Aminopeptidase A (ENPEP) gene polymorphisms and preeclampsia: Descriptive analysis.

OBJECTIVE: The etiology of preeclampsia (PE) remains elusive. Recent genome-wide association studies have identified a number of genetic variants associated with blood pressure variations in east Asians. One of the genetic variants is the aminopeptidase A (ENPEP) gene, which converts angiotensin II to angiotensin III. The C allele of rs6825911 is a risk for hypertension. The current study investigated whether genetic variants of ENPEP play a role in the pathogenesis of preeclampsia. STUDY DESIGN: The study was a descriptive analysis of gene polymorphisms of ENPEP; 602 pregnant women of African ancestry [normotensive (n = 245) and PE (n = 357)] were recruited. The two groups were divided according to their HIV status. The PE group consisted of early- and late-onset sub-categories. A single nucleotide polymorphism of rs6825911 was analyzed using the TaqMan® Probe mix and by means of real time polymerase chain reaction. RESULTS: The risk of C allele for PE was 1.07 (95 % CI 0.83-1.38, P = 0.589) for allele comparison and the risk for preeclampsia CC to CT/TT was 1.33 (95 % CI 0.96-1.85, P = 0.086). The sub analysis for the PE group without HIV infection the risk of C allele was 1.25 (95 % CI 0.838-1.78, P = 0.199) and the risk of PE of CC to CT/TT was 1.51 (95 %CI: 0.96-2.35, P = 0.071). CONCLUSION: This is the first study in a homogenous South African population of African ancestry to show that the variant of ENPEP gene does not play a role in pathogenesis of preeclampsia.

Association of gene polymorphisms of four components of renin-angiotensin-aldosterone system and preeclampsia in South African black women.

OBJECTIVE: To investigate the association of the gene polymorphisms of: angiotensinogen (AGT), renin (REN), angiotensin II receptor 1 (AT1R) and angiotensin II receptor 2 (AT2R), in the pathogenesis of PE in South African Black women. METHODOLOGY (STUDY DESIGN): 603 pregnant women; 246 normotensive and 357 with PE (early-onset=187, late-onset=170), were recruited. Each study group was subdivided into HIV infected and uninfected groups. The distribution and frequencies of gene polymorphisms of AGT (M235T), REN (C-5312T), AT1R (A1166C) and AT2R (C3123A) were determined in purified DNA by Real Time Polymerase Chain Reaction. RESULTS: The distribution of T allele and TT genotype of AGT in PE were significantly higher than the normotensive group (95% vs 91%, OR 1.9, 95%CI 1.2-3.1, p=0.0051; 90% vs 83%, OR 1.84, 95%CI 1.11-3.05, p=0.01) respectively. The distributions of genotypes of REN, AT1R and AT2R were similar in PE and normotensive groups. CONCLUSION: The T allele of AGT may play a role in the pathogenesis of PE. The genotypes of REN, AT1R and AT2R were not associated with the development of PE.

Increased systemic inflammatory interleukin-1ß and interleukin-6 during agitation as predictors of Alzheimer's disease.

OBJECTS: Identification of biomarkers for Alzheimer's disease (AD) is important for its early diagnosis and prevention and a key in advancing our understanding of its pathophysiology. The aim of this study was to determine whether systemic inflammatory interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) as well as hypertension (HT), diabetes mellitus (DM), and body mass index (BMI) are predictors of AD. METHODS: We performed a 10-year follow-up study on 133 elderly who were institutionalized in a nursing home. The associations of IL-1ß and IL-6 at both rest and agitation, as well as HT, DM, and BMI at baseline, were analyzed with the incidences of vascular dementia (VD) and AD during a 10-year follow-up period. RESULTS: The Kaplan-Meier method with log-rank test and Cox regression analyses for the total of 133 subjects showed significantly higher incidences of both VD and AD in subjects with DM or HT at baseline. Resting IL-1ß or IL-6 value, or agitation score, was not significantly associated with the subsequent development of VD or AD. The analyses of 40 subjects who had shown agitation at least once in the previous 3 months demonstrated that IL-1ß and IL-6 values at the agitation stage were significantly associated with AD, but not with VD. CONCLUSION: Our results indicate that systemic inflammatory IL-1ß and IL-6 at the agitation stage are risk factors for the development of AD, but not VD. Inflammatory mechanisms for AD seem to be causal and specific to the development of AD.

An efficient linkage analysis strategy for autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited renal disorders in the world. Mutations in PKD1 are responsible for 80-95% of all autosomal dominant polycystic kidney disease (ADPKD). Although the need for linkage analysis of ADPKD is decreasing after the success of mutation detection at whole exons of PKD1, linkage analysis still has some advantages in detecting non-PKD1 families, thereby avoiding hopeless mutation analysis. METHODS: We evaluated ten microsatellite markers beside or inside PKD1 on chromosome 16p. Allele frequency and heterozygosity of each marker were calculated based on the 100 genotypes obtained from 50 normal Japanese. Automated microsatellite genotyping using ABI Prism 377 and GeneScan software was applied. Markers were mapped using radiation hybrid mapping. Finally, this strategy was applied in the linkage analysis of 6 independent Japanese ADPKD families. RESULTS: D16S3024, D16S3082, D16S3027 and D16S423 showed high heterozygosity (> 0.80) in a normal Japanese population and sufficient proximity to the PKD1 gene for linkage analysis. We could successfully analyze 144 genotypes within 7 hours. This strategy produced theoretically near-maximum LOD scores in 4 independent Japanese families inheriting ADPKD. CONCLUSIONS: Automated genotyping using microsatellite markers, D16S3024, D16S3082, D16S3027 and D16S423 are very useful in the linkage analysis of ADPKD.

A missense mutation, A156T, in the alpha-galactosidase A gene causes typical Fabry disease.

AIMS: Fabry disease is a rare but important cause of end-stage renal disease. Recent molecular investigations on alpha-galactosidase A (alpha-Gal A) have proven the existence of atypical variants in Fabry disease, making genotype assessment of each phenotype indispensable. We report here a missense mutation, which causes a typical form of Fabry disease. MATERIAL AND METHODS: The proband, a 45-year-old man, presented with acroparesthesias, hypohidrosis, left ventricular hypertrophy, renal involvement (proteinuria and renal insufficiency) with typical microscopic findings and extremely reduced plasma alpha-Gal A activity, indicating the typical form of the disease. Total RNA was isolated from the proband's cultured fibroblasts, reverse-transcribed and amplified for direct sequencing of alpha-Gal A. Genomic DNA of the proband's mother and 75 controls (50 males and 25 females) living in the same area as the proband was also examined. RESULTS: Sequencing of the cDNA revealed a substitution of G to A in codon 156 of alpha-Gal A, resulting in a single amino acid change from alanine to threonine (A156T). The mutation can be detected with PCR-RFLP with SfaNI digestion. This technique revealed that the mother was a heterozygote of A156T with no A156T noted in the 100 haplotypes of the controls. With a vigorous search of the same mutation in the literature, no previous description was found other than one case listed in several review papers as a classic phenotype without any other information. In our study, we examined A156T in a pedigree and demonstrated that the mutation was not a polymorphic variant in our area. CONCLUSION: Taken together, the present results strongly suggest that the missense mutation, A156T, in the alpha-Gal A gene causes typical Fabry disease.

Effect of ACE gene polymorphism on age at renal death in polycystic kidney disease in Japan.

Polycystic kidney disease (PKD) is one of the most common genetic disorders and a major cause of renal death or end-stage renal disease (ESRD) requiring regular hemodialysis. The responsible genes recently have been cloned; however, genetic factors influencing the rate of progression to ESRD in patients with PKD have yet to be defined. Several studies have shown increased activity of the renin-angiotensin system (RAS) in patients with PKD. In addition, genetic polymorphisms of the RAS have been associated with the development of cardiovascular diseases. Therefore, these polymorphisms are good candidates for disease-modifying genetic factors or markers in PKD. In two previous reports of white subjects with a cumulative survival analysis, it was suggested that patients with P:KD1 homozygous for the deletion allele of the angiotensin-converting enzyme (ACE) gene are at increased risk for early renal death. To confirm this hypothesis in Japanese subjects, 103 individuals with PKD were genotyped for several components of the RAS, ie, ACE insertion/deletion (I/D) polymorphism, angiotensinogen (AGT) M235T, and angiotensin II type 1 receptor (AT1) A1166C. Seventy-six of the 103 patients (73.8%) reached ESRD at an average age of 52.1 +/- 11.3 years. The frequencies of each genotype of the genes were similar to those expected from Hardy-Weinberg equilibrium. There was a tendency to an excess of patients homozygous for the D allele in patients with ESRD (DD in patients with ESRD, 11.8%; DD in patients without ESRD, 3.7%; chi-square, 1.505; P: = 0.22). Cumulative renal survival was significantly less in those with the DD genotype compared with ID/II genotypes. Estimated mean renal survival was 46.4 years (95% confidence interval, 39.5 to 53.3) in subjects with the DD genotype and 57.2 years (95% confidence interval, 54.2 to 60.2) in ID/II genotypes (chi-square, 7.76; P: = 0.0053). There was no association between age at onset of ESRD and either M235T or A1166C polymorphism. These findings suggest that Japanese patients with PKD homozygous for the D allele of the ACE gene are at increased risk for developing ESRD at an early age.

Nephrotic syndrome due to focal glomerulosclerosis and undifferentiated carcinoma.

In neoplastic disorder-related nephrotic syndrome, focal glomerulosclerosis (FGS) has been reported mainly in hematological disorders like minimal change nephrotic syndrome (MCNS) in association with presumed T lymphocyte dysfunction. The association of FGS with cancer or solid tumor is rare. We report a case of nephrotic syndrome due to FGS in a patient with undifferentiated adenocarcinoma of the cystic duct. Although the underlying mechanism is unclear, the development of FGS seemed to be related to the poor histological differentiation of the cancer in the possibility of production of an active peptide.

Clinical characteristics of polycystic kidney disease with end-stage renal disease. The Kanazawa Renal Disease Study Group.

AIMS: Polycystic kidney disease (PKD) is one of important causes of end-stage renal disease (ESRD). However, there have been few detailed reports on PKD patients with ESRD. The present study was designed to clarify the clinical characteristics of PKD patients with ESRD. SUBJECTS AND METHODS: We collected data from 22 renal divisions in our region, where 63 of 1246 patients with ESRD (male/female: 31/32) were proven to suffer from PKD (5.06%). RESULTS: The average age at the induction of renal replacement therapy was 52.4 +/- 10.0 years. Of these patients, 32 (50.8%) had some family members with apparent PKD. Three (4.8%) and 4 (6.3%) had a history of subarachnoidal hemorrhage and intracerebral hemorrhage, respectively. One (1.6%) suffered from tuberous sclerosis. The prevalence of hypertension treated with antihypertensives, anemia treated with rHuEPO, hepatic cyst, pancreatic cyst, intracranial aneurysm and colonic diverticulum were 66.7%, 58.7%, 85.7%, 16.0%, 33.3% and 50.0%, respectively. CONCLUSION: There was no marked difference in general characteristics or history between the present subjects and those described in previous reports. However, the prevalence of complications seemed to be higher than previously estimated.

Regulation of human renin gene transcription by cAMP.

To delineate the cis-acting elements of the proximal promoter responsible for cAMP-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4 fold when the reporter plasmid contained the promoter region (-582 to +16). Mutation or deletion of the CRE diminished (1.7 fold) but did not abolish cAMP-induced transcription, demonstrating that region containing the CRE and region containing a Pit-1 site were both necessary for cAMP maximal induction. Taken together, these results show that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors (from chorionic and kidney cell origin), different from Pit-1, that interact with the Pit-1 response DNA elements.

Evidence that renal and chorionic tissues contain similar nuclear binding proteins that recognize the human renin promoter.

This study examines whether the human renal cortex, the major renin producing site, contains nuclear factors that bind to the human renin proximal promoter. Footprint analysis of the human renin promoter region showed that human renal cortex cell nuclear extracts interacted with 6 putative cis-elements (the Ets domain-protein, a Pit-1 like binding site, a CRE sequence, an ARP-1 like binding site, an AGE3 like region, and a unknown consensus region, designated element C). Transient DNA transfection studies on chorionic cells implicated the CRE and Pit-1 consensus sites in the regulation of renin gene transcription by cAMP. Electromobility shift assays showed that renal proteins bind specifically to these sequences, and that one of them is CREB; two others seem to be Ets-1 and ARP-1. These results raise the possibility that the human renal cortex and human chorionic cells have the same trans-acting factors that bind to the proximal human renin promoter.

Transcriptional induction of the human renin gene by cyclic AMP requires cyclic AMP response element-binding protein (CREB) and a factor binding a pituitary-specific trans-acting factor (Pit-1) motif.

To delineate the cis-acting elements of the proximal promoter responsible for cyclic AMP (cAMP)-induced human renin gene transcription, 5'-flanking regions of the human renin gene were fused to a luciferase reporter gene and transfected in chorionic cells. Forskolin treatment induced the expression of luciferase by 2.4-fold when the reporter plasmid contained the promoter region (-582 to + 16). Mutation or deletion of the cAMP response element (CRE) diminished (1.7-fold) but did not abolish cAMP-induced transcription, demonstrating that the (-582 to -145) region containing the CRE and the region (-145 to -38) containing a Pit-1 (pituitary-specific trans-acting factor) site were both necessary for cAMP maximal induction. To study the molecular events mediating the cAMP induction, DNase I footprinting and electromobility shift assays (EMSAs) were performed with renin-producing chorionic cell and kidney cortex cell nuclear extracts, showing that the CRE-binding protein (CREB) interacts with the CRE and that tissue-specific factors, distinct from Pit-1, specifically bind the renin Pit-1 motif. Taken together, these results demonstrate that the cAMP response of the human renin gene may involve CREB binding the CRE and tissue-specific factors, different from Pit-1, that interact with the Pit-1 response DNA elements.

Expression of PC2 and PC1/PC3 in human pheochromocytomas.

Expressions of two Kex2-related proteases, Pc2 and PC1/PC3, and of one of their possible substrates, proenkephalin, were examined in normal (n = 7) and various pathological (n = 48) human adrenal tissues. Northern blot analysis detected the expression of these genes in pheochromocytomas only. In the 20 pheochromocytomas studied with this technique, PC2, PC1/PC3 and proenkephalin were expressed in 85%, 50% and 90%, respectively. The presence of PC2 and PC1/PC3 was further confirmed using the sensitive RT/PCR techniques. Other evidence of human tumoral adrenal medullary PC2 expression was provided by in situ hybridization and immunohistochemistry. In addition, proenkephalin was expressed only in the pheochromocytomas expressing PC2 and/or PC1/PC3. These results demonstrate that functional Kex2-related endoproteases are expressed in human pheochromocytomas and may be involved in the processing of proenkephalin.

Co-expression of PC2 and proenkephalin in human tumoral adrenal medullary tissues.

Expression of PC2, a Kex2-related protease, and of one of its possible substrates, proenkephalin, was examined in normal adrenal glands (n = 7) and pheochromocytomas (n = 20). PC2 could only be detected in normal adrenal glands using the sensitive RT/PCR technique. By Northern blot, PC2 and proenkephalin were expressed in 85% and 90% of the 20 pheochromocytomas studied, respectively. Moreover, in situ hybridization and immunohistochemistry confirmed expression of PC2 in human tumoral adrenal medullary tissue. These results show for the first time expression of PC2 in human pheochromocytomas which may be involved in the processing of proenkephalin.

Post-column derivatization of guanidino compounds in high-performance liquid chromatography using ninhydrin.

A new method for the high-performance liquid chromatography of guanidino compounds using ninhydrin as the fluorescence reagent is described. Use of organic solvent is not required since ninhydrin is highly soluble in aqueous media, and the problem of precipitation formation occurring in the phenanthrenequinone method was avoided. Creatine was also assayed in the present procedure. Separation of ten guanidino compounds was completed within 30 min using a small-size (38 X 4.2 mm I.D.) strong cation-exchange column.