Periostin-binding DNA aptamer treatment attenuates renal fibrosis under diabetic conditions.

Diabetic nephropathy, the major cause of chronic kidney disease, is associated with progressive renal fibrosis. Recently, accumulation of periostin, an extracellular matrix protein, was shown to augment renal fibrosis. Aptamers have higher binding affinities without developing the common side effects of antibodies. Thus, we evaluated the effect of periostin inhibition by an aptamer-based inhibitor on renal fibrosis under diabetic conditions. In vitro, transforming growth factor-ß1 (TGF-ß1) treatment significantly upregulated periostin, fibronectin, and type I collagen mRNA and protein expressions in inner medullary collecting duct (IMCD) cells. These increases were attenuated significantly in periostin-binding DNA aptamer (PA)-treated IMCD cells exposed to TGF-ß1. In vivo, PA treatment attenuated the increased blood urea nitrogen levels in the diabetic mice significantly. Fibronectin and type I collagen mRNA and protein expressions increased significantly in the kidneys of diabetic mice: PA administration abrogated these increases significantly. Immunohistochemistry and Sirius Red staining also revealed that fibronectin expression was significantly higher and tubulointersititial fibrosis was significantly worse in diabetic mice kidneys compared with control mice. These changes were ameliorated by PA treatment. These findings suggested that inhibition of periostin using a DNA aptamer could be a potential therapeutic strategy against renal fibrosis in diabetic nephropathy.

Periostin-Binding DNA Aptamer Treatment Ameliorates Peritoneal Dialysis-Induced Peritoneal Fibrosis.

Peritoneal fibrosis is a major complication in peritoneal dialysis (PD) patients, which leads to dialysis discontinuation. Periostin, increased by transforming growth factor ß1 (TGF-ß1) stimulation, induces the expression of extracellular matrix (ECM) genes. Aberrant periostin expression has been demonstrated to be associated with PD-related peritoneal fibrosis. Therefore, the effect of periostin inhibition by an aptamer-based inhibitor on peritoneal fibrosis was evaluated. In vitro, TGF-ß1 treatment upregulated periostin, fibronectin, α-smooth muscle actin (α-SMA), and Snail expression and reduced E-cadherin expression in human peritoneal mesothelial cells (HPMCs). Periostin small interfering RNA (siRNA) treatment ameliorated the TGF-ß1-induced periostin, fibronectin, α-SMA, and Snail expression and restored E-cadherin expression in HPMCs. Similarly, the periostin-binding DNA aptamer (PA) also attenuated fibronectin, α-SMA, and Snail upregulation and E-cadherin downregulation in TGF-ß1-stimulated HPMCs. In mice treated with PD solution for 4 weeks, the expression of periostin, fibronectin, α-SMA, and Snail was significantly increased in the peritoneum, whereas E-cadherin expression was significantly decreased. The thickness of the submesothelial layer and the intensity of Masson's trichrome staining in the PD group were significantly increased compared to the untreated group. These changes were significantly abrogated by the intraperitoneal administration of PA. These findings suggest that PA can be a potential therapeutic strategy for peritoneal fibrosis in PD patients.

The locally activated renin-angiotensin system is involved in albumin permeability in glomerular endothelial cells under high glucose conditions.

Background: Although the diabetic milieu per se , hemodynamic changes, oxidative stress and local growth factors such as angiotensin II (AII) are considered to be mediators in the pathogenesis of diabetic nephropathy, the underlying pathways mediating the changes in glomerular endothelial cells (GECs) are not well understood. Therefore, we investigated changes in the renin-angiotensin system (RAS) components in high glucose (HG)-stimulated GECs and the role of the local RAS in morphological and functional changes in GECs under diabetic conditions. Methods: We stimulated GECs with 5.6 mM glucose or 30 mM glucose with or without an angiotensin II type I receptor blocker (ARB) in vitro and also performed experiments with Sprague-Dawley rats injected with diluent ( n = 16) or streptozotocin [ n = 16, diabetes (DM)]. Eight rats from each group were treated with ARB for 3 months in vivo . Real-time polymerase chain reaction, western blot analysis, enzyme-linked immunosorbent assay and immunofluorescent staining using cultured GECs were performed. The permeability of GECs to macromolecules was assessed by measuring the passage of fluorescein isothiocyanate-labeled bovine serum albumin. Morphological changes were also evaluated by scanning and transmission electron microscopy. Results: There were significant increases in angiotensinogen expression in HG-stimulated GECs along with significant increases in AI and AII levels. Moreover, the expression of heparan sulfate glycosaminoglycans (HS-GAG) assessed by immunofluorescent staining was significantly lower and the permeability to albumin was significantly higher in GECs exposed to HG medium, and ARB treatment significantly abrogated these changes. Upon electron microscopy examination, the mean size of the GEC fenestrae was significantly greater in HG-stimulated GECs and DM rats, and these increases were significantly ameliorated by ARB. Conclusions: The local RAS within GECs was activated under HG conditions, and this activation may be associated with both an alteration in GEC fenestration and a decrease in HS-GAG, resulting in the development of albuminuria in diabetic nephropathy.

Low Dentin Matrix Protein 1 Is Associated With Incident Cardiovascular Events in Peritoneal Dialysis Patients.

Recent reports demonstrated that dentin matrix protein 1 (DMP1) acts as an inhibitor of vascular calcification and might be a potential biomarker for chronic kidney disease-mineral and bone disorder; however, no clinical investigations regarding DMP1 have been performed in dialysis patients. We investigated the prognostic value of DMP1 on cardiovascular outcomes in prevalent peritoneal dialysis patients. We recruited 223 prevalent peritoneal dialysis patients and divided them into high and low DMP1 groups according to log-transformed plasma DMP1 levels. Lateral lumbar spine radiographs were used for measurement of vascular calcification. Major cardiovascular events were compared between the two groups. A Cox proportional hazards analysis determined DMP1 was independently associated with cardiovascular outcomes. In vitro mouse osteocytes were cultured in media containing indoxyl sulfate (IS), and the expressions of DMP1 were examined. The mean age was 52.1 ± 11.8 years, and 116 (52.0%) patients were male. The median value of log DMP1 was 0.91 (0.32-2.81 ng/mL). The multiple logistic regression analysis indicated that DMP1 levels were independently associated with the presence of vascular calcification after adjustment for multiple confounding factors (odds ratio = 0.719; 95% confidence interval [CI] 0.522-0.989; p = 0.043). During a mean follow-up duration of 34.6 months, incident cardiovascular events were observed in 41 (18.4%) patients. A Kaplan-Meier plot showed that the low DMP1 group had a significantly higher rate of incident cardiovascular events compared with the high DMP1 group (log-rank test, p = 0.026). In addition, multiple Cox analysis showed that low DMP1 was significantly associated with incident cardiovascular events (log 1 increase: hazard ratio = 0.855; 95% CI 0.743-0.984; p = 0.029) after adjustment for multiple confounding factors. In IS-stimulated osteocytes, mRNA and protein expression levels of DMP1 were significantly decreased compared with control osteocytes. We showed that low DMP1 levels were significantly associated with presence of vascular calcification and were independently associated with the incident cardiovascular events in prevalent peritoneal dialysis patients. DMP1 might be a potential factor contributing to cardiovascular complications in dialysis patients. © 2016 American Society for Bone and Mineral Research.

Double transduction of a Cre/LoxP lentiviral vector: a simple method to generate kidney cell-specific knockdown mice.

In a lentivirus-based gene delivery system, the incorporated gene is continuously expressed for a long time. In this study, we devised a simple way to knock down a specific gene in a kidney cell-specific pattern in adult mice by lentivirus-assisted transfer of short hairpin RNA (shRNA). Kidney collecting duct (CD)-specific aquaporin-3 (AQP3)-knockdown mice were generated by consecutive injection of Hoxb7-Cre-expressing lentivirus (LV-Hoxb7 Cre) and loxP-AQP3 shRNA-expressing lentivirus (LV-loxP shAQP3) in adult C57BL6/J mice. LV-Hoxb7 Cre was designed to express mCherry, while LV-loxP shAQP3 was designed with a floxed enhanced green fluorescent protein (EGFP)-tagged stop sequence, and thus EGFP would be expressed only in the absence of Cre recombination. In mice treated with LV-Hoxb7 Cre alone, mCherry protein expression, which indicates the presence of Cre recombinase, occurred only in CD cells. However, LV-loxP shAQP3 injection alone resulted in an increase in EGFP expression in all kidney cells, indicating the transcription of the floxed region. When LV-Hoxb7 Cre and LV-loxP shAQP3 were sequentially transduced, EGFP expression was attenuated while mCherry expression was sustained in CD cells, demonstrating a CD cell-specific recombination of the floxed region. AQP3 expression in mice injected with LV-Hoxb7 Cre or LV-loxP shAQP3 alone did not differ, but consecutive injection of LV-Hoxb7 Cre and LV-loxP shAQP3 significantly reduced AQP3 expression in CD cells. However, the expression levels of AQP3 were not altered in other cell types. Double transduction of Cre- and loxP-based lentivirus can easily generate kidney cell-specific knockdown mice, and this method might be applicable to other species.