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

Serial optical coherence microscopy for label-free volumetric histopathology

The observation of histopathology using optical microscope is an essential procedure for examination of tissue biopsies or surgically excised specimens in biological and clinical laboratories. However, slide-based microscopic pathology is not suitable for visualizing the large-scale tissue and native 3D organ structure due to its sampling limitation and shallow imaging depth. Here, we demonstrate serial optical coherence microscopy (SOCM) technique that offers label-free, high-throughput, and large-volume imaging of ex vivo mouse organs. A 3D histopathology of whole mouse brain and kidney including blood vessel structure is reconstructed by deep tissue optical imaging in serial sectioning techniques. Our results demonstrate that SOCM has unique advantages as it can visualize both native 3D structures and quantitative regional volume without introduction of any contrast agents

'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

Metabolic stress promotes renal tubular inflammation by triggering the unfolded protein response

The renal epithelium contributes to the development of inflammation during ischemic injury. Ischemia induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Ischemic tissues generate distress signals and inflammation that activates fibrogenesis and may promote adaptive immunity. Interestingly, the UPR may activate inflammation pathways. Our aim was to test whether the UPR is activated during metabolic stress and mediates a tubular inflammatory response. Glucose deprivation, not hypoxia and amino acids deprivation, activated the UPR in human renal cortical tubular cells in culture. This stress activated NF-κB and promoted the transcription of proinflammatory cytokines and chemokines, including IL-6, IL-8, TNF-α, RANTES and MCP-1. The protein kinase RNA (PKR)-like ER kinase signaling pathway was not required for the induction of inflammation but amplified cytokine. Inositol-requiring enzyme 1 activated NF-κB signaling and was required for the transcription of proinflammatory cytokines and chemokines following metabolic stress. Moreover, acute ischemia activated ER stress and inflammation in rat kidneys. Finally, the ER stress marker GRP78 and NF-κB p65/RelA were coexpressed in human kidney transplants biopsies performed before implantation, suggesting that ER stress activates tubular inflammation in human renal allografts. In conclusion, this study establishes a link between ischemic stress, the activation of the UPR and the generation of a tubular inflammatory response

Inhibiting ERK Activation with CI-1040 Leads to Compensatory Upregulation of Alternate MAPKs and Plasminogen Activator Inhibitor-1 following Subtotal Nephrectomy with No Impact on Kidney Fibrosis

Extracellular-signal regulated kinase (ERK) activation by MEK plays a key role in many of the cellular processes that underlie progressive kidney fibrosis including cell proliferation, apoptosis and transforming growth factor β1-mediated epithelial to mesenchymal transition. We therefore assessed the therapeutic impact of ERK1/2 inhibition using a MEK inhibitor in the rat 5/6 subtotal nephrectomy (SNx) model of kidney fibrosis. There was a twentyfold upregulation in phospho-ERK1/2 expression in the kidney after SNx in Male Wistar rats. Rats undergoing SNx became hypertensive, proteinuric and developed progressive kidney failure with reduced creatinine clearance. Treatment with the MEK inhibitor, CI-1040 abolished phospho- ERK1/2 expression in kidney tissue and prevented phospho-ERK1/2 expression in peripheral lymphocytes during the entire course of therapy. CI-1040 had no impact on creatinine clearance, proteinuria, glomerular and tubular fibrosis, and α-smooth muscle actin expression. However, inhibition of ERK1/2 activation led to significant compensatory upregulation of the MAP kinases, p38 and JNK in kidney tissue. CI-1040 also increased the expression of plasminogen activator inhibitor-1 (PAI-1), a key inhibitor of plasmin-dependent matrix metalloproteinases. Thus inhibition of ERK1/2 activation has no therapeutic effect on kidney fibrosis in SNx possibly due to increased compensatory activation of the p38 and JNK signalling pathways with subsequent upregulation of PAI-1

Patient and caregiver perspectives on blood pressure in children with chronic kidney disease

Background: More than 50% of children with chronic kidney disease (CKD) have uncontrolled hypertension, increasing their long-term risk of cardiovascular disease and progression to kidney failure. Children receiving medications or dialysis may also experience acute blood pressure fluctuations accompanied by debilitating symptoms. We aimed to describe the perspectives of children with CKD and their parental caregivers on blood pressure to inform patient-centered care. / Methods: Secondary thematic analysis was conducted on qualitative data from the Standardized Outcomes in Nephrology—Children and Adolescents initiative, encompassing 16 focus groups, an international Delphi survey and two consensus workshops. We analyzed responses from children with CKD (ages 8–21 years) and caregivers (of children ages 0–21 years) pertaining to blood pressure. / Results: Overall, 120 patients and 250 caregivers from 22 countries participated. We identified five themes: invisibility and normalization (reassured by apparent normotension, absence of symptoms and expected links with CKD), confused by ambiguity (hypertension indistinguishable from cardiovascular disease, questioning the need for prophylactic intervention, frustrated by inconsistent messages and struggling with technical skills in measurement), enabling monitoring and maintaining health (gaging well-being and preventing vascular complications), debilitating and constraining daily living (provoking anxiety and agitation, helpless and powerless and limiting life activities) and burden of medications (overwhelmed by the quantity of tablets and distress from unexpected side effects). / Conclusions: For children with CKD and their caregivers, blood pressure was an important heath indicator, but uncertainty around its implications and treatment hampered management. Providing educational resources to track blood pressure and minimizing symptoms and treatment burden may improve outcomes in children with CKD

Peningkatan aktivitas enzim hemiselulase dengan rekayasa protein untuk pengolahan biomassa berbasis lignoselulosa

The purpose of this research is to investigate the substrate stereospecificity of IlL- arabinofuranosidase GH51 Geobacillus thermoleovorans IT-08 (AbfA) toward Mycobacteria cell wall. In general, there are 3 stages in this research are (1) the production of AbfA recombinant from E. coli BL21 (DE3)/pET-abfA, (2) specific activity assay of AbfA recombinant, (3) substrate stereospecificity analysis of AbfA in silico. In laboratory methods, AbfA has hydrolase activity toward Darabinofuranoside (H37Rv and BFCC). However its activity toward Larabinofuranoside (arabinogalactan, pectin, oat spelt xylan and arabinan) higher than its hydrolase activity toward D-arabinofuranoside (H37Rv and BFCC). On silico analysis, hydrolase activity AbfA toward D-arabinofuranoside may occur due to fingerprint interactions between the ligand and catalytic residue Glu294. Based on in silico analysis, the catalytic mechanism of AbfA toward D-arabinofuranoside was suggested following model catalytic mechanism of a-L-arabinofuranosidase GHSI toward L-arabinofuranoside substrate. Effect of sterie hindrance by Trp99 and Trp298 at the sub-site -1 rationalize the substrate specificity toward D-arabinofuranoside lower than L-arabinofuranoside. This thesis research was the first reported that AbfA has catalytic activity toward D-Arabinofuranoside derived from Mycobacteria cell wall