Ccr2-dependent monocytes exacerbate intestinal inflammation and modulate gut serotonergic signaling following traumatic brain injury.

BACKGROUND: Traumatic brain injury (TBI) leads to acute gastrointestinal dysfunction and mucosal damage, resulting in feeding intolerance. C-C motif chemokine receptor 2 (Ccr2 + ) monocytes are crucial immune cells that regulate the gut's inflammatory response via the brain-gut axis. Using Ccr2 ko mice, we investigated the intricate interplay between these cells to better elucidate the role of systemic inflammation after TBI. METHODS: A murine-controlled cortical impact model was used, and results were analyzed on postinjury days 1 and 3. The experimental groups included (1) sham C57Bl/6 wild type (WT), (2) TBI WT, (3) sham Ccr2 ko , and (4) TBI Ccr2 ko . Mice were euthanized on postinjury days 1 and 3 to harvest the ileum and study intestinal dysfunction and serotonergic signaling using a combination of quantitative real-time polymerase chain reaction, immunohistochemistry, fluorescein isothiocyanate-dextran motility assays, and flow cytometry. Student's t test and one-way analysis of variance were used for statistical analysis, with significance achieved when p < 0.05. RESULTS: Traumatic brain injury resulted in severe dysfunction and dysmotility of the small intestine in WT mice as established by significant upregulation of inflammatory cytokines iNOS , Lcn2 , TNFα , and IL1ß and the innate immunity receptor toll-like receptor 4 ( Tlr4 ). This was accompanied by disruption of genes related to serotonin synthesis and degradation. Notably, Ccr2 ko mice subjected to TBI showed substantial improvements in intestinal pathology. Traumatic brain injury Ccr2 ko groups demonstrated reduced expression of inflammatory mediators ( iNOS , Lcn2 , IL1ß , and Tlr4 ) and improvement in serotonin synthesis genes, including tryptophan hydroxylase 1 ( Tph1 ) and dopa decarboxylase ( Ddc ). CONCLUSION: Our study reveals a critical role for Ccr2 + monocytes in modulating intestinal homeostasis after TBI. Ccr2 + monocytes aggravate intestinal inflammation and alter gut-derived serotonergic signaling. Therefore, targeting Ccr2 + monocyte-dependent responses could provide a better understanding of TBI-induced gut inflammation. Further studies are required to elucidate the impact of these changes on brain neuroinflammation and cognitive outcomes.

2-Mercaptoethanol protects against DNA double-strand breaks after kidney ischemia and reperfusion injury through GPX4 upregulation.

BACKGROUND: Kidney ischemia reperfusion injury (IRI) is characterized by tubular cell death. DNA double-strand breaks is one of the major sources of tubular cell death induced by IRI. 2-Mercaptoethanol (2-ME) is protective against DNA double-strand breaks derived from calf thymus and bovine embryo. Here, we sought to determine whether treatment with 2-ME attenuated DNA double-strand breaks, resulting in reduced kidney dysfunction and structural damage in IRI. METHODS: Kidney IRI or sham-operation in mice was carried out. The mice were treated with 2-ME, Ras-selective lethal 3, or vehicle. Kidney function, tubular injury, DNA damage, antioxidant enzyme expression, and DNA damage response (DDR) kinases activation were assessed. RESULTS: Treatment with 2-ME significantly attenuated kidney dysfunction, tubular injury, and DNA double-strand breaks after IRI. Among DDR kinases, IRI induced phosphorylation of ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR), but IRI reduced phosphorylation of other DDR kinases including ataxia telangiectasia and Rad3 related, checkpoint kinase 1 (Chk1), Chk2, and Chinese hamster cells 1 (XRCC1). Treatment with 2-ME enhanced phosphorylation of ATM and ATM-mediated effector kinases in IRI-subjected kidneys, suggesting that 2-ME activates ATM-mediated DDR signaling pathway. Furthermore, 2-ME dramatically upregulated glutathione peroxidase 4 (GPX4) in IRI-subjected kidneys. Inhibition of GPX4 augmented adverse IRI consequences including kidney dysfunction, tubular injury, DNA double-strand breaks, and inactivation of ATM-mediated DDR signaling pathway after IRI in 2-ME-treated kidneys. CONCLUSIONS: We have demonstrated that exogenous 2-ME protects against DNA double-strand breaks after kidney IRI through GPX4 upregulation and ATM activation.

Modified chitosan for effective renal delivery of siRNA to treat acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden decrease in renal function and impacts growing number of people worldwide. RNA interference (RNAi) showed potential to treat diseases with no or limited conventional therapies, including AKI. Suitable carriers are needed to protect and selectively deliver RNAi to target cells to fully explore this therapeutic modality. Here, we report on the synthesis of chitosan modified with α-cyclam-p-toluic acid (C-CS) as a novel siRNA carrier for targeted delivery to injured kidneys. We demonstrate that conjugation of the α-cyclam-p-toluic acid to chitosan imparts the C-CS polymer with targeting and antagonistic properties to cells overexpressing chemokine receptor CXCR4. In contrast, the parent α-cyclam-p-toluic acid showed no such properties. Self-assembled C-CS/siRNA nanoparticles rapidly accumulate in the injured kidneys and show long retention in renal tubules. Apoptosis and metabolic and inflammatory pathways induced by p53 are important pathological mechanisms in the development of AKI. Nanoparticles with siRNA against p53 (sip53) were formulated and intravenously injected for attenuation of IRI-AKI. Due to the favorable accumulation in injured kidneys, the treatment with C-CS/sip53 decreased renal injury, extent of renal apoptosis, macrophage and neutrophil infiltration, and improved renal function. Overall, our study suggests that C-CS/siRNA nanoparticles have the potential to effectively accumulate and deliver therapeutic siRNAs to injured kidneys through CXCR4 binding, providing a novel way for AKI therapy.

Preferential siRNA delivery to injured kidneys for combination treatment of acute kidney injury.

Acute kidney injury (AKI) is characterized by a sudden loss of renal function and is associated with high morbidity and mortality. Tumor suppressor p53 and chemokine receptor CXCR4 were both implicated in the AKI pathology. Here, we report on the development and evaluation of polymeric CXCR4 antagonist (PCX) siRNA carrier for selective delivery to injured kidneys in AKI. Our results show that PCX/siRNA nanoparticles (polyplexes) provide protection against cisplatin injury to tubule cells in vitro when both CXCR4 and p53 are inhibited. The polyplexes selectively accumulate and are retained in the injured kidneys in cisplatin and bilateral ischemia reperfusion injury models of AKI. Treating AKI with the combined CXCR4 inhibition and p53 gene silencing with the PCX/sip53 polyplexes improves kidney function and decreases renal damage. Overall, our results suggest that the PCX/sip53 polyplexes have a significant potential to enhance renal accumulation in AKI and deliver therapeutic siRNA.

Smooth muscle-generated methylglyoxal impairs endothelial cell-mediated vasodilatation of cerebral microvessels in type 1 diabetic rats.

BACKGROUND AND PURPOSE: Endothelial cell-mediated vasodilatation of cerebral arterioles is impaired in individuals with Type 1 diabetes (T1D). This defect compromises haemodynamics and can lead to hypoxia, microbleeds, inflammation and exaggerated ischaemia-reperfusion injuries. The molecular causes for dysregulation of cerebral microvascular endothelial cells (cECs) in T1D remains poorly defined. This study tests the hypothesis that cECs dysregulation in T1D is triggered by increased generation of the mitochondrial toxin, methylglyoxal, by smooth muscle cells in cerebral arterioles (cSMCs). EXPERIMENTAL APPROACH: Endothelial cell-mediated vasodilatation, vascular transcytosis inflammation, hypoxia and ischaemia-reperfusion injury were assessed in brains of male Sprague-Dawley rats with streptozotocin-induced diabetes and compared with those in diabetic rats with increased expression of methylglyoxal-degrading enzyme glyoxalase-I (Glo-I) in cSMCs. KEY RESULTS: After 7-8 weeks of T1D, endothelial cell-mediated vasodilatation of cerebral arterioles was impaired. Microvascular leakage, gliosis, macrophage/neutrophil infiltration, NF-κB activity and TNF-α levels were increased, and density of perfused microvessels was reduced. Transient occlusion of a mid-cerebral artery exacerbated ischaemia-reperfusion injury. In cSMCs, Glo-I protein was decreased, and the methylglyoxal-synthesizing enzyme, vascular adhesion protein 1 (VAP-1) and methylglyoxal were increased. Restoring Glo-I protein in cSMCs of diabetic rats to control levels via gene transfer, blunted VAP-1 and methylglyoxal increases, cECs dysfunction, microvascular leakage, inflammation, ischaemia-reperfusion injury and increased microvessel perfusion. CONCLUSIONS AND IMPLICATIONS: Methylglyoxal generated by cSMCs induced cECs dysfunction, inflammation, hypoxia and exaggerated ischaemia-reperfusion injury in diabetic rats. Lowering methylglyoxal produced by cSMCs may be a viable therapeutic strategy to preserve cECs function and blunt deleterious downstream consequences in T1D.

Unilateral nephrectomy elongates primary cilia in the remaining kidney via reactive oxygen species.

The length of primary cilia is associated with normal cell and organ function. In the kidney, the change of functional cilia length/mass is associated with various diseases such as ischemia/reperfusion injury, polycystic kidney disease, and congenital solitary kidney. Here, we investigate whether renal mass reduction affects primary cilia length and function. To induce renal mass reduction, mice were subjected to unilateral nephrectomy (UNx). UNx increased kidney weight and superoxide formation in the remaining kidney. Primary cilia were elongated in proximal tubule cells, collecting duct cells and parietal cells of the remaining kidney. Mn(III) Tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), an antioxidant, reduced superoxide formation in UNx-mice and prevented the elongation of primary cilia. UNx increased the expression of phosphorylated ERK, p21, and exocyst complex members Sec8 and Sec10, in the remaining kidney, and these increases were prevented by MnTMPyP. In MDCK, a kidney tubular epithelial cell line, cells, low concentrations of H2O2 treatment elongated primary cilia. This H2O2-induced elongation of primary cilia was also prevented by MnTMPyP treatment. Taken together, these data demonstrate that kidney compensation, induced by a reduction of renal mass, results in primary cilia elongation, and this elongation is associated with an increased production of reactive oxygen species (ROS).

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.

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.

Involvement of hydrogen sulfide and homocysteine transsulfuration pathway in the progression of kidney fibrosis after ureteral obstruction.

Hydrogen sulfide (H2S) produced by cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE) in the transsulfuration pathway of homocysteine plays a number of pathophysiological roles. Hyperhomocysteinemia is involved in kidney fibrosis. However, the role of H2S in kidney fibrosis remains to be defined. Here, we investigated the role of H2S and its acting mechanism in unilateral ureteral obstruction (UO)-induced kidney fibrosis in mice. UO decreased expressions of CBS and CSE in the kidney with decrease of H2S concentration. Treatment with sodium hydrogen sulfide (NaHS, a H2S producer) during UO reduced UO-induced oxidative stress with preservations of catalase, copper-zinc superoxide dismutase (CuZnSOD), and manganese superoxide dismutase (MnSOD) expression, and glutathione level. In addition, NaHS mitigated decreases of CBS and CSE expressions, and H2S concentration in the kidney. NaHS treatment attenuated UO-induced increases in levels of TGF-ß1, activated Smad3, and activated NF-κB. This study provided the first evidence of involvement of the transsulfuration pathway and H2S in UO-induced kidney fibrosis, suggesting that H2S and its transsulfuration pathway may be a potential target for development of therapeutics for fibrosis-related diseases.

Activation of ERK accelerates repair of renal tubular epithelial cells, whereas it inhibits progression of fibrosis following ischemia/reperfusion injury.

Extracellular signal-regulated kinase (ERK) signals play important roles in cell death and survival. However, the role of ERK in the repair process after injury remains to be defined in the kidney. Here, we investigated the role of ERK in proliferation and differentiation of tubular epithelial cells, and proliferation of interstitial cells following ischemia/reperfusion (I/R) injury in the mouse kidney. Mice were subjected to 30min of renal ischemia. Some mice were administered with U0126, a specific upstream inhibitor of ERK, daily during the recovery phase, beginning at 1day after ischemia until sacrifice. I/R caused severe tubular cell damage and functional loss in the kidney. Nine days after ischemia, the kidney was restored functionally with a partial restoration of damaged tubules and expansion of fibrotic lesions. ERK was activated by I/R and the activated ERK was sustained for 9days. U0126 inhibited the proliferation, basolateral relocalization of Na,K-ATPase and lengthening of primary cilia in tubular epithelial cells, whereas it enhanced the proliferation of interstitial cells and accumulation of extracellular matrix. Furthermore, U0126 elevated the expression of cell cycle arrest-related proteins, p21 and phospholylated-chk2 in the post-ischemic kidney. U0126 mitigated the post-I/R increase of Sec10 which is a crucial component of exocyst complex and an important factor in ciliogenesis and tubulogenesis. U0126 also enhanced the expression of fibrosis-related proteins, TGF-ß1 and phosphorylated NF-κB after ischemia. Our findings demonstrate that activation of ERK is required for both the restoration of damaged tubular epithelial cells and the inhibition of fibrosis progression following injury.

Gender-specific role of HDAC11 in kidney ischemia- and reperfusion-induced PAI-1 expression and injury.

Male gender and the male hormone testosterone increase susceptibility to kidney ischemia and reperfusion (I/R) injury, which is associated with inflammatory responses. Possible involvement of histone deacetylase (HDAC) in inflammatory responses has been suggested. We investigated the gender-specific role of HDACs in plasminogen activator inhibitor type-1 (PAI-1) expression and I/R injury. PAI-1 inhibition protected the kidney from I/R-induced inflammation and functional loss. Among HDACs, only HDAC11 negatively regulated PAI-1 expression in I/R-subjected kidney gender specifically and lipopolysaccharide (LPS)-stimulated mouse monocytes/macrophages. HDAC11 gene silencing increased PAI-1 expression. Chromatin immunoprecipitation assay confirmed binding of HDAC11 to the promoter region of PAI-1 and then release by I/R insult or LPS treatment. I/R-induced HDAC11 release was inhibited by orchiectomy and reversed by dihydrotestosterone treatment. Release of HDAC11 increased acetylation of histone H3. In conclusion, male gender and male hormones accelerate I/R-induced decreases in expression and binding of HDAC11, resulting in an increase in PAI-1 expression. These data provide important insight into gender dimorphism offering HDAC11 as a novel target for I/R injury.

Defect in Runx2 gene accelerates ureteral obstruction-induced kidney fibrosis via increased TGF-ß signaling pathway.

Runt-related transcription factor 2 (Runx2) plays an important role in bone formation and de novo synthesis of proteins, including type 1 collagen. Runx2 has a potent effect on signaling of transforming growth factor (TGF)-ß and vice versa, implicating its significant role in fibrosis. Chronic renal failure comprises fibrosis, characterized as an increase in TGF-ß signaling, and expression of α-smooth muscle actin (α-SMA), and extracellular matrix proteins. Here, we evaluated the role of Runx2 in ureteral obstruction (UO)-induced kidney fibrosis using mice whose Runx2 gene expression is genetically down-regulated. UO caused tubular atrophy and dilation, expansion of interstitium, and increased expression of collagens and α-SMA with a concomitant decrease in expression of Runx2. Deficiency of Runx2 gene (Runx2(+/-) mice) showed higher expression of collagens and α-SMA in the kidney following UO compared to wild type (Runx2(+/+)) mice. UO-induced activation of TGF-ß signaling was higher in the Runx2(+/-) kidney than Runx2(+/+) kidney, suggesting an inhibitory effect of Runx2 on TGF-ß signaling in kidney fibrosis. Besides, overexpression of the Runx2 gene using an adenoviral vector in kidney tubule cells resulted in attenuated TGF-ß-induced Smad3 phosphorylation and expressions of α-SMA and collagen I. Furthermore, Runx2 gene deficient mouse embryonic fibroblasts induced greater activation of Smad3 and expression of α-SMA in response to TGF-ß. Collectively, Runx2 plays a protective role in UO-induced kidney fibrosis by inhibition of TGF-ß signaling, suggesting Runx2 as a novel target for protection against fibrosis-related diseases such as chronic renal failure.

Reduction of oxidative stress during recovery accelerates normalization of primary cilia length that is altered after ischemic injury in murine kidneys.

The primary cilium is a microtubule-based nonmotile organelle that extends from the surface of cells, including renal tubular cells. Here, we investigated the alteration of primary cilium length during epithelial cell injury and repair, following ischemia/reperfusion (I/R) insult, and the role of reactive oxygen species in this alteration. Thirty minutes of bilateral renal ischemia induced severe renal tubular cell damage and an increase of plasma creatinine (PCr) concentration. Between 8 and 16 days following the ischemia, the increased PCr returned to normal range, although without complete histological restoration. Compared with the primary cilium length in normal kidney tubule cells, the length was shortened 4 h and 1 day following ischemia, increased over normal 8 days after ischemia, and then returned to near normal 16 days following ischemia. In the urine of I/R-subjected mice, acetylated tubulin was detected. The cilium length of proliferating cells was shorter than that in nonproliferating cells. Mature cells had shorter cilia than differentiating cells. Treatment with Mn(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP), an antioxidant, during the recovery of damaged kidneys accelerated normalization of cilia length concomitant with a decrease of oxidative stress and morphological recovery in the kidney. In the Madin-Darby canine kidney (MDCK) cells, H(2)O(2) treatment caused released ciliary fragment into medium, and MnTMPyP inhibited the deciliation. The ERK inhibitor U0126 inhibited elongation of cilia in normal and MDCK cells recovering from H(2)O(2) stress. Taken together, our results suggest that primary cilia length reflects cell proliferation and the length of primary cilium is regulated, at least, in part, by reactive oxygen species through ERK.

Bone marrow-derived cells play a major role in kidney fibrosis via proliferation and differentiation in the infiltrated site.

Increase of interstitial cell population, resulting in the expansion of interstitium, excessive production of extracellular matrix, and reduction of functioning tubules, is critical in fibrotic progression in the kidney of patients suffering from chronic renal diseases. Here, we investigated the contribution of bone marrow-derived cells (BMDC) in kidney fibrosis caused by ureteral obstruction (UO) using eGFP bone marrow-reconstituted chimeric mice. UO caused dramatic increases in the numbers of interstitial cells and expansion of the interstitium. Most kidney interstitial cells expressed GFP. Twenty nine percent of interstitial cells were cells that had proliferated and approximately 89% among them were BMDCs. Proliferation of fibroblasts differentiated from BMDCs significantly occurred in the interstitium of UO-kidney. Removal of BMDCs by whole body irradiation after UO resulted in reduction of kidney fibrosis, while injection of RAW264.7 cells, monocytes/macrophages, into irradiated mice induced a reversal of this reduction. Treatment with apocynin, an inhibitor of NADPH oxidase, reduced infiltration of BMDCs into the UO-kidney, leading to reduction of kidney fibrosis. In addition, only a few slow-cycling cells were observed in the interstitium of normal kidney. Even after UO, no change in the number of those cells was observed. Our findings demonstrate that BMDCs are a major source for interstitial expansion during kidney fibrosis via infiltration into damaged sites, differentiation to fibroblasts, and subsequent proliferation, contributing kidney fibrosis. These data provide a clear therapeutic target for treatment of chronic kidney disease.

Previous ischemia and reperfusion injury results in resistance of the kidney against subsequent ischemia and reperfusion insult in mice; a role for the Akt signal pathway.

BACKGROUND: Kidneys previously exposed to ischemia and reperfusion (I/R), pre-conditioned by I/R, are less susceptible to subsequent I/R injury. Here, we investigated the role for protein kinase B (Akt) survival signaling pathways including anti-apoptosis pathways in the reduced susceptibility of I/R-pre-conditioned kidneys. METHODS: Mice were exposed to either a single I/R pre-conditioning event (SIRPC, 30 min of bilateral renal ischemia followed by 8 days of reperfusion) or sham-operation (non-SIRPC) and then subjected to either 30 min of bilateral renal ischemia or sham-operation (sham). Some of the mice received intra-peritoneal administrations of wortmannin, which is an inhibitor of phosphatidylinositol-3 kinase, PI3K. RESULTS: Thirty minutes of bilateral renal ischemia in non-SIRPC mice induced a dramatic increase in plasma creatinine (PCr) levels, but this was not observed in the SIRPC mouse. Consistent with the PCr results, tubular damage and apoptotic tubular cell death were more severe in the non-SIRPC mouse kidney than in the SIRPC mice. SIRPC increased the levels of phosphorylated-Akt and -Bad expression as well as the ratio of Bcl-2 to Bax expression in the kidney. I/R resulted in greater increases of phosphorylated-Akt and -Bad, Bcl-xL and Bcl-2, but a lower level of increase of Bax, in the SIRPC mouse kidneys than those in the non-SIRPC-mouse kidneys. Treatment with wortmannin during the SIRPC period inhibited SIRPC-induced increase in phosphorylated-Akt and -Bad expressions and eliminated tolerance of SIRPC mice kidneys to I/R insult. CONCLUSION: Ischemic pre-conditioning confers renal resistance to I/R-induced apoptosis via activation of the Akt signal pathway.

Bone marrow derived cells and reactive oxygen species in hypertrophy of contralateral kidney of transient unilateral renal ischemia-induced mouse.

Renal mass reduction, such as unilateral nephrectomy induces a compensatory hypertrophy of remaining renal mass in response to overload induced by reduction of functional renal parenchyma. In our recent study, we observed that the recovery of ischemic injured kidney following transient unilateral renal ischemia took longer time than that following transient bilateral renal ischemia, indicating that non-damaged kidney may affect the damaged kidney and vice versa. Here, we investigated whether transient and partial renal parenchymal injury by transient unilateral renal ischemia (UI) results in the hypertrophy of its contralateral kidney (CLK) and reactive oxygen species is associated with the hypertrophy. Thirty minutes of UI resulted in gradual increase in CLK weight over time. UI increased superoxide formation, but not lipid peroxidation in the CLK. After UI, a significant increase in the number of NADPH oxidase 2 (Nox2)-expressing cells and the level of Nox2 expression in the CLK was observed. In parallel with the increases in Nox2-expressing cells in CLKs, infiltration of bone marrow-derived cells (BMDC) increased in CLK. Treatments with Mn(III) Tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, a superoxide dismutase (SOD) mimetic) and apocynin (a putative NADPH oxidase inhibitor) inhibited UI-induced hypertrophy of CLK along with reduction in Nox2-positive cell, BMDC, amount of Nox2 expression and superoxide formation. In conclusion, transient and partial renal mass reduction by UI resulted in the hypertrophy of CLK through increased ROS formation by infiltrated cells into the interstitium of CLK.

Orchiectomy attenuates kidney fibrosis after ureteral obstruction by reduction of oxidative stress in mice.

BACKGROUND/AIMS: Men are generally more prone to chronic kidney disease and progression to end-stage renal disease than women. However, the underlying mechanisms remain unclear. In this study, we investigated the role of reactive oxygen species and testosterone in the progression of renal fibrosis in mice with unilateral ureteral obstruction (UUO). METHODS: Mice were subjected to either orchiectomy or sham operation 14 days before either UUO or sham surgery. Harvesting of the kidney was performed 7 days after the UUO surgery to measure the production of reactive oxygen species and expression of antioxidants such as catalase, copper-zinc superoxide dismutase, and manganese superoxide dismutase, as well as fibrosis markers including α-smooth muscle actin (α-SMA) and collagen. RESULTS: UUO resulted in increased expression of α-SMA and collagen deposition in the kidneys of both female and male mice. These increases were significantly greater in males than females. Orchiectomy significantly reduced increases in α-SMA expression and collagen deposition when compared with intact male. UUO increased the production of hydrogen peroxide and lipid peroxidation along with the decreases in expression of manganese superoxide dismutase, copper-zinc superoxide dismutase, and catalase. These changes induced by UUO were significantly attenuated by orchiectomy. CONCLUSION: Males are more susceptible to UUO-induced kidney fibrosis compared with females, and the higher susceptibility of males is obviated by orchiectomy along with reduction in oxidative stress.

Protective role of cytosolic NADP(+)-dependent isocitrate dehydrogenase, IDH1, in ischemic pre-conditioned kidney in mice.

Ischemic pre-conditioning protects the kidney against subsequent ischemia/reperfusion (I/R). This study investigated the role of cytosolic NADP(+)-dependent isocitrate dehydrogenase (IDH1), a producer of NADPH, in the ischemic pre-conditioning. Mice were pre-conditioned by 30 min of renal ischemia and 8 days of reperfusion. In non-pre-conditioned mice 30 min of ischemia had significantly increased the levels of plasma creatinine, BUN, lipid peroxidation and hydrogen peroxide in kidneys, whereas in pre-conditioned mice, the ischemia did not increase them. The reductions of reduced glutathione and NADPH after I/R were greater in non-pre-conditioned mice than in pre-conditioned mice. Ischemic pre-conditioning prevented the I/R-induced decreases in IDH1 activity and expression, but not in glucose-6-phosphate dehydrogenase activity. In conclusion, protection of the kidney afforded by ischemic pre-conditioning may be associated with increased activity of IDH1 which relates to increased levels of NADPH, increased ratios of GSH/total glutathione, less oxidative stress and less kidney injury induced by subsequent I/R insult.

Evaluation of kidney repair capacity using 99mTc-DMSA in ischemia/reperfusion injury models.

Quantitative (99m)Tc-DMSA renal uptake was studied in different renal ischemia/reperfusion (I/R) mice models for the assessment of renal repair capacity. Mice models of nephrectomy, uni- and bi-lateral I/R together with sham-operated mice were established. At 1h, 1d, 4d, 1, 2 and 3 wk after I/R, (99m)Tc-DMSA (27.7 ± 1.3 MBq) was injected via tail vein and after 3h post-injection, the mice were scanned for 30 min with pinhole equipped gamma camera. Higher uptake of (99m)Tc-DMSA was measured in normal kidneys of uni-lateral I/R model and nephrectomized kidney I/R model at 3 wk post-surgery. Comparing the restoration capacities of the affected kidneys of nephrectomy, uni- and bi-lateral I/R models, higher repair capacity was observed in the nephrectomized model followed by bi-lateral then uni-lateral models. The normal kidney may retard the restoration of damaged kidney in uni-lateral I/R model. Moreover, 3 wk after Uni-I/R, the size of injured kidney was significantly smaller than non-ischemic contralateral and sham operated kidneys, while nephrectomy I/R kidneys were significantly enlarged compared to all others at 3 wk post-surgery. Very strong correlation between (99m)Tc-DMSA uptake and weight of dissected kidneys in I/R models was observed. Consistent with (99m)Tc-DMSA uptake results, all histological results indicate that kidney recovery after injury is correlated with the amount of intact tubules and kidney sizes. In summary, our study showed good potentials of (99m)Tc-DMSA scan as a promising non-invasive method for evaluation of kidney restoration after I/R injuries. Interestingly, mice with Bi-I/R injury showed faster repair capacity than those with uni-I/R.

Reactive oxygen species generated by renal ischemia and reperfusion trigger protection against subsequent renal ischemia and reperfusion injury in mice.

Ischemic preconditioning by a single event of ischemia and reperfusion (SIRPC) dramatically protects renal function against ischemia and reperfusion (I/R) induced several weeks later. We recently reported that reactive oxygen species (ROS) and oxidative stress were sustained in a kidney that had functionally recovered from I/R injury, thus suggesting an association between SIRPC and ROS and oxidative stress. However, the role of ROS in SIRPC remains to be clearly elucidated. To assess the involvement of ROS in SIRPC, mice were subjected to SIRPC (30 min of bilateral renal ischemia and 8 days of reperfusion) and then exposed to I/R injury. Thirty minutes of bilateral renal ischemia in the non-SIRPC mice resulted in a marked increase in plasma creatinine levels 4 and 24 h after reperfusion, which was not observed in the I/R in the SIRPC mice. SIRPC resulted in increases in the levels of kidney superoxide. Administrations of manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin [MnTMPyP; a cell-permeable superoxide dismutase (SOD) mimetic] and N-acetylcysteine (NAc; a ROS scavenger) to SIRPC mice blocked the SIRPC-induced increase in superoxide levels and removed approximately 48-64% of the functional protection of the SIRPC kidney. Additionally, these administrations significantly inhibited I/R-induced increases in superoxide formation, hydrogen peroxide production, and lipid peroxidation, along with the inhibition of I/R-induced reductions in the expression and activity of manganese SOD, copper-zinc SOD, and catalase. Furthermore, administrations of MnTMPyP or NAc inhibited the SIRPC-induced increase in inducible nitric oxide synthase expression but did not inhibit the SIRPC-induced increases in heat shock protein-25 expression. In conclusion, the renoprotection afforded by SIRPC was triggered by ROS generated by SIRPC.