The fixation of living skin equivalents.

Histologic evaluations and immunohistochemical characterizations are important in studies of artificial organs such as skin equivalents. However, tissue compact organization is not easy to obtain when the artificial organ is constructed in vitro. Thus, appropriate fixation methods must be selected according to the compactness of the artificial organ and tissue engineering methodologies. The effects of several fixatives-Carnoy, Bouin's solution, formalin, paraformaldehyde, and paraformaldehyde-glutaraldehyde-were examined to select the best fixation method for preserving the structural and molecular markers of skin equivalents. Formalin-based fixatives ware found to be relatively free of the histologic problems (e.g., tissue shrinkage, poor structural preservation, weak stainability, and nonspecific immunolocalization) presented by the soft tissue fixatives (i.e., Carnoy or Bouin's solution). Unfortunately, the standard concentration of formalin induced detachment of epidermis from dermis, but this was prevented by reducing the concentration of the fixative. These findings suggest that fixation procedures should be selected for particular tissue and specific goals; in particular, they show that the paraformaldehyde-glutaraldehyde combination performed best in terms of preserving the histologic features of skin equivalents.

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.

Involvement of ERK AND p38 MAP kinase in AAPH-induced COX-2 expression in HaCaT cells.

Cyclooxygenase-2 (COX-2) appears to play an important role in inflammation and carcinogenesis, and 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) is a hydrophilic azo compound known to generate free radicals. Because reactive oxygen species (ROS) are known to elevate COX-2 expression, we evaluated the effect of AAPH on the expression of COX-2 in a human keratinocyte cell line, HaCaT. When cells were exposed to AAPH, marked COX-2 induction was observed. To clarify the signaling mechanism involved, we next investigated the effects of AAPH upon three major subfamilies of the mitogen-activated protein kinases (MAPKs). AAPH caused an increase in the phosphorylation of extracellular signal-regulated kinase (ERK), p38 and c-Jun NH(2)-terminal kinase (JNK). Furthermore, we found that PD98059, an ERK pathway inhibitor, and SB203580, a p38 MAPK inhibitor, diminished AAPH-induced COX-2 expression and PGE(2) production, whereas JNK inhibitor did not suppress COX-2 expression or PGE(2) production by AAPH. These findings suggest that the ERK and p38 MAPK pathways, but not the JNK pathway, are involved in AAPH-induced inflammatory progression. In addition, we found that both the water-soluble Vitamin E derivative, Trolox, and the green tea constituent, (-)-epigallocatechin gallate (EGCG), diminished AAPH-induced COX-2 expression and p38 activation.

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.

ACE inhibition modulates transforming growth factor-beta receptors in the young rat.

The renin-angiotensin system plays an important role in renal growth and development. Exposure of the neonate to angiotensin converting enzyme (ACE) inhibitors increases mortality and results in growth retardation and abnormal renal development. It has been demonstrated that ACE inhibition in the developing kidney reduces the renal expression of growth factors, which may account for renal growth impairment. This study was designed to investigate the relationship between renal growth impairment and the expression of transforming growth factor-beta1 (TGF-beta1), TGF-beta receptor I [TbetaRI, activin-like kinase (ALK)-1 and ALK-5], and TGF-beta receptor II (TbetaRII). Newborn rat pups were treated with enalapril (30 mg/kg per day) or vehicle for 7 days, and kidneys were removed for Western blotting of TGF-beta1, ALK-1, ALK-5, and TbetaRII, and for RT-PCR of ALK-5 and TbetaRII. TGF-beta1, ALK-1, ALK-5, and TbetaRII were also detected by immunohistochemistry. Enalapril treatment resulted in an increased mortality (30.4%) by day 7, and reduced body weight and kidney weight ( P<0.05 versus vehicle). Enalapril decreased renal TGF-beta1, ALK-1, and ALK-5 protein expression ( P<0.05). Also, enalapril decreased ALK-5 mRNA expression ( P<0.05). TbetaRII expression was not changed by enalapril treatment. These results indicate that ACE inhibition in the developing kidney decreases TGF-beta1, ALK-1, and ALK-5 expression, which may account for renal growth impairment. TbetaRII may not be modulated by ACE inhibition in the developing kidney.

Effect of intrauterine growth retardation on the progression of nephrotic syndrome.

BACKGROUND/AIM: Neonates with intrauterine growth retardation (IUGR) experience higher morbidity and mortality rates than appropriate-for-gestational-age (AGA) neonates. The purpose of our study was to clarify whether IUGR has any influences on the progression of nephrotic syndrome in children. METHODS: We performed a retrospective review of 56 children with nephrotic syndrome. IUGR was defined as a birth weight less than the tenth percentile for gestational age. Among 56 patients having nephrotic syndrome, 8 had IUGR, and 48 were AGA. RESULTS: The 24-hour urinary protein level in children with IUGR was significantly higher than that in children who were AGA (7.61 +/- 6.75 vs. 3.92 +/- 3.70 g/day, p < 0.05). There was a statistically significant difference in the incidence of steroid resistance (62.5 vs. 10.4%, p < 0.05) and in the time to remission (median 60 vs. 13 days, p < 0.05) between the children with IUGR and those being AGA. Also, there was a significant difference in the incidences of treatment with cytotoxic agents (75 vs. 29.2%, p < 0.05) and complications such as hypertension. CONCLUSIONS: Our report indicates that IUGR predicts an unfavorable progression of nephrotic syndrome. So, it is important for nephrologists to pay attention to the clinical course of nephrotic syndrome in neonates with IUGR.

Angiotensin converting enzyme inhibition decreases cell turnover in the neonatal rat heart.

The renin angiotensin system plays an important role in growth and development. Exposure of the neonate to an ACE inhibitor increases mortality and results in growth retardation and abnormal development. We have demonstrated that ACE inhibition in the developing kidney increases apoptosis and decreases cell proliferation, which may account for renal growth impairment. To evaluate the role of endogenous angiotensin in cardiac development, the relationship between ACE inhibition, cell proliferation, apoptosis, several modulators of apoptosis (bcl-2, bcl-xl, and clusterin) was examined in the developing rat heart. Thirty-five newborn rat pups were treated with enalapril (30 mg/kg/d) or a vehicle (control group) for 7 d, and hearts were removed for rt-PCR and Western blotting of bcl-2, bcl-xl, and clusterin. An additional 10 rat pups were treated with hydralazine (10 mg/kg/d) or a vehicle, to serve as a hypotensive control. Cell proliferation was determined by PCNA immunostaining, and apoptosis was detected using the total TUNEL technique. Enalapril treatment resulted in a 24% mortality, reduced body weight, and decreased heart weight (p < 0.05). Enalapril decreased proliferating myocytes by 23%, and reduced proliferating cardiac interstitial cells by 8.1% (p < 0.05). Enalapril also decreased myocytes apoptosis by 60%, but the proportion of myocytes undergoing apoptosis was 10-fold less than that of proliferating cells. Cardiac bcl-2 mRNA, clusterin mRNA, bcl-2 protein, and bcl-xl protein content were not changed, but clusterin protein expression was decreased by enalapril treatment. Hydralazine did not alter cardiac cell proliferation or apoptosis. We conclude that ACE inhibition decreases cell turnover in the developing rat heart, which may contribute to cardiac growth impairment. The loss of myocytes may lead to greater myocyte hypertrophy and myocardial damage during later life.