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Hyperglycemia: GDNF-EGR1 pathway target renal epithelial cell migration and apoptosis in diabetic renal embryopathy.

Lin CY, Lin TY, Lee MC, Chen SC, Chang JS - PLoS ONE (2013)

Bottom Line: Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment.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.

View Article: PubMed Central - PubMed

Affiliation: Clinical Immunology Center, China Medical University Hospital, Taichung, Taiwan. cylin@mail.cmuh.org.tw

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.

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DM newborn (Day 1) mouse kidney showed nephrogenesis period between 3rd and 4th stages, revealing less demarcated cortical labyrinth and medullary rays, as well as decreased fetal glomeruli and convoluted tubules in deeper cortical and juxtamedullary regions with scattered ‘-shaped’bodies, residual advancing ampullae of ureteric buds, diminished branching of developing convolution (100×) (Fig.4a).Normal newborn (Day 1) mouse kidney showed nephrogenesis period near 4th (last) stage, plus clearly discernible cortical labyrinth and medullary rays with fetal glomeruli and well-developed tubules in the deeper cortical and juxtamedullary regions, developing glomeruli (100×)(Fig.4b). TUNEL assay revealed apoptosis cells increasing in metanephric mesenchyme (dark brown dots) and collapsed nephron region in newborn kidneys of the hyperglycemic group (200×) (Fig.4c). With maternal diabetes circumstances, large amounts of nephrons collapsed (HE stain, Fig. 4d 200× arrow, Fig. 4e 600× arrow). 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. At E12, week expression of EGR-1, GDNF and ERK-2 in intermediate mesoderm of fetal kidney tissue from hyperglycemic mothers (Figs.3f, 3h and 3j). Ureteric buds unable to invade metanephric mesenchyme correlated with reduced immunoreactivity in the tubular cell basement membrane of fetal kidney tissue from hyperglycemic mother on E15 (Figs. 3g, 3i and 3k, 600×; similar results noted in six independent experiments) (Figs.4f–k).
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pone-0056731-g004: DM newborn (Day 1) mouse kidney showed nephrogenesis period between 3rd and 4th stages, revealing less demarcated cortical labyrinth and medullary rays, as well as decreased fetal glomeruli and convoluted tubules in deeper cortical and juxtamedullary regions with scattered ‘-shaped’bodies, residual advancing ampullae of ureteric buds, diminished branching of developing convolution (100×) (Fig.4a).Normal newborn (Day 1) mouse kidney showed nephrogenesis period near 4th (last) stage, plus clearly discernible cortical labyrinth and medullary rays with fetal glomeruli and well-developed tubules in the deeper cortical and juxtamedullary regions, developing glomeruli (100×)(Fig.4b). TUNEL assay revealed apoptosis cells increasing in metanephric mesenchyme (dark brown dots) and collapsed nephron region in newborn kidneys of the hyperglycemic group (200×) (Fig.4c). With maternal diabetes circumstances, large amounts of nephrons collapsed (HE stain, Fig. 4d 200× arrow, Fig. 4e 600× arrow). 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. At E12, week expression of EGR-1, GDNF and ERK-2 in intermediate mesoderm of fetal kidney tissue from hyperglycemic mothers (Figs.3f, 3h and 3j). Ureteric buds unable to invade metanephric mesenchyme correlated with reduced immunoreactivity in the tubular cell basement membrane of fetal kidney tissue from hyperglycemic mother on E15 (Figs. 3g, 3i and 3k, 600×; similar results noted in six independent experiments) (Figs.4f–k).

Mentions: We explored effect of hyperglycemia on tubulogenesis in developing fetal renal tissue. Comparing normal newborn (Day 1) mouse kidney to hyperglycemic mouse kidney, histological analysis found newborn kidneys of the diabetic group showing nephrogenesis between 3rd and 4th period, revealing less demarcated cortical labyrinth and medullary rays, and decreased number of fetal glomeruli and developed convoluted tubules in the deeper cortical and juxtramedullary regions with scattered ‘-shaped’bodies, residual advancing ampullae of ureteric buds, diminished branching of developing convoluted tubules, and presence of adjacent metanephric mesenchyme in subcapsular superficial cortex or cortical labyrinth suggestive of abnormal nephrogenesis in both quantity and quality (Fig. 4a). Careful counting revealed newborn nephron number in offspring of diabetic mothers as significantly lower than in normoglycemic mothers (hyperglycemic vs normoglycemic newborn: 1974±131 vs 2985±124 in number [both n = 6; p<0.001]).


Hyperglycemia: GDNF-EGR1 pathway target renal epithelial cell migration and apoptosis in diabetic renal embryopathy.

Lin CY, Lin TY, Lee MC, Chen SC, Chang JS - PLoS ONE (2013)

DM newborn (Day 1) mouse kidney showed nephrogenesis period between 3rd and 4th stages, revealing less demarcated cortical labyrinth and medullary rays, as well as decreased fetal glomeruli and convoluted tubules in deeper cortical and juxtamedullary regions with scattered ‘-shaped’bodies, residual advancing ampullae of ureteric buds, diminished branching of developing convolution (100×) (Fig.4a).Normal newborn (Day 1) mouse kidney showed nephrogenesis period near 4th (last) stage, plus clearly discernible cortical labyrinth and medullary rays with fetal glomeruli and well-developed tubules in the deeper cortical and juxtamedullary regions, developing glomeruli (100×)(Fig.4b). TUNEL assay revealed apoptosis cells increasing in metanephric mesenchyme (dark brown dots) and collapsed nephron region in newborn kidneys of the hyperglycemic group (200×) (Fig.4c). With maternal diabetes circumstances, large amounts of nephrons collapsed (HE stain, Fig. 4d 200× arrow, Fig. 4e 600× arrow). 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. At E12, week expression of EGR-1, GDNF and ERK-2 in intermediate mesoderm of fetal kidney tissue from hyperglycemic mothers (Figs.3f, 3h and 3j). Ureteric buds unable to invade metanephric mesenchyme correlated with reduced immunoreactivity in the tubular cell basement membrane of fetal kidney tissue from hyperglycemic mother on E15 (Figs. 3g, 3i and 3k, 600×; similar results noted in six independent experiments) (Figs.4f–k).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585314&req=5

pone-0056731-g004: DM newborn (Day 1) mouse kidney showed nephrogenesis period between 3rd and 4th stages, revealing less demarcated cortical labyrinth and medullary rays, as well as decreased fetal glomeruli and convoluted tubules in deeper cortical and juxtamedullary regions with scattered ‘-shaped’bodies, residual advancing ampullae of ureteric buds, diminished branching of developing convolution (100×) (Fig.4a).Normal newborn (Day 1) mouse kidney showed nephrogenesis period near 4th (last) stage, plus clearly discernible cortical labyrinth and medullary rays with fetal glomeruli and well-developed tubules in the deeper cortical and juxtamedullary regions, developing glomeruli (100×)(Fig.4b). TUNEL assay revealed apoptosis cells increasing in metanephric mesenchyme (dark brown dots) and collapsed nephron region in newborn kidneys of the hyperglycemic group (200×) (Fig.4c). With maternal diabetes circumstances, large amounts of nephrons collapsed (HE stain, Fig. 4d 200× arrow, Fig. 4e 600× arrow). 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. At E12, week expression of EGR-1, GDNF and ERK-2 in intermediate mesoderm of fetal kidney tissue from hyperglycemic mothers (Figs.3f, 3h and 3j). Ureteric buds unable to invade metanephric mesenchyme correlated with reduced immunoreactivity in the tubular cell basement membrane of fetal kidney tissue from hyperglycemic mother on E15 (Figs. 3g, 3i and 3k, 600×; similar results noted in six independent experiments) (Figs.4f–k).
Mentions: We explored effect of hyperglycemia on tubulogenesis in developing fetal renal tissue. Comparing normal newborn (Day 1) mouse kidney to hyperglycemic mouse kidney, histological analysis found newborn kidneys of the diabetic group showing nephrogenesis between 3rd and 4th period, revealing less demarcated cortical labyrinth and medullary rays, and decreased number of fetal glomeruli and developed convoluted tubules in the deeper cortical and juxtramedullary regions with scattered ‘-shaped’bodies, residual advancing ampullae of ureteric buds, diminished branching of developing convoluted tubules, and presence of adjacent metanephric mesenchyme in subcapsular superficial cortex or cortical labyrinth suggestive of abnormal nephrogenesis in both quantity and quality (Fig. 4a). Careful counting revealed newborn nephron number in offspring of diabetic mothers as significantly lower than in normoglycemic mothers (hyperglycemic vs normoglycemic newborn: 1974±131 vs 2985±124 in number [both n = 6; p<0.001]).

Bottom Line: Preliminary study found EGR-1 persistently expressed with GDNF in hyperglycemic environment.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.

View Article: PubMed Central - PubMed

Affiliation: Clinical Immunology Center, China Medical University Hospital, Taichung, Taiwan. cylin@mail.cmuh.org.tw

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.

Show MeSH
Related in: MedlinePlus