Asociación del polimorfismo rs5186 del gen AGTR1 con disminución de la TFGe en pacientes con diabetes tipo 2 de la Ciudad de México / Association of the rs5186 polymorphism of the AGTR1 gene with decreased eGFR in patients with type 2 diabetes from Mexico City

Antecedentes: La búsqueda de biomarcadores tempranos de enfermedad renal diabética (ERD) en pacientes con diabetes mellitus tipo 2 (DMT2), como los marcadores genéticos para identificar pacientes vulnerables de la enfermedad, incluso antes de la presencia de una disminución de la estimación de tasa de filtrado glomerular (TFGe) o presencia de microalbuminuria ha cobrado importancia en los últimos años. El polimorfismo rs5186 (A1166C) presente en el gen receptor tipo 1 de la angiotensina II (AGTR1) ha sido asociado a distintos efectos del riesgo de daño renal que suelen estar presentes en pacientes con diabetes mellitus (DM). Se ha descrito que el rs5186 podría influir en la estabilidad de las proteínas que conforman al receptor de la angiotensina II tipo 1 (AT1) alterando su actividad, por lo que podría ser considerado como un factor de riesgo a enfermedad renal crónica (ERC) caracterizada por una disminución progresiva de la TFG. Sin embargo, la asociación del polimorfismo rs5186 del gen AGTR1 con ERD en pacientes con DMT2 ha sido controversial, no concluyente, incluso nula. Las controversias podrían ser por los estudios de asociación y estimación del riesgo del rs5186 previamente reportados incluyen distintos fenotipos clínicos considerados como inductores y potenciadores de ERC, además, los tamaños de las muestras analizadas en pacientes con DMT2 eran pequeñas y no tenían un control estricto en su inclusión, careciendo incluso de marcadores bioquímicos o estadificación KDOQI que han dificultado su análisis. Objetivo: Determinar la asociación del rs5186 del gen AGTR1 con la disminución de TFGe considerada como riesgo al desarrollo de ERD en pacientes con DMT2.(AU)

Background: Early biomarkers search for Diabetic Kidney Disease (DKD) in patients with Type 2 Diabetes Mellitus (T2DM), as genetic markers to identify vulnerable carriers of the disease even before Glomerular Filtration Rate (GFR) decline or microalbuminuria development, has been relevant during the last few years. The rs5186 (A116C) polymorphism of the Angiotensin II Receptor Type I gene (AGTR1), has been associated to multiple effects of renal injury risk, commonly detected in patients with Diabetes Mellitus (DM). It has been described that rs5186 could have an effect in stability proteins that assemble Angiotensin II Receptor Type I (AT1), modifying its action, which is why it should be considered as a risk factor for Chronic Kidney Disease (CKD), characterized by a GFR progressive reduction. Even though, the association between rs5186 AGTR1 gene polymorphism and DKD in patients with T2DM has been controversial, inconclusive, and even absent. This disputable issue might be as a result of association studies in which many and varied clinical phenotypes included are contemplated as CKD inductors and enhancers. Although, the sample sizes studied in patients with T2DM are undersized and did not have a strict inclusion criteria, lacking of biochemical markers or KDOQI classification, which have hindered its examination.Objective: The aim of our study was to establish an association between rs5186 AGTR1 gene polymorphism and GFR depletion, assessed as a risk factor to DKD development in patients with T2DM. (AU)

Selection of a GPER1 Ligand via Ligand-based Virtual Screening Coupled to Molecular Dynamics Simulations and Its Anti-proliferative Effects on Breast Cancer Cells.

BACKGROUND: Recent reports have demonstrated the role of the G Protein-Coupled Estrogen Receptor 1 (GPER1) on the proliferation of breast cancer. The coupling of GPER1 to estrogen triggers cellular signaling pathways related to cell proliferation. OBJECTIVE: Develop new therapeutic strategies against breast cancer. METHOD: We performed in silico studies to explore the binding mechanism of a set of G15 /G1 analogue compounds. We included a carboxyl group instead of the acetyl group from G1 to form amides with several moieties to increase affinity on GPER1. The designed ligands were submitted to ligand-based and structure-based virtual screening to get insights into the binding mechanism of the best designed compound and phenol red on GPER1. RESULTS: According to the in silico studies, the best molecule was named G1-PABA ((3aS,4R,9bR)-4-(6- bromobenzo[d][1,3]dioxol-5-yl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-carboxylic acid). It was synthesized and assayed in vitro in breast cancer (MCF-7 and MDA-MB-231) and normal (MCF-10A) cell lines. Experimental studies showed that the target compound was able to decrease cell proliferation, IC50 values of 15.93 µM, 52.92 µM and 32.45 µM in the MCF-7, MDA-MB-231 and MCF-10A cell lines, respectively, after 72 h of treatment. The compound showed better IC50 values without phenol red, suggesting that phenol red interfere with the G1-PABA action at GPER1, as observed through in silico studies, which is present in MCF-7 cells according to PCR studies and explains the cell proliferation effects. CONCLUSION: Concentration-dependent inhibition of cell proliferation occurred with G1-PABA in the assayed cell lines and could be due to its action on GPER1.

Sodium and epithelial sodium channels participate in the regulation of the capacitation-associated hyperpolarization in mouse sperm.

In a process called capacitation, mammalian sperm gain the ability to fertilize after residing in the female tract. During capacitation the mouse sperm plasma membrane potential (E(m)) hyperpolarizes. However, the mechanisms that regulate sperm E(m) are not well understood. Here we show that sperm hyperpolarize when external Na(+) is replaced by N-methyl-glucamine. Readdition of external Na(+) restores a more depolarized E(m) by a process that is inhibited by amiloride or by its more potent derivative 5-(N-ethyl-N-isopropyl)-amiloride hydrochloride. These findings indicate that under resting conditions an electrogenic Na(+) transporter, possibly involving an amiloride sensitive Na(+) channel, may contribute to the sperm resting E(m). Consistent with this proposal, patch clamp recordings from spermatogenic cells reveal an amiloride-sensitive inward Na(+) current whose characteristics match those of the epithelial Na(+) channel (ENaC) family of epithelial Na(+) channels. Indeed, ENaC-alpha and -delta mRNAs were detected by reverse transcription-PCR in extracts of isolated elongated spermatids, and ENaC-alpha and -delta proteins were found on immunoblots of sperm membrane preparations. Immunostaining indicated localization of ENaC-alpha to the flagellar midpiece and of ENaC-delta to the acrosome. Incubations known to produce capacitation in vitro or induction of capacitation by cell-permeant cAMP analogs decreased the depolarizing response to the addition of external Na(+). These results suggest that increases in cAMP content occurring during capacitation may inhibit ENaCs to produce a required hyperpolarization of the sperm membrane.

KATP channels in mouse spermatogenic cells and sperm, and their role in capacitation.

Mammalian sperm must undergo a series of physiological changes after leaving the testis to become competent for fertilization. These changes, collectively known as capacitation, occur in the female reproductive tract where the sperm plasma membrane is modified in terms of its components and ionic permeability. Among other events, mouse sperm capacitation leads to an increase in the intracellular Ca(2+) and pH as well as to a hyperpolarization of the membrane potential. It is well known that ion channels play a crucial role in these events, though the molecular identity of the particular channels involved in capacitation is poorly defined. In the present work, we report the identification and potential functional role of K(ATP) channels in mouse spermatogenic cells and sperm. By using whole-cell patch clamp recordings in mouse spermatogenic cells, we found K(+) inwardly rectifying (K(ir)) currents that are sensitive to Ba(2+), glucose and the sulfonylureas (tolbutamide and glibenclamide) that block K(ATP) channels. The presence of these channels was confirmed using inhibitors of the ATP synthesis and K(ATP) channel activators. Furthermore, RT-PCR assays allowed us to detect transcripts for the K(ATP) subunits SUR1, SUR2, K(ir)6.1 and K(ir)6.2 in total RNA from elongated spermatids. In addition, immunoconfocal microscopy revealed the presence of these K(ATP) subunits in mouse spermatogenic cells and sperm. Notably, incubation of sperm with tolbutamide during capacitation abolished hyperpolarization and significantly decreased the percentage of AR in a dose-dependent fashion. Together, our results provide evidence for the presence of K(ATP) channels in mouse spermatogenic cells and sperm and disclose the contribution of these channels to the capacitation-associated hyperpolarization.

Tep22, a novel testicular expressed gene, is involved in the biogenesis of the acrosome and the midpiece of the sperm tail.

To identify new genes that could be involved in the differentiation and function of male germ cells, we have screened a murine testis cDNA library and isolated a clone that was named Tep22. The gene encoding Tep22 consists of three exons and is localized in the telomeric region of mouse chromosome 12. Expression analyses with RNA from different adult tissues revealed that Tep22 is predominantly expressed in spermatocytes and spermatids of the murine testis. Four Tep22 transcript sizes ranging from 647 to 1122 nucleotides were detected in testes of 15-day-old mice due to variable 5' UTRs, while the open reading frame of Tep22 has a length of 567bp in all transcript forms. Specific antibodies against Tep22 detected an approximately 22kDa band in testicular protein extracts, which was first observed in 18-day-old mice, indicating that Tep22 is translationally repressed for several days. Indirect immunofluorescence and immunoelectron microscopy experiments demonstrate that Tep22 is localized in the acrosomal region of early elongating spermatids, while the surrounding cytoplasm is barely labeled. During further germ cell development, the intensity of the staining in the acrosomal region decreases and is no longer detectable in late stages of elongating spermatids, whereas the amount of the Tep22 protein increases in the cytoplasm. Finally, Tep22 is incorporated into the midpiece of spermatids and is also present in the mitochondrial sheath of mature spermatozoa. Taken together, our results suggest that Tep22 is involved in the biogenesis of the acrosome as well as in the function of the midpiece of murine spermatozoa.

The Hook1 gene is non-functional in the abnormal spermatozoon head shape (azh) mutant mouse.

In mice carrying the autosomal recessive mutation 'abnormal spermatozoon head shape' (azh) all spermatozoa display a highly abnormal head morphology that differs drastically from the compact and hook-shaped head of the normal murine sperm. Moreover, the azh mutation causes tail abnormalities often resulting in coiled sperm tails or in the decapitation of the sperm head from the flagellum. We have isolated and characterized murine Hook1 cDNA and analyzed the corresponding genomic structure. Furthermore, the Hook1 gene was mapped to the same region on chromosome 4 to which the azh locus was previously linked. The Hook1 gene is predominantly expressed in haploid male germ cells, and immunohistochemical analysis revealed that Hook1 is responsible for the linkage of the microtubular manchette and the flagellum to cellular structures. Here, we report that the azh mutation is due to a deletion of exons 10 and 11 in the murine Hook1 gene leading to a non-functional protein. Our results indicate that loss of Hook1 function results in ectopic positioning of microtubular structures within the spermatid and causes the azh phenotype. Therefore, the human HOOK1 gene could serve as a candidate gene for male infertility due to teratozoospermia or decapitation defects.