lement and generates C3a and C5a in vitro/ex vivo in urine from healthy persons when exogenous, inactive, plasminogen, and complement factors are added. Amiloride inhibits uPA and attenuates complement activation in vitro and in vivo. In conditional podocin knockout (KO) mice with severe proteinuria, blocking of uPA with monoclonal antibodies significantly reduces the urine excretion of C3a and C5a and lowers tissue NLRP3-inflammasome protein without major changes in early fibrosis markers. This mechanism provides a link to proinflammatory signaling in proteinuria with possible long-term consequences for kidney function. Background Persistent proteinuria is associated with tubular interstitial inflammation and predicts progressive kidney injury. In proteinuria, plasminogen is aberrantly filtered and activated by urokinase-type plasminogen activator (uPA), which promotes kidney fibrosis. We hypothesized that plasmin activates filtered complement factors C3 and C5 directly in tubular fluid, generating anaphylatoxins, and that this is attenuated by amiloride, an off-target uPA inhibitor. Methods Purified C3, C5, plasminogen, urokinase, and urine from healthy humans were used for in vitro/ex vivo studies. Complement activation was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblotting, and ELISA. Urine and plasma from patients with diabetic nephropathy treated with high-dose amiloride and from mice with proteinuria (podocin knockout [KO]) treated with amiloride or inhibitory anti-uPA antibodies were analyzed. Results The combination of uPA and plasminogen generated anaphylatoxins C3a and C5a from intact C3 and C5 and was inhibited by amiloride. Addition of exogenous plasminogen was sufficient for urine from healthy humans to activate complement. Conditional podocin KO in mice led to severe proteinuria and C3a and C5a urine excretion, which was attenuated reversibly by amiloride treatment for 4 days and reduced by >50% by inhibitory anti-uPA antibodies without altering proteinuria. NOD-, LRR- and pyrin domain-containing protein 3-inflammasome protein was reduced with no concomitant effect on fibrosis. In patients with diabetic nephropathy, amiloride reduced urinary excretion of C3dg and sC5b-9 significantly. Conclusions In conditions with proteinuria, uPA-plasmin generates anaphylatoxins in tubular fluid and promotes downstream complement activation sensitive to amiloride. This mechanism links proteinuria to intratubular proinflammatory signaling. In perspective, amiloride could exert reno-protective effects beyond natriuresis and BP reduction. Clinical Trial registry name and registration number: Increased Activity of a Renal Salt Transporter (ENaC) in Diabetic Kidney Disease, NCT01918488 and Increased Activity of ENaC in Proteinuric Kidney Transplant Recipients, NCT03036748....

中文翻译：

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阿米洛利减少肾小球蛋白尿中尿激酶/纤溶酶原驱动的管内补体激活

当添加外源性、无活性、纤溶酶原和补体因子时，在健康人的尿液中体外/离体产生 C3a 和 C5a。阿米洛利在体外和体内抑制 uPA 并减弱补体激活。在患有严重蛋白尿的条件性 Podocin 敲除 (KO) 小鼠中，用单克隆抗体阻断 uPA 显着减少了 C3a 和 C5a 的尿液排泄，并降低了组织 NLRP3 炎症体蛋白，而早期纤维化标志物没有发生重大变化。这种机制与蛋白尿中的促炎信号传导有关，可能对肾功能产生长期影响。背景 持续性蛋白尿与肾小管间质炎症相关，可预测进行性肾损伤。在蛋白尿中，纤溶酶原被尿激酶型纤溶酶原激活剂（uPA）异常过滤和激活，从而促进肾脏纤维化。我们假设纤溶酶直接激活肾小管液中滤过的补体因子 C3 和 C5，产生过敏毒素，并且这种现象可以被脱靶 uPA 抑制剂阿米洛利减弱。方法 使用来自健康人的纯化 C3、C5、纤溶酶原、尿激酶和尿液进行体外/离体研究。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳、免疫印迹和 ELISA 评估补体激活。对接受高剂量阿米洛利治疗的糖尿病肾病患者和接受阿米洛利或抑制性抗 uPA 抗体治疗的蛋白尿（podocin 敲除 [KO]）小鼠的尿液和血浆进行了分析。结果 uPA 和纤溶酶原结合，从完整的 C3 和 C5 中产生过敏毒素 C3a 和 C5a，并被阿米洛利抑制。添加外源性纤溶酶原足以使健康人的尿液激活补体。条件性 Podocin KO 小鼠会导致严重的蛋白尿以及 C3a 和 C5a 尿液排泄，阿米洛利治疗 4 天可逆地减弱这种蛋白尿，并通过抑制性抗 uPA 抗体减少> 50%，而不会改变蛋白尿。含有 NOD-、LRR- 和 Pyrin 结构域的蛋白 3-炎性体蛋白减少，但对纤维化没有伴随作用。在糖尿病肾病患者中，阿米洛利显着减少 C3dg 和 sC5b-9 的尿排泄。结论 在蛋白尿情况下，uPA-纤溶酶在肾小管液中产生过敏毒素，并促进对阿米洛利敏感的下游补体激活。这种机制将蛋白尿与肾小管内促炎信号传导联系起来。从长远来看，阿米洛利除了利尿钠和降低血压外，还可以发挥肾脏保护作用。临床试验注册名称和注册号：糖尿病肾病中肾盐转运蛋白 (ENaC) 的活性增加，NCT01918488 和蛋白尿肾移植受者中 ENaC 的活性增加，NCT03036748....