Molecular Physiology Unit, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico.
Department of Nephrology and Mineral Metabolism, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, Mexico.
FASEB J. 2022 Mar;36(3):e22190. doi: 10.1096/fj.202101912R.
We demonstrated that serpinA3c/k relocates from the cytoplasm to the apical tubular membrane (ATM) in chronic kidney disease (CKD), suggesting its secretion in luminal space in pathophysiological contexts. Here, we studied serpinA3c/k expression and secretion under different stressful conditions in vitro and in vivo. HEK-293 cells were transfected with a FLAG-tagged serpinA3c/k clone and exposed to H O or starvation. Both stressors induced serpinA3c/k secretion but with a higher molecular weight. Glycanase treatment established that serpinA3c/k is glycosylated. Site-directed mutagenesis for each of the four glycosylation sites was performed. During cellular stress, serpinA3c/k secretion increased with each mutant except in the quadruple mutant. In rats and patients suffering acute kidney injury (AKI), an atypical urinary serpinA3c/k excretion (uSerpinA3c/k) was observed. In rats with AKI, the greater the induced kidney damage, the greater the uSerpinA3 c/k, together with relocation toward ATM. Our findings show that: (1) serpinA3c/k is glycosylated and secreted, (2) serpinA3c/k secretion increases during cellular stress, (3) its appearance in urine reveals a pathophysiological state, and (4) urinary serpinA3 excretion could become a potential biomarker for AKI.
我们证明，丝氨酸蛋白酶抑制剂 A3c/k 在慢性肾脏病（CKD）中从细胞质重新定位到顶端管状膜（ATM），表明其在病理生理环境中在腔空间分泌。在这里，我们研究了丝氨酸蛋白酶抑制剂 A3c/k 在体外和体内不同应激条件下的表达和分泌。用 FLAG 标记的丝氨酸蛋白酶抑制剂 A3c/k 克隆转染 HEK-293 细胞，并暴露于 H 2 O 2 或饥饿。这两种应激源都诱导丝氨酸蛋白酶抑制剂 A3c/k 分泌，但分子量更高。糖基酶处理证实丝氨酸蛋白酶抑制剂 A3c/k 是糖基化的。对每个四个糖基化位点进行了定点突变。在细胞应激期间，除四重突变体外，每个突变体的丝氨酸蛋白酶抑制剂 A3c/k 分泌都增加。在急性肾损伤（AKI）的大鼠和患者中，观察到一种非典型的尿丝氨酸蛋白酶抑制剂 A3c/k 排泄（uSerpinA3c/k）。在 AKI 大鼠中，诱导的肾脏损伤越大，uSerpinA3 c/k 越大，同时向 ATM 重新定位。我们的研究结果表明：（1）丝氨酸蛋白酶抑制剂 A3c/k 是糖基化和分泌的，（2）丝氨酸蛋白酶抑制剂 A3c/k 在细胞应激期间分泌增加，（3）其在尿液中的出现揭示了一种病理生理状态，（4）尿丝氨酸蛋白酶抑制剂 A3 的排泄可能成为 AKI 的潜在生物标志物。
