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Abstract：
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.
摘要：
随着减肥手术率的上升，减肥手术后的骨质疏松是一个日益增加的健康问题。在模仿减肥程序的动物研究中，骨病,随着血清Ca2+水平的降低，描述了Mg2和胃激素Ghrelin。Ghrelin通过结合并激活也在肾脏中表达的生长激素促分泌素受体（GHSR）来调节代谢。因为钙和镁是骨骼的关键成分，我们检验了Ghrelin缺乏通过降低肾钙通道TRPV5和异聚镁通道TRPM6/7的上调而导致骨质疏松症的假设。我们用TRPV5或TRPM6/7通道在HEK293细胞中表达GHSR并用纯化的Ghrelin处理它们。通过膜片钳记录分析全细胞电流密度。肾单位特异性基因表达通过管状显微切割进行，然后在野生型（WT）小鼠中进行qPCR，和GHSR-eGFP小鼠的免疫荧光成像。在基线和热量限制后，在GHSR无效和WT小鼠中分析了管状镁稳态。Ghrelin暴露后,TRPV5的全细胞电流密度没有变化,但TRPM6/7的全细胞电流密度呈剂量依赖性增加.应用Ghrelin模拟物（D-Trp7，Ala8，D-Phe10）-α-MSH（6-11）酰胺，不使用和使用GHSR拮抗剂（D-Lys3）-GHRP6，我们证实了Ghrelin对TRPM6/7的刺激作用。当GHSR通过蛋白激酶A（PKA）启动下游信号时，我们发现PKA抑制剂H89消除了Ghrelin对TRPM6/7的刺激.同样，转染的Gαs，但不是Gαs突变体Q227L,也没有Gαi2，Gαq，或Gα13上调TRPM6/7电流密度。在显微解剖的TAL和DCT中，检测到相似的GHSRmRNA水平。相比之下,与DCT相比,TRPM6mRNA在DCT中表达,并且在TAL中也以25%的表达检测到。使用报告基因GHSR-eGFP小鼠的免疫荧光研究显示TAL中的强eGFP信号,但令人惊讶地在DCT中没有显示eGFP信号。在3-，6-，和9个月大的GHSR-null和WT小鼠，基线血清镁没有显着差异，但9月龄GHSR空小鼠24小时尿镁排泄升高。在热量受限的GHSR无效小鼠中，与WT小鼠相比，我们检测到尿镁排泄过多和血清镁水平降低。来自受热量限制的WT小鼠的肾脏显示磁化基因Hnf1b的基因表达上调，Cldn-16,Cldn-19,Fxyd-2b,和小白蛋白与GHSR-null小鼠相比。我们的体外研究表明Ghrelin通过GHSR和Gαs-PKA信号传导刺激TRPM6/7。小鼠研究与Ghrelin-GHSR信号诱导减少的尿镁排泄一致，特别是在Ghrelin水平升高的热量受限小鼠中。这种作用可能是由TAL中TRPM6的Ghrelin上调和/或其他磁化基因的上调介导的。我们假设饥饿时Ghrelin水平的升高有助于肾脏Mg2重吸收的增加，以弥补肠内Mg2摄取的不足。
