In Sun Bae

Genetic control of VEGF and TGF-β1 gene polymorphisms in childhood urinary tract infection and vesicoureteral reflux

We investigated whether genetic polymorphisms of vascular endothelial growth factor (VEGF) and transforming growth factor-β1 (TGF-β1), potential candidate genes in the pathogenesis of urinary tract infection (UTI) and vesicoureteral reflux (VUR), are associated with the susceptibility to UTI and VUR, and renal scarring. We recruited 89 controls and 86 UTI and 58 VUR children. The UTI group was subdivided into two groups according to renal scarring. Two polymorphisms of VEGF and three of TGF-β1 genes were investigated by using PCR-restriction fragment length polymorphism analysis. In both UTI and VUR groups, there was an increase in frequency of the VEGF –460 CC (control, 4.3; UTI, 15.9; VUR, 17.8%; p < 0.05), TGF-β1 –509 CC (control, 8.7; UTI, 34.6; VUR, 35.1%; p < 0.001), and TGF-β1 –800 GG genotypes (control, 19.1; UTI, 40.5; VUR, 40.4%; p < 0.05). An increase in the TGF-β1 +869 CC (scar-positive, 35.4; scar-negative, 10.3%; p < 0.05) and a decrease in the +869 TC genotype (scar-positive, 29.2; scar-negative, 55.2%; p < 0.05) were observed in the scar-positive subjects. There were no differences in +405 VEGF genotype frequencies. The VEGF T-460C and the TGF-β1 C-509T, G-800A, and T869C polymorphisms could be genetic markers of the process of UTI and VUR.

Aldosterone regulates cellular turnover and mitogen‐activated protein kinase family expression in the neonatal rat kidney

Growing evidence indicates that aldosterone is a potent mitogenic signal regulating genes involved in antiapoptosis, cell proliferation and growth. We investigated the role of endogenous aldosterone in renal development, cell proliferation and apoptosis, and mitogen‐activated protein kinase (MAPK) family expression. Newborn rats were treated with either spironolactone (200 mg/kg/d) in olive oil or only olive oil for 7 days. TUNEL assay and proliferating cell nuclear antigen (PCNA) stain were performed on kidney sections. Immunoblots, immunohistochemical (IHC) stain, and reverse transcriptase‐PCR for MAPKs were performed. PCNA‐positive proliferating cells decreased and apoptotic cells increased significantly with spironolactone (P < 0.05). In the spironolactone‐treated group, c‐jun N‐terminal kinase (JNK)‐2 expression increased, whereas extracellular signal regulated kinase (ERK)‐2 and p38 expressions decreased in immunoblots (P < 0.05) and IHC stain. ERK‐2 and p38 mRNA expressions increased in the spironolactone‐treated group (P < 0.05). This study demonstrates that aldosterone blockade in the developing kidney decreases cellular proliferation, increases apoptosis, and modulates the expressions of JNK‐2, ERK‐2, and p38. Aldosterone possibly participates in renal development and MAPK family may serve as, in part, the signaling intermediate through the mineralocorticoid receptor (MR) in the developing kidney. J. Cell. Physiol. 219: 724–733, 2009.

Angiotensin-converting enzyme inhibition modulates mitogen-activated protein kinase family expressions in the neonatal rat kidney.

Among the mitogen-activated protein kinase (MAPK) family members, extracellular signal–regulated kinase (ERK) promotes cell proliferation or differentiation, whereas c-jun N terminal kinase (JNK) and p38 MAPK are thought to inhibit cell growth and induce apoptosis. The MAPK family may plays some role during kidney development, when large-scale proliferation and apoptosis have been observed to occur. Also, in this period, the renin-angiotensin system is markedly activated. We have demonstrated that angiotensin-converting enzyme inhibition in the developing rat kidney increases apoptosis and decreases cell proliferation, which may account for renal growth impairment. The aim of this study, therefore, was to examine the relationship between the MAPK family and renin-angiotensin system during neonatal renal development. Newborn rat pups were treated with enalapril (30 mg · kg−1 · d−1) or normal saline for 7 d. Right kidneys of both groups were selected for immunohistochemical stains of MAPKs and activating transcription factor-2 (ATF-2), and left kidneys were selected for reverse transcriptase–PCR and immunoblot analysis of MAPKs, phospho-MAPKs, and ATF-2. To determine whether apoptosis is involved in the same tubules that highly expressed JNK and p38, we performed terminal deoxynucleotide transferase-mediated nick-end labeling stain for apoptotic cells and immunohistochemical stains for JNK-2, p38, and ATF-2 expression in the serial sections from the same kidney of the enalapril-treated group. In the enalapril-treated group, JNK-2, p38, phospho-JNK-2, phospho-p38, and ATF-2 protein expressions were significantly increased, and their immunoactivities were strongly detected in the proximal tubular epithelial cells in the cortex, compared with the control group. Especially JNK-2 and p38 expressions were highly activated and were spatially in accordance with the occurrence of apoptosis. ERK1/2 and phospho-ERK expressions were not changed by enalapril. These results suggest that the expressions of the MAPK family are modulated by angiotensin-converting enzyme inhibition in the developing kidney. JNK and p38 may be implicated to participate in angiotensin II–related intracellular signaling pathways of renal apoptosis in the developing kidney.

Genetic polymorphism of the renin-angiotensin system on the development of primary vesicoureteral reflux

Background: The familial clustering of vesicoureteral reflux (VUR) has suggested a genetic basis. This study was designed to investigate the genetic polymorphism of the renin-angiotensin system (RAS) in Korean children. Methods: Genetic polymorphism of angiotensin-converting enzyme (ACE) and angiotensin II receptor genes was evaluated in 67 primary VUR patients and compared to 58 controls with no urological abnormalities. To detect the relation of the risk factors of primary VUR with the genetic polymorphism, the distribution of ACE, AT1 and AT2 genotypes after stratification by risk factors was also studied in the primary VUR patients. Results: The incidence of AT2 A-1332G transition was significantly lower in primary VUR patients (p = 0.047). Furthermore, in the case of combination of ACE and AT2 gene, a significantly lower incidence of primary VUR was seen with II genotype of ACE and A-1332G transition in the AT2 receptor gene (p = 0.003). Concerning the risk factors of primary VUR, there were no biologically significant results. Conclusions: These findings indicate that a lower incidence of AT2 A-1332G transition is seen in primary VUR patients, at least in the Korean population. Also, in the case of combination of ACE and AT2 gene, the combination of ACE II genotype and AT2 A-1332G transition occurs infrequently in primary VUR.

Postnatal early overnutrition dysregulates the intrarenal renin–angiotensin system and extracellular matrix-linked molecules in juvenile male rats

Overnutrition during the perinatal period has been associated with susceptibility to obesity and related comorbidities. We examined the effects of postnatal early overnutrition on the development of juvenile obesity and the associated renal pathophysiological changes. Three or 10 pups per mother from rat pup litters were assigned to either the overnutrition or control groups during the first 21 days of life. The effects of overfeeding were measured at 28 days. The smaller male litter pups were heavier than the controls between 4 and 28 days after birth (P<.05). By 28 days of age, the kidney weight per body weight ratio decreased in the small litter group (P<.05). Circulating leptin levels increased in the small litter rats (P<.05). Overnutrition had no effect on renal cell proliferation, apoptosis, macrophages and glomerulosclerosis. In the immunoblots and immunohistochemistry, renin and angiotensin II type (AT) 2 receptor expression increased in the overfed rats (P<.05). By contrast, the plasminogen activator inhibitor (PAI)-1 and matrix metalloproteinase (MMP)-9 expression decreased in the overnutrition group (P<.05). The AT 1 receptor, tissue inhibitor of MMP-1, monocyte chemoattractant protein-1, tumor necrosis factor-α, osteopontin and adiponectin expression was not changed. Our data showed that postnatal early overfeeding led to hyperleptinemia, juvenile obesity and the acquired reset of renal maturation. Up-regulation of renin and AT2 and down-regulation of PAI-1 and MMP-9 might contribute to abnormal programming of renal growth in rats exposed to postnatal early overnutrition.

Aldosterone Modulates Cell Proliferation and Apoptosis in the Neonatal Rat Heart

In the present study, we investigated whether and how the mineralocorticoid receptor antagonist spironolactone affects cardiac growth and development through apoptosis and cell proliferation in the neonatal rat heart. Newborn rat pups were treated with spironolactone (200 mg/kg/d) for 7 days. The cell proliferation was studied by PCNA immunostaining. The treatment with spironolactone decreased proliferating myocytes by 32% (P<0.05), and reduced myocytes apoptosis by 29% (P<0.05). Immunoblot and immunohistochemistry for the expression of p38, p53, clusterin, TGF-β2, and extracellular signal-regulated kinase were performed. In the spironolactone group, p38, p53, clusterin, and TGF-β2 protein expression was significantly decreased (P<0.05). These results indicate that aldosterone inhibition in the developing rat heart induces cardiac growth impairment by decreasing proliferation and apoptosis of myocytes.

Overweight, hypertension and renal dysfunction in adulthood of neonatally overfed rats

Accelerated growth in early infancy has been associated with later cardiovascular and metabolic diseases. We investigated the influence of overnutrition during neonatal periods on the development of renal pathophysiological changes in adult offspring rats. Three or 10 male pups per mother were assigned to either the small litter (SL) or normal litter (NL) control groups during the first 21 days of life. The effects of early postnatal overnutrition on body weight, blood pressure and renal changes were determined at 3 and 6 months. Pups in the SL group weighed more than controls between 7 days and 6 months of age (P<.05). In the SL group, serum creatinine levels were higher at 3 and 6 months (P<.05), and at 6 months, blood pressure levels were higher than those of the controls (P<.05). The number of ED-1 positive macrophages in renal cortex and glomerulosclerosis index increased in the SL group at 3 and 6 months (P<.05). Additionally, cortical apoptotic cells increased in the SL group at 6 months (P<.05). Immunoblotting and immunohistochemistry showed that matrix metalloproteinase (MMP)-9 protein expressions decreased and tissue inhibitor of MMP-1, tumor necrosis factor-α, osteopontin and adiponectin expressions increased in the SL group at 3 months (P<.05). However, at 6 months, MMP-9 expression was elevated, and osteopontin expression remained elevated in the SL group (P<.05). Early postnatal overfeeding can lead to lasting overweight, hypertension and renal dysfunction and place a greater burden on the kidney.

Postnatal early overnutrition causes long-term renal decline in aging male rats

Background:We evaluated the influence of postnatal early overnutrition on renal pathophysiological changes in aging rats.Methods:Three or 10 male pups per mother were assigned to either the small litter (SL) or normal litter (control) groups, respectively, during the first 21 d of life. The effects of early postnatal overnutrition were determined at 12 mo.Results:SL rats weighed more than controls between 4 d and 6 mo of age (P < 0.05). However, between 6 and 12 mo, body weights in both groups were not different. In the SL group, at 12 mo, systolic blood pressure was higher and creatinine clearance was lower than the same in controls (P < 0.05). Numbers of CD68 (ED1)-positive macrophages and apoptotic cells in renal cortex were higher in SL rats (P < 0.05). Furthermore, index scores for glomerulosclerosis and tubulointerstitial fibrosis were higher in the SL group (P < 0.05). Significantly less glomeruli per section area were found in aging SL rats (P < 0.05). Immunoblotting and immunohistochemistry showed decreased intrarenal renin expression in SL rats (P < 0.05).Conclusion:Early postnatal overnutrition can potentiate structural and functional abnormalities in the aging kidney and can lead to systolic hypertension with reduced intrarenal renin activity.

Endothelin A Receptor Blockade Influences Apoptosis and Cellular Proliferation in the Developing Rat Kidney

Endothelin systems are believed to play important roles in the emergence and maintenance of functions of various organs during perinatal development, including the kidney. The present study was designed to investigate the roles of endothelin systems on physiologic renal growth and development. Newborn rat pups were treated with either Bristol-Myers Squibb-182874 (30 mg/kg/day), a selective endothelin A receptor (ETAR) antagonist, or vehicle for 7 days. To identify cellular changes, kidneys were examined for apoptotic cells by terminal deoxynucleotide transferase-mediated nick-end labeling stain and proliferating cell nuclear antigen (PCNA) by immunohistochemical (IHC) stain. To clarify the molecular control of these processes, immunoblots and reverse transcriptase-polymerase chain reaction for Clusterin, Bcl-2, Bcl-XL, Bax, and p53 were performed. ETAR antagonist treatment resulted in reduced kidney weights, decreased PCNA-positive proliferating cells, and increased apoptotic cells. The protein expressions of renal Bcl-XL and Bax in the ETAR antagonist-treated group were significantly decreased, whereas the mRNA expressions of these genes were not changed. There were no significant differences in the expressions of Clusterin, Bcl-2, and p53. In conclusion, inhibition of endogenous endothelins impairs renal growth, in which decreased cellular proliferation, increased apoptosis and decreased expressions of renal Bcl-XL and Bax are possibly implicated.

Spironolactone and enalapril differentially up-regulate the expression of VEGF and heme oxygenase-1 in the neonatal rat kidney.

Both the renin-angiotensin-aldosterone system (RAAS) and hypoxia are vital physiological factors involved in the control of nephrogenesis and vascularization. We investigated the relationship between RAAS and hypoxia in the developing kidney. The expression of VEGF and heme oxygenase (HO)-1 related with the oxygen was analyzed in the enalapril- or spironolactone-treated neonatal rat kidneys. Enalapril (30 mg/kg/d) or spironolactone (200 mg/kg/d) was administered to newborn rat pups for 7 d. The newborn rats were injected i.p. with pimonidazole (200 mg/kg), a marker of severe tissue hypoxia, 1 h before killing. VEGF and HO-1 protein expression was significantly increased by immunoblots and immunohistochemistry in both the enalapril- and spironolactone-treated kidneys, compared with the controls (p < 0.05). HO-1 mRNA expression was increased in the spironolactone-treated group (p < 0.05). The immunoactivity of pimonidazole was not different from that of the controls in the enalapril-treated group, whereas it was increased in the spironolactone-treated group. The results of this study indicate that aldosterone blockade or angiotensin II inhibition in the developing rat kidney up-regulated renal VEGF and HO-1 expression regardless of the hypoxic conditions and may differentially modulate VEGF and HO-1 production.