Stehberger Paul A, Schulz Nicole, Finberg Karin E, Karet Fiona E, Giebisch Gerhard, Lifton Richard P, Geibel John P, Wagner Carsten A
Departments of Cellular and Molecular Physiology, Genetics, and Surgery, Yale University School of Medicine, New Haven, Connecticut, USA.
J Am Soc Nephrol. 2003 Dec;14(12):3027-38. doi: 10.1097/01.asn.0000099375.74789.ab.
Vacuolar-type H(+)-ATPases (V-H(+)-ATPases) are the major H(+)-secreting protein in the distal portion of the nephron and are involved in net H(+) secretion (bicarbonate generation) or H(+) reabsorption (net bicarbonate secretion). In addition, V-H(+)-ATPases are involved in HCO(3)(-) reabsorption in the proximal tubule and distal tubule. V-H(+)-ATPases consist of at least 13 subunits, the functions of which have not all been elucidated. Mutations in the accessory ATP6V0A4 (a4 isoform) subunit have recently been shown to cause an inherited form of distal renal tubular acidosis in humans. Here, the localization of this subunit in human and mouse kidney was studied and the regulation of expression and localization of this subunit in mouse kidney in response to acid-base and electrolyte intake was investigated. Reverse transcription-PCR on dissected mouse nephron segments amplified a4-specific transcripts in proximal tubule, loop of Henle, distal convoluted tubule, and cortical and medullary collecting duct. a4 protein was localized by immunohistochemistry to the apical compartment of the proximal tubule (S1/S2 segment), the loop of Henle, the intercalated cells of the distal convoluted tubule, the connecting segment, and all intercalated cells of the entire collecting duct in human and mouse kidney. All types of intercalated cells expressed a4. NH(4)Cl or NaHCO(3) loading for 24 h, 48 h, or 7 d as well as K(+) depletion for 7 and 14 d had no influence on a4 protein expression levels in either cortex or medulla as determined by Western blotting. Immunohistochemistry, however, demonstrated a subcellular redistribution of a4 in response to the different stimuli. NH(4)Cl and K(+) depletion led to a pronounced apical staining in the connecting segment, cortical collecting duct, and outer medullary collecting duct, whereas NaHCO(3) loading caused a stronger bipolar staining in the cortical collecting duct. Taken together, these results demonstrate a4 expression in the proximal tubule, loop of Henle, distal tubule, and collecting duct and suggest that under conditions in which increased V-H(+)-ATPase activity is required, a4 is regulated by trafficking but not protein expression. This may allow for the rapid adaptation of V-H(+)-ATPase activity to altered acid-base intake to achieve systemic pH homeostasis. The significance of a4 expression in the proximal tubule in the context of distal renal tubular acidosis will require further clarification.
液泡型H(+)-ATP酶（V-H(+)-ATP酶）是肾单位远端主要的H(+)分泌蛋白，参与净H(+)分泌（碳酸氢盐生成）或H(+)重吸收（净碳酸氢盐分泌）。此外，V-H(+)-ATP酶还参与近端小管和远端小管中的HCO(3)(-)重吸收。V-H(+)-ATP酶至少由13个亚基组成，其功能尚未完全阐明。最近研究表明，辅助ATP6V0A4（a4亚型）亚基的突变会导致人类遗传性远端肾小管酸中毒。在此，研究了该亚基在人和小鼠肾脏中的定位，并研究了小鼠肾脏中该亚基在酸碱和电解质摄入反应中的表达和定位调控。对解剖的小鼠肾单位节段进行逆转录PCR，在近端小管、髓袢、远端曲管以及皮质和髓质集合管中扩增出a4特异性转录本。通过免疫组织化学将a4蛋白定位到人和小鼠肾脏近端小管（S1/S2节段）的顶端区室、髓袢、远端曲管的闰细胞、连接段以及整个集合管的所有闰细胞。所有类型的闰细胞均表达a4。通过蛋白质印迹法测定，给予NH(4)Cl或NaHCO(3) 24小时、48小时或7天以及给予K(+)耗竭7天和14天，对皮质或髓质中的a4蛋白表达水平均无影响。然而，免疫组织化学显示，a4会因不同刺激而发生亚细胞重新分布。NH(4)Cl和K(+)耗竭导致连接段、皮质集合管和外髓集合管出现明显的顶端染色，而NaHCO(3)负荷则导致皮质集合管出现更强的双极染色。综上所述，这些结果表明a4在近端小管、髓袢、远端小管和集合管中表达，并表明在需要增加V-H(+)-ATP酶活性的情况下，a4通过运输而非蛋白质表达进行调控。这可能使V-H(+)-ATP酶活性能够快速适应酸碱摄入的变化，以实现全身pH稳态。a4在近端小管中的表达在远端肾小管酸中毒背景下的意义尚需进一步阐明。
