Citations

Scattered tubular-like cells (STCs), dedifferentiated renal tubular epithelial cells, contribute to renal self-healing, but severe injury might blunt their effectiveness. We hypothesized that ischemic renovascular disease (RVD) induces senescence in STC and impairs their reparative potency. CD24+/CD133+ STCs were isolated from swine kidneys after 16 weeks of RVD or healthy controls. To test their reparative capabilities in injured kidneys, control or RVD-STC (5×10 5 ) were prelabeled and injected into the aorta of 2 kidneys, 1-clip (2k,1c) mice 2 weeks after surgery. Murine renal function and oxygenation were studied in vivo 2 weeks after injection using micro-magnetic resonance imaging, and fibrosis, tubulointerstitial injury, capillary density, and expression of profibrotic and inflammatory genes ex vivo. STC isolated from swine RVD kidneys showed increased gene expression of senescence and senescence-associated secretory phenotype markers and positive SA-β-gal staining. Delivery of normal pig STCs in 2k,1c mice improved murine renal perfusion, blood flow, and glomerular filtration rate, and downregulated profibrotic and inflammatory gene expression. These renoprotective effects were blunted using STC harvested from RVD kidneys, which also failed to attenuate hypoxia, fibrosis, tubular injury, and capillary loss in injured mouse 2k,1c kidneys. Hence, RVD may induce senescence in endogenous STC and impair their reparative capacity. These observations implicate cellular senescence in the pathophysiology of ischemic kidney disease and support senolytic therapy to permit self-healing of senescent kidneys.

AbstractBackgroundThe pathological features of AD are neurofibrillary tangles (NFTs) composed of highly phosphorylated tau and senile plaques (SPs) composed of amyloid β protein (Aβ). It was suggested that Cu2 + induced hyperphosphorylation of tau and Zn2 + deposits on SPs. Clioquinol (CQ) has a mild chelating effect on Zn2 + and Cu2 +. In addition, CQ has been shown to reduce Aβ aggregation by removing Zn2+ from SPs. However, the effect of CQ on tau aggregation is not fully understood. The purpose of our study is to examine the effect of CQ on tau metabolism by using a cell culture model of tauopathy.MethodsHuman neuroblastoma cell line M1C cells that express wild‐type tau protein (4R0N) via tetracycline (Tet) off induction was used. M1C cells were exposed to 0.1‐10 μM CQ on day 4 of Tet‐Off induction, and harvested at the end of Day 5. Total tau detected by Tau5, and phosphorylated tau detected by PS199/202, AT180, AT270, CP13, and PHF‐1 antibodies, were examined using Western blot analysis. Immunocytochemical studies, fractional analysis and immunoprecipitation of ubiquitinated tau were also performed. Tau kinases including c‐Jun N‐terminal kinase (JNK), and P38 MAPK and tau phosphatase, protein phosphatase 2A (PP2A) were also examined. C‐terminal truncated pathological tau was detected by using the antibody TauC3. In addition, autophagy and proteasome activity were also examined.ResultPhosphorylated tau levels were reduced by 1‐10 μM CQ treatment. Tau kinases, JNK and p38 MAPK were inactivated by CQ treatment. Interestingly, activation of the tau phosphatase PP2A was also observed with CQ treatment. Fractionation studies showed that CQ treatment reduced AT180 and AT270 positive high molecular weight tau in the Tris‐insoluble, sarkosyl‐soluble fraction. CQ also reduced tau oligomer specific antibody TOC1‐positive oligomeric tau. CQ reduced caspase‐cleaved tau detected by antibody TauC3. P62 levels were reduced by CQ treatment, suggesting that CQ activates autophagy. In addition, CQ slightly reduced high molecular weight ubiquitinated tau. This implies activation of the ubiquitin‐proteasome by CQ.ConclusionCQ could provide beneficial effects against pathogenic forms of tau via multiple mechanisms and may represent a viable therapeutic approach for AD and other tauopathies.

AbstractBackgroundNeurofibrillary tangle (NFT), composed of highly phosphorylated tau is one of the pathological hallmarks of Alzheimer’s disease (AD). Spleen tyrosine kinase (Syk) is non receptor type tyrosine kinase, and has various physiological functions, including histamine release in mast cells, phagocytosis in macrophage, and B cell differentiation. The possibility of Syk inhibitor for clinical application was proposed in allergic rhinitis, rheumatoid arthritis, and immunologic thrombocytopenia. The possible relationship between Syk and AD has been reported recently. We have examined the effects of Syk specific inhibitor BAY61‐3606 on phosphorylation levels of tau protein and oligomeric tau by using cell culture model of tauopathy, M1C cells.MethodsWe utilized human neuroblastoma cell line M1C cells, which expresses wild type tau protein (4R0N) via tetracycline off induction (Tet Off induction). Reduction of phosphorylated tau protein and oligomeric tau protein by Syk inhibitor was examined by using various phospho‐tau antibodies, and tau oligomer specific antibody. Influences of activity of autophagy by Syk inhibitor was also examined by western blot analysis.ResultsIn this cell line, 72kDa of Syk protein was identified by Western blot analysis. Phosphorylated tau detected by various phospho‐tau antibodies, including PHF‐1, CP13, and AT270 was decreased by 1, 10 μM of Syk inhibitor. One of the patholoigical form of tau, caspase cleaved tau detected by TauC3 was also decreased by Syk inhibitor. Oligomeric tau, which detected by tau oligomer specific antibody, TOC1 was also decreased by Syk inhibitor. Syk inhibitor also inactivated glycogen synthase kinase 3β (GSK3β). Syk inhibitor upregulated autophagy detected by increase of LC3‐II, and reduction of P62. In summary, Syk inhibitor decreased phosphorylated tau as well as total tau by upregulating autophagy, and inactivating tau kinases, including GSK3β. Finally, oligomeric tau was reduced by Syk inhibitor.ConclusionSyk inhibitor may have promising for the treatment of tauopathy, including AD.

Abstract BACKGROUND Renovascular hypertension (RVH) often manifest with metabolic syndrome (MetS) as well. Coexisting MetS and hypertension increases cardiovascular morbidity and mortality, but the mechanisms underlying cardiac injury remain unknown. We hypothesized that superimposition of MetS induces myocardial mitochondrial damage, leading to cardiac injury and dysfunction in swine RVH. METHODS Pigs were studied after 16 weeks of diet-induced MetS with or without RVH (unilateral renal artery stenosis), and Lean controls (n = 6 each). Systolic and diastolic cardiac function were assessed by multidetector CT, and cardiac mitochondrial morphology (electron microscopy) and myocardial function in tissue and isolated mitochondria. RESULTS Body weight was similarly higher in MetS groups vs. Lean. RVH groups achieved significant stenosis and developed hypertension. Mitochondrial matrix density and adenosine triphosphate production were lower and H2O2 production higher in RVH groups vs. Lean and MetS. Lean + RVH (but not MetS + RVH) activated mitophagy, which was associated with decreased myocardial expression of mitophagy-related microRNAs. MetS groups exhibited higher numbers of intermitochondrial junctions, which could have prevented membrane depolarization/activation of mitophagy in MetS + RVH. Cardiac fibrosis, hypertrophy (increased left ventricular muscle mass), and diastolic function (decreased E/A ratio) were greater in MetS + RVH vs. Lean + RVH. CONCLUSIONS MetS+RVH induces myocardial mitochondrial damage and dysfunction. MetS + RVH failed to activate mitophagy, resulting in greater cardiac remodeling, fibrosis, and diastolic dysfunction. Mitochondrial injury and impaired mitophagy may constitute important mechanisms and therapeutic targets to ameliorate cardiac damage and dysfunction in patients with coexisting MetS and RVH.

Background. Coexisting metabolic syndrome (MetS) and renal artery stenosis (RAS) are linked to poor renal outcomes. Mesenchymal stem/stromal cell- (MSC-) derived extracellular vesicles (EVs) from lean animals show superior ability to repair the experimental MetS+RAS kidney compared to EVs from MetS pig MSCs. We hypothesized that MetS leads to selective packaging in porcine EVs of microRNAs capable of targeting mitochondrial genes, interfering with their capacity to repair the MetS+RAS kidney. Methods. Five groups of pigs ( n = 7 each) were studied after 16 weeks of diet-induced MetS and RAS (MetS+RAS) and MetS+RAS 4 weeks after a single intrarenal delivery of EVs harvested from allogeneic adipose tissue-derived MSCs isolated from Lean or MetS pigs, and Lean or MetS sham controls. Single-kidney blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector CT, whereas EV microRNA cargo, renal tubular mitochondrial structure and bioenergetics, and renal injury pathways were assessed ex vivo. Results. microRNA sequencing revealed 19 dysregulated microRNAs capable of targeting several mitochondrial genes in MetS-EVs versus Lean-EVs. Lean- and MetS-EVs were detected in the stenotic kidney 4 weeks after administration. However, only MetS-EVs failed to improve renal mitochondrial density, structure, and function or attenuate oxidative stress, tubular injury, and fibrosis. Furthermore, Lean-EVs but not MetS-EVs restored RBF and GFR in MetS+RAS. Conclusion. MetS alters the cargo of mitochondria-related microRNAs in swine MSC-derived EVs, which might impair their capacity to repair the poststenotic kidney in MetS+RAS. These observations may contribute to develop approaches to improve the efficacy of MSC-EVs for patients with MetS.

AbstractPercutaneous transluminal renal angioplasty (PTRA) has been used to treat renovascular disease (RVD), a chronic condition characterized by renal ischemia and metabolic abnormalities. Mitochondrial injury has been implicated as a central pathogenic mechanism in RVD, but whether it can be reversed by PTRA remains uncertain. We hypothesized that PTRA attenuates mitochondrial damage, renal injury, and dysfunction in pigs with coexisting renal artery stenosis (RAS) and metabolic syndrome (MetS). Four groups of pigs (n = 6 each) were studied after 16 weeks of diet‐induced MetS and RAS (MetS + RAS), MetS + RAS treated 4 weeks earlier with PTRA, and Lean and MetS Sham controls. Single‐kidney renal blood flow (RBF) and glomerular filtration rate (GFR) were assessed in vivo with multidetector computed tomography, and renal tubular mitochondrial structure and function and renal injury ex vivo. PTRA successfully restored renal artery patency, but mean arterial pressure remained unchanged. Stenotic kidney RBF and GFR, which fell in MetS + RAS compared to MetS, rose after PTRA. PTRA attenuated MetS + RAS‐induced mitochondrial structural abnormalities in tubular cells and peritubular capillary endothelial cells, decreased mitochondrial H202 production, and increased renal cytochrome‐c oxidase‐IV activity and ATP production. PTRA also improved cortical microvascular and peritubular capillary density and ameliorated tubular injury and tubulointerstitial fibrosis in the poststenotic kidney. Importantly, renal mitochondrial damage correlated with poststenotic injury and dysfunction. Renal revascularization attenuated mitochondrial injury and improved renal hemodynamics and function in swine poststenotic kidneys. This study suggests a novel mechanism by which PTRA might be relatively effective in ameliorating mitochondrial damage and improving renal function in coexisting MetS and RAS.

Abstract Background: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. Methods: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. Results: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10−5). We found seven loci associated with risk for both cancers (PBonferroni < 2.4 × 10−9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P < 5 × 10−7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. Conclusions: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. Impact: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.

Abstract Objectives To test the hypothesis that lymph node yield will vary by pathology assistant (PA) in patients undergoing radical cystectomy (RC) with pelvic lymph node dissection (PLND). Methods This is a single-institution retrospective review that included patients who underwent an RC with PLND for bladder cancer from January 1, 2007, to January 1, 2018. Predicted mean lymph node counts were generated using multivariable regression analysis. Results In a total of 430 patients who underwent RC with PLND, the median lymph node count (interquartile range) was 15.0 (11.0-21.0). The frequency of the limits of lymphadenectomy was as follows: external iliac, internal iliac, and obturator (true pelvis) (33.3%); true pelvis plus common iliac to the level of the aortic bifurcation (47.9%); and inferior mesenteric artery (18.8%). On descriptive analysis, there were differences in lymph node yield when evaluating the following variables: level of dissection, clinical stage, neoadjuvant chemotherapy, surgical approach, surgeon, pathologist, and PA (P < .05). On multivariable analysis, adjusted lymph node counts varied between surgeons, pathologists, clinical stage, and level of dissection but not by PA (P = .18). Conclusions Lymph node yield after RC varies on several known levels, including surgeon, extent of lymphadenectomy, clinical stage, and pathologist. This study found no significant variation in lymph node yield according to PA.

Abstract A data-independent acquisition (DIA) approach is being increasingly adopted as a promising strategy for identification and quantitation of proteomes. As most DIA data sets are acquired with wide isolation windows, highly complex MS/MS spectra are generated, which negatively impacts obtaining peptide information through classical protein database searches. Therefore, the analysis of DIA data mainly relies on the evidence of the existence of peptides from prebuilt spectral libraries. Consequently, one major weakness of this method is that it does not account for peptides that are not included in the spectral library, precluding the use of DIA for discovery studies. Here, we present a strategy termed Precursor ion And Small Slice-DIA (PASS-DIA) in which MS/MS spectra are acquired with small isolation windows (slices) and MS/MS spectra are interpreted with accurately determined precursor ion masses. This method enables the direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was observed to be superior to both data-dependent acquisition (DDA) and conventional DIA experiments with 69 and 48% additional protein identifications, respectively. Application of PASS-DIA for the analysis of post-translationally modified peptides again highlighted its superior performance in characterizing phosphopeptides (77% more), N-terminal acetylated peptides (56% more), and N-glycopeptides (83% more) as compared to DDA alone. Finally, the use of PASS-DIA to characterize a rare proteome of human fallopian tube organoids enabled 34% additional protein identifications than DDA alone and revealed biologically relevant pathways including low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool that outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that the PASS-DIA method is an important strategy for discovery-type studies when deeper proteome characterization is required.