1,25-(OH)2-D PRODUCTION BY KIDNEY CELLS

观察到维生素D代谢异常的两名患者都患有 X连锁低磷血症性软骨病，以及与此类似的小鼠 人类疾病，C57BL6/J小鼠患有x连锁低磷血症。在……里面 血清培养肾皮质细胞的初步研究 来自受感染雄性小鼠(Hyp/y)及其正常小鼠的游离培养液 (+/y)，我们观察到表观基础活性 肾脏25-OH-D维生素D31α-羟基酶在Hyp/y细胞中降低 与+/y细胞相比。酶活性的评估由 25-羟基-D3转化为1，25-(OH)2-D3的测定 放射受体分析。我们现在建议研究 可能是1a-羟基酶基础酶活性降低的原因。 在培养的Hyp/y细胞中。我们将探索：1)文化是否 Hyp/y和Of+/y细胞包含大致相同数量的近端 肾小管上皮细胞，用α-甲基葡萄糖苷摄取法测定 结合，其他酶的活性，以及超微结构的外观。2) 酶活性的明显差异是否存在于较低和较低的 生理浓度更高的25-OH-D3。3)线粒体是否 从Hyp/y细胞中分离的细胞也显示1α-羟基酶活性降低 活动。4)Hyp/y和+/y的线粒体酶活性是否相似 细胞，我们将确定酶活性是否在整体上降低 细胞可能与25-OH-D3进入细胞的异常有关，或者 Hyp/y细胞含有线粒体酶胞质抑制物 活动。5)此外，由于已知1，25-(OH)2-D3自身调节 生产，我们将评估变化在 1，25-(OH)2-D3与Hyp/y结合，与+/y细胞质结合。6)至 确定降低的基础1α-羟基酶活性是否 当施加已知的酶刺激时，Hyp/y细胞持续存在，我们将 评估甲状旁腺激素的作用及对 Hyp/y与+/y培养基磷浓度的比较 细胞。我们预计，这些研究将为 肾组织维生素D代谢调节异常的发病机制 小鼠X连锁低磷血症性软骨病。这类数据可能 有助于更好地了解本病的发病机制和治疗方法 人类的低磷血症性软骨病。

Abnormal vitamin D metabolism has been observed in both patients with x-linked hypophosphatemic rickets, as well as in the murine analog of this human disease, the C57BL6/J mouse with x-linked hypophosphatemia. In preliminary studies of cultured kidney cortical cells, cultured in serum free medium, from affected male mice (Hyp/y) and from their normal littermates (+/y), we have observed that the apparent basal activity of the renal 25-OH-D vitamin D3 1Alpha-hydroxylase is reduced in the Hyp/y cells as compared to the +/y cells. Enzyme activity is assessed by the conversion of 25-OH-D3 to 1,25-(OH)2-D3 with measurement of the latter by radioreceptor assay. We now propose to investigate the mechanisms which may account for the reduced basal enzyme activity of the 1Alpha-hydroxylase in the cultured Hyp/y cells. We will explore: 1) Whether the cultures of Hyp/y and of +/y cells contain approximately equivalent numbers of proximal tubular cells, as measured by Alpha-methylglucoside uptake, phlorizin binding, activity of other enzymes, and by ultrastructural appearance. 2) Whether the apparent difference in enzyme activity is present at lower and more physiological concentrations of 25-OH-D3. 3) Whether mitochondria isolated from Hyp/y cells also exhibit reduced 1Alpha-hydroxylase activity. 4) If mitochondrial enzyme activity is similar in Hyp/y and +/y cells, we will determine whether the reduced enzyme activity in the whole cells may be related to abnormalities in cell entry of 25-OH-D3 or whether Hyp/y cells contain a cytosolic inhibitor of mitochondrial enzyme activity. 5) In addition, since 1,25-(OH)2-D3 is known to regulate its own production, we will evaluate the possible role of alteration in 1,25-(OH)2-D3 binding to Hyp/y as compared to +/y cytoplasm. 6) To determine whether the reduced basal 1Alpha-hydroxylase activity in the Hyp/y cells persists when known stimuli of the enzyme are applied, we will evaluate the effects of parathyroid hormone and alterations in the phosphate concentration in culture media in Hyp/y as compared to +/y cells. We anticipate that these studies will provide new insights into the pathogenesis of abnormal regulation of renal vitamin D metabolism in x-linked hypophosphatemic rickets in mice. Such data are potentially relevant to a better understanding of the pathogenesis and treatment of hypophosphatemic rickets in human beings.