An intriguing local epithelial enzyme contribution to renal medullary urea accumulation and ischemic tolerance was uncovered in a study by Ansermet et al1 published in the present issue of Acta Physiologica . It was shown in vivo in mice with tubular epithelial‐specific deletion of the enzyme L‐arginase type 2 (ARG2) that ARG2 contributed to medullary urea accumulation, through its hydrolysis of L‐arginine into urea and L‐ornithine, and to tissue protection after ischemia‐reperfusion injury. The ability of the S3 segment to generate urea ex vivo has been known for decades and was now proven to contribute to systemic plasma urea after ischemic insult and to medullary accumulation of urea. The ability of the proximal tubule S3 segment that resides in the renal outer medulla, a region with a narrow margin between sufficient oxygen supply and metabolic oxygen consumption, to employ ARG2‐facilitated anerobic metabolism was hypothesized to exert protection during ischemia.

Two organ‐specific isoforms of arginase are recognized, liver arginase 1, found in the cytosol, and kidney‐dominant arginase 2 (ARG2) associated with mitochondria. ARG2 is detected in proximal tubule cells of the straight descending part situated in cortex with terminal part in the outer stripe of the outer medulla, and in inner medullary collecting ducts, where protein localization corresponded with arginase activity.2, 3 What roles for arginase 2 are established in kidney? Even before arginase isoforms were cloned, the ability to form urea locally within isolated medullary segments prompted Levillain et al4 in 1989 to suggest a role in formation and maintenance of the cortical‐medullary urea concentration gradient necessary for urine concentration. Second, because of the presence of ornithine aminotransferase in these cells, a coupling to glutamine synthesis and alpha ketoglutarate (α‐KG) formation was proposed.5 Why is this relevant? The kidney outer medulla is very vulnerable to ischemic insults and S3‐proximal tubule cells in outer medulla may anaerobically generate ATP by intramitochondrial phosphorylation and such anaerobic pathways of mitochondrial energy metabolism may limit mitochondrial dysfunction upon ischemic/hypoxic insults.6 Therefore, ARG2 could exert a protective influence by generating indirectly α‐KG. Ansermet et al1 addressed both roles experimentally in the study published in Acta .

Authors employed inducible, epithelial‐specific, deletion in kidney and showed that ARG2‐deleted kidneys had lower arginase activity but with no overt morphological or functional renal phenotype. A 12h water deprivation challenge did not unravel any difference in urine volume and osmolality and no significant change in medullary aquaporin 2 protein abundance.1 This observation was in sharp contrast to published observations by Huang et al7 who showed enhanced urine concentration in absence of ARG2. Why this apparent difference since both studies used 12 hours of water deprivation in adult C57BL6 mice? Whereas Ansermet et al1 used male mice and inducible disruption of ARG2, Huang et al7 did not report mice sex and used germline global deletion of ARG2. At baseline, kidney medulla tissue urea was decreased by ARG2 deletion.1 However, Ansermet et al did not present tissue urea concentration after water deprivation. It is technically challenging to extract and determine urea in dissected inner medulla from mice, so the issue of ARG2 contribution to maximal urine concentration by local urea formation remains an intriguing but unresolved issue. The other part of the study tested the potential protective action of ARG2, which is a refreshing, provocative idea since most data point to a detrimental action of ARG2 activity in kidney disease. Arginine serves as a precursor for nitric oxide (NO) synthesis and arginine can be provided either through dietary intake or de novo synthesis in the urea cycle from citrulline. In the urea cycle, arginase catalyses cleavage of arginine into urea and ornithine. Thus, arginase competes with NO synthases for the common substrate L‐arginine and although K_m for arginase is much higher than for NOS3 (eNOS, abundant in kidney medulla), the V _max is much higher for arginase which explains competition. In 2 experimental models of kidney injury, mass reduction and diabetes, ARG2 deletion had protective effects. ARG2 was increased in experimental diabetic nephropathy8 along with albuminuria, inflammation and a decline in blood flow, effects that were attenuated by pharmacological arginase inhibition and tubular‐specific deletion of arginase 2.8 Also in the uni‐nephrectomy model, there was a decrease in systemic L‐arginine and a small blood pressure increase that was prevented by dietary arginine supplementation and by ARG2 deletion.9 Thus, in these 2 models, ARG2 aggravated injury.

In the present study, authors proposed that ARG2 activity, potentially through providing α‐KG, exerted a protective influence after transient kidney ischemia. Authors showed significant upregulation of ARG2 similar to previous studies8, 9 and, at 24 hours, there were aggravated tissue injury parameters, greater diuresis and electrolyte losses and lower urine concentration albeit with no change in tissue urea concentration.1 Fourteen days after the insult, in the chronic phase, ARG2 expression remained enhanced in ischemic kidney and tissue fibrosis markers, GFR and diuresis were not different. Plasma urea and arginine changed inversely in ischemic control mice and this response was abolished in ARG2‐deficient mice at a time where GFR was similar and not different from control. This documented that renal tubular ARG2 contributed to systemic arginine metabolism and urea formation after renal ischemia. Furthermore, authors showed that ARG2 deletion had no impact on tissue ornithine metabolites (polyamines putrescine and spermine) and on mitochondria number while the oxidative phosphorylation machinery was more impaired in ARG2‐/‐. What are the perspectives? First, when kidney function is stressed by diabetes, mass reduction or ischemia/hypoxia, this uniformly stimulated expression and activity of ARG2 in S3 segment and led to urea synthesis reflected in systemic levels. Thus, if applicable to humans, then plasma urea concentration in states of acute kidney injury not only reflects GFR decline but also proximal tubule stress. Second, if stimulated ARG2 activity in kidney can lower systemic L‐arginine, the substrate of NO synthases, then oral supplementation with L‐arginine could be relevant. A caveat in the published studies on kidney fibrosis and injury after systemic or local arginase deletion or pharmacologic inhibition is the confounding factor of blood pressure. Hypertension aggravates most chronic kidney disease conditions while lowering of blood pressure has the opposite effect. Blood pressure should be considered in the light of potential competition between arginase and NO synthases. Any aggravating or protective effect of ARG2 deletion or inhibition at kidney tissue level should be interpreted along with available data on arterial blood pressure.

Ansermet 等人1发表在本期生理学学报上的一项研究揭示了一种有趣的局部上皮酶对肾髓质尿素积累和缺血耐受性的贡献. 在体内研究表明，在肾小管上皮特异性缺失 L-精氨酸酶 2 (ARG2) 的小鼠中，ARG2 通过将 L-精氨酸水解为尿素和 L-鸟氨酸，促进髓质尿素积累，并有助于组织保护缺血再灌注损伤后。S3 片段离体产生尿素的能力已为人所知，现在已被证明有助于缺血性损伤后的全身性血浆尿素和尿素的髓质积聚。假设位于肾外髓质的近端小管 S3 段的能力，该区域在足够的氧气供应和代谢性氧气消耗之间有一个狭窄的界限，利用 ARG2 促进的无氧代谢的能力被假设为在缺血期间发挥保护作用。

精氨酸酶有两种器官特异性同工型，即在胞质溶胶中发现的肝脏精氨酸酶 1 和与线粒体相关的肾脏优势精氨酸酶 2 (ARG2)。ARG2 在位于皮质的直降部分的近端小管细胞中检测到，末端部分在外髓的外条纹中，以及在内髓集合管中，其中蛋白质定位与精氨酸酶活性相对应。2, 3精氨酸酶 2 在肾脏中的作用是什么？甚至在精氨酸酶亚型被克隆之前，在分离的髓质节段内局部形成尿素的能力促使 Levillain 等人41989 年提出了在形成和维持尿液浓缩所必需的皮质-髓质尿素浓度梯度中的作用。其次，由于这些细胞中存在鸟氨酸氨基转移酶，因此提出了与谷氨酰胺合成和α-酮戊二酸 (α-KG) 形成的偶联。5为什么这是相关的？肾脏外髓质非常容易受到缺血性损伤，外髓质中的 S3 近端小管细胞可能通过线粒体内磷酸化厌氧产生 ATP，线粒体能量代谢的这种厌氧途径可能会限制缺血/缺氧损伤时的线粒体功能障碍。6因此，ARG2 可以通过间接产生 α-KG 来发挥保护作用。Ansermet 等人1在Acta上发表的研究中通过实验解决了这两个角色。

作者在肾脏中采用了可诱导的、上皮特异性的缺失，并表明 ARG2 缺失的肾脏精氨酸酶活性较低，但没有明显的形态学或功能性肾脏表型。12 小时禁水挑战没有发现尿量和渗透压的任何差异，髓质水通道蛋白 2 蛋白丰度也没有显着变化。1这一观察结果与 Huang 等人7发表的观察结果形成鲜明对比，后者显示在没有 ARG2 的情况下尿液浓度增加。既然两项研究都在成年 C57BL6 小鼠中禁水 12 小时，为什么会有这种明显的差异？Ansermet 等人1使用雄性小鼠和 ARG2 的诱导性破坏，而 Huang 等人7没有报告小鼠性别并使用 ARG2 的种系全局缺失。在基线时，肾髓质组织尿素因 ARG2 缺失而减少。1然而，Ansermet 等人在缺水后没有呈现组织尿素浓度。从小鼠解剖的内髓质中提取和测定尿素在技术上具有挑战性，因此 ARG2 通过局部尿素形成对最大尿液浓度的贡献仍然是一个有趣但未解决的问题。该研究的另一部分测试了 ARG2 的潜在保护作用，这是一个令人耳目一新、具有挑衅性的想法，因为大多数数据都表明 ARG2 活性对肾脏疾病的有害作用。精氨酸是一氧化氮 (NO) 合成的前体，精氨酸可以通过饮食摄入或从瓜氨酸在尿素循环中从头合成来提供。在尿素循环中，精氨酸酶催化精氨酸裂解为尿素和鸟氨酸。因此，_{精氨酸酶的m}远高于 NOS3（eNOS，肾髓质中丰富），精氨酸酶的V _max高得多，这解释了竞争。在肾损伤、质量减少和糖尿病的2个实验模型中，ARG2缺失具有保护作用。ARG2 在实验性糖尿病肾病8中随着蛋白尿、炎症和血流量下降而增加，这些影响被药物精氨酸酶抑制和精氨酸酶 2 肾小管特异性缺失所减弱。8同样在单肾切除模型中，也有减少在全身性 L-精氨酸和通过膳食精氨酸补充和 ARG2 缺失防止的小幅血压升高。9因此，在这 2 个模型中，ARG2 加重了损伤。

在本研究中，作者提出 ARG2 活性可能通过提供 α-KG 在短暂性肾缺血后发挥保护作用。作者显示 ARG2 的显着上调与之前的研究8、9 相似，并且在 24 小时时，组织损伤参数加重，利尿和电解质损失更大，尿浓度降低，尽管组织尿素浓度没有变化。1损伤后 14 天，在慢性期，缺血性肾脏和组织纤维化标志物 ARG2 表达保持增强，GFR 和利尿没有差异。缺血对照小鼠的血浆尿素和精氨酸发生相反的变化，而这种反应在 ARG2 缺陷小鼠中被消除，此时 GFR 与对照相似且无差异。这证明肾小管 ARG2 有助于肾缺血后的全身精氨酸代谢和尿素形成。此外，作者表明，ARG2 缺失对组织鸟氨酸代谢物（多胺腐胺和精胺）和线粒体数量没有影响，而 ARG2-/- 中的氧化磷酸化机制受到的损害更大。有哪些观点？首先，当肾功能因糖尿病、体重减轻或缺血/缺氧而受到压力时，这一致刺激了 S3 片段中 ARG2 的表达和活性，并导致尿素合成反映在全身水平。因此，如果适用于人类，则急性肾损伤状态下的血浆尿素浓度不仅反映 GFR 下降，还反映近端小管压力。其次，如果刺激肾脏中的 ARG2 活性可以降低全身 L-精氨酸（NO 合酶的底物），那么口服补充 L-精氨酸可能是相关的。已发表的关于全身或局部精氨酸酶缺失或药物抑制后肾纤维化和损伤的研究中的一个警告是血压的混杂因素。高血压会加重大多数慢性肾脏疾病，而降低血压则会产生相反的效果。鉴于精氨酸酶和 NO 合酶之间的潜在竞争，应考虑血压。ARG2 缺失或肾组织水平抑制的任何加重或保护作用应与动脉血压的可用数据一起解释。