Infiltration of M1, but not M2, macrophages is impaired after unilateral ureter obstruction in Nrf2-deficient mice

Chronic inflammation can be a major driver of the failure of a variety of organs, including chronic kidney disease (CKD). The NLR family pyrin domain-containing 3 (NLRP3) inflammasome has been shown to play a pivotal role in inflammation in a mouse kidney disease model. Nuclear factor erythroid 2-related factor 2 (Nrf2), the master transcription factor for anti-oxidant responses, has also been implicated in inflammasome activation under physiological conditions. However, the mechanism underlying inflammasome activation in CKD remains elusive. Here, we show that the loss of Nrf2 suppresses fibrosis and inflammation in a unilateral ureter obstruction (UUO) model of CKD in mice. We consistently observed decreased expression of inflammation-related genes NLRP3 and IL-1β in Nrf2-deficient kidneys after UUO. Increased infiltration of M1, but not M2, macrophages appears to mediate the suppression of UUO-induced CKD symptoms. Furthermore, we found that activation of the NLRP3 inflammasome is attenuated in Nrf2-deficient bone marrow–derived macrophages. These results demonstrate that Nrf2-related inflammasome activation can promote CKD symptoms via infiltration of M1 macrophages. Thus, we have identified the Nrf2 pathway as a promising therapeutic target for CKD

マウス横紋筋融解症誘発性急性腎障害モデルにおけるNrf2活性化の意義の検討

生体は親電子性物質，活性酸素種によって生成される酸化ストレスから生体を保護する応答システムを有している．Keap1（Kelch-like ECH-associated protein 1） -Nrf2（NF-E2 related factor2）システムがこの応答機構において重要な役割を果たす．核内移行したNrf2は転写因子として，NQO1，HO-1などの抗酸化遺伝子群の発現を制御する．　横紋筋融解症による急性腎障害（AKI: Acute Kidney Injury）の機序として，酸化ストレスが尿細管障害に大きく関与する．それ故，横紋筋融解症誘発性AKI においてもNrf2活性化による腎保護効果が期待される．横紋筋融解症誘発性AKI におけるNrf2活性化の意義と治療標的としての可能性を検討した．ヒト近位尿細管上皮細胞（hPTECs）を用いhemin 刺激に対するNrf2活性化の意義を検討した．hemin 刺激によりhPTECs におけるNrf2関連抗酸化遺伝子群の上昇，細胞障害を認めた．Nrf2-siRNA によるNrf2ノックダウン（KD）を行うことでhemin 刺激に対する抗酸化遺伝子群の発現上昇は抑制され，細胞障害が有意に増悪した．野生型マウス （WT），Nrf2欠損マウス（Nrf2KO）を用い，グリセロール筋注による横紋筋融解症モデルを作成した．（１）WT/Cont，（２）WT/ 横紋筋融解症（RM），（３）Nrf2KO/Cont，（４）Nrf2KO/RM の４群で比較検討した．結果は，WT/Cont に比べWT/RM 群で腎機能障害，尿細管障害，マクロファージ浸潤を認め，Nrf2KO/RM 群で有意に増悪した．抗酸化遺伝子群の発現はNrf2KO/RM 群で低下していた．　横紋筋融解症誘発性AKI において，Nrf2活性化が腎保護効果を有する事が示された．横紋筋融解症によるAKI に対して，Nrf2活性化が新たな治療標的となり得ることが明らかとなった．Cells are equipped with cytoprotective systems against oxidative stress caused by reactive oxygen species and electrophilic stress. The Keap1-Nrf2 pathway plays a central role in such mechanisms against oxidative and xenobiotic damage. Nrf2, as a transcription factor, activates a series of genes including NQO1 and HO-1.As the mechanism of acute kidney injury (AKI) due to rhabdomyolysis, renal tubule injury due to oxidative stress is the major component of the pathology. Therefore, in rhabdomyolysisinduced AKI, reno-protective effect of Nrf2 activation is expected. In the present study, the role of Nrf2 activation in rhabdomyolysis-induced AKI was investigated. In vitro, human proximal tubular epithelial cells (hPTECs) were used to determine the significance of Nrf2 for hemin stimulation. Hemin stimulation revealed elevation of Nrf2-related antioxidant gene group and cytotoxicity. Nrf2 knockdown (KD) with Nrf2-siRNA suppressed the rise of the expression of the antioxidant genes against hemin stimulation, and the cell damage was significantly exacerbated. A model of rhabdomyolysis by glycerol intramuscular injection was also prepared in vivo using wild type mice (WT) and Nrf2-deficient mice (Nrf2 KO). These mice were of the C57BL/6J background. We divided them into four groups: (1) WT/Cont, (2) WT/rhabdomyolysis (RM), (3) Nrf2 KO/Cont, and (4) Nrf2 KO/RM. Renal dysfunction and macrophage infiltration occurred more often in the WT/RM than in the WT/Cont, and it significantly worsened in the Nrf2 KO/RM group compared to the WT/RM. The expression of the antioxidant gene group was suppressed more in the Nrf2 KO/RM group compared with the WT/RM.These results indicate that Nrf2 activation exerts reno-protective effect in rhabdomyolysisinduced AKI. Nrf2 activation may be a new therapeutic target for rhabdomyolysis-induced AKI

The eNOS-NO pathway attenuates kidney dysfunction via suppression of inflammasome activation in aldosterone-induced renal injury model mice.

Hypertension causes vascular complications, such as stroke, cardiovascular disease, and chronic kidney disease (CKD). The relationship between endothelial dysfunction and progression of kidney disease is well known. However, the relationship between the eNOS-NO pathway and chronic inflammation, which is a common pathway for the progression of kidney disease, remains unexplored. We performed in vivo experiments to determine the role of the eNOS-NO pathway by using eNOS-deficient mice in a hypertensive kidney disease model. All mice were unilateral nephrectomized (Nx). One week after Nx, the mice were randomly divided into two groups: the aldosterone infusion groups and the vehicle groups. All mice also received a 1% NaCl solution instead of drinking water. The aldosterone infusion groups were treated with hydralazine to correct blood pressure differences. After four weeks of drug administration, all mice were euthanized, and blood and kidney tissue samples were collected. In the results, NLRP3 inflammasome activation was elevated in the kidneys of the eNOS-deficient mice, and tubulointerstitial fibrosis was accelerated. Suppression of inflammasome activation by knocking out ASC prevented tubulointerstitial injury in the eNOS knockout mice, indicating that the eNOS-NO pathway is involved in the development of kidney dysfunction through acceleration of NLRP3 inflammasome in macrophages. We revealed that endothelial function, particularly the eNOS-NO pathway, attenuates the progression of renal tubulointerstitial injury via suppression of inflammasome activation. Clinically, patients who develop vascular endothelial dysfunction have lifestyle diseases, such as hypertension or diabetes, and are known to be at risk for CKD. Our study suggests that the eNOS-NO pathway could be a therapeutic target for the treatment of chronic kidney disease associated with endothelial dysfunction

Dipeptidyl peptidase-4 inhibitor linagliptin reduces urinary albumin excretion through the protection of glomerular endothelial function

　Background: In most developed countries, diabetic kidney disease is the most common cause of chronic kidney disease, leading to end-stage renal disease, and it is also associated with cardiovascular diseases, including heart failure, and a higher risk of other microvascular complications. A recent clinical trial indicated that the dipeptidyl peptidase-4 inhibitor linagliptin prevents the occurrence and progression of albuminuria in patients with type 2 diabetes. Thus, this study aimed to elucidate the molecular mechanism underlying the inhibitory effect of linagliptin on albuminuria in diabetic kidney disease. Methods: Control C57BL/6 mice and diabetic Ins2+/Akita mice were orally administered linagliptin (5 mg/kg/ day) every day for 8 weeks. Results: Compared to control mice, Ins2+/Akita mice had markedly elevated blood glucose and HbA1c levels, but there were no significant changes after linagliptin treatment. Furthermore, albuminuria and urinary 8-OHdG levels were significantly increased and glomerular mesangial area was significantly expanded in Ins2+/Akita mice compared to those in control mice; these changes were ameliorated by linagliptin treatment, which also improved the degradation of glomerular endothelial glycocalyx and enhancement of glomerular permeability of macromolecules. The activity of AMP-activated protein kinase and the expression of guanosine 5\u27-triphosphate cyclohydrolase I in human glomerular endothelial cells were significantly lower in high glucose conditions and were improved by linagliptin or GLP-1 administration. Discussion: These results together suggest that linagliptin reduced albuminuria in a blood glucose-independent manner via the reduction of oxidative stress and maintenance of the glycocalyx in endothelial cells. Thus, earlier treatment with linagliptin may slow the progression of diabetic kidney disease

Essential role and therapeutic targeting of the glomerular endothelial glycocalyx in lupus nephritis

Lupus nephritis (LN) is a major organ complication and cause of morbidity and mortality in patients with systemic lupus erythematosus (SLE). There is an unmet medical need for developing more efficient and specific, mechanism-based therapies, which depends on improved understanding of the underlying LN pathogenesis. Here we present direct visual evidence from high-power intravital imaging of the local kidney tissue microenvironment in mouse models showing that activated memory T cells originated in immune organs and the LN-specific robust accumulation of the glomerular endothelial glycocalyx played central roles in LN development. The glomerular homing of T cells was mediated via the direct binding of their CD44 to the hyaluronic acid (HA) component of the endothelial glycocalyx, and glycocalyx-degrading enzymes efficiently disrupted homing. Short-course treatment with either hyaluronidase or heparinase III provided long-term organ protection as evidenced by vastly improved albuminuria and survival rate. This glycocalyx/HA/memory T cell interaction is present in multiple SLE-affected organs and may be therapeutically targeted for SLE complications, including LN