MiR-21 alleviates renal tubular epithelial cells injury induced by ischemia by targeting TLR4.

Renal ischemia is the initial stage of kidney damage, leading to mitochondrial metabolism disorders and cell necrosis. In this study, we aimed to investigate the biological functions and potential mechanisms of miR-21 in protecting renal tubular epithelial cells from oxidative stress and apoptosis following oxygen glucose deprivation (OGD). Following an OGD injury, miR-21 levels increased in HK-2 renal tubular epithelial cells. Overexpression of miR-21 decreased the protein expressions of cleaved caspase-3, BAX, P53, cell apoptosis and increased Bcl-2 expression in HK-2 cells with OGD injury. In vivo studies found that miR-21 agomir reduced renal tissue apoptosis, while miR-21 antagomir increased it. In addition, overexpression of miR-21 reduced levels of reactive oxygen species (ROS), malondialdehyde (MDA) and lactate dehydrogenase (LDH) in HK-2 cells with OGD injury. However, miR-21 inhibition exhibited the opposite effect. A dual-luciferase reporter assay demonstrated that miR-21 directly regulates Toll-like receptor 4 (TLR4) by targeting the 3'-UTR of TLR4 mRNA. Overexpression of miR-21 led to decreased TLR4 protein expression, and TLR4 knockdown was shown to greatly increase AKT activity in HK-2 cells by in vitro kinase assay. Additionally, TLR4 knockdown promoted AKT phosphorylation and hypoxia-inducible factor-1α (HIF-1α) expression, while TLR4 overexpression inhibited these processes. Furthermore, AKT activation abolished the effect of TLR4 on HIF-1α, while AKT inhibition decreased the expression of TLR4 on HIF-1α in TLR4 knockdown HK-2 cells. Further study revealed that HIF-1α inhibition abolished the protective effect of miR-21 overexpression on ROS, LDH levels and cell apoptosis in HK-2 cells after OGD injury, which is indicated by increased levels of ROS and LDH, as well as increased cell apoptosis after HIF-1α inhibition in miR-21-treated HK-2 cells. In conclusion, miR-21 defends OGD-induced HK-2 cell injury via the TLR4/AKT/HIF-1α axis.

Roles of pattern recognition receptors in diabetic nephropathy.

Diabetic nephropathy (DN) is currently the most common complication of diabetes. It is considered to be one of the leading causes of end-stage renal disease (ESRD) and affects many diabetic patients. The pathogenesis of DN is extremely complex and has not yet been clarified; however, in recent years, increasing evidence has shown the important role of innate immunity in DN pathogenesis. Pattern recognition receptors (PRRs) are important components of the innate immune system and have a significant impact on the occurrence and development of DN. In this review, we classify PRRs into secretory, endocytic, and signal transduction PRRs according to the relationship between the PRRs and subcellular compartments. PRRs can recognize related pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), thus triggering a series of inflammatory responses, promoting renal fibrosis, and finally causing renal impairment. In this review, we describe the proposed role of each type of PRRs in the development and progression of DN.

BRAIN FUNCTION ALTERATIONS IN PATIENTS WITH DIABETIC NEPHROPATHY COMPLICATED BY RETINOPATHY UNDER RESTING STATE CONDITIONS ASSESSED BY VOXEL-MIRRORED HOMOTOPIC CONNECTIVITY.

Objective: The voxel-mirrored homologous connection (VHMC) technique was applied to detect resting brain function alterations in patients with diabetic nephropathy and retinopathy (DNR), and their relationships with clinical manifestations in the kidneys and eyes are discussed. Methods: Twenty-two patients with DNR and 22 healthy controls (HCs) similarly matched in age, sex, and educational background were recruited. Resting-state functional magnetic resonance imaging scans were performed for all subjects. Retinal fundus photography and renal biopsy were employed to observe the clinical features of the kidney and retina. Pearson correlation analysis was used to analyze the relationship between clinical manifestations and experimental results. Results: Compared with the HCs, patients with DNR showed decreased mean VMHC values in the bilateral middle temporal gyrus, bilateral middle occipital gyrus (BMOG), and bilateral medial frontal gyrus. The receiver operating characteristic curve analysis of each brain region confirmed that the accuracy of the area under the curve was excellent. The results showed that the average VHMC value of BMOG signals was positively correlated with the urinary protein to creatinine ratio in female subjects (r = 0.626; P<.05). Nonetheless, no such correlation was noted among the male subjects. Conclusion: There were significant changes in brain function in DNR patients compared to the control group. Changes in the central nervous system in patients with DNR were mainly due to the dual negative effects of kidney function and diabetes mellitus. Abbreviations: ACR = albumin/creatinine ratio; BMFG = bilateral medial frontal gyrus; BMOG = bilateral middle occipital gyrus; BMTG = bilateral middle temporal gyrus; DN = diabetic nephropathy; DNR = diabetic nephropathy complicated by retinopathy; DR = diabetic retinopathy; fMRI = functional magnetic resonance imaging; HC = healthy control; MRI = magnetic resonance imaging; PCR = protein to creatinine ratio; ROC = receiver operating characteristic; VHMC = voxel-mirrored homologous connection.

The predictive potential of altered spontaneous brain activity patterns in diabetic retinopathy and nephropathy.

OBJECTIVE: The amplitude of low-frequency fluctuation (ALFF) fMRI technique was used to study the changes of spontaneous brain activity in patients with diabetic retinopathy and nephropathy (DRN), and to explore the application of ALFF technique in the potential prediction and the targeted prevention of diabetic microangiopathy. METHODS: Nineteen patients with diabetic retinopathy and nephropathy and 19 healthy controls (HCs) were matched for age and gender. Spontaneous cerebral activity variations were investigated using the ALFF technique. The average ALFF values of the DRN patients and the HCs were classified utilizing receiver operating characteristic (ROC) curves. RESULTS: In contrast to the results in the HCs, the patients with DRN had significantly higher ALFF values in the cerebellum (bilaterally in the posterior and anterior lobes) and the left inferior temporal gyrus, but the ALFF values of the bilateral medial frontal gyrus, right superior temporal gyrus, right middle frontal gyrus, left middle/inferior frontal gyrus, bilateral precuneus, and left inferior parietal lobule were lower. ROC curve analysis of each brain region showed the accuracy of AUC was excellent. However, the mean ALFF values in the different regions did not correlate with clinical performance. The subjects showed abnormal neuronal synchronization in many areas of the brain, which is consistent with cognitive and visual functional deficits. CONCLUSION: Abnormal spontaneous activity was detected in many areas of the brain, which may provide useful information for understanding the pathology of DRN. Abnormal ALFF values of these brain regions may be of predictive value in the development of early DRN and be a targeted intervention indicator for individualized treatment of diabetic microvascular diseases.

Evidence of altered brain network centrality in patients with diabetic nephropathy and retinopathy: an fMRI study using a voxel-wise degree centrality approach.

BACKGROUND: Over recent years, some researchers believe that diabetic nephropathy (DN) and diabetic retinopathy (DR) both independently increase the incidence of brain diseases, such as stroke, cerebral infarction, and cerebral hemorrhage. In the present study, we used the voxel-wise degree centrality (DC) method to investigate potential changes of functional network brain activity in patients with DN and retinopathy (DNR). METHODS: Twenty DNR patients (9 men, 11 women) and 20 healthy controls (HCs; 9 men, 11 women) were recruited; the controls were matched for age, sex, and educational background. All subjects underwent resting-state functional magnetic resonance imaging. Ophthalmoscopy, renal biopsy and single-photon emission computed tomography were used to evaluate microvascular lesions in the eye and kidney. Data were categorized using receiver operating characteristic curves, and correlation analysis was performed using Pearson's correlation analysis. RESULTS: Compared with HCs, DNR patients showed reduced mean DC values in the right inferior temporal gyrus (RITG) and left subcallosal gyrus regions (LSG) and increased mean DC values in the bilateral precuneus (BP). Moreover, mean DC in the BP was correlated with renal estimated glomerular filtration rate (eGFR; r = 0.762). The area under the curve (AUC) value was 0.829 for BP and 0.839 for RITG and LSG. CONCLUSION: DNR patients showed dysfunction in three different brain regions. The linear correlation between eGFR and mean brain DC values indicates the presence of common diabetic microangiopathy in the brain and kidney, which may provide new ideas for multiorgan microvascular lesions of diabetics.

The role of pirfenidone in alkali burn rat cornea.

To evaluate the effects of pirfenidone in the treatment of HUVEC using an in vitro model and on rat corneal wound healing, edema, cornea neovascularization (CNV) and inflammation after alkali burn in vivo model. In vitro, CCK-8 assay was used to detect the effect of pirfenidone on the viability of HUVECs. The effects of pirfenidone on migration and tube formation of HUVEC were evaluated by HUVEC cell wound closure and tube formation assay. In vivo, Eye drops containing pirfenidone or phosphate buffered saline (PBS) were administered to an alkali-burn-induced corneal inflammatory and neovascularization model four times daily. The clinical evaluations, including fluorescent staining and cornea edema, were performed on days 1, 4, 7 and 14 using slit lamp microscopy. Global specimens were collected on day 7 and processed for immunofluorescent staining Collagen IV, α-smooth muscle actin (α-SMA), vascular endothelial growth factor (VEGF), pigment epithelium derived factor (PEDF) and cluster of differentiation34 (CD34). The levels of α-SMA, VEGF, PEDF, CD34, CD31 and nuclear factor-kappa B (NF-κB) proteins in the corneas were determined by western blot. Pirfenidone affects HUVEC viability, migration and tube formation in a dose-dependent manner. High concentration of pirfenidone can inhibit HUVEC viability, migration and tube formation in vitro and reduce alkali burn rat cornea edema, promote corneal wound healing, inhibit CNV and inflammation after alkali burn in vivo. Pirfenidone promotes corneal wound healing, and inhibits cornea neovascularization and inflammation after alkali burn in vitro and in vivo. Pirfenidone may be the potential anti-inflammation agent for the clinical treatment of CNV.

Relationship of angiotensin I-converting enzyme (ACE) and bradykinin B2 receptor (BDKRB2) polymorphism with diabetic nephropathy.

PURPOSE: To determine whether ACE2 I/D and BDKRB23 +9/-9 polymorphism causatively affect diabetic nephropathy progression RESULTS: STZ-induced metabolic disorder, as well as inflammatory responses, was significantly aggravated in ACE II-B2R4+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp diabetic mice but not ACE II-B2R-9bp, indicating the genetic susceptibility of ACE DD or B2R+9bp to diabetic nephropathy. Furthermore, ACE II-B2R+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp rather than ACE II-B2R-9bp, worsened renal performance and enhanced pathological alterations induced by STZ. Markedly elevated monocyte chemoattractant protein-1(MCP-1), podocin, osteopontin (OPN), transforming growth factor-ß1 (TGF-ß1), and reduced nephrin, podocin were also detected both in diabetic mice and podocytes under hyperglycemic conditions in response to ACE II-B2R+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp, versus ACE II-B2R-9bp. In addition, high glucose-induced mitochondrial oxidative stress and cell apoptosis were observably increased in response to ACE II-B2R+9bp, ACE DD-B2R+9bp, or ACE DD-B2R-9bp but not ACE II-B2R-9bp. CONCLUSIONS: We provide first evidence indicating the causation between ACE DD or B2R+9bp genotype and the increased risk for diabetic nephropathy, broadening our horizon about the role of genetic modulators in this disease.

The role of high mobility group box 1 (HMGB-1) in the diabetic retinopathy inflammation and apoptosis.

Diabetic Retinopathy (DR) is one of the most common complications of the late phase diabetes, and also a common cause of blindness. High mobility group box 1 (HMGB-1) is considered to be an inflammatory mediator in the late phase that promotes inflammation and neovascularization in diabetes. Therefore, this paper discussed the role of HMGB-1 in diabetic retinopathy inflammation and neovascularization. 96 adult SD rats were randomly divided into control and diabetes group. The diabetic rat model was established by intraperitoneal injection of streptomycin (0.1 mol/L). Western blot was applied to determine HMGB-1 and its receptor RAGE and TLR2 protein expression in the serum. TUNEL was used to detect retinal apoptosis. Immunofluorescence was performed to test HMGB1 protein expression in retina. HBGM-1 and RAGE expression in diabetic rat retina was significantly higher than the control (P < 0.05), while TLR2 expression was lower (P < 0.05). TUNEL detection showed that diabetic rat retinal cells presented obviously higher apoptosis rate (P < 0.05). Immunofluorescence test revealed that HMGB1 largely expressed in the diabetic rat retinal cells (P < 0.05). HMGB1 may involve in the pathogenesis of diabetic retinopathy by binding with RAGE receptor to accelerate rat retinal cells apoptosis.

Cross-talk between angiotensin-II and toll-like receptor 4 triggers a synergetic inflammatory response in rat mesangial cells under high glucose conditions.

Toll-like Receptor 4 (TLR4) may play an important role in the pathogenesis of diabetic nephropathy (DN). In this study, We observed the TLR4 signal and the release of inflammation factors after angiotensin II (Ang II) stimulation in rat mesangial cells (MCs) under high glucose conditions, this revealed the innate immune mechanism of injury by Ang II in DN. Our data showed that TLR4 and MyD88 were up-regulated significantly in high glucose and AngII-induced MCs; meanwhile, NF-κB as well as MCP-1, IL-6 were also highly expressed. In cells that were transfected with TLR4 SiRNA，the parameters were greatly inhibited; similar effects were detected in cells that were treated with Irbesartan. We concluded that Ang II synergized with high glucose in the release of pro-inflammatory factors mainly through the upregulation of TLR4 signaling in MCs, Cross-talk between Ang II and TLR4 contributed to the MC inflammatory injury under high glucose conditions.

HSP47 regulates ECM accumulation in renal proximal tubular cells induced by TGF-ß1 through ERK1/2 and JNK MAPK pathways.

Heat shock protein (HSP)47 is a collagen-specific molecular chaperone that is essential for the biosynthesis of collagen molecules. It is likely that increased levels of HSP47 contribute to the assembly of procollagen and thereby cause an excessive accumulation of collagens in disease processes associated with fibrosis. Although HSP47 promotes renal fibrosis, the underlying mechanism and associated signaling events have not been clearly delineated. We examined the role of HSP47 in renal fibrosis using a rat unilateral ureteral obstruction model and transforming growth factor (TGF)-ß(1)-treated human proximal tubular epithelial (HK-2) cells. An upregulation of HSP47 in both in vivo and in vitro models was observed, which correlated with the increased synthesis of extracellular matrix (ECM) proteins and expression of tissue-type plasminogen activator inhibitor (PAI)-1. Blockade of HSP47 by short interfering RNA suppressed the expression of ECM proteins and PAI-1. In addition, TGF-ß(1)-induced HSP47 expression in HK-2 cells was attenuated by ERK1/2 and JNK MAPK inhibitors. These data suggest that ERK1/2 and JNK signaling events are involved in modulating the expression of HSP47, the chaperoning effect of which on TGF-ß(1) would ultimately contribute to renal fibrosis by enhancing the synthesis and deposition of ECM proteins.

Candesartan attenuates Angiotensin II-induced mesangial cell apoptosis via TLR4/MyD88 pathway.

Angiotensin II (Ang II) can stimulate Toll-like receptor 4 (TLR4) expression in mesangial cells (MCs), but the role of TLR4 in the Ang II-induced apoptosis and the effect of candesartan on TLR4 expression remain unclear. Here, we report that Ang II-induced MC apoptosis in a time-dependent manner and up-regulated TLR4/MyD88 expression, and that the intracellular ROS was subsequently increased. We also show that candesartan attenuated the Ang II-induced MC apoptosis, and that this protective effect was dependent on decreased TLR4/MyD88 expression as well as reduced intracellular ROS formation. Furthermore, Ang II increased the apoptosis inducing factor protein level, while candesartan markedly reduced this increase. These results demonstrate that TLR4/MyD88 pathway was involved in the Ang II promoted MC apoptosis, which was related to TLR4/MyD88 mediated oxidative stress. These data also suggest that candesartan exerted anti-apoptotic effect as an antioxidant by modulating this pathway.