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1.
Am J Physiol Renal Physiol ; 316(6): F1124-F1132, 2019 06 01.
Article in English | MEDLINE | ID: mdl-30785352

ABSTRACT

Inhibition of p53 has been shown to be an efficient strategy for ameliorating kidney ischemia-reperfusion (I/R) injury in experimental models. The therapeutic value of p53 siRNA-based inhibition for I/R in renal transplantation is currently being evaluated in clinical studies. While the major rationale for these studies is the suppression of proapoptotic properties, there are more equally important injury response pathways regulated by p53. A p53-dependent pathway shown to be crucial for renal long-term outcome is cellular senescence. In this study, we tested the hypothesis that p53 siRNA reduces I/R-induced senescence and thereby improves kidney outcome. By comparing the impact of different treatment durations in a mouse model of renal I/R, we found that repetitive administration of p53 siRNA during the first 14 days after I/R reduced the senescence load and ameliorated the postischemic phenotype. Prolonged application of p53 siRNA over a 26-day period after I/R, however, did not provide any additional benefit for senescence reduction but reversed some of the renoprotective effects of the early treatment. These data suggest a time-dependent role of p53 activity supporting the current therapeutic concept of a short-term inhibition, while advocating against a prolonged treatment after I/R.


Subject(s)
Acute Kidney Injury/therapy , Cellular Senescence , Kidney Tubules, Proximal/metabolism , RNA, Small Interfering/administration & dosage , RNAi Therapeutics , Reperfusion Injury/therapy , Tumor Suppressor Protein p53/metabolism , Acute Kidney Injury/genetics , Acute Kidney Injury/metabolism , Acute Kidney Injury/pathology , Animals , Apoptosis , Disease Models, Animal , Kidney Tubules, Proximal/pathology , Male , Mice, Inbred C57BL , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , RNA, Small Interfering/toxicity , RNAi Therapeutics/adverse effects , Reperfusion Injury/genetics , Reperfusion Injury/metabolism , Reperfusion Injury/pathology , Signal Transduction , Time Factors , Tumor Suppressor Protein p53/genetics
2.
Cell Mol Neurobiol ; 38(7): 1369-1382, 2018 Oct.
Article in English | MEDLINE | ID: mdl-29992390

ABSTRACT

Cerebral cavernous malformations (CCM) are vascular malformations associated with abnormally dilated blood vessels and leaky capillaries that often result in hemorrhages. Despite recent advances, precise understanding of the cellular and molecular mechanism leading to the pathogenesis of CCM remains elusive. Emerging evidence indicates that small nucleolar RNAs (snoRNAs), belonging to the class of non-coding RNAs, may play a significant role as diagnostic markers in human diseases. However, there is no report till date that studied the role of snoRNAs in CCM biology. The objective of the current study was to identify snoRNAs associated with CCM pathogenesis. Using genome-wide small RNA sequencing, we identified a total of 271 snoRNAs reliably expressed in CCM. By applying additional statistical stringency, three snoRNAs (SNORD115-32, SNORD114-22, and SNORD113-3) were found to be significantly downregulated in CCM patient tissue samples (n = 3) as compared to healthy brains (n = 3). Deregulation of the selected snoRNAs was further validated by qRT-PCR. Further, cellular localization via in situ hybridization also confirmed robust reduction in the expression of SNORD115-32 and SNORD114-22 in CCM tissues as compared to the healthy controls. By applying high-throughput sequencing and cellular localization analyses, we report here for the first time the genome-wide expression profile of snoRNAs in CCM tissues and a robust downregulation of candidate snoRNAs in CCM conditions. Future studies should warrant the screening in large CCM patient cohorts and will be helpful in the development of potential biomarkers and improved clinical diagnosis.


Subject(s)
Down-Regulation/genetics , Genome, Human , Hemangioma, Cavernous, Central Nervous System/genetics , RNA, Small Nucleolar/genetics , Sequence Analysis, DNA , Adult , Animals , Cluster Analysis , Female , Gene Expression Profiling , Hemangioma, Cavernous, Central Nervous System/pathology , Humans , Magnetic Resonance Imaging , Male , Middle Aged , RNA, Small Nucleolar/metabolism , Reproducibility of Results
3.
J Nephropathol ; 6(2): 90-96, 2017 Mar.
Article in English | MEDLINE | ID: mdl-28491859

ABSTRACT

BACKGROUND: Mammalian target of rapamycin (mTOR) inhibitors are increasingly used as immunosuppressive agents in kidney transplantation. In the experimental setting it has been shown that mTOR inhibitors promote autophagy, but the concept that this might also occur in transplant patients has not been addressed. OBJECTIVES: This study was designed to investigate the association between mTOR inhibition and autophagy in renal transplants under routine clinical conditions. MATERIALS AND METHODS: Protocol transplant biopsies of patients receiving sirolimus were compared to biopsies of patients treated without mTOR inhibitor. Electron microscopy was used for quantitative stereological analysis of autophagosomal volume fractions. Ultrastructural analysis was focused on podocytes to avoid cell type bias. Autophagy-related gene products were profiled by QPCR from laser assisted microdissected glomeruli and by immunohistochemistry for semiquantitative evaluation. RESULTS: By electron microscopy, we observed a significant > 50% increase in podocytic autophagosomal volume fractions in patients treated with sirolimus. Evaluation of biopsy material from the same patients using transcriptional profiling of laser capture microdissected glomeruli revealed no differences in autophagy-related gene expressions. Immunohistochemical evaluation of autophagic degradation product p62 was also unaltered whereas a significant increase was observed in podocytic LC3 positivity in biopsies of sirolimus treated patients. CONCLUSIONS: These results indicate an association of sirolimus treatment and autophagosome formation in transplant patients. However, they might reflect autophagosomal buildup rather than increased autophagic flux. Further research is needed to investigate the potential functional consequences in short- and long-term outcome of patients treated with mTOR inhibitors.

4.
J Mol Neurosci ; 61(2): 178-188, 2017 Feb.
Article in English | MEDLINE | ID: mdl-28181149

ABSTRACT

Cerebral cavernous malformations (CCM) are vascular lesions associated with loss-of-function mutations in one of the three genes encoding KRIT1 (CCM1), CCM2, and PDCD10. Recent understanding of the molecular mechanisms that lead to CCM development is limited. The role of microRNAs (miRNAs) has been demonstrated in vascular pathologies resulting in loss of tight junction proteins, increased vascular permeability and endothelial cell dysfunction. Since the relevance of miRNAs in CCM pathophysiology has not been elucidated, the primary aim of the study was to identify the miRNA-mRNA expression network associated with CCM. Using small RNA sequencing, we identified a total of 764 matured miRNAs expressed in CCM patients compared to the healthy brains. The expression of the selected miRNAs was validated by qRT-PCR, and the results were found to be consistent with the sequencing data. Upon application of additional statistical stringency, five miRNAs (let-7b-5p, miR-361-5p, miR-370-3p, miR-181a-2-3p, and miR-95-3p) were prioritized to be top CCM-relevant miRNAs. Further in silico analyses revealed that the prioritized miRNAs have a direct functional relation with mRNAs, such as MIB1, HIF1A, PDCD10, TJP1, OCLN, HES1, MAPK1, VEGFA, EGFL7, NF1, and ENG, which are previously characterized as key regulators of CCM pathology. To date, this is the first study to investigate the role of miRNAs in CCM pathology. By employing cutting edge molecular and in silico analyses on clinical samples, the current study reports global miRNA expression changes in CCM patients and provides a rich source of data set to understand detailed molecular machinery involved in CCM pathophysiology.


Subject(s)
Down-Regulation , Hemangioma, Cavernous, Central Nervous System/genetics , MicroRNAs/genetics , Adult , Case-Control Studies , Female , Gene Regulatory Networks , Humans , Male , Middle Aged , Transcriptome
5.
Cell Cycle ; 15(21): 2973-2979, 2016 Nov.
Article in English | MEDLINE | ID: mdl-27715411

ABSTRACT

Autophagy and senescence are 2 distinct pathways that are importantly involved in acute kidney injury and renal repair. Recent data indicate that the 2 processes might be interrelated. To investigate the potential link between autophagy and senescence in the kidney we isolated primary tubular epithelial cells (PTEC) from wild-type mice and monitored the occurrence of cellular senescence during autophagy activation and inhibition. We found that the process of cell isolation and transfer into culture was associated with a strong basal autophagic activation in PTEC. Specific inhibition of autophagy by silencing autophagy-related 5 (Atg5) counteracted the occurrence of senescence hallmarks under baseline conditions. Reduced senescent features were also observed in Atg5 silenced PTEC after γ-irradiation and during H-Ras induced oncogenic senescence, but the response was less uniform in these stress models. Senescence inhibition was paralleled by better preservation of a mature epithelial phenotype in PTEC. Interestingly, treatment with rapamycin, which acts as an activator of autophagy, also counteracted the occurrence of senescence features in PTEC. While we interpret the anti-senescent effect of rapamycin as an autophagy-independent effect of mTOR-inhibition, the more specific approach of Atg5 silencing indicates that overactivated autophagy can have pro-senescent effects in PTEC. These results highlight the complex interaction between cell culture dependent stress mechanisms, autophagy and senescence.


Subject(s)
Autophagy , Cellular Senescence , Epithelial Cells/cytology , Kidney Tubules/cytology , Animals , Biomarkers/metabolism , Cells, Cultured , Epithelial Cells/metabolism , Epithelial Cells/ultrastructure , Mice, Inbred C57BL , Phenotype , Stress, Physiological
6.
Neurosurg Rev ; 39(4): 581-9, 2016 Oct.
Article in English | MEDLINE | ID: mdl-26779617

ABSTRACT

Cerebral cavernous malformations (CCM) commonly known as cavernous hemangioma are associated with abnormally enlarged thin-walled blood vessels. As a result, these dilated capillaries are prone to leakage and result in hemorrhages. Clinically, such hemorrhages lead to severe headaches, focal neurological deficits, and epileptic seizures. CCM is caused by loss of function mutations in one of the three well-known CCM genes: Krev interaction trapped 1 (KRIT1), OSM, and programmed cell death 10 (PDCD10). Loss of CCM genes have been shown to be synergistically related to decreased Notch signaling and excessive angiogenesis. Despite recent evidences indicating that Notch signaling plays a pivotal role in regulating angiogenesis, the role of Notch in CCM development and progression is still not clear. Here, we provide an update literature review on the current knowledge of the structure of Notch receptor and its ligands, its relevance to angiogenesis and more precisely to CCM pathogenesis. In addition to reviewing the current literatures, this review will also focus on the cross talk between Delta-Notch and vascular endothelial growth factor (VEGF) signaling in angiogenesis and in CCM pathogenesis. Understanding the role of Notch signaling in CCM development and progression might help provide a better insight for novel anti-angiogenic therapies.


Subject(s)
Apoptosis Regulatory Proteins/metabolism , Carrier Proteins/metabolism , Hemangioma, Cavernous, Central Nervous System/metabolism , Receptors, Notch/metabolism , Apoptosis Regulatory Proteins/genetics , Carrier Proteins/genetics , Hemangioma, Cavernous, Central Nervous System/pathology , Humans , Signal Transduction/physiology , Vascular Endothelial Growth Factor A/metabolism
7.
Autophagy ; 12(3): 596-7, 2016.
Article in English | MEDLINE | ID: mdl-26761120

ABSTRACT

Antagonizing the strongly activated pathway of autophagy in renal ischemic injury has been associated with poor outcome. In our recent study we used mice with a selective deletion of Atg5 in the S3 proximal tubule segment, which is most susceptible to ischemic damage. In line with the notion that autophagy is a prosurvival mechanism our studies revealed an early accelerated cell death of heavily damaged tubular cells in the S3 segment of these mice. Interestingly, this expedited loss of cells was associated with better long-term outcome as reflected by less inflammation, improved tubular repair, and function and reduced accumulation of senescent cells. While these data confirm the role of tubular autophagy as a prosurvival mechanism in ischemic kidney injury, they also show that autophagy may enable severely damaged cells to persist and exert deleterious effects. Such ambivalent effects might be of relevance if modulating autophagy is considered as a therapeutic option.


Subject(s)
Acute Kidney Injury/pathology , Autophagy , Animals , Autophagy-Related Protein 5/metabolism , Kidney Tubules, Proximal/pathology , Mice , Models, Biological , Reperfusion Injury/pathology
8.
J Am Soc Nephrol ; 27(6): 1609-16, 2016 Jun.
Article in English | MEDLINE | ID: mdl-26487561

ABSTRACT

Evidence suggests that autophagy promotes the development of cellular senescence. Because cellular senescence contributes to renal aging and promotes the progression from AKI to CKD, we investigated the potential effect of tubular autophagy on senescence induction. Compared with kidneys from control mice, kidneys from mice with conditional deletion of autophagy-related 5 (Atg5) for selective ablation of autophagy in proximal tubular S3 segments (Atg5(Δ) (flox/) (Δ) (flox)) presented with significantly less tubular senescence, reduced interstitial fibrosis, and superior renal function 30 days after ischemia/reperfusion injury. To correlate this long-term outcome with differences in the early injury process, kidneys were analyzed 2 hours and 3 days after reperfusion. Notably, compared with kidneys of control mice, Atg5(Δ) (flox/) (Δ) (flox) kidneys showed more cell death in outer medullary S3 segments at 2 hours but less tubular damage and inflammation at day 3. These data suggest that the lack of autophagy prevents early survival mechanisms in severely damaged tubular cells. However, if such compromised cells persist, then they may lead to maladaptive repair and proinflammatory changes, thereby facilitating the development of a senescent phenotype and CKD.


Subject(s)
Autophagy , Cellular Senescence , Kidney Tubules, Proximal/cytology , Animals , Male , Mice
9.
J Am Soc Nephrol ; 26(11): 2659-68, 2015 Nov.
Article in English | MEDLINE | ID: mdl-25788525

ABSTRACT

Zinc-α2-glycoprotein (AZGP1) is a secreted protein synthesized by epithelial cells and adipocytes that has roles in lipid metabolism, cell cycling, and cancer progression. Our previous findings in AKI indicated a new role for AZGP1 in the regulation of fibrosis, which is a unifying feature of CKD. Using two models of chronic kidney injury, we now show that mice with genetic AZGP1 deletion develop significantly more kidney fibrosis. This destructive phenotype was rescued by injection of recombinant AZGP1. Exposure of AZGP1-deficient mice to cardiac stress by thoracic aortic constriction revealed that antifibrotic effects were not restricted to the kidney but were cardioprotective. In vitro, recombinant AZGP1 inhibited kidney epithelial dedifferentiation and antagonized fibroblast activation by negatively regulating TGF-ß signaling. Patient sera with high levels of AZGP1 similarly attenuated TGF-ß signaling in fibroblasts. Taken together, these findings indicate a novel role for AZGP1 as a negative regulator of fibrosis progression, suggesting that recombinant AZGP1 may have translational effect for treating fibrotic disease.


Subject(s)
Kidney Failure, Chronic/genetics , Kidney/metabolism , Myocardium/metabolism , Seminal Plasma Proteins/metabolism , Adipokines , Animals , Aorta/pathology , Carrier Proteins/metabolism , Cell Differentiation , Epithelium/pathology , Fibroblasts/metabolism , Fibrosis/pathology , Gene Deletion , Glycoproteins/metabolism , HEK293 Cells , Humans , Kidney/pathology , Kidney Diseases/metabolism , Kidney Failure, Chronic/metabolism , Male , Mice , Myocardium/pathology , Phosphorylation , Protein Biosynthesis , Rats , Recombinant Proteins/chemistry , Signal Transduction , Transforming Growth Factor beta/metabolism , Ureteral Obstruction/pathology , Zn-Alpha-2-Glycoprotein
10.
PLoS One ; 9(2): e88071, 2014.
Article in English | MEDLINE | ID: mdl-24505380

ABSTRACT

Acute kidney injury is a major clinical problem and advanced age is associated with ineffective renal regeneration and poor functional outcome. Data from kidney injury models suggest that a loss of tubular epithelial proliferation contributes to a decrease in renal repair capacity with aging, but aging can also lead to a higher severity of inflammation and damage which may influence repair. In this study we tested intrinsic age-dependent changes in tubular epithelial proliferation in young and old mice, by injecting low-dose lead acetate as a non-injurious mitogen. In parallel, we explored in vitro techniques of studying cellular senescence in primary tubular epithelial cells (PTEC). Lead acetate induced tubular epithelial proliferation at a significantly higher rate in young as compared to old mice. Old kidneys showed significantly more senescence as demonstrated by increased p16 (INK4a), senescence associated ß-galactosidase, and γH2AX(+)/Ki-67(-) cells. This was paralleled in old kidneys by a higher number of Cyclin D1 positive tubular cells. This finding was corroborated by a positive correlation between Cyclin D1 positivity and age in human renal biopsies. When tubular cells were isolated from mouse kidneys they rapidly lost their age-associated differences under culture conditions. However, senescence was readily induced in PTEC by γ-irradiation representing a future model for study of cellular senescence in the renal epithelium. Together, our data indicate that the tubular epithelium of aged kidney has an intrinsically reduced proliferative capacity probably due to a higher load of senescent cells. Moreover, stress induced models of cellular senescence are preferable for study of the renal epithelium in vitro. Finally, the positive correlation of Cyclin D1 with age and cellular senescence in PTEC needs further evaluation as to a functional role of renal epithelial aging.


Subject(s)
Aging/physiology , Cellular Senescence/physiology , Epithelial Cells/physiology , Kidney Tubules/physiology , Acute Kidney Injury/genetics , Acute Kidney Injury/physiopathology , Aging/genetics , Animals , Cell Proliferation , Cells, Cultured , Cellular Senescence/genetics , Cyclin D1/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Epithelium/physiology , Histones/genetics , Ki-67 Antigen/genetics , Male , Mice , Mice, Inbred C57BL , Regeneration/genetics , Regeneration/physiology , beta-Galactosidase/genetics
11.
J Am Soc Nephrol ; 23(9): 1467-73, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22797186

ABSTRACT

Long-term graft survival after kidney transplantation remains unsatisfactory and unpredictable. Interstitial fibrosis and tubular atrophy are major contributors to late graft loss; features of tubular cell senescence, such as increased p16(INK4a) expression, associate with these tubulointerstitial changes, but it is unknown whether the relationship is causal. Here, loss of the INK4a locus in mice, which allows escape from p16(INK4a)-dependent senescence, significantly reduced interstitial fibrosis and tubular atrophy and associated with improved renal function, conservation of nephron mass, and transplant survival. Compared with wild-type controls, kidneys from INK4a(-/-) mice developed significantly less interstitial fibrosis and tubular atrophy after ischemia-reperfusion injury. Consistently, mice that received kidney transplants from INK4a/ARF(-/-) donors had significantly better survival 21 days after life-supporting kidney transplantation and developed less tubulointerstitial changes. This correlated with higher proliferative rates of tubular cells and significantly fewer senescent cells. Taken together, these data suggest a pathogenic role of renal cellular senescence in the development of interstitial fibrosis and tubular atrophy and kidney graft deterioration by preventing the recovery from injury. Inhibiting premature senescence could have therapeutic benefit in kidney transplantation but has to be balanced against the risks of suspending antitumor defenses.


Subject(s)
Cellular Senescence/physiology , Graft Survival/physiology , Kidney Transplantation/physiology , Kidney/physiology , Regeneration/physiology , Animals , Atrophy , Cyclin-Dependent Kinase Inhibitor p16/deficiency , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/physiology , Fibrosis , Kidney/pathology , Kidney Transplantation/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Reperfusion Injury/pathology , Transplantation, Homologous/physiology
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