'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

Characterisation of feline renal cortical fibroblast cultures and their transcriptional response to transforming growth factor beta 1

Chronic kidney disease (CKD) is common in geriatric cats, and the most prevalent pathology is chronic tubulointerstitial inflammation and fibrosis. The cell type predominantly responsible for the production of extra-cellular matrix in renal fibrosis is the myofibroblast, and fibroblast to myofibroblast differentiation is probably a crucial event. The cytokine TGF-β1 is reportedly the most important regulator of myofibroblastic differentiation in other species. The aim of this study was to isolate and characterise renal fibroblasts from cadaverous kidney tissue of cats with and without CKD, and to investigate the transcriptional response to TGF-β1

Transglutaminase inhibition ameliorates experimental diabetic nephropathy

Diabetic nephropathy is characterized by excessive extracellular matrix accumulation resulting in renal scarring and end-stage renal disease. Previous studies have suggested that transglutaminase type 2, by formation of its protein crosslink product epsilon-(gamma-glutamyl)lysine, alters extracellular matrix homeostasis, causing basement membrane thickening and expansion of the mesangium and interstitium. To determine whether transglutaminase inhibition can slow the progression of chronic experimental diabetic nephropathy over an extended treatment period, the inhibitor NTU281 was given to uninephrectomized streptozotocin-induced diabetic rats for up to 8 months. Effective transglutaminase inhibition significantly reversed the increased serum creatinine and albuminuria in the diabetic rats. These improvements were accompanied by a fivefold decrease in glomerulosclerosis and a sixfold reduction in tubulointerstitial scarring. This was associated with reductions in collagen IV accumulation by 4 months, along with reductions in collagens I and III by 8 months. This inhibition also decreased the number of myofibroblasts, suggesting that tissue transglutaminase may play a role in myofibroblast transformation. Our study suggests that transglutaminase inhibition ameliorates the progression of experimental diabetic nephropathy and can be considered for clinical application

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-beta, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-beta 1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, beta 1-integrin, calponin and a-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-beta signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in trans-endothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-beta-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-beta-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation

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

CRIM1 is localized to the podocyte filtration slit diaphragm of the adult human kidney

Background. CRIM1 is a plasma membrane bound protein containing six cysteine-rich repeats (CRR). Through these, CRIM1 has been shown to interact with a subgroup of the TGF-β superfamily, the bone morphogenic proteins (BMP) isoforms 2, 4 and 7. The probable action is to modulate the signalling properties of these factors. CRIM1 has also been shown to regulate the release of VEGFA by podocytes during renal organogenesis. Knock-out studies in mice have shown that CRIM1 is critically involved in the development of the central nervous system, eye and kidney. Replacement of CRIM1 with a defective version leads to renal dysgenesis and perinatal death. We have analysed the distribution of CRIM1 in adult human renal tissue

Tissue Microenvironments Define and Get Reinforced by Macrophage Phenotypes in Homeostasis or during Inflammation, Repair and Fibrosis

Current macrophage phenotype classifications are based on distinct in vitro culture conditions that do not adequately mirror complex tissue environments. In vivo monocyte progenitors populate all tissues for immune surveillance which supports the maintenance of homeostasis as well as regaining homeostasis after injury. Here we propose to classify macrophage phenotypes according to prototypical tissue environments, e.g. as they occur during homeostasis as well as during the different phases of (dermal) wound healing. In tissue necrosis and/or infection, damage- and/or pathogen-associated molecular patterns induce proinflammatory macrophages by Toll-like receptors or inflammasomes. Such classically activated macrophages contribute to further tissue inflammation and damage. Apoptotic cells and antiinflammatory cytokines dominate in postinflammatory tissues which induce macrophages to produce more antiinflammatory mediators. Similarly, tumor-associated macrophages also confer immunosuppression in tumor stroma. Insufficient parenchymal healing despite abundant growth factors pushes macrophages to gain a profibrotic phenotype and promote fibrocyte recruitment which both enforce tissue scarring. Ischemic scars are largely devoid of cytokines and growth factors so that fibrolytic macrophages that predominantly secrete proteases digest the excess extracellular matrix. Together, macrophages stabilize their surrounding tissue microenvironments by adapting different phenotypes as feed-forward mechanisms to maintain tissue homeostasis or regain it following injury. Furthermore, macrophage heterogeneity in healthy or injured tissues mirrors spatial and temporal differences in microenvironments during the various stages of tissue injury and repair. Copyright (C) 2012 S. Karger AG, Base

Regulation of endothelial cell plasticity by TGF-β

Recent evidence has demonstrated that endothelial cells can have a remarkable plasticity. By a process called Endothelial-to-Mesenchymal Transition (EndMT) endothelial cells convert to a more mesenchymal cell type that can give rise to cells such as fibroblasts, but also bone cells. EndMT is essential during embryonic development and tissue regeneration. Interestingly, it also plays a role in pathological conditions like fibrosis of organs such as the heart and kidney. In addition, EndMT contributes to the generation of cancer associated fibroblasts that are known to influence the tumor-microenvironment favorable for the tumor cells. EndMT is a form of the more widely known and studied Epithelial-to-Mesenchymal Transition (EMT). Like EMT, EndMT can be induced by transforming growth factor (TGF)-β. Indeed many studies have pointed to the important role of TGF-β receptor/Smad signaling and downstream targets, such as Snail transcriptional repressor in EndMT. By selective targeting of TGF-β receptor signaling pathological EndMT may be inhibited for the therapeutic benefit of patients with cancer and fibrosis

Proteinase-activated receptor 4 stimulation-induced epithelial-mesenchymal transition in alveolar epithelial cells

BACKGROUND: Proteinase-activated receptors (PARs; PAR(1–4)) that can be activated by serine proteinases such as thrombin and neutrophil catepsin G are known to contribute to the pathogenesis of various pulmonary diseases including fibrosis. Among these PARs, especially PAR(4), a newly identified subtype, is highly expressed in the lung. Here, we examined whether PAR(4 )stimulation plays a role in the formation of fibrotic response in the lung, through alveolar epithelial-mesenchymal transition (EMT) which contributes to the increase in myofibroblast population. METHODS: EMT was assessed by measuring the changes in each specific cell markers, E-cadherin for epithelial cell, α-smooth muscle actin (α-SMA) for myofibroblast, using primary cultured mouse alveolar epithelial cells and human lung carcinoma-derived alveolar epithelial cell line (A549 cells). RESULTS: Stimulation of PAR with thrombin (1 U/ml) or a synthetic PAR(4 )agonist peptide (AYPGKF-NH(2), 100 μM) for 72 h induced morphological changes from cobblestone-like structure to elongated shape in primary cultured alveolar epithelial cells and A549 cells. In immunocytochemical analyses of these cells, such PAR(4 )stimulation decreased E-cadherin-like immunoreactivity and increased α-SMA-like immunoreactivity, as observed with a typical EMT-inducer, tumor growth factor-β (TGF-β). Western blot analyses of PAR(4)-stimulated A549 cells also showed similar changes in expression of these EMT-related marker proteins. Such PAR(4)-mediated changes were attenuated by inhibitors of epidermal growth factor receptor (EGFR) kinase and Src. PAR(4)-mediated morphological changes in primary cultured alveolar epithelial cells were reduced in the presence of these inhibitors. PAR(4 )stimulation increased tyrosine phosphorylated EGFR or tyrosine phosphorylated Src level in A549 cells, and the former response being inhibited by Src inhibitor. CONCLUSION: PAR(4 )stimulation of alveolar epithelial cells induced epithelial-mesenchymal transition (EMT) as monitored by cell shapes, and epithelial or myofibroblast marker at least partly through EGFR transactivation via receptor-linked Src activation