The Effect Of Intrauterine Growth Restriction And Postnatal High-protein Diet On Renal Proteomic Expression In Rats

Intrauterine growth restriction (IUGR) has long-term effects on various organisms through fetal programming. IUGR causes hormone imbalance and metabolic disorders, affects growth and development in many organs and is associated with many diseases that can jeopardize human health such as hypertension, coronary heart disease, diabetes and chronic kidney disease. Human studies of the association between IUGR and renal diseases indicate that, in contrast to the normal fetal kidney, the IUGR fetal kidney has less volume, with a significant reduction in the number of glomeruli. In addition, long-term follow-up after birth shows significantly lower glomerular filtration and greater incidence of proteinuria in the IUGR group compared to the control group. Our previous animal studies have also shown a decrease in the number of glomeruli in IUGR newborn rats. At the same time, the cortex thickness in IUGR group was significantly thinner than that in control group, while the thickness of nephrogenic zone in IUGR group was significantly larger than that in control group, with an increased incidence of proteinuria and hypertension in the postnatal follow-up period. However, the mechanism underlying IUGR-induced abnormal nephrogenesis and the pathogenesis of IUGR-induced postnatal kidney diseases have not been fully clarified.Previous theories have suggested that a high protein diet for infants in whom IUGR is diagnosed, at an early postnatal stage may assist rapid postnatal growth, but the high protein diet may further exacerbate kidney injury. Our previous animal studies have shown that postnatal overnutrition following IUGR causes more severe hypertension and proteinuria than IUGR itself. Currently, there is no definite conclusion on the proper postnatal nutritional intervention for IUGR fetuses.In the past few years, proteomic technology has been used extensively in basic medical research. It has been used to study systemic and quantitative proteomic changes in tissues and cells at different disease progression stages. This is very important when studying disease pathogenesis and when searching for new drug targets. Therefore, we investigated the differences of proteomic profiles between normal and IUGR neonatal rats, the differences of proteomic profiles between normal and IUGR adult rats and the effects of postnatal high-protein diet on the proteomic profiles of kidney by a comparative proteomic method to identify the possible mechanism of nephrogenesis in IUGR rats, the key proteins that are associated with IUGR-induced kidney injury and the effects of high-protein diet intervention on the kidney.In order to explore the possible mechanism of abnormal nephrogenesis in IUGR rats, a low-protein isocaloric diet consisting of 6% protein was provided to the study group throughout the entire pregnancy period until natural labor. The resulting newborn rats with body weight 2 standard deviations below the average were assigned to the neonatal IUGR rats (IUGR group). The normal control group was supplied during the gestation period with conventional feed (22% protein) until natural labor. The resulting neonatal rats were assigned to the control group. Eight neonatal IUGR kidneys (average weight 48±4 mg/kidney, total weight 385 mg) and six neonatal normal kidneys (average weight 64±4 mg/kidney, total weight 383 mg) were mixed for total protein extraction. A series methods including two-dimensional gel electrophoresis, silver staining, mass spectrometry and database searching were used. The differential expression and mass spectrometry analysis found that eleven protein spots were expressed only in the IUGR group (incuding non-specific dipeptidase, methylmalonate semialdehyde dehydrogenase, transketolase, uridine monophosphate synthetase, vimentin, apolipoprotein A-IV, cytokeratin 10,26S protease regulatory subunit-7, delta-1-pyrroline-5-carboxylate dehydrogenase, retinal dehydrogenase-1 and Rho GDP dissociation inhibitor alpha) and one spot only in the control group, which was splicing factor (arginine/serine-rich 9). Seven protein spots were up-regulated more than fivefold (including uroporphyrinogen decarboxylase,75kDa glucose regulated protein, heterogeneous nuclear ribonucleoprotein K,227kDa spindle and centromere associated protein, disulfide-isomerase A3, cell division protein kinase-2 and alpha-electron transfer flavoprotein) and two spots were down-regulated more than fivefold (including gamma-actin and perlecan) in the IUGR group compared with those in the control group. These proteins are involved primarily in energy metabolism, oxidation and reduction, signal transduction, cell proliferation and apoptosis. Meanwhile, there were four structural molecules which were vimentin, perlecan, gamma-actin and cytokeratin 10. Confirmation of vimentin and perlecan expression by western blot and immunohistochemistry showed that vimentin was expressed primarily in glomeruli, and its expression was significantly increased in the IUGR group compared to the control group, while perlecan was expressed both in the glomeruli and the tubules, and the level of perlecan was significantly reduced in the IUGR group compared to the control group. These data were consistent with the findings using the proteomic approach.In order to identify the key proteins that are associated with IUGR-induced kidney injury and the effects of high-protein diet intervention on the kidney, IUGR pups were divided into two groups, fed with either a conventional diet (containing 22% protein, IUGR group) or a high-protein diet (containing 30% protein, HP group) until 12 weeks after birth. The normal control group was fed with a conventional pregnancy diet (22% protein) until natural delivery, and the newborn rats were fed with a conventional diet (22% protein, control group) until 12 weeks after birth. At 12 weeks of age,6 kidneys were selected from each group and were mixed for total protein extraction. A series methods including two-dimensional gel electrophoresis, coomassie brilliant blue staining, mass spectrometry and database searching were used. The differential expression and mass spectrometry analysis found that, compared with control group, a total of eleven proteins showed the same trend of expression changes in IUGR group and HP group. These eleven proteins included glutathione-S-transferase alpha-1, transketolase, fructose-bisphosphate aldolase A and aconitate hydratase that participate in body metabolism; long-chain specific acyl-CoA dehydrogenase, hydroxyacid oxidase-2, retinal dehydrogenase-1 and glutamate dehydrogenase-1 that participate in oxidation-reduction; alpha-enolase, alpha-crystallin and nucleoside diphosphate kinase B that participate in transcriptional and apoptosis regulation. These differentially expressed proteins probably participate in postnatal kidney disease in IUGR rats. Other eight proteins showed differential expression only after high-protein nutritional intervention. These included chloride intracellular channel-1 that participates in apoptosis regulation; isocitrate dehydrogenase, disulfide-isomerase A3 and cytochrome b-c1 complex that participate in oxidation-reduction; GM2 ganglioside activator protein, aspartoacylase-2, aminoacylase-1 and 5,10-methenyltetrahydrofolate synthetase that participate in body metabolism. These differential expression proteins may be related to the effects of high-protein diet intervention on the kidneys of IUGR rats. Meanwhile, it was found that compared with control group, two proteins which were capping protein (actin filament) and prohibitin had consecutive changes among three groups (the ratio of control group to IUGR group>5 and no obvious expression in HP group). These two proteins may be involved both in postnatal kidney disease in IUGR and the effects of high-protein diet intervention on the kidneys of IUGR. Confirmation of prohibitin expression by western blot and immunohistochemistry showed that prohibitin was primarily expressed in renal tubular epithelial cells. Its expression in IUGR group was lower than control group and the expression in HP group was even lower. This data was consistent with our findings using the proteomic approach.In conclusion, data from this study may provide, at least partly, evidence that abnormality of metabolism, imbalance of redox and apoptosis, and disorder of cellular signal and cell proliferation may be the major mechanisms responsible for abnormal nephrogenesis in IUGR. Some key proteins including glutathione-S-transferase alpha-1, alpha-enolase, alpha-crystallin and nucleoside diphosphate kinase B probably are associated with IUGR-induced kidney injury and some other key proteins including chloride intracellular channel-1, isocitrate dehydrogenase are involved in the effects of high-protein diet intervention on the kidney. Meanwhile, capping protein (actin filament) and prohibitin may be involved both in postnatal kidney disease in IUGR and the effects of high-protein diet intervention on the kidneys of IUGR. The comparative proteomic approach has provided new avenues for future research to explore the pathogenesis of abnormal nephrogenesis in IUGR, IUGR-induced kidney injury and the effects of high-protein diet intervention on the kidney.