María de los Ángeles Granados-Silvestre

Garlic ameliorates gentamicin nephrotoxicity: relation to antioxidant enzymes

Reactive oxygen species are involved in gentamicin (GM) nephrotoxicity, and garlic is effective in preventing or ameliorating oxidative stress. Therefore, the effect of garlic on GM nephrotoxicity was investigated in this work. Four groups of rats were studied: (i) fed normal diet (CT), (ii) treated with GM (GM), (iii) fed 2% garlic diet (GA), and (iv) treated with GM and 2% garlic diet (GM + GA). Rats were placed in metabolic cages and GM nephrotoxicity was induced by injections of GM (75 mg/kg every 12 h) for 6 d. Lipoperoxidation and enzyme determinations were made in renal cortex on day 7. GM nephrotoxicity was made evident on day 7 by (i) tubular histological damage, (ii) enhanced BUN and urinary excretion of N-acetyl-beta-D-glucosaminidase, and (iii) decreased creatinine clearance. These alterations were prevented or ameliorated in GM + GA group. The rise in lipoperoxidation and the decrease in Mn-SOD and glutathione peroxidase (GPx) activities observed in the GM group, were prevented in the GM + GA group. Cu, Zn-SOD activity and Mn-SOD and Cu,Zn-SOD content did not change. CAT activity and content decreased in the GM, GA, and GM + GA groups. CAT mRNA levels decreased in the GM group. The protective effect of garlic is associated with the prevention of the decrease of Mn-SOD and GPx activities and with the rise of lipoperoxidation in renal cortex.

Post-transcriptional control of catalase expression in garlic-treated rats.

Regulation of catalase (CAT) expression, a major antioxidant enzyme that detoxifies H2O2, is very complex. Garlic is effective to prevent or ameliorate oxidative stress probably through its intrinsic antioxidant properties and/or to its ability to modify antioxidant enzyme expression. In this paper we studied the effect of a 2% garlic diet on the renal and hepatic CAT expression (mRNA levels, and enzyme activity, content, synthesis, and degradation). The study was made 2 weeks after feeding rats with a 2% garlic diet. CAT activity and content were measured by a spectrophotometric method and Western blot, respectively. CAT mRNA levels and CAT synthesis (ks) and degradation (kD) in vivo were measured by Northern blot and kinetic of reappearance of CAT activity after aminotriazole injection, respectively. Garlic-treatment decreased CAT activity and content, and CAT mRNA levels were unchanged in both tissues. ks decreased and kD remained unchanged in kidney and liver. The decrease in ks without changes in kD and CAT mRNA levels could explain the low CAT expression in garlic-fed rats. In vivo H2O2 generation in kidney and liver was markedly decreased in garlic-fed rats which could be due to a direct antioxidant effect of garlic. This may be the initial event in the garlic-fed rats that leads to the decreased CAT expression. Our data strongly suggest that the diminished renal and hepatic CAT expression in garlic-fed rats is mediated by post-transcriptional changes (mainly low translational efficiency) which could be an adaptation to the low H2O2.

Effect of the in vivo catalase inhibition on aminonucleoside nephrosis

Reactive oxygen species have been involved in the pathophysiology of puromycin aminonucleoside (PAN)-nephrosis. The role of H2O2 in these rats may be studied modulating the amount or activity of catalase, which breakdowns H2O2 to water and oxygen. To explore the role of H2O2 in this experimental model, we studied the effect of the in vivo catalase inhibiton with 3-amino-1,2,4-triazole (ATZ) on the course of PAN-nephrosis. Four groups of rats were studied: control rats (CT group), PAN-injected rats (PAN group), ATZ-injected rats (ATZ group), and ATZ- and PAN-injected rats (ATZPAN group). Rats were placed in metabolic cages to collect 24 h urine along the study, ATZ (1 g/kg) was given 24 h before PAN injection (75 mg/kg), and the proteinuria was measured on days 0, 2, 4, 6, 8, and 10. Proteinuria started before (day 4) and was significantly higher on days 6, 8, and 10 in the ATZPAN group than in the PAN group. On day 10, hypercholesterolemia was significantly higher in the ATZPAN group than in the PAN group. These data indicate that the in vivo catalase inhibition magnifies PAN-nephrosis, suggesting that H2O2 is produced in vivo and involved in the renal damage in this experimental disease.

Developmental programming of neonatal pancreatic β-cells by a maternal low-protein diet in rats involves a switch from proliferation to differentiation

Maternal low-protein diets (LP) impair pancreatic β-cell development, resulting in later-life failure and susceptibility to type 2 diabetes (T2D). We hypothesized that intrauterine and/or postnatal developmental programming seen in this situation involve altered β-cell structure and relative time course of expression of genes critical to β-cell differentiation and growth. Pregnant Wistar rats were fed either control (C) 20% or restricted (R) 6% protein diets during pregnancy (1st letter) and/or lactation (2nd letter) in four groups: CC, RR, RC, and CR. At postnatal days 7 and 21, we measured male offspring β-cell fraction, mass, proliferation, aggregate number, and size as well as mRNA level for 13 key genes regulating β-cell development and function in isolated islets. Compared with CC, pre- and postnatal LP (RR) decreased β-cell fraction, mass, proliferation, aggregate size, and number and increased Hnf1a, Hnf4a, Pdx1, Isl1, Rfx6, and Slc2a2 mRNA levels. LP only in pregnancy (RC) also decreased β-cell fraction, mass, proliferation, aggregate size, and number and increased Hnf1a, Hnf4a, Pdx1, Rfx6, and Ins mRNA levels. Postnatal LP offspring (CR) showed decreased β-cell mass but increased β-cell fraction, aggregate number, and Hnf1a, Hnf4a, Rfx6, and Slc2a2 mRNA levels. We conclude that LP in pregnancy sets the trajectory of postnatal β-cell growth and differentiation, whereas LP in lactation has smaller effects. We propose that LP promotes differentiation through upregulation of transcription factors that stimulate differentiation at the expense of proliferation. This results in a decreased β-cell reserve, which can contribute to later-life predisposition to T2D.

Diabetes susceptibility in Mayas: Evidence for the involvement of polymorphisms in HHEX, HNF4α, KCNJ11, PPARγ, CDKN2A/2B, SLC30A8, CDC123/CAMK1D, TCF7L2, ABCA1 and SLC16A11 genes.

Association of type 2 diabetes (T2D) with common variants in HHEX, HNF4α, KCNJ11, PPARγ, CDKN2A/2B, SLC30A8, CDC123/CAMK1D, TCF7L2, ABCA1 and SLC16A11 genes have been reported, mainly in populations of European and Asian ancestry and to a lesser extent in Latin Americans. Thus, we aimed to investigate the contribution of rs1111875 (HHEX), rs1800961 (HNF4α), rs5219 (KCNJ11), rs1801282 (PPARγ), rs10811661 (CDKN2A/2B), rs13266634 (SLC30A8), rs12779790 (CDC123/CAMK1D), rs7903146 (TCF7L2), rs9282541 (ABCA1) and rs13342692 (SLC16A11) polymorphisms in the genetic background of Maya population to associate their susceptibility to develop T2D. This is one of the first studies designed specifically to investigate the inherited component of T2D in the indigenous population of Mexico. SNPs were genotyped by allelic discrimination method in 575 unrelated Maya individuals. Two SNPs rs10811661 and rs928254 were significantly associated with T2D after adjusting for BMI; rs10811661 in a recessive and rs9282541 in a dominant model. Additionally, we found phenotypical alterations associated with genetic variants: HDL to rs9282541 and insulin to rs13342692. In conclusion, these findings support an association of genetic polymorphisms to develop T2D in Maya population.

Garlic ameliorates hyperlipidemia in chronic aminonucleoside nephrosis.

Nephrotic syndrome (NS) is characterized by proteinuria, oxidative stress and endogenous hyperlipidemia. Hyperlipidemia and oxidative stress may be involved in coronary heart disease and the progression of renal damage in these patients. Garlic has been suggested to be beneficial in various disease states. Some of the beneficial effects of garlic may be secondary to its hypolipidemic and antioxidant properties. Therefore, the effect of a 2% garlic diet on acute and chronic experimental NS induced by puromycin aminonucleoside (PAN) was studied in this work. Acute NS was induced by a single injection of PAN to rats which were sacrificed 10 days later. Chronic NS was induced by repeated injections of PAN to rats which were sacrificed 84 days after the first injection. Garlic treatment was unable to modify proteinuria in either acute or chronic NS, and hypercholesterolemia and hypertriglyceridemia in acute NS. However, garlic treatment diminished significantly total-cholesterol, LDL-cholesterol and triglycerides, but not HDL-cholesterol in chronic NS. Garlic induced no change in the percentage of sclerotic glomeruli in chronic NS and a significative decrease on the percentage of sclerotic area of these glomeruli (33 ± 3% in NS+Garlic group vs. 47 ± 4% in NS group, p = 0.0126). The enhanced in vivo renal H2O2 production and the diminished renal Cu, Zn-SOD and catalase activities in acute NS, and the decreased renal catalase activity in chronic NS were not prevented by garlic treatment. These data indicate that garlic treatment ameliorates hyperlipidemia and renal damage in chronic NS which is unrelated to proteinuria or antioxidant enzymes.

High frequency of T130I mutation of HNF4A gene in Mexican patients with early-onset type 2 diabetes

To the Editor: HNF4A is a member of the nuclear hormone receptor family (1), which regulates the expression of a large number of genes in the liver, pancreas, kidney, intestine and other tissues (2). In liver, HNF4A regulates gluconeogenesis, whereas in pancreas it directs the expression and secretion of insulin (3). In mouse, the complete embryonic disruption of HNF4A is lethal (4). HNF4A was the first transcription factor where mutations of the gene were associated with maturity-onset diabetes of the young (5) and presently, more than 20 mutations in HNF4A have been reported (6). The T130I functional variant has been found in Japanese (7), as well as in Danish (8) populations, where it has been associated to type 2 diabetes (T2D). The mutation T130I affects the DNA binding domain, resulting in the reduction of the transactivation function of HNF4A protein. Two studies have revealed the impact of T130I mutation upon the transcriptional process. Zhu et al. (9) showed a decreased activity of T130I as compared with the wild type in primary culture of mouse hepatocyte and in HepG2 cell line, but not in the MIN6 cell line. In a different study, Ek et al. (10) demonstrated a decreased transcriptional activity of T130I in Cos6 cells. The aim of the present work was to study the contribution of HNF4A gene variants in the pathogenesis of early-onset type 2 diabetes in Mexican subjects. The entire coding region of the gene was studied to identify gene variants that could be further tested in a case-control association study. The T2D group consists of 100 early-onset (,35 years of age), non-obese diabetic patients recruited at Hospital Juárez de México in México City. Diagnosis of type 2 diabetes was based on the American Diabetes Association Criteria (11). The protocol and informed consent forms were approved by the Human Research Ethical Committee of the Hospital Juárez de México. DNA was extracted from peripheral blood lymphocytes. Polymorphic screening of the coding region of HNF4A gene was performed by polymerase chain reaction–single-strand conformation polymorphism and sequence analysis. Analysis of the whole coding region HNF4A in both T2D and non-diabetic subjects resulted in identification of the T130I sequence change at exon 4 as the only variant. Despite the reduced number of individuals, the genotype distribution among groups revealed a significant association of T130I to early-onset T2D (T130I genotypes, 16% for diabetic subjects vs 5.6% for non-diabetic subjects, odds ratio 1⁄4 3.38, 95% confidence interval 1⁄4 1.14–10.03, p 1⁄4 0.028). Different studies of Finnish, Swedish, USA-Caucasians and Japanese populations have documented the presence of T130I mutation, and its association to late-onset diabetes in Danish (allelic frequency of diabetics 1⁄4 4.7% and controls 1⁄4 1.9%) and in Japanese (allelic frequency of diabetics 1⁄4 1.7% and controls 1⁄4 0.4%) populations. In this regard, the frequency of I130 allele found among Mexican individuals represents the highest reported so far (8%), and the increased frequency of T130I genotype among early-onset T2D cases strongly suggest the participation of T130I functional variant as major susceptibility allele for the development of T2D in Mexicans. The anthropometric results (Table 1) demonstrate that T2D heterozygotes carriers of I130 allele presented a significantly lower body mass index (BMI) and waist-hip ratio compared with the homozygotes T130T carriers. Additionally, T130I carriers displayed an earlier development of complications. From the 16 patients who carried the T130I mutation, all of them presented chronic complications. Particularly, they had two times more nephropathy than the T homozygotes carriers. This finding in addition with a lower

LOC387761 polymorphism is associated with type 2 diabetes in the Mexican population.

Worldwide researchers have invested time, effort, and money during the last years to find new genes associated with diabetes susceptibility, such as LOC387761, HHEX, EXT2, and SLC30A8. The aim of the present study was to evaluate whether single-nucleotide polymorphisms (SNPs) of these genes are associated with type 2 diabetes (T2D) and metabolic traits in the Mexican population. We also assessed these SNPs in Mexican indigenous groups to identify a possible inherited susceptibility. Seven SNPs were analyzed in 789 Mexicans (234 control subjects, 455 type 2 diabetic patients, and 100 of indigenous origin), using the KASPar assay (KBioscience Company). Analysis of the data showed an association of the LOC387761 SNP rs7480010 with T2D (p = 0.019). The risk allele A of rs7480010 increased body mass index in diabetic patients (p = 0.01). In addition, there was no association between T2D and the SNPs of HHEX, EXT2, and SLC30A8. Our findings suggest that the SNP rs7480010 (LOC387761) can contribute to a failure in insulin secretion, thus increasing the susceptibility to T2D in Mexicans.

Síndrome metabólico en niños mexicanos: poca efectividad de las definiciones diagnósticas

BACKGROUND Early identification of children with metabolic syndrome (MS) is essential to decrease the risk of developing diabetes and cardiovascular disease in adulthood. Detection of MS is however challenging because of the different definitions for diagnosis; as a result, preventive actions are not taken in some children at risk. The study objective was therefore to compare prevalence of MS in children according to the IDF, NCEP-ATP-III, Cook, de Ferranti and Weiss definitions, considering insulin resistance (IR) markers such as HOMA-IR and/or metabolic index (MI). METHODS A total of 508 Mexican children (aged 9 to 13 years) from seven schools were enrolled in a cross-sectional study. Somatometric, biochemical, and hormonal measurements were evaluated. RESULTS Frequency of MS was 2.4-45.9% depending on the definition used. Frequency of IR in children not diagnosed with MS was 12.4-25.2% using HOMA-IR and 4.0-16.3% using MI. When HOMA-IR or MI was included in each of the definitions, frequency of MS was 8.5-50.2% and 7.7-46.9% respectively. The kappa value including HOMA-IR and/or MI was greater than 0.8. CONCLUSIONS This study demonstrated the poor effectiveness of the current criteria used to diagnose MS in Mexican children, as shown by the variability in the definitions and by the presence of IR in children who not diagnosed with MS. Inclusion of HOMA-IR and/or MI in definitions of MS (thus increasing agreement between them) decreases the chance of excluding children at risk and allows for MS prevalence between populations.

Genetic variability of CYP2C9*2 and CYP2C9*3 in seven indigenous groups from Mexico

AIM CYP2C9 is one of the major drug metabolizing enzymes, however, little is known about polymorphisms in CYP2C9 gene and pharmacological implications in Mexican indigenous populations. Thus, frequencies of CYP2C9*2 and CYP2C9*3 alleles were evaluated in indigenous groups located in northwest (Cora), center (Mazahua and Teenek), south (Chatino and Mixteco) and southeast (Chontal and Maya) regions Mexico. MATERIALS & METHODS Allelic discrimination was performed by real-time PCR. RESULTS CYP2C9*2 allele was found only in Chontal and Maya groups, despite the low contribution of Caucasian component in these populations. CYP2C9*3 allele was present in all populations except in Mazahua, showing a wide genetic variability in the studied populations. Interestingly, we found significant differences between indigenous groups in CYP2C9*3 allele, even in groups located at the same region and belonging to the same linguistic family. CONCLUSION These results contribute to laying the pharmacogenetic bases in Mexico, in addition to improving treatment, taking into account the genetic interethnic differences.