CRISPR/Cas Derivatives as Novel Gene Modulating Tools:Possibilities and In Vivo Applications

The field of genome editing started with the discovery of meganucleases (e.g., the LAGLIDADG family of homing endonucleases) in yeast. After the discovery of transcription activator-like effector nucleases and zinc finger nucleases, the recently discovered clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) system has opened a new window of applications in the field of gene editing. Here, we review different Cas proteins and their corresponding features including advantages and disadvantages, and we provide an overview of the different endonuclease-deficient Cas protein (dCas) derivatives. These dCas derivatives consist of an endonuclease-deficient Cas9 which can be fused to different effector domains to perform distinct in vitro applications such as tracking, transcriptional activation and repression, as well as base editing. Finally, we review the in vivo applications of these dCas derivatives and discuss their potential to perform gene activation and repression in vivo, as well as their potential future use in human therapy

Proteomic analysis of embryonic kidney development: Heterochromatin proteins as epigenetic regulators of nephrogenesis

Elucidation of the mechanisms underlying the nephrogenesis will boost enormously the regenerative medicine. Here we performed 2-D gel-based comparative proteome analyses of rat embryonic kidney from different developmental stages. Out of 288 non-redundant identified proteins, 102 were common in all developmental stages. 86% of the proteins found in E14 and E16 were identical, in contrast only 37% of the identified proteins overlap between E14 and P1. Bioinformatics analysis suggests developmental stage-specific pathway activation and highlighted heterochromatin protein 1 (Cbx1, Cbx3, Cbx5) and Trim28 as potential key players in nephrogenesis. These are involved in the epigenetic regulation of gene silencing and were down-regulated in the course of kidney development. Trim28 is a potential epigenetic regulator of the branching inhibitor Bmp4. Silencing of Trim28 in cultured kidneys resulted in branching arrest. In contrast knockdown of Cbx5 was associated with abnormal ureteric bud growth and slight impairment of branching. ChIP analysis showed that the H3K9me3 distribution on Bmp4 promoters at E14 and E19 inversely correlate with mRNA expression levels. The concentrated expression-pattern of heterochromatin proteins and the negative impact of their silencing on kidney development, suggest an important role in reciprocal and inductive signaling between the ureteric bud and the metanephric mesenchyme

The secretome analysis of activated human renal fibroblasts revealed beneficial effect of the modulation of the secreted peptidyl-prolyl cis-trans isomerase A in kidney fibrosis

The secretome is an important mediator in the permanent process of reciprocity between cells and their environment. Components of secretome are involved in a large number of physiological mechanisms including differentiation, migration, and extracellular matrix modulation. Alteration in secretome composition may therefore trigger cell transformation, inflammation, and diseases. In the kidney, aberrant protein secretion plays a central role in cell activation and transition and in promoting renal fibrosis onset and progression. Using comparative proteomic analyses, we investigated in the present study the impact of cell transition on renal fibroblast cells secretome. Human renal cell lines were stimulated with profibrotic hormones and cytokines, and alterations in secretome were investigated using proteomic approaches. We identified protein signatures specific for the fibrotic phenotype and investigated the impact of modeling secretome proteins on extra cellular matrix accumulation. The secretion of peptidyl-prolyl cis-trans isomerase A (PPIA) was demonstrated to be associated with fibrosis phenotype. We showed that the in-vitro inhibition of PPIA with ciclosporin A (CsA) resulted in downregulation of PPIA and fibronectin (FN1) expression and significantly reduced their secretion. Knockdown studies of PPIA in a three-dimensional (3D) cell culture model significantly impaired the secretion and accumulation of the extracellular matrix (ECM), suggesting a positive therapeutic effect on renal fibrosis progression

3D virtual histology of murine kidneys-high resolution visualization of pathological alterations by micro computed tomography

The increasing number of patients with end stage chronic kidney disease not only calls for novel therapeutics but also for pioneering research using convincing preclinical disease models and innovative analytical techniques. The aim of this study was to introduce a virtual histology approach using micro computed tomography (mu CT) for the entire murine kidney in order to close the gap between single slice planar histology and a 3D high resolution dataset. An ex vivo staining protocol based on phosphotungstic acid diffusion was adapted to enhance renal soft tissue x-ray attenuation. Subsequent CT scans allowed (i) the detection of the renal cortex, medulla and pelvis in greater detail, (ii) the analysis of morphological alterations, (iii) the quantification of the volume as well as the radio-opacity of these portions and (iv) the quantification of renal fibrotic remodeling based on altered radio-opacity using the unilateral ureteral obstruction model. Thus, virtual histology based on PTA contrast enhanced CT will in future help to refine the outcome of preclinical research on kidney associated murine disease models

Restoration of Podocyte Structure and Improvement of Chronic Renal Disease in Transgenic Mice Overexpressing Renin

Proteinuria is a major marker of the decline of renal function and an important risk factor of coronary heart disease. Elevated proteinuria is associated to the disruption of slit-diaphragm and loss of podocyte foot processes, structural alterations that are considered irreversible. The objective of the present study was to investigate whether proteinuria can be reversed and to identify the structural modifications and the gene/protein regulation associated to this reversal.We used a novel transgenic strain of mouse (RenTg) that overexpresses renin at a constant high level. At the age of 12-month, RenTg mice showed established lesions typical of chronic renal disease such as peri-vascular and periglomerular inflammation, glomerular ischemia, glomerulosclerosis, mesangial expansion and tubular dilation. Ultrastructural analysis indicated abnormal heterogeneity of basement membrane thickness and disappearance of podocyte foot processes. These structural alterations were accompanied by decreased expressions of proteins specific of podocyte (nephrin, podocin), or tubular epithelial cell (E-cadherin and megalin) integrity. In addition, since TGFbeta is considered the major pro-fibrotic agent in renal disease and since exogenous administration of BMP7 is reported to antagonize the TGFbeta-induced phenotype changes in kidney, we have screened the expressions of several genes belonging in the TGFbeta/BMP superfamily. We found that the endogenous inhibitors of BMPs such as noggin and Usag-1 were several-fold activated inhibiting the action of BMPs and thus reinforcing the deleterious action of TGFbeta.Treatment with an AT1 receptor antagonist, at dose that did not decrease arterial pressure, gradually reduced albuminuria. This decrease was accompanied by re-expression of podocin, nephrin, E-cadherin and megalin, and reappearance of podocyte foot processes. In addition, expressions of noggin and Usag-1 were markedly decreased, permitting thus activation of the beneficial action of BMPs.These findings show that proteinuria and alterations in the expression of proteins involved in the integrity and function of glomerular and renal epithelial phenotype are reversible events when the local action of angiotensin II is blocked, and provide hope that chronic renal disease can be efficiently treated

'Special K' and a loss of cell-to-cell adhesion in proximal tubule-derived epithelial cells: modulation of the adherens junction complex by ketamine

Ketamine, a mild hallucinogenic class C drug, is the fastest growing ‘party drug’ used by 16–24 year olds in the UK. As the recreational use of Ketamine increases we are beginning to see the signs of major renal and bladder complications. To date however, we know nothing of a role for Ketamine in modulating both structure and function of the human renal proximal tubule. In the current study we have used an established model cell line for human epithelial cells of the proximal tubule (HK2) to demonstrate that Ketamine evokes early changes in expression of proteins central to the adherens junction complex. Furthermore we use AFM single-cell force spectroscopy to assess if these changes functionally uncouple cells of the proximal tubule ahead of any overt loss in epithelial cell function. Our data suggests that Ketamine (24–48 hrs) produces gross changes in cell morphology and cytoskeletal architecture towards a fibrotic phenotype. These physical changes matched the concentration-dependent (0.1–1 mg/mL) cytotoxic effect of Ketamine and reflect a loss in expression of the key adherens junction proteins epithelial (E)- and neural (N)-cadherin and β-catenin. Down-regulation of protein expression does not involve the pro-fibrotic cytokine TGFβ, nor is it regulated by the usual increase in expression of Slug or Snail, the transcriptional regulators for E-cadherin. However, the loss in E-cadherin can be partially rescued pharmacologically by blocking p38 MAPK using SB203580. These data provide compelling evidence that Ketamine alters epithelial cell-to-cell adhesion and cell-coupling in the proximal kidney via a non-classical pro-fibrotic mechanism and the data provides the first indication that this illicit substance can have major implications on renal function. Understanding Ketamine-induced renal pathology may identify targets for future therapeutic intervention

High-fidelity CRISPR/Cas9-based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model

miR-132-3p and KLF7 as novel regulators of aortic stiffening-associated EndMT in type 2 diabetes mellitus

Background: The prevalence of diabetes mellitus has risen considerably and currently affects more than 422 million people worldwide. Cardiovascular diseases including myocardial infarction and heart failure represent the major cause of death in type 2 diabetes (T2D). Diabetes patients exhibit accelerated aortic stiffening which is an independent predictor of cardiovascular disease and mortality. We recently showed that aortic stiffness precedes hypertension in a mouse model of diabetes (db/db mice), making aortic stiffness an early contributor to cardiovascular disease development. Elucidating how aortic stiffening develops is a pressing need in order to halt the pathophysiological process at an early time point. Methods: To assess EndMT occurrence, we performed co-immunofluorescence staining of an endothelial marker (CD31) with mesenchymal markers (α-SMA/S100A4) in aortic sections from db/db mice. Moreover, we performed qRT-PCR to analyze mRNA expression of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. To identify the underlying mechanism by which EndMT contributes to aortic stiffening, we used aortas from db/db mice and diabetic patients in combination with high glucose-treated human umbilical vein endothelial cells (HUVECs) as an in vitro model of diabetes-associated EndMT. Results: We demonstrate robust CD31/α-SMA and CD31/S100A4 co-localization in aortic sections of db/db mice which was almost absent in control mice. Moreover, we demonstrate a significant upregulation of EndMT transcription factors in aortic sections of db/db mice and diabetic patients. As underlying regulator, we identified miR-132-3p as the most significantly downregulated miR in the micronome of db/db mice and high glucose-treated HUVECs. Indeed, miR-132-3p was also significantly downregulated in aortic tissue from diabetic patients. We identified Kruppel-like factor 7 (KLF7) as a target of miR-132-3p and show a significant upregulation of KLF7 in aortic sections of db/db mice and diabetic patients as well as in high glucose-treated HUVECs. We further demonstrate that miR-132-3p overexpression and KLF7 downregulation ameliorates EndMT in high glucose-treated HUVECs. Conclusions: We demonstrate for the first time that EndMT contributes to aortic stiffening in T2D. We identified miR-132-3p and KLF7 as novel EndMT regulators in this context. Altogether, this gives us new insights in the development of aortic stiffening in T2D.</p

Dynamic Monitoring of Cellular Remodeling Induced by the Transforming Growth Factor-β1

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate