Cytoglobin (Cygb), a novel member of the globin superfamily, is expressed by fibroblasts in various organs. However, its function remains unknown. Because of its localization, we speculated that a biological role of Cygb may be related to fibrogenesis. To clarify the role of Cygb in kidney fibrosis, we employed the remnant kidney model of in rats. Immunohistochemical analysis showed an increase in Cygb expression in parallel with disease progression. To investigate the functional consequence of Cygb up-regulation, we established transgenic rats overexpressing rat Cygb. Overexpression of Cygb improved histological injury, preserved renal function, and ameliorated fibrosis, as estimated by the accumulation of collagen I and IV as well as Masson's trichrome staining. These protective effects of Cygb were associated with a decrease in of nitrotyrosine deposition in the kidney and urinary 8-OHdG excretion as a marker of oxidative stress. We also performed in vitro studies utilizing a rat kidney fibroblast cell line transiently overexpressing Cygb, an inducible kidney cell transfectant with Cygb, and primary cultured fibroblasts isolated from the kidneys of the transgenic rats. These different experimental systems consistently showed that Cygb inhibited collagen synthesis. Further, mutant disruption of heme in Cygb which impaired its anti-oxidant properties led to the loss of anti-fibrotic effects, suggesting that Cygb reduces fibrosis via a radical scavenging function. In conclusion, we showed that Cygb plays an important role in protection of the kidney against fibrosis via the amelioration of oxidative stress both in vitro and in vivo. Cygb might represent a good therapeutic target in chronic kidney disease.

Obesity is one of several factors implicated in the genesis of diabetic nephropathy (DN). Obese, hypertensive, type 2 diabetic rats SHR/NDmcr-cp were given, for 12 weeks, either a normal, middle-carbohydrate/middle-fat diet (MC/MF group) or a high-carbohydrate/low-fat diet (HC/LF group). Daily caloric intake was the same in both groups. Nevertheless, the HC/LF group gained less weight. Despite equivalent degrees of hypertension, hyperglycemia, hyperlipidemia, hyperinsulinemia, and even a poorer glycemic control, the HC/LF group had less severe renal histological abnormalities and a reduced intrarenal advanced glycation and oxidative stress. Mediators of the renoprotection, specifically linked to obesity and body weight control, include a reduced renal inflammation and TGF-beta expression, together with an enhanced level of adiponectin. Altogether, these data identify a specific role of body weight control by a high-carbohydrate/low-fat diet in the progression of DN. Body weight control thus impacts on local intrarenal advanced glycation and oxidative stress through inflammation and adiponectin levels.

Uremic toxins can deteriorate renal function, but little is known about its mechanism. Because tubular injury is central to progression of chronic kidney disease (CKD), we investigated the effects of a representative uremic toxin indoxyl sulfate (IS) on tubular cells. IS induced endoplasmic reticulum (ER) stress in cultured human proximal tubular cells, demonstrated by the increase in C/EBP homologous protein (CHOP) in the immunoblots. Moreover, administration of an oral adsorbent AST-120 reduced serum IS concentration and decreased tubular expression of CHOP in immunohistochemistry in 5/6-nephretomized, CKD model, rats. Further, we disclosed that IS inhibited proliferation of tubular cells in MTS and 5-bromo-2'-deoxy-uridine assay, while the results of trypan blue exclusion and lactate dehydrogenase assay showed that IS did not promote cell death. This inhibition was mitigated by small interfering (si)RNA against CHOP. Furthermore, IS increased a cyclin-dependent kinase inhibitor p21 (WAF1/CIP1) (p21). Surprisingly, this was mediated by an inflammatory cytokine interleukin (IL)-6, the expression of which was decreased by siRNA against ATF4, another ER stress marker; however, the induction IL-6 and p21 by IS was not suppressed by siRNA targeted to CHOP, suggesting that they were downstream of ER stress, but independent of CHOP. Moreover, we found that their up-regulation was dependent on ERK, using an ERK pathway inhibitor U0126. Collectively, we demonstrated that IS induced ER stress in tubular cells and inhibited cell proliferation via two pathways downstream of ER stress; namely CHOP, and ERK - IL-6 - p21. These are possible targets for suppressing progression of CKD.

In addition to causing uremic symptoms, uremic toxins accelerate the progression of renal failure. In order to elucidate the pathophysiology of uremic states, we investigated the effect of indoxyl sulfate (IS), a representative uremic toxin, on oxygen metabolism in tubular cells. We demonstrated an increase in oxygen consumption by IS in freshly isolated rat and human proximal tubules. Studies utilizing ouabain, the Na/K-ATPase inhibitor, and apocynin, the NADPH oxidase inhibitor, as well as the in vivo gene silencing approach to knock down p22(phox) showed that the increase in tubular oxygen consumption by IS is dependent on Na/K-ATPase and oxidative stress. We investigated whether the enhanced oxygen consumption led to subsequent hypoxia of the kidney. An increase in serum IS concentrations in rats administered with indole was associated with a decrease in renal oxygenation (8 hours). The remnant kidney in rats developed hypoxia at 16 weeks. Treatment of the rats with AST-120, an oral adsorbent that removes uremic toxins, reduced serum IS levels and improved oxygenation of the kidney. Amelioration of hypoxia in the remnant kidney was associated with better renal functions and less histological injury. Reduction of serum IS levels also led to a decrease in oxidative stress in the kidney. Our ex vivo and in vivo studies implicated that uremic states may deteriorate renal dysfunction via dysregulating oxygen metabolism in tubular cells. The abnormal oxygen metabolism in tubular cells by uremic toxins was, at least in part, mediated by oxidative stress.

Aim: Autoimmune pancreatitis (AIP) is a rare subtype of chronic pancreatitis. AIP has been suggested to be complicated by tubulointerstitial nephritis or glomerulonephritis, implying that the kidney is involved as a phenotype of IgG4-positive multi-organ lymphoproliferative syndrome; however, the clinical significance of this novel entity is not well-defined. Methods: We conducted a retrospective cohort analysis of 47 (male, 39; female, 8) AIP patients. Results: The patients (mean age, 70.3 +/- 9.5 years) had a mean observation period of 4.1 years. Before treatment, renal dysfunction with an eGFR of 30 and 15 ml/min/1.73 m2 developed only in 10.6% (5/47) and 2.1% (1/47) of the patients, respectively. Nevertheless, urinary N-acetyl-beta-D-glucosaminidase and alpha1-microglobulin levels were elevated in 78.6% (11/14) and 30.8% (4/13) of the patients, respectively. Renal involvement in contrast-enhanced CT imaging was present in 18.2% (8/44) of the patients and was associated with proteinuria (p = 0.04) and a decrease in eGFR (p < 0.01). Furthermore, a follow-up CT study (mean, 545 days) revealed improved kidney lesions in 80.0% (4/5) of the patients after oral corticosteroid administration. In contrast, first-time kidney involvements appeared newly in 3.6% (1/28) of the patients after steroid therapy for nonrenal AIP symptoms, and in 14.3% (1/7) of the patients under no specific therapy (p = 0.02). Conclusion: Although severe renal failure develops rarely in AIP patients, renal abnormalities have been significantly detected by biochemical and radiological tests. Oral corticosteroid administration, even when not targeting symptomatic nephropathy, can treat and prevent kidney involvements in AIP.

Methylglyoxal (MG), a highly reactive alpha-oxoaldehyde generated by oxidation of carbohydrate and glycolysis, binds to proteins and forms advanced glycation end products (AGE). MG and MG adducts have been implicated in oxidative stress-related diseases, therefore, MG detoxifying system such as the glyoxalase system (glyoxalase I) also contributes to progression of these diseases. Recent papers have emphasized the pathophysiological effects of MG and the glyoxalase system in acute hypoxic injury, which is associated with acute oxidative stress. We investigated the kinetics of MG level and glyoxalase I activity in renal acute hypoxic injury induced by ischemia-reperfusion (I/R). I/R induced tubulointerstitial injury and the histological changes were associated with a significant decrease in renal glyoxalase I activity and an increase in MG level in the damaged tubular cells. Of note, rats over expressing human glyoxalase I showed amelioration of I/R-induced histological and functional damages and it was associated with a decrease in MG level in the lesion resulting in reduction of oxidative stress and tubular cell apoptosis. In conclusion, glyoxalase I has renoprotective effects in renal hypoxia such as I/R injury via a reduction in cytotoxic MG level in tubular cells.

PURPOSE OF REVIEW: Tubulointerstitial hypoxia in the kidney has been considered a hallmark of injury and mediator of disease progression. This review focuses on hypoxia-inducible factor (HIF)-1, a master transcription factor in cellular adaptation to hypoxia. RECENT FINDINGS: HIF-1alpha is expressed in the hypoxic tubular epithelium as well in as the papillary interstitium and glomerular epithelial cells. Although HIF-1 plays a protective role in a number of acute kidney injury models when overexpressed, its activation in chronic kidney disease results in multiple phenotypic changes, depending on the pathological context. SUMMARY: Hypoxia, especially HIF-1, is a critical mediator in the pathogenesis of chronic kidney disease. Underlying molecular mechanisms, together with responsible HIF target genes, are currently under investigation.

BACKGROUND: Autophagy, an intracellular self-degradation system conserved throughout eukaryotes, plays an important role in a variety of biological processes, including cell death, development, cancer, defence against infection and neurodegeneration. However, little information about autophagy in renal tubular cells is available. We investigated the relationship of autophagy with endoplasmic reticulum (ER) stress in proximal tubular cells. METHODS: Immortalized rat proximal tubular cells were exposed to the classic ER stress inducers tunicamycin or brefeldin A. Autophagy was detected mainly by immunoblot analysis of LC3, a widely used marker of autophagy, and also by immunofluorescent cytochemistry of LC3 and electron microscopy. Biological significance of the phenomenon was studied using bafilomycin A1, an inhibitor of autophagosome degradation. Signal transduction pathways following ER stress were also investigated using inhibitors of the MAPK pathway. RESULTS: Both ER stress inducers significantly increased LC3-II as a marker of autophagy in immunoblot analysis. Immunocytochemistry of LC3 and electron microscopy also showed activation of autophagy by ER stress. Inhibition by bafilomycin A1 showed that autophagy following ER stress fulfilled its intrinsic function, namely degradation of cytoplasmic components. Further, use of the MEK 1/2 inhibitor U0126, which inhibits ER stress-induced autophagy induction and ERK activation, showed that ERK, a MAPK family member, was necessary to the induction of autophagy. CONCLUSIONS: For the first time, we demonstrate the induction of autophagy following ER stress in renal tubules, and clarify its mechanism. These findings serve as the foundation for further investigation into autophagy in renal diseases.

Genome-wide screening using a small interfering RNA (siRNA) library has revealed novel molecules that are involved in a wide range of physiological responses. The expression of vascular endothelial growth factor (VEGF) is increased under hypoxic conditions, and plays a crucial role in tumor angiogenesis and tissue responses to ischemia. Here, we used a siRNA expression vector library to elucidate molecules that modify VEGF expression. Screening using an siRNA library revealed that MAPKKK6 (MEKK6/MAP3K6) regulates VEGF expression under both normoxic and hypoxic conditions in vitro, although the biological function of MAP3K6 remains unknown. Attenuation of VEGF expression by MAP3K6 inhibition was demonstrated by transient transfection of double-stranded RNA as well as by stable transfection of short hairpin RNA-expressing vectors against MAP3K6. Conditioned medium of MAP3K6-knocked down cells attenuated both endothelial proliferation and capillary network formation in a VEGF-dependent manner in vitro. In addition, tumor cells with down-regulation of MAP3K6 expression showed significant suppression of tumor growth in vivo, which was accompanied by significant repression of vessel formation and VEGF expression in these tumors. The results of this study suggest that MAP3K6 regulates VEGF expression in both normoxia and hypoxia, and that regulation of VEGF by MAP3K6 may play a crucial role in both angiogenesis and tumorigenesis.

Recent studies emphasize the role of chronic hypoxia in the tubulointerstitium as a final common pathway to end-stage renal disease. Therefore, therapeutic approaches which target the chronic hypoxia should prove effective against a broad range of renal diseases. Many of hypoxia-triggered protective mechanisms are hypoxia inducible factor (HIF)-dependent. Although HIF-1 alpha and HIF-2 alpha share both structural and functional similarity, they have different localization and can contribute in a non-redundant manner. While gene transfer of constitutively active HIF has been shown effective, pharmacological approaches to activate HIF are more desirable. Oxygen-dependent activation of prolyl hydroxylases (PHD) regulates the amount of HIF by degradation of this transcription factor. Therefore, PHD inhibitors have been the focus of recent studies on novel strategies to stabilize HIF. Cobalt is one of the inhibitors of PHD, and stimulation of HIF with cobalt is effective in a variety of kidney disease models. Furthermore, crystal structures of the catalytic domain of human prolyl hydroxylase 2 have been clarified recently. The structure aids in the design of PHD selective inhibitors for the treatment of hypoxic tissue injury. Current advance has elucidated the detailed mechanism of hypoxia-induced transcription, giving hope for the development of novel therapeutic approaches against hypoxia.

Methylglyoxal (MG), a highly reactive carbonyl compound generated by carbohydrate oxidation and glycolysis, is the major precursor of protein glycation and induces cytotoxicity leading to apoptosis. Although recent studies have emphasized that MG accumulates in not only chronic oxidative stress-related diseases but also acute hypoxic conditions, the pathogenic contribution of MG in acute diseases is unclear. MG is efficiently metabolized by the glyoxalase system, namely, glyoxalase I. We investigated the pathophysiological role of glyoxalase I as an MG detoxifier in rat renal ischemia-reperfusion (I/R) injury. I/R-induced tubulointerstitial injury was associated with a deterioration in renal glyoxalase I activity independent of its cofactor, GSH, as well as an increase in renal MG level. In in vitro studies, knockdown of glyoxalase I by small interference RNA transfection in rat tubular cells exacerbated cell death by hypoxia-reoxygenation compared with control cells. We also examined whether glyoxalase I overexpression prevented renal I/R damage in rats overexpressing human glyoxalase I with enzyme activity in the kidney 17-fold higher than in wild-type. The histological and functional manifestations of I/R in these rats were significantly ameliorated in association with a decrease in intracellular MG adduct accumulation, oxidative stress, and tubular cell apoptosis. In conclusion, glyoxalase I exerts renoprotective effects in renal I/R injury via a reduction in MG accumulation in tubular cells.

In the conversion of an arthrodesed hip to a total hip arthroplasty, the osteotomy of the femoral neck and the placement of the acetabular socket are difficult procedures as anatomical abnormalities hamper identification of the femoral neck and of the original center of the acetabulum. A 59-year-old woman who had a hip arthrodesis for dysplastic osteoarthritis at 21 years of age underwent total hip arthroplasty for relief of back pain, achievement of good gait function, and improvement of activities of daily living. In this report, we introduce a technical solution, using a computed tomography-based navigation system to determine the site and direction of the femoral neck osteotomy and the positioning of the acetabular socket.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-dependent alpha-subunit and constitutively expressed beta subunit, which plays a central role in cellular adaptation to hypoxia by transcriptionally upregulating its target genes involved in angiogenesis, erythropoiesis, glycolysis, and so on. Recent studies demonstrated that hypoxia in the tubulointerstitium is involved in the pathology of progressive renal diseases and that HIF, which is activated in experimental kidney diseases, may serve to protect tubulointerstitium from the ischemic insult. The expression of HIF alpha-chains is post-translationally regulated and hydroxylation at one or two of the conserved proline residues by prolyl-hydroxylase domains (PHDs) is a critical step for the oxygen-dependent recruitment of the von Hippel-Lindau gene product (pVHL), a recognition component of the E3 ubiquitin ligase complex, and degradation of HIF-alpha. Conversely, modalities to inhibit the enzymatic activities of PHDs have been shown to activate HIF irrespective of oxygenation status and are regarded as candidate targets of pharmacological approaches against chronic kidney diseases characterized by hypoxia.

Recent studies underscore that chronic hypoxia in the tubulointerstitium is a final common pathway to progression to end-stage renal failure regardless of etiology. We used microarray analysis of rat kidneys made hypoxic by unilateral renal artery stenosis to measure transcriptomic events and clarify pathophysiological mechanisms of renal injury induced by chronic hypoxia. Many genes were upregulated in the kidney by chronic hypoxia, but we focused on metallothionein due to its antioxidative properties. Using tubular epithelial cells transfected with a reporter construct of luciferase, driven by the hypoxia-responsive elements (HRE), we found that addition of metallothionein to the culture media increased luciferase activity. This was associated with upregulation of the target genes of hypoxia-inducible factor (HIF), such as vascular endothelial growth factor and glucose transporter-1. Stimulation of the HIF-HRE pathway by metallothionein was confirmed by metallothionein overexpression. Hypoxia and exogenous metallothionein increased HIF-1alpha protein without changes in its mRNA levels, suggesting protein stabilization. Upregulation of the HIF-HRE system by metallothionein was associated with phosphorylation of ERK but not Akt. MEK inhibition and rapamycin decreased metallothionein-induced HIF activity. Our study shows that upregulation of metallothionein expression by hypoxia activates the HIF-HRE system through the ERK/mTOR pathway and may be a novel defense against hypoxia.

BACKGROUND: This study aimed to elucidate the early involvement of T lymphocytes in renal ischemia/reperfusion injury. METHODS: Athymic nude rats (F344/N_Jcl-nu) and control F344/Jcl were subjected to 45 min unilateral renal ischemia. To determine whether the observed differences might be derived from the T lymphocyte presence, T lymphocytes from the spleens of F344/Jcl were injected into F344/N_Jcl-nu via tail vein at the initiation of reperfusion. Immunohistochemical analysis was performed for CD3, the proliferative cell nuclear antigen (PCNA), vimentin, and E-cadherin. T lymphocytes were obtained from the green fluorescent protein transgenic (GFP) rats, and transplanted to F344/N_Jcl-nu 10 min before reperfusion. The animals were euthanized 15 min after reperfusion. RESULTS: F344/N_Jcl-nu showed less retention of both Cr and BUN at 24 and 48 h after reperfusion, compared with F344/Jcl. F344/N_Jcl-nu received T lymphocyte transplantation showed significantly higher retention of both Cr and BUN 24, 48, and 72 h after reperfusion than those without T lymphocyte. A rapid infiltration of T lymphocytes into proximal tubular epithelial cells and tubular lumen was observed using T lymphocytes with green fluorescent protein. In contrast, T lymphocytes were observed with much less frequency 24 h after ischemia. The number of PCNA-positive proximal tubular cells 24 h after the initiation of reperfusion was significantly smaller in the T lymphocyte transplantation group compared with the non-transplantation group. The vimentin positivity and cytoplasmic staining of E-cadherin were also more prominent in the transplantation group. CONCLUSION: These findings demonstrate a rapid renal T lymphocyte infiltration, which propagate renal functional deterioration.

Representative remodeling of the kidney in CKD includes development of glomerulosclerosis and tubulointerstitial fibrosis. Injury to glomerular endothelium, mesangium, or podocyte can induce glomerulosclerosis, although recent studies have focused on a role of podocytes due to its lack of proliferation. Development of tubulointerstitial fibrosis is multi-factorial, and includes proteinuria, chronic hypoxia, activation of the intra-renal renin-angiotensin system, imbalance of matrix production and protease activity, and epithelial-mesenchymal transdifferentiation. Optimal remodeling may induce restoration of normal kidney structure, and some potential candidates of resident renal stem cells have been proposed. Development of therapeutic approaches targeting final common pathways will open a new avenue in management of patients with CKD.

Tissue hypoxia occurs when local metabolism is disturbed by an imbalance between oxygen supply and consumption. In patients with chronic kidney disease, chronic hypoxia in the kidneys is the end result of multiple processes and mechanisms. Once established, however, accumulating evidence points to this chronic hypoxia as the central player and final common pathway to end-stage renal disease. The cellular response to hypoxia is centered on hypoxia-inducible factor, HIF. This factor is composed of two subunits, an oxygen-sensitive HIF-alpha subunit and a constitutively expressed HIF-beta subunit. Intracellular accumulation of HIF induces the coordinated expression of a number of adaptive genes against hypoxic insult.Three isoforms of HIF-alpha subunits have been identified, HIF-1alpha, HIF-2alpha, and HIF-3alpha, of which HIF-2alpha is involved in the regulation of erythropoietin as well as oxidative stress. HIF is regulated by prolyl hydroxylation and asparaginyl hydroxylation of the HIF-alpha subunit at the protein level. Because HIF is activated only to suboptimal levels in various pathogenic states, therapeutic activation holds promise as a novel and effective approach to the future care of end-stage renal disease.

BACKGROUND: Antihypertensive angiotensin II receptor blockers (ARBs) protect the kidney, at least in part, independently of blood pressure lowering. Still, the extent to which blood pressure lowering is related to renoprotection remains unclear. METHODS AND RESULTS: 139 newly synthesized ARB-derivatives were assayed for inhibition of advanced glycation (AGEs). The 9 most powerful compounds were then tested for transition metal chelation, angiotensin II type 1 receptor (AT1R) affinity, and pharmacokinetic parameters. R-147176 was eventually selected as it strongly inhibits advanced glycation but is 6700 times less effective than olmesartan in AT1R binding. It is orally bioavailable and toxicologically safe. Despite a minimal blood pressure lowering effect, it provides significant renoprotection in 3 experimental rat models with renal injury, ie, obese, hypertensive, type 2 diabetic rats (SHR/NDmcr-cp), normotensive type 2 diabetic rats (Zucker diabetic fatty), and remnant kidney rats. CONCLUSIONS: R-147176 retains renal protective properties despite a minimal blood pressure-lowering effect. Clearly, the renal benefits of ARBs do not necessarily depend on blood pressure lowering and AT1R affinity, but rather on the inhibition of AGEs and oxidative stress inherent to their chemical structure. R-147176 opens new avenues in the treatment of cardiovascular and kidney diseases.