RESUMO
Osteoporosis after bariatric surgery is an increasing health concern as the rate of bariatric surgery has risen. In animal studies mimicking bariatric procedures, bone disease, together with decreased serum levels of Ca2+, Mg2+ and the gastric hormone Ghrelin were described. Ghrelin regulates metabolism by binding to and activating the growth hormone secretagogue receptor (GHSR) which is also expressed in the kidney. As calcium and magnesium are key components of bone, we tested the hypothesis that Ghrelin-deficiency contributes to osteoporosis via reduced upregulation of the renal calcium channel TRPV5 and the heteromeric magnesium channel TRPM6/7. We expressed GHSR with TRPV5 or TRPM6/7 channel in HEK293 cells and treated them with purified Ghrelin. Whole-cell current density was analyzed by patch-clamp recording. Nephron-specific gene expression was performed by tubular microdissection followed by qPCR in wild-type (WT) mice, and immunofluorescent imaging of GHSR-eGFP mice. Tubular magnesium homeostasis was analyzed in GHSR-null and WT mice at baseline and after caloric restriction. After Ghrelin exposure, whole-cell current density did not change for TRPV5 but increased for TRPM6/7 in a dose-dependent fashion. Applying the Ghrelin-mimetic (D-Trp7, Ala8,D-Phe10)-α-MSH (6-11) amide without and with the GHSR antagonist (D-Lys3)-GHRP6, we confirmed the stimulatory role of Ghrelin towards TRPM6/7. As GHSR initiates downstream signaling via protein kinase A (PKA), we found that the PKA inhibitor H89 abrogated TRPM6/7 stimulation by Ghrelin. Similarly, transfected Gαs, but not the Gαs mutant Q227L, nor Gαi2, Gαq, or Gα13 upregulated TRPM6/7 current density. In microdissected TALs and DCTs similar levels of GHSR mRNA were detected. In contrast, TRPM6 mRNA was expressed in the DCT and also detected in the TAL at 25% expression compared to DCT. Immunofluorescent studies using reporter GHSR-eGFP mice showed a strong eGFP signal in the TAL but surprisingly displayed no eGFP signal in the DCT. In 3-, 6-, and 9-month-old GHSR-null and WT mice, baseline serum magnesium was not significantly different, but 24-h urinary magnesium excretion was elevated in 9-month-old GHSR-null mice. In calorically restricted GHSR-null mice, we detected excess urinary magnesium excretion and reduced serum magnesium levels compared to WT mice. The kidneys from calorically restricted WT mice showed upregulated gene expression of magnesiotropic genes Hnf1b, Cldn-16, Cldn-19, Fxyd-2b, and Parvalbumin compared to GHSR-null mice. Our in vitro studies show that Ghrelin stimulates TRPM6/7 via GHSR and Gαs-PKA signaling. The murine studies are consistent with Ghrelin-GHSR signaling inducing reduced urinary magnesium excretion, particularly in calorically restricted mice when Ghrelin levels are elevated. This effect may be mediated by Ghrelin-upregulation of TRPM6 in the TAL and/or upregulation of other magnesiotropic genes. We postulate that rising Ghrelin levels with hunger contribute to increased renal Mg2+ reabsorption to compensate for lack of enteral Mg2+ uptake.
RESUMO
Objective: Human papillomavirus (HPV) 6 and 11 are the two most common low-risk HPV subtypes, accounting for more than 90% of condyloma acuminatum. A simple, accurate and rapid screening method to be applied in community-level hospitals is in high demand. Methods: Endogenous internally controlled recombinase-assisted amplification (EIC-RAA) assays for HPV6 and 11 were performed in a single closed-tube at 39 °C within 30 min. The sensitivity and specificity of EIC-RAA were examined using recombinant plasmids and pre-tested HPV DNA. A total of 233 clinical samples were collected, and the DNA was extracted by traditional multi-step extraction, or sample releasing agent, before analysis by EIC-RAA. For comparison, HPV detection via Quantitative real-time PCR (qPCR) was also performed. Results: The sensitivity of EIC-RAA analysis was 10 copies/reaction for HPV6, 100 copies/reaction for HPV11, and 100 copies/reaction for the human ß-globin gene. No cross-reaction was observed with other HPV subtypes. Clinical performance of the EIC-RAA assay achieved a 100% of concordance rate with the commercial HPV qPCR kit. Further, the EIC-RAA assay achieved a 100% of concordance rate when using multi-step extracted DNA and sample releasing agent-processed DNA. Summary: The EIC-RAA assay for HPV6 and 11 detection possesses the advantages of accuracy, simplicity and rapidity, and demonstrates great potential to be used in community-level hospitals for field investigation.
RESUMO
Recombinase aided amplification (RAA) is an emerging isothermal amplification method used for detecting various pathogens. However, RAA requires a complex and long probe to ensure high sensitivity during fluorescence assay. TaqMan probe used for quantitative PCR (qPCR) is simple and universal. Herein, we developed a new approach for detecting nucleic acids of pathogens, known as RAP (Recombinase aided PCR). The method combines RAA and qPCR to ensure a rapid and highly sensitive detection using a conventional qPCR device. RAP is a two-stage amplification process performed in a single tube within 1 hour. The method involves an RAA reaction for 10 min at 39°C (first stage) followed by 15 cycles of qPCR (second stage). Using human adenovirus 3 (HADV3) and human adenovirus 7 (HADV7) plasmids, the sensitivities of RAP assays for detecting HADV3 and HADV7 were 6 and 17 copies per reaction, respectively. The limit of RAP detection was at least 16-fold lower than the corresponding qPCR, and no-cross reaction with other respiratory viruses was observed. The results of RAP analysis revealed 100% consistency with qPCR assay. This study shows that RAP assay is a rapid, specific, and highly sensitive detection method with a potential for clinical and laboratory application.