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Telomeres are specialized chromatin structures that prevent the degradation and instability of the ends of linear chromosomes. While telomerase maintains long stretches of the telomeric repeat, the majority of telomeric DNA is duplicated by conventional DNA replication. A fundamental step in eukaryotic DNA replication involves chromatin binding of the origin recognition complex (ORC). In human cells, telomeric repeat binding factor 2 (TRF2) is thought to play a role in the recruitment of ORC onto telomeres. To better understand the mechanism of TRF2-mediated ORC recruitment, we utilized a lacO-LacI protein tethering system in U2OS cells and found that ectopically targeted TRF2, but not TRF1, can recruit ORC onto the lacO array. We further found that the TRF homology (TRFH) dimerization domain of TRF2, but not its mutant defective in dimerization, is sufficient for ORC and minichromosome maintenance (MCM) recruitment. Mutations impairing the dimerization also compromised ORC recruitment by full-length TRF2. Similar results were obtained using immunoprecipitation and GST pull-down assays. Together, these results suggest that dimerized TRF2 recruits ORC and stimulates pre-replication complex (pre-RC) formation at telomeres through the TRFH domain., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The protective effects of Rho kinase inhibitor fasudil against renal diseases have recently been reported. We compared the therapeutic effects of fasudil on the spontaneously hypercholesterolemic (SHC) rat, a model of chronic kidney disease (CKD) with proteinuria, with those of the angiotensin receptor blocker olmesartan (OL) by paying attention to the proteinuria and the macrophage phenotype. SHC rats were allocated to six treatment groups: a vehicle (Ve) group, a low-dose fasudil (FL) group, a high-dose fasudil (FH) group, an OL group, a combination of low-dose fasudil and OL (CL) group, and a combination of high-dose fasudil and OL (CH) group. Sprague-Dawley rats treated with vehicle served as a control (n = 7/each). The rats were treated for 24 wk. Compared with the Ve group, proteinuria was significantly decreased in the FH, OL, and CL groups, and it completely disappeared in the CH group. Glomerular stainings of nephrin and F-actin were focally impaired in the Ve group but were restored in the CH group. Western blotting showed that the CH group had significantly increased renal nephrin expression compared with the Ve group. Interstitial infiltration of macrophages was significantly increased in the Ve group, which was significantly attenuated in all treatment groups. The ratio of CD206 (M2 macrophage marker) to CD68 mRNA was significantly greater in the CH group than in the Ve group. These results indicate that fasudil with OL reduces proteinuria by protecting podocyte integrity and alters the interstitial macrophage density/phenotype, thereby exerting renoprotective effects against CKD.

Aim: Renal interstitial fibrosis is the final common pathway determining long-term prognosis of chronic kidney diseases, but its repair process is scarcely understood. Because recent reports indicate that M2 macrophages play important roles in the repair of various tissues, special attention was paid to the phenotypes of infiltrating macrophages in the present study when the histological changes occurring in mouse kidneys after the release of unilateral ureteral obstruction (UUO) inducing renal fibrosis were analyzed., Methods: The left ureter of male mice was obstructed for 10 days by using a vascular clamp, and that kidney was removed for analysis either on the day when the clamp was removed or after the kidney had been allowed to recover for 3, 7 or 21 days., Results: Interstitial fibrosis assessed by picrosirius red staining decreased with time after the release, and this decrease was paralleled by a decrease in the interstitial area positive for α-smooth muscle actin. Macrophage infiltration assessed by F4/80 staining also significantly decreased from day 3. In contrast, real-time reverse transcription polymerase chain reaction revealed that the ratios of mRNA for the macrophage scavenger receptor (CD204) and the mannose receptor (CD206), both of which are preferentially expressed on M2 macrophages, to CD68 (a general macrophage marker) were significantly greater on day 7 than on day 0 in the UUO-released mice., Conclusion: Although the total number of infiltrating myofibroblasts and macrophages decreased after UUO release, the ratios of macrophages expressing CD204 and CD206 increased, suggesting that M2 macrophages play an important role in the repair of renal fibrosis., (© 2011 The Authors. Nephrology © 2011 Asian Pacific Society of Nephrology.)

A 16-year-old male adolescent who had fallen and scratched his knee developed cold-like symptoms. Gross hematuria, oliguria, and peripheral edema appeared about 2 weeks after his fall. Tests showed hypocomplementemia, increased antistreptolysin O titers, and severe kidney failure with hematuria/proteinuria. Kidney biopsy showed endocapillary proliferative glomerulonephritis visible using light microscopy and deposits of complement C3 on capillary walls, detectable using immunofluorescence microscopy. Although these findings suggested that he had acute poststreptococcal glomerulonephritis (APSGN), electron microscopy failed to detect subepithelial humps, a feature typical of APSGN; instead, massive electron-dense deposits were visible in the subendothelial space. A second biopsy performed 3 months later showed mesangial cell proliferation without endocapillary proliferation using light microscopy and the disappearance of subendothelial electron-dense deposits in electron micrographs, suggesting that APSGN had resolved. Glomerular deposition of nephritis-associated plasmin receptor, a nephritogenic streptococcal antigen, was observed in the first biopsy specimen, but not the second. The patient required 4 hemodialysis treatments, and within 1 year of supportive therapy only, kidney function and urinalysis had normalized. This is an interesting case of APSGN presenting with acute kidney injury and atypical massive subendothelial deposits, but no subepithelial deposits on electron microscopy, a pattern resembling that in membranoproliferative glomerulonephritis type I., (Copyright © 2011 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.)

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows the selective pharmacological suppression of effector memory T cells (T(EM)) without affecting the function of naïve T cells (T(N)) and central memory T cells (T(CM)). We found that Kv1.3 was expressed on glomeruli and some tubules in rats with anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). A flow cytometry analysis using kidney cells revealed that most of the CD4(+) T cells and some of the CD8(+) T cells had the T(EM) phenotype (CD45RC(-)CD62L(-)). Double immunofluorescence staining using mononuclear cell suspensions isolated from anti-GBM GN kidney showed that Kv1.3 was expressed on T cells and some macrophages. We therefore investigated whether the Kv1.3 blocker Psora-4 can be used to treat anti-GBM GN. Rats that had been given an injection of rabbit anti-rat GBM antibody were also injected with Psora-4 or the vehicle intraperitoneally. Rats given Psora-4 showed less proteinuria and fewer crescentic glomeruli than rats given the vehicle. These results suggest that T(EM) and some macrophages expressing Kv1.3 channels play a critical role in the pathogenesis of crescentic GN and that Psora-4 will be useful for the treatment of rapidly progressive glomerulonephritis.

Interaction between epithelial cells and mesenchymal cells is essential in normal organ morphogenesis and in tissue repair after injury. Epimorphin, a mesenchymal protein that regulates epithelial morphogenesis through epithelial-mesenchymal interactions, has recently attracted attention as an important modulator of tissue repair. In this study we analyzed the role of epimorphin in renal fibrosis. We first found a progressive increase in epimorphin expression corresponding to the progression of renal fibrosis in mice with unilateral ureteral obstruction (UUO). To determine whether this expression has a role in the repair or progression of renal fibrosis, we analyzed a model of renal fibrosis repair, the UUO-release (UUO-R) model. Epimorphin expression was increased at 3 and 7 days after the UUO-R rather than on the day of release, but was decreased at 21 days after the release. Inhibition of endogenous epimorphin with anti-epimorphin antibody (MC-1) significantly delayed the repair of fibrosis. When compared with normal-IgG-injected mice, MC-1-injected mice showed significantly decreased renal matrix metalloproteinase (MMP)-2 and MMP-9 expressions by western blotting and increased expression of TGF-beta and collagen-I mRNA by real-time RT-PCR. Recombinant epimorphin induced prominent increases in MMP-2 and MMP-9 activities in the culture media of renal interstitial fibroblasts in vitro. These findings indicate that epimorphin has a pivotal role in the repair of renal fibrosis by modulating both extracellular matrix (ECM) degradation and its production.

Aim: To examine the additive protective effects of the peroxisome proliferator-activated receptor-gamma agonist pioglitazone (Pio) and the angiotensin II receptor blocker candesartan (Cand) in a murine model of renal fibrosis: mice with unilateral ureteral obstruction (UUO)., Methods: Mice were randomly assigned into four groups that after UUO received i.p. injections of either Pio (10 mg/kg/day), Cand (1 mg/kg/day), Cand + Pio or vehicle for 10 days. Physiological parameters, the degree of renal fibrosis and molecules related to renal fibrosis were analysed, and sham-operated mice were used as controls., Results: Total collagen assay showed prominent renal fibrosis in the vehicle-treated mice, significantly attenuated renal fibrosis in the Cand-treated and the Pio-treated mice, and further attenuated renal fibrosis in the (Cand + Pio)-treated mice. Real-time reverse transcription polymerase chain reaction revealed that this attenuation pattern was also evident in the expression of the mRNA for transforming growth factor-beta, collagens I and III, and plasminogen activator inhibitor-1., Conclusion: Pioglitazone and candesartan have additive protective effects on renal fibrosis due to UUO in mice, suggesting that their use in combination would be an effective treatment for chronic kidney disease.

The aims of treating hypertension in patients with chronic kidney disease including diabetes are to prevent both cardiovascular events and end stage renal failure, and goal of blood pressure is less than 130/80 mmHg. The first choice is angiotensin II receptor blocker (ARB) or angiotensin converting enzyme inhibitor. If the patient is salt-sensitive and hypervolemic, the second choice is diuretic. When the patient has some risk factors for cardiovascular events, the second choice is calcium channel blocker. In treating patient whose serum creatinine more than 2 mg/dL, we should start the ARB with lower dose and examine serum creatinine and potassium every 2 week.

The mechanisms of hypertensive nephrosclerosis are not fully understood. In experimental models of the disease, inflammatory reactions such as macrophage infiltration play an important role. In human hypertensive nephrosclerosis, however, there have been few studies examining the role of inflammation histologically. We investigated whether the number of infiltrating macrophages was increased in human hypertensive nephrosclerosis, and evaluated the effects of a blockade of the renin-angiotensin system on clinical and histological findings. We examined macrophage infiltration using immunohistochemistry in renal biopsy specimens obtained from 16 patients with hypertensive nephrosclerosis, 5 patients with IgA nephropathy, 5 patients with membranous nephropathy, and 5 patients with minimal change nephrotic syndrome. The number of infiltrating macrophages in glomeruli was significantly larger in the patients with hypertensive nephrosclerosis than in those with minimal change nephrotic syndrome. The patients with hypertensive nephrosclerosis were divided into groups based on their use of antihypertensive agents at the time of renal biopsy. We investigated the effects of antihypertensive agents on clinical findings, macrophage infiltration, and monocyte chemoattractant protein-1 expression. There was no difference in clinical findings between the hypertensive groups. The numbers of infiltrating macrophages and monocyte chemoattractant protein-1-positive cells in glomeruli were significantly smaller in patients treated with an angiotensin-converting enzyme inhibitor or angiotensin II type 1 receptor blocker, whereas calcium channel blockers had no influence on histological findings. In conclusion, inflammation is involved in the progression of human hypertensive nephrosclerosis and the inflammatory process is inhibited by blocking the renin-angiotensin system.

Fasudil, a Rho-kinase inhibitor, may improve insulin signaling. However, its long-term effect on metabolic abnormalities and its preventive effect on diabetic nephropathy are still unknown. We assessed these effects of fasudil in insulin-resistant diabetic rats, comparing them with those of an angiotensin II receptor blocker, olmesartan. Male Otsuka Long-Evans Tokushima fatty (OLETF) and Long-Evans Tokushima Otsuka, non-diabetic control, rats at 15 weeks of age were used. OLETF rats were randomized to receive a low or a high dose of fasudil or olmesartan for 25 weeks. To examine the therapeutic effects after the development of diabetes, OLETF rats at 30 weeks of age were given fasudil for 10 weeks. Administration of high-dose fasudil completely suppressed the development of diabetes, obesity, and dyslipidemia and increased serum adiponectin levels in OLETF rats. High-dose olmesartan also decreased hemoglobin A1c and increased serum adiponectin. There was a significant correlation between hemoglobin A1c and serum adiponectin or free fatty acid levels. The treatment with high-dose fasudil ameliorated proteinuria, glomerulosclerosis, renal interstitial fibrosis, and macrophage infiltration in OLETF rats. Olmesartan, even at the low dose, suppressed renal complications. The treatment with fasudil after the development of diabetes improved the metabolic abnormalities in OLETF rats, but could not suppress the progression of nephropathy. We conclude that the long-term treatment with fasudil prevents the development of diabetes, at least in part, by improving adipocyte differentiation in insulin-resistant diabetic rats. Early use of fasudil may prevent diabetic nephropathy.

Background: We previously reported that fractalkine was upregulated in streptozotocin-induced diabetic kidneys. Fractalkine in diabetic kidneys was detected on glomerular capillaries and the mesangium. This upregulation was suppressed by treatment with angiotensin-converting enzyme inhibitor (ACE-I) or aminoiguanidine. We examined what factors induce fractalkine upregulation in normal rat glomeruli., Methods: Glomeruli were collected from the kidneys of normal Sprague-Dawley rats by a microdissection method. Ten glomeruli were incubated in a solution with glucose, mannitol, angiotensin II, tumour necrosis factor (TNF)-alpha and advanced glycation end-product (AGE)-bovine serum albumin (BSA) for 1, 2 and 4 h. Fractalkine mRNA expression in glomeruli was examined by reverse transcription-polymerase chain reaction., Results: Fractalkine mRNA levels in the 30 mM glucose solution significantly increased (121%) compared with those in the control or 30 mM mannitol solution at 1 h. Fractalkine mRNA levels in the 15 mM glucose solution showed no significant differences at 1 or 2 h, but significantly increased (106%) after 4 h incubation. Fractalkine mRNA levels in 10(-6)-10(-8) M angiotensin II solution showed no significant differences. Fractalkine mRNA levels in the 5 or 10 ng/ml TNF-alpha solution significantly increased compared with those in the control in a time- and dose-dependent manner (by 94 to 253%). Fractalkine mRNA levels in the 50-200 microg/ml AGE-BSA solution also increased compared with those in BSA solution in a time- and dose-dependent manner (by 119 to 261%). By pre-incubation with MG132, a nuclear factor-kappaB inhibitor, fractalkine upregulation by AGE-BSA or 30 mM glucose was completely suppressed., Conclusions: High glucose levels, AGE formation and cytokine activation in diabetes may induce fractalkine upregulation in the kidneys and lead to progression of diabetic nephropathy.