Deletion of podocyte Rho-associated, coiled-coil-containing protein kinase 2 protects mice from focal segmental glomerulosclerosis.

Focal segmental glomerulosclerosis (FSGS) shares podocyte damage as an essential pathological finding. Several mechanisms underlying podocyte injury have been proposed, but many important questions remain. Rho-associated, coiled-coil-containing protein kinase 2 (ROCK2) is a serine/threonine kinase responsible for a wide array of cellular functions. We found that ROCK2 is activated in podocytes of adriamycin (ADR)-induced FSGS mice and cultured podocytes stimulated with ADR. Conditional knockout mice in which the ROCK2 gene was selectively disrupted in podocytes (PR2KO) were resistant to albuminuria, glomerular sclerosis, and podocyte damage induced by ADR injection. In addition, pharmacological intervention for ROCK2 significantly ameliorated podocyte loss and kidney sclerosis in a murine model of FSGS by abrogating profibrotic factors. RNA sequencing of podocytes treated with a ROCK2 inhibitor proved that ROCK2 is a cyclic nucleotide signaling pathway regulator. Our study highlights the potential utility of ROCK2 inhibition as a therapeutic option for FSGS.

Pro-Calcifying Role of Enzymatically Modified LDL (eLDL) in Aortic Valve Sclerosis via Induction of IL-6 and IL-33.

One of the contributors to atherogenesis is enzymatically modified LDL (eLDL). eLDL was detected in all stages of aortic valve sclerosis and was demonstrated to trigger the activation of p38 mitogen-activated protein kinase (p38 MAPK), which has been identified as a pro-inflammatory protein in atherosclerosis. In this study, we investigated the influence of eLDL on IL-6 and IL-33 induction, and also the impact of eLDL on calcification in aortic valve stenosis (AS). eLDL upregulated phosphate-induced calcification in valvular interstitial cells (VICs)/myofibroblasts isolated from diseased aortic valves, as demonstrated by alizarin red staining. Functional studies demonstrated activation of p38 MAPK as well as an altered gene expression of osteogenic genes known to be involved in vascular calcification. In parallel with the activation of p38 MAPK, eLDL also induced upregulation of the cytokines IL-6 and IL-33. The results suggest a pro-calcifying role of eLDL in AS via induction of IL-6 and IL-33.

SDF-1α promotes subchondral bone sclerosis and aggravates osteoarthritis by regulating the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

BACKGROUND: Subchondral bone sclerosis is a major feature of osteoarthritis (OA), and bone marrow mesenchymal stem cells (BMSCs) are presumed to play an important role in subchondral bone sclerosis. Accumulating evidence has shown that stromal cell-derived factor-1α (SDF-1α) plays a key role in bone metabolism-related diseases, but its role in OA pathogenesis remains largely unknown. The purpose of this study was to explore the role of SDF-1α expressed on BMSCs in subchondral bone sclerosis in an OA model. METHODS: In the present study, C57BL/6J mice were divided into the following three groups: the sham control, destabilization of the medial meniscus (DMM), and AMD3100-treated DMM (DMM + AMD3100) groups. The mice were sacrificed after 2 or 8 weeks, and samples were collected for histological and immunohistochemical analyses. OA severity was assessed by performing hematoxylin and eosin (HE) and safranin O-fast green staining. SDF-1α expression in the OA model was measured using an enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (q-PCR), and immunohistochemistry. Micro-CT was used to observe changes in subchondral bone in the OA model. CD44, CD90, RUNX2, and OCN expression in subchondral bone were measured using q-PCR and immunohistochemistry. In vitro, BMSCs were transfected with a recombinant lentivirus expressing SDF-1α, an empty vector (EV), or siRNA-SDF-1α. Western blot analysis, q-PCR, and immunofluorescence staining were used to confirm the successful transfection of BMSCs. The effect of SDF-1α on BMSC proliferation was evaluated by performing a CCK-8 assay and cell cycle analysis. The effect of SDF-1α on the osteogenic differentiation of BMSCs was assessed by performing alkaline phosphatase (ALP) and alizarin red S (ARS) staining. Cyclin D1, RUNX2 and OCN expression were measured using Western blot analysis, q-PCR, and immunofluorescence staining. RESULTS: SDF-1α expression in the DMM-induced OA model increased. In the DMM + AMD3100 group, subchondral bone sclerosis was alleviated, OA was effectively relieved, and CD44, CD90, RUNX2, and OCN expression in subchondral bone was decreased. In vitro, high levels of SDF-1α promoted BMSC proliferation and increased osteogenic differentiation. Cyclin D1, RUNX2, and OCN expression increased. CONCLUSION: The results of this study reveal a new molecular mechanism underlying the pathogenesis of OA. The targeted regulation of SDF-1α may be clinically effective in suppressing OA progression.

Inflammation-Mediated Aberrant Glucose Metabolism in Subchondral Bone Induces Osteoarthritis.

Osteoarthritis (OA) is an entire joint disease with pathological alteration in both articular cartilage and subchondral bone. It has been recognized recently the association between metabolic syndrome and OA, particularly glucose metabolism in regulation of articular cartilage homeostasis and joint integrity. Whereas the role of glucose metabolism in subchondral bone sclerosis remains largely unknown during pathogenesis of OA. Consistent with common OA features, we observed subchondral bone sclerosis and abnormal bone remodeling in human OA joints and murine OA joints as reflected by hyperactive bone resorption and overall bone formation which was measured via dynamic histomorphometry. Osx-CreER;tdTomato mice also displayed the similar overall bone formation under injury-induced OA condition. Immunohistochemistry further revealed increased IL-1ß expression in human and murine OA subchondral bone. Given the inflammatory environment in joints under OA condition, we treated MC3T3-E1 cell, a pre-osteoblast cell line, with IL-1ß in this study and demonstrated that IL-1ß treatment could stimulate the cell osteogenic differentiation and meanwhile upregulate glycolysis and oxidative phosphorylation in cell cultures. More importantly, intraperitoneal injection of 2-deoxy-D-glucose (2-DG) and oligomycin (OGM), respectively, suppressed the subchondral bone glycolysis and oxidative phosphorylation in mice. Consequently, 2-DG and OGM treatment attenuated abnormal osteoblast differentiation and protected against aberrant bone formation in subchondral bone and articular cartilage degradation in wildtype mice following with joint injury. Collectively, these data strongly suggest glycolysis and oxidative may serve as important therapeutic targets for OA treatment.

Translocation of High Mobility Group Box 1 From the Nucleus to the Cytoplasm in Depressed Patients With Epilepsy.

Depression is a common psychiatric comorbidity in patients with epilepsy, especially those with temporal lobe epilepsy (TLE). The aim of this study was to assess changes in high mobility group box protein 1 (HMGB1) expression in epileptic patients with and without comorbid depression. Sixty patients with drug-resistant TLE who underwent anterior temporal lobectomy were enrolled. Anterior hippocampal samples were collected after surgery and analyzed by immunofluorescence (n = 7/group). We also evaluated the expression of HMGB1 in TLE patients with hippocampal sclerosis and measured the level of plasma HMGB1 by enzyme-linked immunosorbent assay. The results showed that 28.3% of the patients (17/60) had comorbid depression. HMGB1 was ubiquitously expressed in all subregions of the anterior hippocampus. The ratio of HMGB1-immunoreactive neurons and astrocytes was significantly increased in both TLE patients with hippocampal sclerosis and TLE patients with comorbid depression compared to patients with TLE only. The ratio of cytoplasmic to nuclear HMGB1-positive neurons in the hippocampus was higher in depressed patients with TLE than in nondepressed patients, which suggested that more HMGB1 translocated from the nucleus to the cytoplasm in the depressed group. There was no significant difference in the plasma level of HMGB1 among patients with TLE alone, TLE with hippocampal sclerosis, and TLE with comorbid depression. The results of the study revealed that the translocation of HMGB1 from the nucleus to the cytoplasm in hippocampal neurons may play a previously unrecognized role in the initiation and amplification of epilepsy and comorbid depression. The direct targeting of neural HMGB1 is a promising approach for anti-inflammatory therapy.

Salinomycin alleviates osteoarthritis progression via inhibiting Wnt/ß-catenin signaling.

Osteoarthritis (OA) is the most prevalent degenerative whole-joint disease characterized by cartilage degeneration, synovial hyperplasia, osteophyte formation, and subchondral bone sclerosis. Currently there are no disease-modifying treatments available for OA because its etiology and pathogenesis are largely unknown. Here we report that a natural carboxylic polyether ionophore that is used as an anti-tumor drug, salinomycin (SAL), may be a promising therapeutic drug for OA in the future. We found that SAL showed no cytotoxicity on mouse chondrocytes and displayed a protective effect against interleukin-1ß (IL-1ß), in cultured mouse chondrocytes and cartilage explants. Treatment with low SAL concentrations directly upregulated the anabolism factors collagen II and aggrecan, while it inhibited the catabolic factors matrix metalloproteinase-13 (MMP13) and metalloproteinase with thrombospondin motifs-5 (ADAMTS5) to protect against extracellular matrix (ECM) degradation, and also suppressed inflammatory responses in mouse chondrocytes. Furthermore, SAL reduced the severity of OA-associated changes and delayed cartilage destruction, subchondral bone sclerosis, and osteophyte formation in a destabilized medial meniscus (DMM) surgery-induced mouse OA model. Mechanistically, a low SAL concentration induced anabolism and inhibited catabolism in chondrocytes via inhibiting Lrp6 phosphorylation and Wnt/ß-catenin signaling. Our results suggested that SAL may serve as a potential disease-modifying therapeutic against OA pathogenesis.

Activation of necroptosis pathway in podocyte contributes to the pathogenesis of focal segmental glomerular sclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is characterized by podocyte damage and severe proteinuria. The exact mechanism of podocyte damage and loss remains unclear. Necroptosis, a lytic form of programmed cell death mediated by RIP3 and MLKL, has emerged as an important cell death pattern in multiple tissues and cell types. Necroptosis in FSGS has not been investigated. METHODS: Public GEO data regarding podocyte treated with vehicle or adriamycin (ADR) was identified and analyzed. Cultured human podocytes were used to explore the activation of necroptosis upon ADR stimulation. The expression of necroptosis pathway molecules, p-RIP3 and p-MLKL, was examined in the glomeruli and defoliated urinary podocytes of patients with FSGS. The effect of necroptosis inhibition was assessed in ADR-induced glomerulopathy mice using GSK872. RESULTS: Publicly available RNA-sequencing data analysis showed that both necroptosis and NLRP3 inflammasome pathway were up-regulated in ADR-injured podocyte. Immunofluorescent staining showed increased expression of p-RIP3 and p-MLKL, the active forms of RIP3 and MLKL, in podocytes of FSGS patients and ADR-induced glomerulopathy mice but not in the normal control. GSK872, an RIP3 kinase inhibitor, significantly inhibited the expression of p-RIP3, p-MLKL and activation of NLRP3 in cultured podocytes treated with ADR. GSK872 treatment of mice with ADR-induced nephropathy resulted in the reduced expression of p-RIP3, p-MLKL, NLRP3 and caspase-1 p20. GSK872 also significantly inhibited the expression of p-MLKL in the podocytes of ADR-induced nephropathy, resulting in the attenuation of proteinuria and renal histological lesions. CONCLUSION: Necroptosis pathway might be a valuable target for the treatment of FSGS.

Reorganization of the Brain Extracellular Matrix in Hippocampal Sclerosis.

The extracellular matrix (ECM) is an important regulator of excitability and synaptic plasticity, especially in its highly condensed form, the perineuronal nets (PNN). In patients with drug-resistant mesial temporal lobe epilepsy (MTLE), hippocampal sclerosis type 1 (HS1) is the most common histopathological finding. This study aimed to evaluate the ECM profile of HS1 in surgically treated drug-resistant patients with MTLE in correlation to clinical findings. Hippocampal sections were immunohistochemically stained for aggrecan, neurocan, versican, chondroitin-sulfate (CS56), fibronectin, Wisteria floribunda agglutinin (WFA), a nuclear neuronal marker (NeuN), parvalbumin (PV), and glial-fibrillary-acidic-protein (GFAP). In HS1, besides the reduced number of neurons and astrogliosis, we found a significantly changed expression pattern of versican, neurocan, aggrecan, WFA-specific glycosylation, and a reduced number of PNNs. Patients with a lower number of epileptic episodes had a less intense diffuse WFA staining in Cornu Ammonis (CA) fields. Our findings suggest that PNN reduction, changed ECM protein, and glycosylation expression pattern in HS1 might be involved in the pathogenesis and persistence of drug-resistant MTLE by contributing to the increase of CA pyramidal neurons' excitability. This research corroborates the validity of ECM molecules and their modulators as a potential target for the development of new therapeutic approaches to drug-resistant epilepsy.

MicroRNAs miR-629-3p, miR-1202 and miR-1225-5p as potential diagnostic and surgery outcome biomarkers for mesial temporal lobe epilepsy with hippocampal sclerosis.

BACKGROUND: Mesial temporal lobe epilepsy (MTLE) is a symptomatic epilepsy syndrome clinically characterized by high prevalence, pharmacoresistance, good surgical prognosis and hippocampal sclerosis (HS); however, no singular criteria can be considered sufficient for the MTLE-HS diagnosis. MicroRNAs (miRNAs) are small non-coding molecules that act as important gene-expression regulators at post-transcriptional level. Evidences on the involvement of miRNAs in epilepsy pathogenesis as well as their potential to be employed as biomarkers claim for investigations on miRNAs' applicability as epilepsy diagnosis and prognosis biomarkers. Consequently, the present study aimed to evaluate the applicability of three specific miRNAs as biomarkers of diagnosis and surgical outcomes in adult patients with MTLE-HS. METHOD: Hippocampus, amygdala and blood samples from 20 patients with MTLE-HS were analyzed, 10 with favorable surgical prognosis (Engel I) and 10 with unfavorable surgical prognosis (Engel III-IV). For the control groups, hippocampus and amygdala from necropsy and blood samples from healthy individuals were adopted. The miRNAs expression analysis was performed using Real-Time Quantitative Polymerase Chain Reaction for miRNAs highlighted from microarray as being involved in GABAergic neurotransmission. RESULTS: The miRNAs miR-629-3p, miR-1202 and miR-1225-5p were found to be hyper-expressed in MTLE-HS patients' blood. CONCLUSIONS: Our data suggest the existence of three circulating miRNAs (miR-629-3p, miR-1202 and miR-1225-5p) that could possibly act as additional tools in the set of factors that contribute to MTLE-HS diagnose.

Mast cells and basic fibroblast growth factor in physiological aging of rat heart and kidney.

Age-related morphological and physiological changes occur in cells, tissues and organs with high metabolic or mitotic activity; these changes decrease their regenerative capacity. One such change is interstitial fibrosis. Mast cells contain basic fibroblast growth factor and have been related to pro-fibrotic activity. We investigated the role of mast cells in physiological aging of the heart and kidney. We analyzed changes in mast cell number and compared the left and right heart ventricles and kidneys of 6- and 12-month-old Wistar rats. We also evaluated the immunohistochemical expression of basic fibroblast growth factor. Finally, we analyzed changes in the extent of interstitial fibrosis and in the glomerular sclerosis index as nonspecific markers of aging and correlated these parameters with of mast cells. Mast cells were visualized by toluidine blue staining and specific immunohistochemical expression of tryptase. The expression of basic fibroblast growth factor was assessed semiquantitatively. The extent of interstitial fibrosis was investigated using Mallory's trichrome staining. Glomerular sclerosis was evaluated using periodic acid-Schiff staining. We found that the number of mast cells increased significantly in the older rats. We also found that the number of mast cells was greatest in the left ventricle followed by the right ventricle, then the kidney. The immunoreactivity of basic fibroblast growth factor also increased in older animals. Correlations between the number of mast cells and immunoreactivity of basic fibroblast growth factor, extent of interstitial fibrosis and glomerular sclerosis index demonstrated the association between mast cells and age-related tissue remodeling of the heart and kidney.

Antigen-presenting innate lymphoid cells orchestrate neuroinflammation.

Pro-inflammatory T cells in the central nervous system (CNS) are causally associated with multiple demyelinating and neurodegenerative diseases1-6, but the pathways that control these responses remain unclear. Here we define a population of inflammatory group 3 innate lymphoid cells (ILC3s) that infiltrate the CNS in a mouse model of multiple sclerosis. These ILC3s are derived from the circulation, localize in proximity to infiltrating T cells in the CNS, function as antigen-presenting cells that restimulate myelin-specific T cells, and are increased in individuals with multiple sclerosis. Notably, antigen presentation by inflammatory ILC3s is required to promote T cell responses in the CNS and the development of multiple-sclerosis-like disease in mouse models. By contrast, conventional and tissue-resident ILC3s in the periphery do not appear to contribute to disease induction, but instead limit autoimmune T cell responses and prevent multiple-sclerosis-like disease when experimentally targeted to present myelin antigen. Collectively, our data define a population of inflammatory ILC3s that is essential for directly promoting T-cell-dependent neuroinflammation in the CNS and reveal the potential of harnessing peripheral tissue-resident ILC3s for the prevention of autoimmune disease.

Mitochondrial Dysfunction in Podocytes Caused by CRIF1 Deficiency Leads to Progressive Albuminuria and Glomerular Sclerosis in Mice.

Recent studies have implicated mitochondrial disruption in podocyte dysfunction, which is a characteristic feature of primary and diabetic glomerular diseases. However, the mechanisms by which primary mitochondrial dysfunction in podocytes affects glomerular renal diseases are currently unknown. To investigate the role of mitochondrial oxidative phosphorylation (OxPhos) in podocyte dysfunction, glomerular function was examined in mice carrying a loss of function mutation of the gene encoding CR6-interacting factor-1 (CRIF1), which is essential for intramitochondrial production and the subsequent insertion of OxPhos polypeptides into the inner mitochondrial membrane. Homozygotic deficiency of CRIF1 in podocytes resulted in profound and progressive albuminuria from 3 weeks of age; the CRIF1-deficient mice also developed glomerular and tubulointerstitial lesions by 10 weeks of age. Furthermore, marked glomerular sclerosis and interstitial fibrosis were observed in homozygous CRIF1-deficient mice at 20 weeks of age. In cultured mouse podocytes, loss of CRIF1 resulted in OxPhos dysfunction and marked loss or abnormal aggregation of F-actin. These findings indicate that the OxPhos status determines the integrity of podocytes and their ability to maintain a tight barrier and control albuminuria. Analyses of the glomerular function of the podocyte-specific primary OxPhos dysfunction model mice demonstrate a link between podocyte mitochondrial dysfunction, progressive glomerular sclerosis, and tubulointerstitial diseases.

Contribution of podocyte inflammatory exosome release to glomerular inflammation and sclerosis during hyperhomocysteinemia.

The nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3) inflammasome has been implicated in podocyte injury and glomerular sclerosis in response to hyperhomocysteinemia (hHcy). However, it remains unknown how the products of NLRP3 inflammasome in cytoplasm are secreted out of podocytes. In the present study, we tested whether exosome release serves as a critical mechanism to mediate the action of NLRP3 inflammasome activation in hHcy-induced glomerular injury. By various approaches, we found that hHcy induced NLRP3 inflammasome activation and neutrophil infiltration in glomeruli of WT/WT mice. Lysosome-MVB interaction in glomeruli remarkably decreased in WT/WT mice fed with FF diet, leading to elevation of urinary exosome excretion of these mice. Podocyte-derived exosomes containing pro-inflammatory cytokines increased in urine of WT/WT mice in response to hHcy. The release of inflammatory exosomes from podocytes was prevented by Smpd1 gene deletion but enhanced by podocyte-specific Smpd1 gene overexpression (Smpd1 encodes Asm in mice). Pathologically, hHcy-induced podocyte injury and glomerular sclerosis were blocked by Smpd1 gene knockout but amplified by podocyte-specific Smpd1 gene overexpression. Taken together, our results suggest that Asm-ceramide signaling pathway contributes to NLRP3 inflammasome activation and robust release of inflammatory exosomes in podocytes during hHcy, which together trigger local glomerular inflammation and sclerosis.

Divergent effects of distinct perivascular cell subsets for intra-articular cell therapy in posttraumatic osteoarthritis.

Intra-articular injection of mesenchymal stem cells has shown benefit for the treatment of osteoarthritis (OA). However, mesenchymal stem/stromal cells at the origin of these clinical results are heterogenous cell populations with limited cellular characterization. Here, two transgenic reporter mice were used to examine the differential effects of two precisely defined perivascular cell populations (Pdgfrα+ and Pdgfrß+ cells) from white adipose tissue for alleviation of OA. Perivascular mesenchymal cells were isolated from transgenic Pdgfrα-and Pdgfrß-CreERT2 reporter animals and delivered as a one-time intra-articular dose to C57BL/6J mice after destabilization of the medial meniscus (DMM). Both Pdgfrα+ and Pdgfrß+ cell preparations improved metrics of cartilage degradation and reduced markers of chondrocyte hypertrophy. While some similarities in cell distribution were identified within the synovial and perivascular spaces, injected Pdgfrα+ cells remained in the superficial layers of articular cartilage, while Pdgfrß+ cells were more widely dispersed. Pdgfrß+ cell therapy prevented subchondral sclerosis induced by DMM, while Pdgfrα+ cell therapy had no effect. In summary, while both cell therapies showed beneficial effects in the DMM model, important differences in cell incorporation, persistence, and subchondral sclerosis were identified.

Evaluation of patients with late-onset and medically refractory temporal lobe epilepsy with mesial temporal sclerosis.

OBJECTIVES: Our study aims to compare demographics, clinical features and postsurgical outcomes between early and late-onset patients with medically refractory temporal lobe epilepsy (TLE) related to mesial temporal sclerosis (MTS). PATIENTS AND METHODS: Seventy-one patients admitting to the Epilepsy Clinic of Istanbul University-Cerrahpasa, Cerrahpasa Faculty of Medicine between 1995-2015, who were diagnosed with refractory TLE related to MTS, were included in our study. All of these patients were resistant to medical treatment, and thus candidates for, or underwent surgery, and had no pathology other than MTS in their cranial magnetic resonance imaging (MRI). Based on previous studies, those patients were divided into two categories as "early-onset" and "late-onset", according to the age-onset of afebrile recurrent seizures, where the cutoff was determined as 20 years. Demographics, clinical features, and postsurgical outcomes were compared between both groups. RESULTS: Fifty-three patients included in our study had early-onset MTS-TLE, and 18 patients had late-onset MTS-TLE. Demographics, clinical features, characteristics of electroencephalography (EEG), MRI, PET MRI/CT, neuropsychometric test (NPT) and postsurgical outcomes were similar in both groups. CONCLUSION: In both the early-onset and late-onset groups, the presence of similar demographics, clinical features, and postoperative outcomes have suggested that the course of the disease and the success of surgical treatment were not associated with the age-onset of seizures in TLE related to MTS.

Inhibition of mTOR delayed but could not prevent experimental collapsing focal segmental glomerulosclerosis.

Anti-Thy1.1 transgenic mice develop glomerular lesions that mimic collapsing focal segmental glomerulosclerosis (FSGS) in humans with collapse of the glomerular tuft and marked hyperplasia of the parietal epithelial cells (PECs). Immunostaining of phosphor-S6 ribosomal protein (pS6RP) revealed high mTOR activity in PECs of the FSGS lesions of these mice. In this study we questioned whether the mTOR inhibitor rapamycin (sirolimus) could attenuate the development and progression of glomerulosclerotic lesions in the anti-Thy1.1 transgenic mice. We observed reduced mTOR signalling and proliferation in human parietal epithelial cells after rapamycin treatment. Experiments with anti-Thy1.1. mice showed that early treatment with sirolimus reduced the development of glomerular lesions and glomerular cell proliferation at day 4. Levels of albuminuria, podocyte injury and podocyte number were similar in the sirolimus and vehicle treated groups. The initial beneficial effects of sirolimus treatment were not observed at day 7. Late sirolimus treatment did not reduce albuminuria or the progression of glomerulosclerosis. Taken together, rapamycin attenuated PEC proliferation and the formation of early FSGS lesions in experimental FSGS and reduced human PEC proliferation in vitro. However, the initial inhibition of PEC proliferation did not translate into a decline of albuminuria nor in a sustained reduction in sclerotic lesions.

Osteocyte TGFß1­Smad2/3 is positively associated with bone turnover parameters in subchondral bone of advanced osteoarthritis.

Subchondral sclerosis is considered the main characteristic of advanced osteoarthritis, in which bone remodeling mediated by transforming growth factor ß (TGFß) signaling plays an indispensable role in the metabolism. Osteocytes have been identified as pivotal regulators of bone metabolism, due to their mechanosensing and endocrine function. Therefore, the aim of the present study was to investigate the association between osteocyte TGFß signal and subchondral sclerosis. Knee tibia plateau samples were collected from osteoarthritic patients and divided into three groups: The full cartilage, partial cartilage and full defect groups. Next, changes in osteocyte TGFß signaling and subchondral bone structure underlying various types of cartilage erosion were detected. Bone mineral density (BMD) assay, histology [hematoxylin and eosin, Safranin­O/Fast green, and tartrate resistant acid phosphatase (TRAP) staining], and reverse transcription­quantitative PCR mainly detected structural alterations, osteogenic and osteoclastic activity in the cartilage and subchondral bone. The activation of the TGFß signaling pathway in the subchondral bone was detected by immunohistochemistry and western blotting. The association between osteocyte TGFß and the regulation of bone metabolism was analyzed by correlation analysis, and further proven in vitro. It was confirmed that the BMD of the subchondral bone increased and underwent sclerosis in the partial cartilage and full defect groups. Additional observation included the thinning of the area of calcified cartilage, in which a bone island formed locally, with subchondral bone plate thickening and increased trabecular bone volume. TRAP staining suggested an increase in bone resorption in subchondral underlying areas of the partial cartilage and full defect groups. Immunohistochemistry results confirmed the activation of osteocyte TGFß in subchondral underlying areas with severe cartilage erosion. Moreover, osteocyte phosphorylated­Smad2/3 was positively correlated with subchondral BMD, alkaline phosphatase and osteopontin mRNA expression, but it was negatively correlated with TRAP+ cells. Furthermore, it was confirmed in vitro that osteocyte TGFß signaling could regulate the osteogenic and osteoclastic activity of the mesenchymal stem cells. This study illustrated that osteocyte TGFß signaling is positively associated with the remodeling of subchondral bone in advanced osteoarthritis and provides a preliminary theoretical basis for further investigations of the role and mechanism of osteocyte TGFß in subchondral of osteoarthritis.

Aerobic Endurance Exercise Ameliorates Renal Vascular Sclerosis in Aged Mice by Regulating PI3K/AKT/mTOR Signaling Pathway.

Renal vascular sclerosis caused by aging plays an important role in the occurrence and development of chronic kidney disease. Clinical studies have confirmed that endurance exercise is able to delay the aging of skeletal muscle and brain tissue. However, to date, few studies have assessed whether endurance exercise is able to improve the occurrence of renal vascular sclerosis caused by natural aging and its related mechanisms. In this study, we investigated the protective effect of aerobic endurance exercise on renal vascular sclerosis in aged mice and its effect on the phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway. The results suggested that aerobic endurance exercise preserved kidney morphology and renal function. Glomerular basement membrane thickness was evidently increased, podocyte foot processes were effaced in aged mice, and aerobic endurance exercise significantly ameliorated the overall lesion range. The protein expression of vascular endothelial growth factor (VEGF) and JG12 was lower in the senile control group (OC group). The protein expression of VEGF and JG12 was significantly increased after aerobic endurance exercise. Furthermore, aerobic endurance exercise resulted in downregulation of Bax, Caspase 3, IL-6, and senescent cells and upregulation of Bcl-2. The upregulation of PI3K and its downstream signal molecules AKT and mTOR after aerobic endurance exercise was further observed. Our observations indicated that aerobic endurance exercise may inhibit renal vascular sclerosis in aged mice by regulating the PI3K/AKT/mTOR signaling pathway.

Silencing of keratin 1 inactivates the Notch signaling pathway to inhibit renal interstitial fibrosis and glomerular sclerosis in uremia.

Renal interstitial fibrosis is a key factor in the development of chronic renal diseases, possibly leading to uremia. The present study conducted aimed to assess the hypothesis whether keratin 1 (KRT1) silencing could suppress kidney interstitial fibrosis and glomerular sclerosis via the Notch pathway to alleviate uremic symptoms. Differentially expressed genes associated with uremia were identified using the gene expression omnibus (GEO) database. Uremic rat models were established, in which short hairpin-RNA against KRT1, activators, and inhibitors of the Notch pathway were transfected. To further validate the mechanism of KRT1 in uremia, KRT1 expression, cell apoptosis, glomerular area (GA), and glomerular capillary volume (GV), the score of glomerular sclerosis, and tubulointerstitial injury were assayed and investigated. GEO database revealed that KRT1 was upregulated in uremia and regulated the Notch pathway. GA, GV, cell apoptosis, glomerular sclerosis, and tubulointerstitial injury were typically located in more elevated levels of uremia in rats. KRT1 silencing and Notch pathway inhibition decreased the expression of Jagged1, Notch1, NICD1, Hey1, Hes1, α-SMA, and FN, which further resulted in decreased cell apoptosis, GA, GV, the score of glomerular sclerosis, and tubulointerstitial injury. Subsequently, the effect of KRT1 silencing on uremia was no longer evident once the Notch pathway was activated. The co-localization of high expression KRT1 and Notch1 was found in uremia. In summary, the results identified KRT1 as a key regulator in uremia progression, and KRT1 silencing can suppress glomerular sclerosis and tubulointerstitial injury via inactivation of the Notch pathway in uremic rats.

Phosphoproteomic analysis reveals Akt isoform-specific regulation of cytoskeleton proteins in human temporal lobe epilepsy with hippocampal sclerosis.

Akt is one of the most important downstream effectors of phosphatidylinositol 3-kinase/mTOR pathway. Hyperactivation and expression of this pathway are seen in a variety of neurological disorders including human temporal lobe epilepsy with hippocampal sclerosis (TLE-HS). Nevertheless, the expression and activation profiles of the Akt isoforms, Akt1, Akt2, and Akt3 and their functional roles in human TLE-HS have not been studied. We examined the protein expression and activation (phosphorylation) patterns of Akt and its isoforms in human hippocampal tissue from TLE and non-TLE patients. A phosphoproteomic approach followed by interactome analysis of each Akt isoform was used to understand protein-protein interactions and their role in TLE-HS pathology. Our results demonstrated activation of the Akt/mTOR pathway as well as activation of Akt downstream substrates like GSK3ß, mTOR, and S6 in TLE-HS samples. Akt1 isoform levels were significantly increased in the TLE-HS samples as compared to the non-TLE samples. Most importantly, different isoforms were activated in different TLE-HS samples, Akt2 was activated in three samples, Akt2 and Akt1 were simultaneously activated in one sample and Akt3 was activated in two samples. Our phosphoproteomic screen across six TLE-HS samples identified 183 proteins phosphorylated by Akt isoforms, 29 of these proteins belong to cytoskeletal modification. Also, we were able to identify proteins of several other classes involved in glycolysis, neuronal development, protein folding and excitatory amino acid transport functions as Akt substrates. Taken together, our data offer clues to understand the role of Akt and its isoforms in underlying the pathology of TLE-HS and further, modulation of Akt/mTOR pathway using Akt isoforms specific inhibitors may offer a new therapeutic window for treatment of human TLE-HS.