Excessive recruitment of monocytes and progression of fibrosis are hallmarks of chronic kidney disease (CKD). Recently we reported that the expression of connexin 43 (Cx43) was upregulated in the kidney during experimental nephropathy. To investigate the role of Cx43 in the progression of CKD, we interbred RenTg mice, a genetic model of hypertension-induced CKD, with Cx43+/- mice. The renal cortex of 5-month-old RenTgCx43+/- mice showed a marked decrease of cell adhesion markers leading to reduced monocyte infiltration and interstitial renal fibrosis compared with their littermates. In addition, functional and histological parameters such as albuminuria and glomerulosclerosis were ameliorated in RenTgCx43+/- mice. Interestingly, treatment with Cx43 antisense produced remarkable improvement of renal function and structure in 1-year-old RenTg mice. Similar results were found in Cx43+/- or wild-type mice treated with Cx43 antisense after obstructive nephropathy. Furthermore, in these mice, Cx43 antisense attenuated E-cadherin downregulation and phosphorylation of the transcription factor Sp1 by the ERK pathway resulting in decreased transcription of type I collagen gene. Interestingly, Cx43-specific blocking peptide inhibited monocyte adhesion in activated endothelium and profibrotic pathways in tubular cells. Cx43 was highly increased in biopsies of patients with CKD. Thus, Cx43 may represent a new therapeutic target against the progression of CKD.

Transcriptome analysis by RNA-seq technology allows novel insights into gene expression and regulatory networks in health and disease. To better understand the molecular basis of renal fibrosis, we performed RNA-seq analysis in the Unilateral Ureteric Obstruction (UUO) mouse model. We analysed sham operated, 2-and 8-day post-ligation renal tissues. Thousands of genes with statistical significant changes in their expression were identified and classified into cellular processes and molecular pathways. Many novel protein-coding genes were identified, including critical transcription factors with important regulatory roles in other tissues and diseases. Emphasis was placed on long non-coding RNAs (lncRNAs), a class of molecular regulators of multiple and diverse cellular functions. Selected lncRNA genes were further studied and their transcriptional activity was confirmed. For three of them, their transcripts were also examined in other mouse models of nephropathies and their up-or downregulation was found similar to the UUO model. In vitro experiments confirmed that one selected lncRNA is independent of TGFβ or IL1b stimulation but can influence the expression of fibrosis-related proteins and the cellular phenotype. These data provide new information about the involvement of protein-coding and lncRNA genes in nephropathies, which can become novel diagnostic and therapeutic targets in the near future.Chronic kidney disease (CKD) is a frequent condition, causing severe long-term effects with devastating personal and societal consequences 1-3 . There is a need for novel approaches to prevent the decline in renal function during progression of CKD. Considering that the structural basis for this decline is the development of fibrosis, we believe that understanding the molecular basis of renal fibrosis, could offer valuable insights for the improvement of monitoring techniques and therapeutic interventions.To address this question, we combined a systems biology approach in animal models for renal fibrosis, focusing on (but not limited to) the unilateral ureteric obstruction (UUO) model 4,5 . We identified the full transcriptome of renal tissue, using the RNA-seq methodology, during early and late time intervals of kidney fibrosis. This methodology allows the identification of new protein-coding transcripts and novel non-coding RNA transcripts 6 . This is an exciting new direction, since about 75% of the mammalian genome (including human) is transcribed but not translated into proteins, and certain types of non-coding RNAs, especially long non coding RNAs (lncRNAs), play critical regulatory roles in many biological processes 7,8 . However, no data are currently available on the full transcriptome analysis of renal tissue from the UUO model in mice. By performing whole transcriptome sequencing and thorough bioinformatics analysis, we gathered novel information regarding up-regulated and down-regulated genes, pathways and biological processes, and we made lists of differentially expressed genes not suspected so far to be i...

Notch3 expression is found in the glomerular podocytes of patients with lupus nephritis or focal segmental GN but not in normal kidneys. Here, we show that activation of the Notch3 receptor in the glomeruli is a turning point inducing phenotypic changes in podocytes promoting renal inflammation and fibrosis and leading to disease progression. In a model of rapidly progressive GN, Notch3 expression was induced by several-fold in podocytes concurrently with disease progression. By contrast, mice lacking Notch3 expression were protected because they exhibited less proteinuria, uremia, and inflammatory infiltration. Podocyte outgrowth from glomeruli isolated from wild-type mice during the early phase of the disease was higher than outgrowth from glomeruli of mice lacking Notch3. In vitro studies confirmed that podocytes expressing active Notch3 reorganize their cytoskeleton toward a proliferative/migratory and inflammatory phenotype. We then administered antisense oligodeoxynucleotides targeting Notch3 or scramble control oligodeoxynucleotides in wild-type mice concomitant to disease induction. Both groups developed chronic renal disease, but mice injected with Notch3 antisense had lower values of plasma urea and proteinuria and inflammatory infiltration. The improvement of renal function was accompanied by fewer deposits of fibrin within the glomeruli and by decreased peritubular inflammation. Finally, abnormal Notch3 staining was observed in biopsy samples of patients with crescentic GN. These results demonstrate that abnormal activation of Notch3 may be involved in the progression of renal disease by promoting migratory and proinflammatory pathways. Inhibiting Notch3 activation could be a novel, promising approach to treat GN.

Tissue damage following injury leads to inflammation and fibrosis. To understand the molecular mechanisms and the proteins involved in the fibrotic process, we used the well-established unilateral ureteric obstruction rat model and we analyzed the alterations at early and late time intervals using a classical proteomic approach. Data analysis demonstrates a correlation between calreticulin up-regulation and progression of fibrosis. Calreticulin is involved in Ca++ homeostasis but has not been previously implicated in animal models of fibrosis. Proteomic analysis consistently revealed up-regulation of calreticulin in both early and late time intervals. These findings were further confirmed by biochemical and morphological approaches. Next, animal models of lung fibrosis (bleomycin-induced) and heart fibrosis (desmin-null) were examined. In the lung model, calreticulin expression was up-regulated from early time intervals, whereas in the heart model no change in the expression of calreticulin was observed. In addition, TGF-beta, a well known major contributing factor in several fibrotic processes, was found to up-regulate calreticulin in cultured human proximal tubule epithelial cells. The above observations suggest that calreticulin might be involved in fibrotic processes; however the mechanism(s) underlying its possible involvement are yet unresolved.

Background: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannelmediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. Methods: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-β1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43 +/− mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. Results: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-β1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43 +/− mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. Conclusion: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD.

De novo expression in the kidney of periostin, a protein involved in odontogenesis and osteogenesis, has been suggested as a biomarker of renal disease. In this study, we investigated the mechanism(s) of induction and the role of periostin in renal disease. Using a combination of bioinformatics, reporter assay, and chromatin immunoprecipitation analyses, we found that NFkB and other proinflammatory transcription factors induce periostin expression in vitro and that binding of these factors on the periostin promoter is enriched in glomeruli during experimental GN. Mice lacking expression of periostin displayed preserved renal function and structure during GN. Furthermore, delayed administration of periostin antisense oligonucleotides in wild-type animals with GN reversed already established proteinuria, diminished tissue inflammation, and improved renal structure. Lack of periostin expression also blunted the de novo renal expression of integrin-b3 and phosphorylation of focal adhesion kinase and AKT, known mediators of integrin-b3 signaling that affect cell motility and survival, observed during GN in wild-type animals. In vitro, recombinant periostin increased the expression of integrin-b3 and the concomitant phosphorylation of focal adhesion kinase and AKT in podocytes. Notably, periostin and integrin-b3 were highly colocalized in biopsy specimens from patients with inflammatory GN. These results demonstrate that interplay between periostin and renal inflammation orchestrates inflammatory and fibrotic responses, driving podocyte damage through downstream activation of integrin-b3 signaling. Targeting periostin may be a novel therapeutic strategy for treating CKD.

GN refers to a variety of renal pathologies that often progress to ESRD, but the molecular mechanisms underlying this progression remain incompletely characterized. Here, we determined whether dysregulated expression of the gap junction protein connexin 43, which has been observed in the progression of renal disease, contributes to GN progression. Immunostaining revealed expression of connexin 43 in damaged glomeruli in patients with glomerular diseases as well as in mice after induction of experimental GN. Notably, 2 weeks after the induction of GN with nephrotoxic serum, mice with a heterozygous deletion of the connexin 43 gene () had proteinuria, BUN, and serum creatinine levels significantly lower than those of wild-type animals. Additionally, the connexin 43+/- mice showed less crescent formation, tubular dilation, monocyte infiltration, and interstitial renal fibrosis. Treatment of cultured podocytes with connexin 43-specific blocking peptides attenuated TGF--induced cytoskeletal and morphologic changes and apoptosis as did treatment with the purinergic blocker suramin. Finally, therapeutic treatment of GN mice with connexin 43-specific antisense oligodeoxynucleotide improved functional and structural renal parameters. These findings suggest that crosstalk between connexin 43 and purinergic signaling contributes to podocyte damage in GN. Given that this protein is highly induced in individuals with glomerular diseases, connexin 43 may be a novel target for therapeutic treatment of GN.

The vasculature of the kidney is a heterogeneous structure, whose functional integrity is essential for the regulation of renal function. Owing to the importance of the endothelium in vascular biology, chronic endothelial alterations are therefore susceptible to impair multiple aspects of renal physiology and, in turn, to contribute to renal fibrosis. Although systemic endothelial dysfunction is undoubtedly associated with chronic kidney disease, the role of the renal endothelium in the initiation and the progression of renal fibrosis remains largely elusive. In this article, we critically review recent evidence supporting direct and indirect contributions of renal endothelial alterations to fibrosis in the kidney. Specifically, the potential implications of renal endothelial dysfunction and endothelial paucity in parenchymal hypoxia, in the regulation of local inflammation, and in the generation of renal mesenchymal cells are reviewed. We thereafter discuss therapeutic perspectives targeting renal endothelial alterations during the initiation and the progression of renal fibrogenesis.