Rac1 inhibition protects the kidney against kidney ischemia/reperfusion through the inhibition of macrophage migration.

Kidney ischemia/reperfusion (I/R) injury, a common cause of acute kidney injury (AKI), is associated with the migration of inflammatory cells into the kidney. Ras-related C3 botulinum toxin substrate 1 (Rac1), a member of the Rho family of small GTPase, plays an important role in inflammatory cell migration by cytoskeleton rearrangement. Here, we investigated the role of Rac1 on kidney I/R injury and macrophage migration. Male mice were subjected to either 25 min of bilateral ischemia followed by reperfusion (I/R) or a sham operation. Some mice were administrated with either NSC23766, an inhibitor of Rac1, or 0.9% NaCl (vehicle). Kidney damage and Rac1 activity and expression were measured. The migration and lamellipodia formation of RAW264.7 cells, mouse monocyte/macrophage, induced by monocyte chemoattractant protein-1 (MCP-1, a chemokine) were determined using transwell migration assay and phalloidin staining, respectively. In sham-operated kidneys, Rac1 was expressed in tubular cells and interstitial cells. In I/R-injured kidneys, Rac1 expression was decreased in tubule cells in correlation with the damage of tubular cells, whereas Rac1 expression increased in the interstitium in correlation with an increased population of F4/80 cells, monocytes/macrophages. I/R increased Rac1 activity without changing total Rac1 expression in the whole kidney lysates. NSC23766 administration blocked Rac1 activation and protected the kidney against I/R-induced kidney damage and interstitial F4/80 cell increase. NSC23766 suppressed monocyte MCP-1-induced lamellipodia and filopodia formation and migration of RAW 264.7 cells. These results indicate Rac1 inhibition protects the kidney against I/R via inhibition of monocytes/macrophages migration into the kidney.

A nonbiodegradable scaffold-free cell sheet of genome-engineered mesenchymal stem cells inhibits development of acute kidney injury.

Cell therapy using genome-engineered stem cells has emerged as a novel strategy for the treatment of kidney diseases. By exploiting genome editing technology, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) secreting an angiogenic factors or an anti-inflammatory factor were generated for therapeutic application in acute kidney injury. Junction polymerase chain reaction analysis verified zinc finger nucleases-assisted integration of the desired gene into the hUC-MSCs. Flow cytometry and differentiation assays indicated that genome editing did not affect the differentiation potential of these mesenchymal stem cells. Protein measurement in conditioned media with the use of ELISA and immunoblotting revealed the production and secretion of each integrated gene product. For cell therapy in the bilateral ischemia-reperfusion mouse model of acute kidney injury, our innovative scaffold-free cell sheets were established using a non-biodegradable temperature-responsive polymer. One of each type of scaffold-free cell sheets of either the angiogenic factor vascular endothelial grown factor or angiopoietin-1, or the anti-inflammatory factor erythropoietin, or α-melanocyte-stimulating hormone-secreting hUC-MSCs was applied to the decapsulated kidney surface. This resulted in significant amelioration of kidney dysfunction in the mice with acute kidney injury, effects that were superior to intravenous administration of the same genome-engineered hUC-MSCs. Thus, our scaffold-free cell sheets of genome-engineered mesenchymal stem cells provides therapeutic effects by inhibiting acute kidney injury via angiogenesis or anti-inflammation.

IDH2 gene deficiency accelerates unilateral ureteral obstruction-induced kidney inflammation through oxidative stress and activation of macrophages.

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) produces NADPH, which is known to inhibit mitochondrial oxidative stress. Ureteral obstruction induces kidney inflammation and fibrosis via oxidative stress. Here, we investigated the role and underlying mechanism of IDH2 in unilateral ureteral obstruction (UUO)-induced kidney inflammation using IDH2 gene deleted mice (IDH2-/-). Eight- to 10-week-old female IDH2-/- mice and wild type (IDH2+/+) littermates were subjected to UUO and kidneys were harvested 5 days after UUO. IDH2 was not detected in the kidneys of IDH2-/- mice, while UUO decreased IDH2 in IDH2+/+ mice. UUO increased the expressions of markers of oxidative stress in both IDH2+/+ and IDH2-/- mice, and these changes were greater in IDH2-/- mice compared to IDH2+/+ mice. Bone marrow-derived macrophages of IDH2-/- mice showed a more migrating phenotype with greater ruffle formation and Rac1 distribution than that of IDH2+/+ mice. Correspondently, UUO-induced infiltration of monocytes/macrophages was greater in IDH2-/- mice compared to IDH2+/+ mice. Taken together, these data demonstrate that IDH2 plays a protective role against UUO-induced inflammation through inhibition of oxidative stress and macrophage infiltration.

Mitochondrial NADP+-Dependent Isocitrate Dehydrogenase Deficiency Exacerbates Mitochondrial and Cell Damage after Kidney Ischemia-Reperfusion Injury.

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) catalyzes the oxidative decarboxylation of isocitrate to α-ketoglutarate, synthesizing NADPH, which is essential for mitochondrial redox balance. Ischemia-reperfusion (I/R) is one of most common causes of AKI. I/R disrupts the mitochondrial redox balance, resulting in oxidative damage to mitochondria and cells. Here, we investigated the role of IDH2 in I/R-induced AKI. I/R injury in mice led to the inactivation of IDH2 in kidney tubule cells. Idh2 gene deletion exacerbated the I/R-induced increase in plasma creatinine and BUN levels and the histologic evidence of tubule injury, and augmented the reduction of NADPH levels and the increase in oxidative stress observed in the kidney after I/R. Furthermore, Idh2 gene deletion exacerbated I/R-induced mitochondrial dysfunction and morphologic fragmentation, resulting in severe apoptosis in kidney tubule cells. In cultured mouse kidney proximal tubule cells, Idh2 gene downregulation enhanced the mitochondrial damage and apoptosis induced by treatment with hydrogen peroxide. This study demonstrates that Idh2 gene deletion exacerbates mitochondrial damage and tubular cell death via increased oxidative stress, suggesting that IDH2 is an important mitochondrial antioxidant enzyme that protects cells from I/R insult.

Macrophages promote Fibrinogenesis during kidney injury.

Macrophages (Mø) are widely considered fundamental in the development of kidney fibrosis since Mø accumulation commonly aggravates kidney fibrosis, while Mø depletion mitigates it. Although many studies have aimed to elucidate Mø-dependent mechanisms linked to kidney fibrosis and have suggested various mechanisms, the proposed roles have been mostly passive, indirect, and non-unique to Mø. Therefore, the molecular mechanism of how Mø directly promote kidney fibrosis is not fully understood. Recent evidence suggests that Mø produce coagulation factors under diverse pathologic conditions. Notably, coagulation factors mediate fibrinogenesis and contribute to fibrosis. Thus, we hypothesized that kidney Mø express coagulation factors that contribute to the provisional matrix formation during acute kidney injury (AKI). To test our hypothesis, we probed for Mø-derived coagulation factors after kidney injury and uncovered that both infiltrating and kidney-resident Mø produce non-redundant coagulation factors in AKI and chronic kidney disease (CKD). We also identified F13a1, which catalyzes the final step of the coagulation cascade, as the most strongly upregulated coagulation factor in murine and human kidney Mø during AKI and CKD. Our in vitro experiments revealed that the upregulation of coagulation factors in Mø occurs in a Ca2 + -dependent manner. Taken together, our study demonstrates that kidney Mø populations express key coagulation factors following local injury, suggesting a novel effector mechanism of Mø contributing to kidney fibrosis.

Shortening of primary cilia length is associated with urine concentration in the kidneys.

BACKGROUND: The primary cilium, a microtubule-based cellular organelle present in certain kidney cells, functions as a mechano-sensor to monitor fluid flow in addition to various other biological functions. In kidneys, the primary cilia protrude into the tubular lumen and are directly exposed to pro-urine flow and components. However, their effects on urine concentration remain to be defined. Here, we investigated the association between primary cilia and urine concentration. METHODS: Mice either had free access to water (normal water intake, NWI) or were not allowed access to water (water deprivation, WD). Some mice received tubastatin, an inhibitor of histone deacetylase 6 (HDAC6), which regulates the acetylation of α-tubulin, a core protein of microtubules. RESULTS: WD decreased urine output and increased urine osmolality, concomitant with apical plasma membrane localization of aquaporin 2 (AQP2) in the kidney. After WD, compared with after NWI, the lengths of primary cilia in renal tubular epithelial cells were shortened and HDAC6 activity increased. WD induced deacetylation of α-tubulin without altering α-tubulin levels in the kidney. Tubastatin prevented the shortening of cilia through increasing HDAC6 activity and consequently increasing acetylated α-tubulin expression. Furthermore, tubastatin prevented the WD-induced reduction of urine output, urine osmolality increase, and apical plasma membrane localization of AQP2. CONCLUSIONS: WD shortens primary cilia length through HDAC6 activation and α-tubulin deacetylation, while HDAC6 inhibition blocks the WD-induced changes in cilia length and urine output. This suggests that cilia length alterations are involved, at least in part, in the regulation of body water balance and urine concentration.

High water intake induces primary cilium elongation in renal tubular cells.

Background: The primary cilium protrudes from the cell surface and functions as a mechanosensor. Recently, we found that water intake restriction shortens the primary cilia of renal tubular cells, and a blockage of the shortening disturbs the ability of the kidneys to concentrate urine. Here, we investigate whether high water intake (HWI) alters primary cilia length, and if so, what is its underlying mechanism and its role on kidney urine production. Methods: Experimental mice were given free access to normal water (normal water intake) or 3% sucrose-containing water for HWI for 2 days. Some mice were administered with U0126 (10 mg/kg body weight), an inhibitor of MEK kinase, from 2 days before HWI, daily. The primary cilium length and urine amount and osmolality were investigated. Results: HWI-induced diluted urine production and primary cilium elongation in renal tubular cells. HWI increased the expression of α-tubulin acetyltransferase 1 (αTAT1), leading to the acetylation of α-tubulins, a core protein of the primary cilia. HWI also increased phosphorylated ERK1/2 (p-ERK1/2) and exocyst complex component 5 (EXOC5) expression in the kidneys. U0126 blocked HWI-induced increases in αTAT1, p-ERK1/2, and EXOC5 expression. U0126 inhibited HWI-induced α-tubulin acetylation, primary cilium elongation, urine amount increase, and urine osmolality decrease. Conclusion: These results show that increased water intake elongates the primary cilia via ERK1/2 activation and that ERK inhibition prevents primary cilium elongation and diluted urine production. These data suggest that the elongation of primary cilium length is associated with the production of diluted urine.

Short-term control of diet affects cisplatin-induced acute kidney injury through modulation of mitochondrial dynamics and mitochondrial GSH.

Obesity affects acute kidney injury (AKI) induced by various clinical settings, including transplantation and cisplatin-cancer therapy. However, the effect of short-term food intake change remains to be defined. Here, we investigated the effects of short-term high-fat diet intake and food restriction on cisplatin-induced AKI. Mice were fed either a high-fat diet (HFD) or a low-fat diet (LFD) for 11 days or were not fed for 40 hh (fasting), before cisplatin administration. Cisplatin-induced functional and structural damages to kidneys in both HFD- and LFD-fed mice, with greater damages in HFD-fed mice than LFD-fed mice. HFD decreased mitochondrial total glutathione (tGSH) level, along with increases in the plasma and kidney cholesterol levels. Cisplatin caused the increase of kidney cholesterol levels and oxidative stress, along with the decrease of mitochondrial tGSH levels. In addition, cisplatin-induced mitochondrial damage and apoptosis of tubular cells in both HFD- and LFD-fed mice. An increase of Fis1 (mitochondria fission 1 protein), whereas a decrease of Opa1 (mitochondria fusion 1 protein) occurred by cisplatin. These cisplatin effects were greater in HFD-fed mice than in LFD-fed mice. Administration of mitochondria-specific antioxidant treatment during HFD feeding inhibited these cisplatin-induced changes. Fasting for 40 h also significantly reduced the cisplatin-induced changes mentioned above. These data demonstrate that short-term HFD intake worsens cisplatin-induced oxidative stress by the reduction of mitochondrial tGSH, resulting in increased cisplatin-induced nephrotoxicity. These data newly indicate that the control of calorie intake, even for a short period, affects kidney susceptibility to injury. Although most studies described the effects of a long-term high-fat diet on the kidneys, in this study, we found that even if a high-fat diet was consumed for a short-term, physiological changes and mitochondria tGSH decrease in the kidneys, and consequently increased cisplatin-nephrotoxic susceptibility. These data suggest the association of calorie intake with kidney susceptibility to cisplatin.

In Vivo Evaluation of Dendropanax morbifera Leaf Extract for Anti-Obesity and Cholesterol-Lowering Activity in Mice.

Metabolic syndrome is a worldwide health problem, and obesity is closely related to type 2 diabetes, cardiovascular disease, hypertension, and cancer. According to WHO in 2018, the prevalence of obesity in 2016 tripled compared to 1975. D. morbifera reduces bad cholesterol and triglycerides levels in the blood and provides various antioxidant nutrients and germicidal sub-stances, as well as selenium, which helps to remove active oxygen. Moreover, D. morbifera is useful for treating cardiovascular diseases, hypertension, hyperlipidemia, and diabetes. Therefore, we study in vivo efficacy of D. morbifera to investigate the prevention effect of obesity and cholesterol. The weight and body fat were effectively reduced by D. morbifera water (DLW) extract administration to high-fat diet-fed C57BL/6 mice compared to those of control mice. The group treated with DLW 500 mg∙kg-1∙d-1 had significantly lower body weights compared to the control group. In addition, High-density lipoprotein (HDL) cholesterol increased in the group treated with DLW 500 mg∙kg-1∙d-1. The effect of DLW on the serum lipid profile could be helpful to prevent obesity. DLW suppresses lipid formation in adipocytes and decreases body fat. In conclusion, DLW can be applied to develop anti-obesity functional foods and other products to reduce body fat.

Isocitrate dehydrogenase 2 deficiency aggravates prolonged high-fat diet intake-induced hypertension.

The development of hypertension is associated with mitochondrial redox balance disruptions. NADP+-dependent isocitrate dehydrogenase 2 (IDH2) plays an important role in the maintenance of mitochondrial redox balance by producing mitochondrial NADPH, which is an essential cofactor in the reduction of glutathione (from GSSG to GSH) to reduced form of glutathione (GSH). We investigated the association of IDH2 between the development of prolonged high-fat diet (HFD)-induced hypertension. Idh2 gene-deleted (Idh2-/-) male mice and wild-type (Idh2+/+) littermates were fed either HFD or low-fat diet (LFD). Some mice were administrated with Mito-TEMPO, a mitochondria-specific antioxidant. HFD feeding increased blood pressure (BP) in both Idh2-/- mice and Idh2+/+ mice. HFD-induced BP increase was greater in Idh2-/- than Idh2+/+ mice. HFD intake decreased IDH2 activity, NADPH levels, and the GSH/(GSH + GSSG) ratio in the renal mitochondria. However, HFD intake increased mitochondrial ROS levels, along with the accompanying oxidative stress and damage. HFD intake increased angiotensin II receptor 1 type 1 mRNA levels in the kidneys and plasma renin and angiotensin II concentrations. These HFD-induced changes were more prominent in Idh2-/- mice than Idh2+/+ mice. Mito-TEMPO mitigated the HFD-induced changes in both Idh2-/- and Idh2+/+ mice, with greater effects in Idh2-/- mice than Idh2+/+ mice. These results indicate that prolonged HFD intake disrupts the IDH2-NADPH-GSH-associated antioxidant system and activates the renin-angiotensin system in the kidney, leading to increased BP, suggesting that IDH2 is a critical enzyme in the development of hypertension and that the IDH2-associated antioxidant system could serve as a potential hypertension treatment target.

Fragmentation of kidney epithelial cell primary cilia occurs by cisplatin and these cilia fragments are excreted into the urine.

The primary cilium, which protrudes from the cell surface, is associated with the pathogenesis of various diseases, including acute kidney injury (AKI). Primary cilium length dynamically changes during the progression of diseases. However, its relevance in disease and the underlying mechanism are largely unknown. In this study, we investigated the role of primary cilia in AKI induced by cisplatin, an effective anticancer drug, and the underlying mechanisms. In addition, we evaluated the usefulness of length alteration and deciliation of primary cilia into the urine for the diagnosis of AKI. Cisplatin induced shortening, elongation, and normalization of the primary cilia in kidney epithelial cells over time. During shortening, primary cilia fragments and ciliary proteins were excreted into the urine. During deciliation, cell proliferation and the expression of cyclin-dependent kinase inhibitor and proliferating cell nuclear antigen were not significantly changed. Shortening and deciliation of primary cilia were observed before significant increases in plasma creatinine and blood urea nitrogen concentration occurred. Pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, prevented cisplatin-induced primary cilium shortening and inhibited the increases in superoxide formation, lipid peroxidation, blood urea nitrogen, and tissue damage. In contrast, isocitrate dehydrogenase 2 (Idh2) gene deletion, which results in defect of the NADPH-associated mitochondrial antioxidant system, exacerbated cisplatin-induced changes in mice. Taken together, our findings demonstrate that cisplatin induces deciliation into the urine and antioxidant treatment prevents this deciliation, renal dysfunction, and tissue damage after cisplatin injection. These results suggest that cisplatin-induced AKI is associated with primary cilia and urine primary cilia proteins might be a non-invasive biomarker of kidney injury.

Mitochondrial NADP+-dependent isocitrate dehydrogenase deficiency increases cisplatin-induced oxidative damage in the kidney tubule cells.

Mitochondrial NADP+-dependent isocitrate dehydrogenase (IDH2) plays an important role in the formation of NADPH, which is critical for the maintenance of mitochondrial redox balance. Cis-diamminedichloroplatinum II (cisplatin), an effective anticancer drug, induces oxidative stress-related nephrotoxicity, limiting its use. Therefore, we investigated whether IDH2, which is a critical enzyme in the NADPH-associated mitochondrial antioxidant system, is involved in cisplatin nephrotoxicity. Idh2 gene-deleted (Idh2-/-) mice and wild-type (Idh2 +/+ ) littermates were treated with cisplatin, with or without 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (Mito-T), a mitochondria-specific antioxidant. Cisplatin-induced renal functional and morphological impairments were greater in Idh2-/- mice than in Idh2 +/+ mice. Mito-T mitigated those impairments in both Idh2-/- and Idh2 +/+ mice and this mitigation was greater in Idh2-/- than in Idh2 +/+ mice. Cisplatin impaired IDH2 function in the mitochondria, decreasing mitochondrial NADPH and GSH levels and increasing H2O2 generation; protein, lipid, and DNA oxidation; mitochondrial damage; and apoptosis. These cisplatin-induced changes were much more severe in Idh2-/- mice than in Idh2 +/+ mice. Mito-T treatment attenuated cisplatin-induced alterations in both Idh2-/- and Idh2 +/+ mice and this mitigation was greater in Idh2-/- than in Idh2 +/+ mice. Altogether, these data demonstrate that cisplatin induces the impairment of the mitochondrial IDH2-NADPH-GSH antioxidant system and IDH2 deficiency aggravates cisplatin-induced mitochondrial oxidative damage, inducing more severe nephrotoxicity. This suggests that the mitochondrial IDH2-NADPH-GSH antioxidant system is a target for the prevention of cisplatin-induced kidney cell death.