenesis of ureteric bud branching, Glial cell line-derived neurotrophilic factor (GDNF) is a key regulator of the initiation of ureteric branching. Early growth response gene-1 (EGR-1) is an immediate early gene. Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment. To evaluate the potential relationship of hyperglycemia-GDNF-EGR-1 pathway, in vitro human renal proximal tubular epithelial (HRPTE) cells as target and in vivo streptozotocin-induced mice model were used. Our in vivo microarray, real time-PCR and confocal morphological observation confirmed apoptosis in hyperglycemia-induced fetal nephropathy via activation of the GDNF/MAPK/EGR-1 pathway at E12-E15. Detachment between ureteric branch and metanephrons, coupled with decreasing number and collapse of nephrons on Day 1 newborn mice indicate hyperglycemic environment suppress ureteric bud to invade metanephric rudiment. In vitro evidence proved that high glucose suppressed HRPTE cell migration and enhanced GDNF-EGR-1 pathway, inducing HRPTE cell apoptosis. Knockdown of EGR-1 by siRNA negated hyperglycemic suppressed GDNF-induced HRPTE cells. EGR-1 siRNA also reduced GDNF/EGR-1-induced cRaf/MEK/ERK phosphorylation by 80%. Our findings reveal a novel mechanism of GDNF/MAPK/EGR-1 activation playing a critical role in HRPTE cell migration, apoptosis and fetal hyperglycemic nephropathy.
Highlights
Clinical and experimental evidence demonstrate that maternal diabetes induces a broad array of congenital malformation [1,2], risk of birth defect in diabetic pregnancy ranging from two to six times above normal [3]
The developing kidney seems sensitive to high-glucose milieu [4,5,6,7]; with the fetus exposed to sustained high glucose ambience, damage affects multiple organs, a condition known as diabetic embryopathy [3,8]
We found a group of genes involving Glial cell line-derived neurotrophilic factor (GDNF)-Early growth response gene-1 (EGR-1) pathway upregulated on E12 down-regulated on E13 and progressively up-regulated in hyperglycemic renal tissue, results identical to realtime PCR
Summary
Clinical and experimental evidence demonstrate that maternal diabetes induces a broad array of congenital malformation [1,2], risk of birth defect in diabetic pregnancy ranging from two to six times above normal [3]. To confirm GDNF, EGR-1, Ras, cRaf, MEK and ERK mRNA expression differing significantly between hyperglycemic and normoglycemic fetal kidney tissues observed using microarray analysis, we performed quantitative RT-PCR and compared expression patterns, as shown in Figs.2a, b, c, d, e, f, g. We found that GDNF, EGR-1, Ras, cRaf, MEK and ERK gene expression differed between hyperglycemic and normoglycemic fetal kidney tissue. Day 1 hyperglycemic mice showed detachment between ureteric branch and metanephros (blue arrow), as well as collapse of nephrons (yellow arrow) (Figs.4d–4e).Serial changes of GDNF, EGR-1 and ERK-2 expression on branching morphogenesis: Upper parts were fetal kidney tissue from hyperglycemic versus lower parts from normoglycemic mothers. By comparing fluorescent intensity in fetal kidneys of hyperglycemic and normoglycemic mothers, we observed E12 weaker expression of
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