Jee In Kim

C/EBP homologous protein (CHOP) gene deficiency attenuates renal ischemia/reperfusion injury in mice.

C/EBP homologous protein (CHOP), a transcription factor for the expression of apoptosis-related genes, plays an important role in endoplasmic reticulum (ER) stress-related organ diseases, including diseases of the kidney. Here, we investigated the role of CHOP in ischemia/reperfusion (I/R)-induced acute kidney injury using CHOP-knockout (CHOP(-/-)) and wild type (CHOP(+/+)) mice. Fifteen or thirty minutes of bilateral renal ischemia (I/R) insult resulted in necrotic and apoptotic tubular epithelial cell death, together with increases in plasma creatinine (PCr) and blood urea nitrogen (BUN) concentrations. After I/R, BiP/GRP78 and CHOP expressions in the kidney gradually increased over time. CHOP expression was greater in the outer medulla than that in the cortex and localized intensely in the nucleus. I/R caused apoptosis of tubular epithelial cells in both CHOP(-/-) and CHOP(+/+) mice. The number of apoptotic cells after I/R was lower in CHOP(-/-) mice than that in CHOP(+/+) mice. Consistent with the degree of apoptosis, I/R-induced kidney morphological and functional damages were milder in CHOP(-/-) than that in CHOP(+/+) mice. The cleavage of procaspase-3 and the induction of Bax protein after I/R were lower in CHOP(-/-) than that in CHOP(+/+) mice. In contrast, the expression levels of Bcl-2, Bcl-xL, cIAP2, Mcl-1, and XIAP were higher in CHOP(-/-) than that in CHOP(+/+) mice. These results indicate that I/R induces ER stress, leading to the activation of CHOP-associated apoptosis signals, resulting in renal functional and histological damages.

Recruitment and subsequent proliferation of bone marrow-derived cells in the postischemic kidney are important to the progression of fibrosis

Acute kidney injury (AKI) is an independent risk factor of the development of chronic kidney disease. Kidney fibrosis is a typical feature of chronic kidney disease and is characterized as an expansion of the interstitium due to increases in extracellular matrix molecules and interstitial cells caused by accumulations of extrarenal cells and by the proliferation or differentiation of intrarenal cells. However, the role of bone marrow-derived cells (BMDCs) in AKI-induced kidney fibrosis remains to be defined. Here, we investigated the role of BMDCs in kidney fibrosis after ischemia-reperfusion injury (IRI)-induced AKI in green fluorescent protein (GFP)-expressing bone marrow chimeric mice. IRI resulted in severe fibrotic changes in kidney tissues and dramatically increased interstitial cell numbers. Furthermore, GFP-expressing BMDCs accounted for >80% of interstitial cells in fibrotic kidneys. Interstitial GFP-expressing cells expressed α-smooth muscle actin (a myofibroblast marker), fibroblast-specific protein-1 (a fibroblast marker), collagen type III, and F4/80 (a macrophage marker). Over 20% of interstitial cells were bromodeoxyuridine-incorporating (proliferating) cells, and of these, 80% cells were GFP-expressing BMDCs. Daily treatment of IRI mice with apocynin (a NADPH oxidase inhibitor that functions as an antioxidant) from the day after surgery until euthanization slightly inhibited these changes with a small reduction of fibrosis. Taken together, our findings show that BMDCs make a major contribution to IRI-induced fibrosis due to their infiltration, subsequent differentiation, and proliferation in injured kidneys, suggesting that BMDCs be considered an important target for the treatment of kidney fibrosis.

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.

Regulator of G protein signaling 2 (RGS2) deficiency accelerates the progression of kidney fibrosis

The regulator of G protein signaling 2 (RGS2) is a potent negative regulator of Gq protein signals including the angiotensin II (AngII)/AngII receptor signal, which plays a critical role in the progression of fibrosis. However, the role of RGS2 on the progression of kidney fibrosis has not been assessed. Here, we investigated the role of RGS2 in kidney fibrosis induced by unilateral ureteral obstruction (UUO) in mice. UUO resulted in increased expression of RGS2 mRNA and protein in the kidney along with increases of AngII and its type 1 receptor (AT1R) signaling and fibrosis. Furthermore, UUO increased the levels of F4/80, Ly6G, myeloperoxidase, and CXCR4 in the kidneys. RGS2 deficiency significantly enhanced these changes in the kidney. RGS2 deletion in the bone marrow-derived cells by transplanting the bone marrow of RGS2 knock-out mice into wild type mice enhanced UUO-induced kidney fibrosis. Overexpression of RGS2 in HEK293 cells, a human embryonic kidney cell line, and RAW264.7 cells, a monocyte/macrophage line, inhibited the AngII-induced activation of ERK and increase of CXCR4 expression. These findings provide the first evidence that RGS2 negatively regulates the progression of kidney fibrosis following UUO, likely by suppressing fibrogenic and inflammatory responses through the inhibition of AngII/AT1R signaling.

Hepatic ischemia/reperfusion injury disrupts the homeostasis of kidney primary cilia via oxidative stress

Acute kidney injury (AKI) is a major complication of hepatic surgeries. The primary cilium protrudes to the lumen of kidney tubules and plays an important role in renal functions. Disruption of primary cilia homeostasis is highly associated with human diseases including AKI. Here, we investigated whether transient hepatic ischemia induces length change and deciliation of kidney primary cilia, and if so, whether reactive oxygen species (ROS)/oxidative stress regulates those. HIR induced damages to the liver and kidney with increases in ROS/oxidative stress. HIR shortened the cilia of kidney epithelial cells and caused them to shed into the urine. This shortening and shedding of cilia was prevented by Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, an antioxidant). The urine of patient undergone liver resection contained ciliary proteins. These findings indicate that HIR induces shortening and deciliation of kidney primary cilia into the urine via ROS/oxidative stress, suggesting that primary cilia is associated with HIR-induced AKI and that the presence of ciliary proteins in the urine could be a potential indication of kidney injury.

Methionine sulfoxide reductase A deficiency exacerbates progression of kidney fibrosis induced by unilateral ureteral obstruction

Methionine sulfoxide reductase A (MsrA), which stereospecifically catalyzes the reduction of methionine-S-sulfoxide, is an important reactive oxygen species (ROS) scavenger. Tissue fibrosis is a maladaptive repair process following injury, associated with oxidative stress. In this study, we investigated the role of MsrA in unilateral ureteral obstruction (UUO)-induced kidney fibrosis and its underlying mechanisms by using MsrA gene-deleted mice (MsrA(-/-)). MsrA deletion increased collagen deposition in the interstitium and the expression of collagen III and α-smooth muscle actin in the UUO kidneys, indicating that MsrA deficiency exacerbated the progression of UUO-induced kidney fibrosis. UUO reduced the kidney expression of MsrA, MsrB1, and MsrB2, thereby decreasing MsrA and MsrB activity. UUO increased hydrogen peroxide and lipid peroxidation levels and the ratio of oxidized glutathione (GSSG) to total glutathione (GSH) in the kidneys. The UUO-induced elevations in the levels of these oxidative stress markers and leukocyte markers were much higher in the MsrA(-/-) than in the MsrA(+/+) kidneys, the latter suggesting that the exacerbated kidney fibrosis in MsrA(-/-) mice was associated with enhanced inflammatory responses. Collectively, our data suggest that MsrA plays a protective role in the progression of UUO-induced kidney fibrosis via suppression of fibrotic responses caused by oxidative stress and inflammation.

Hydrogen sulfide-producing cystathionine γ-lyase is critical in the progression of kidney fibrosis

Cystathionine γ-lyase (CSE), the last key enzyme of the transsulfuration pathway, is involved in the production of hydrogen sulfide (H2S) and glutathione (GSH), which regulate redox balance and act as important antioxidant molecules. Impairment of the H2S- and GSH-mediated antioxidant system is associated with the progression of chronic kidney disease (CKD), characterized by kidney fibrosis and dysfunction. Here, we evaluated the role of CSE in the progression of kidney fibrosis after unilateral ureteral obstruction (UUO) using mice deficient in the Cse gene. UUO of wild-type mice reduced the expression of H2S-producing enzymes, CSE, cystathionine β-synthase, and 3-mercaptopyruvate sulfurtransferase in the obstructed kidneys, resulting in decreased H2S and GSH levels. Cse gene deletion lowered H2S and GSH levels in the kidneys. Deleting the Cse gene exacerbated the decrease in H2S and GSH levels and increase in superoxide formation and oxidative damage to proteins, lipids, and DNA in the kidneys after UUO, which were accompanied by greater kidney fibrosis, deposition of extracellular matrixes, expression of α-smooth muscle actin, tubular damage, and infiltration of inflammatory cells. Furthermore, Cse gene deletion exacerbated mitochondrial fragmentation and apoptosis of renal tubule cells after UUO. The data provided herein constitute in vivo evidence that Cse deficiency impairs renal the H2S- and GSH-producing activity and exacerbates UUO-induced kidney fibrosis. These data propose a novel therapeutic approach against CKD by regulating CSE and the transsulfuration pathway.

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.

IDH2 deficiency increases the liver susceptibility to ischemia-reperfusion injury via increased mitochondrial oxidative injury

Mitochondrial NADP+-dependent isocitrate dehydrogenase 2 (IDH2) is a major producer of mitochondrial NADPH, required for glutathione (GSH)-associated mitochondrial antioxidant systems including glutathione peroxidase (GPx) and glutathione reductase (GR). Here, we investigated the role of IDH2 in hepatic ischemia-reperfusion (HIR)-associated mitochondrial injury using Idh2-knockout (Idh2-/-) mice and wild-type (Idh2+/+) littermates. Mice were subjected to either 60 min of partial liver ischemia or sham-operation. Some mice were administered with 2-(2,2,6,6-tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (mito-TEMPO, a mitochondria-targeting antioxidant). HIR induced severe histological and functional damages of liver in both Idh2+/+ mice and Idh2-/- mice and those damages were more severe in Idh2-/- mice than in wild-type littermates. HIR induces dysfunction of IDH2, leading to the decreases of NADPH level and mitochondrial GR and GPx functions, consequently resulting in mitochondrial and cellular oxidative injury as reflected by mitochondrial cristae loss, mitochondrial fragmentation, shift in mitochondrial fission, cytochrome c release, and cell death. These HIR-induced changes were greater in Idh2-/- mice than wild-type mice. The mito-TEMPO supplement significantly attenuated the aforementioned changes, and these attenuations were much greater in Idh2-/- mice when compared with wild-type littermates. Taken together, results have demonstrated that HIR impairs in the IDH2-NADPH-GSH mitochondrial antioxidant system, resulting in increased mitochondrial oxidative damage and dysfunction, suggesting that IDH2 plays a critical role in mitochondrial redox balance and HIR-induced impairment of IDH2 function is associated with the pathogenesis of ischemia-reperfusion-induced liver failure.

Ablation of C/EBP homologous protein attenuates renal fibrosis after ureteral obstruction by reducing autophagy and microtubule disruption

Fibrosis is an undesirable consequence of injury and a critical problem in many diseases. Recent studies have demonstrated an association of C/EBP homologous protein (CHOP) with fibrosis. We investigated the mechanism of CHOP in kidney fibrosis progression after unilateral ureteral obstruction (UUO) using Chop gene-deleted (Chop-/-) mice and their wild-type littermates (Chop+/+). UUO-induced kidney fibrosis was reduced in the Chop-/- than Chop+/+ mice. After UUO, CHOP expression was detected in the cytosol and nucleus of distal tubule cells and collecting duct cells of the kidney. UUO formed the autophagosome and increased the expression of autophagy proteins, Beclin-1, LC3-I and II, and p62 in the kidneys. These UUO-induced changes were significantly reduced in Chop-/- mice. Furthermore, Chop gene deletion attenuated mitochondrial fragmentation with lower expression of Fis-1, a mitochondrial fission protein, but higher expression of Opa-1, a mitochondrial fusion protein, than that seen in the wild-type mice. UUO disrupted the microtubule, which is involved in autophagosome formation, and this disruption was milder in the Chop-/- than Chop+/+ mouse kidney, with less reduction of histone deacetylase 6 and α‑tubulin acetyl transferase, which acetylates tubulin, a component of the microtubule. After UUO, apoptosis, a consequence of autophagy and mitochondrial damage, was reduced in the Chop-/- mouse kidney cells than in Chop+/+ mice. Thus, the ablation of Chop attenuates renal fibrosis, accompanied by reduced autophagy, mitochondrial fragmentation, microtubule disruption, and apoptosis. Overall, these results suggest that CHOP plays a critical role in the progression of kidney fibrosis, likely through regulation of autophagy and apoptosis.