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INTRODUCTION

Nephrolithiasis is a painful and costly healthcare complication. The most common kidney stones are composed of calcium oxalate and thus renal handling of oxalate is an important facet of understanding the pathogenesis of nephrolithiasis. Recently, the Malpighian tubule (MT) has emerged as a robust model of trans-epithelial ion transport and nephrolithiasis as MTs readily form luminal calcium-oxalate crystals in the presence of oxalate. Prestin (dPrestin, Slc26a6) transports oxalate across the apical surface of the MT into the lumen but a full model of the trans-epithelial movement of oxalate (Ox) in the MT has been lacking as the basolateral oxalate transporter has remained uncharacterized.

METHODS

The objective of this work was to identify and characterize the basolateral Ox transporter through real-time quantification of intracellular pH (pH) and oocyte transport assays.

RESULTS

A putative basolateral oxalate transporter CG5002 ("Neat") was identified through sequence homology and displayed robust Cl-independent Ox transport and electroneutral Ox transport in oocytes. pH in extracted fly MTs was monitored by using the GAL4/UAS system to selectively express pHerry, a pseudo-ratiometric genetically-encoded pH indicator (GEpHI) in the cytosol of the principal cells of the MT. Basolateral perfusion of MTs in CO/HCO -buffered solution produced a large acidification followed by rapid recovery in the transitional segment of the anterior MT. Recovery was interrupted by basolateral application of 1 mM Ox or 1 mM SO . Tissue specific knock-down of Neat with interference RNA (RNAi) reduced the rate of acid-loading in the transitional segment of the MT with regard to Ox and SO . Knockdown of Neat in the MT also significantly reduced luminal calcium oxalate crystal formation in a fly model of calcium oxalate nephrolithiasis.

DISCUSSION

These data indicate Neat is a significant basolateral MT oxalate transporter and the basolateral movement of oxalate is functionally coupled to movement of acid equivalents, potentially as Ox/HCO exchange, Ox/OH exchange, or Ox:H co-transport.

INTRODUCTION

METHODS

The objective of this work was to identify and characterize the basolateral Ox transporter through real-time quantification of intracellular pH (pH) and oocyte transport assays.

RESULTS

DISCUSSION

引言

肾结石是一种痛苦且代价高昂的医疗并发症。最常见的肾结石由草酸钙组成，因此肾脏对草酸盐的处理是理解肾结石发病机制的一个重要方面。最近，马尔皮基氏小管（MT）已成为跨上皮离子转运和肾结石形成的一个强大模型，因为在草酸盐存在的情况下，MT很容易形成管腔草酸钙晶体。 Prestin（dPrestin，Slc26a6）将草酸盐跨MT的顶端表面转运到管腔中，但由于基底外侧草酸盐转运体尚未得到表征，因此缺乏MT中草酸盐（Ox）跨上皮运动的完整模型。

方法

这项工作的目的是通过实时定量细胞内pH值（pH）和卵母细胞转运试验来鉴定和表征基底外侧Ox转运体。

结果

通过序列同源性鉴定出一个假定的基底外侧草酸盐转运体CG5002（“Neat”），它在卵母细胞中表现出强大的不依赖Cl的Ox转运和电中性Ox转运。通过使用GAL4/UAS系统在MT主细胞的细胞质中选择性表达pHerry（一种伪比率基因编码pH指示剂（GEpHI））来监测提取的果蝇MT中的pH值。在CO/HCO缓冲溶液中对MT进行基底外侧灌注会在前MT的过渡段产生大量酸化，随后迅速恢复。基底外侧应用1 mM Ox或1 mM SO会中断恢复。用干扰RNA（RNAi）对Neat进行组织特异性敲低会降低MT过渡段中关于Ox和SO的酸加载速率。在草酸钙肾结石果蝇模型中，敲低MT中的Neat也会显著减少管腔草酸钙晶体的形成。