Identifying disease progression in chronic kidney disease using proton magnetic resonance spectroscopy.

Chronic kidney disease (CKD) affects approximately 10% of the world population, higher still in some developing countries, and can cause irreversible kidney damage eventually leading to kidney failure requiring dialysis or kidney transplantation. However, not all patients with CKD will progress to this stage, and it is difficult to distinguish between progressors and non-progressors at the time of diagnosis. Current clinical practice involves monitoring estimated glomerular filtration rate and proteinuria to assess CKD trajectory over time; however, there remains a need for novel, validated methods that differentiate CKD progressors and non-progressors. Nuclear magnetic resonance techniques, including magnetic resonance spectroscopy and magnetic resonance imaging, have the potential to improve our understanding of CKD progression. Herein, we review the application of magnetic resonance spectroscopy both in preclinical and clinical settings to improve the diagnosis and surveillance of patients with CKD.

PAR2-Induced Tissue Factor Synthesis by Primary Cultures of Human Kidney Tubular Epithelial Cells Is Modified by Glucose Availability.

Coagulopathies common to patients with diabetes and chronic kidney disease (CKD) are not fully understood. Fibrin deposits in the kidney suggest the local presence of clotting factors including tissue factor (TF). In this study, we investigated the effect of glucose availability on the synthesis of TF by cultured human kidney tubular epithelial cells (HTECs) in response to activation of protease-activated receptor 2 (PAR2). PAR2 activation by peptide 2f-LIGRLO-NH2 (2F, 2 µM) enhanced the synthesis and secretion of active TF (~45 kDa) which was blocked by a PAR2 antagonist (I-191). Treatment with 2F also significantly increased the consumption of glucose from the cell medium and lactate secretion. Culturing HTECs in 25 mM glucose enhanced TF synthesis and secretion over 5 mM glucose, while addition of 5 mM 2-deoxyglucose (2DOG) significantly decreased TF synthesis and reduced its molecular weight (~40 kDa). Blocking glycosylation with tunicamycin also reduced 2F-induced TF synthesis while reducing its molecular weight (~36 kDa). In conclusion, PAR2-induced TF synthesis in HTECs is enhanced by culture in high concentrations of glucose and suppressed by inhibiting either PAR2 activation (I-191), glycolysis (2DOG) or glycosylation (tunicamycin). These results may help explain how elevated concentrations of glucose promote clotting abnormities in diabetic kidney disease. The application of PAR2 antagonists to treat CKD should be investigated further.

PAR2 Activation on Human Kidney Tubular Epithelial Cells Induces Tissue Factor Synthesis, That Enhances Blood Clotting.

Coagulation abnormalities and increased risk of atherothrombosis are common in patients with chronic kidney diseases (CKD). Mechanisms that alter renal hemostasis and lead to thrombotic events are not fully understood. Here we show that activation of protease activated receptor-2 (PAR2) on human kidney tubular epithelial cells (HTECs), induces tissue factor (TF) synthesis and secretion that enhances blood clotting. PAR-activating coagulation-associated protease (thrombin), as well as specific PAR2 activators (matriptase, trypsin, or synthetic agonist 2f-LIGRLO-NH2 (2F), induced TF synthesis and secretion that were potently inhibited by PAR2 antagonist, I-191. Thrombin-induced TF was also inhibited by a PAR1 antagonist, Vorapaxar. Peptide activators of PAR1, PAR3, and PAR4 failed to induce TF synthesis. Differential centrifugation of the 2F-conditoned medium sedimented the secreted TF, together with the exosome marker ALG-2 interacting protein X (ALIX), indicating that secreted TF was associated with extracellular vesicles. 2F-treated HTEC conditioned medium significantly enhanced blood clotting, which was prevented by pre-incubating this medium with an antibody for TF. In summary, activation of PAR2 on HTEC stimulates synthesis and secretion of TF that induces blood clotting, and this is attenuated by PAR2 antagonism. Thrombin-induced TF synthesis is at least partly mediated by PAR1 transactivation of PAR2. These findings reveal how underlying hemostatic imbalances might increase thrombosis risk and subsequent chronic fibrin deposition in the kidneys of patients with CKD and suggest PAR2 antagonism as a potential therapeutic strategy for intervening in CKD progression.

Progress curve analysis of microtitre plate plasma clotting assays. Assessment of tissue factor levels.

MTP plasma clotting assays monitor the time course of fibrin formation in re-calcified plasma by absorbance measurements and are increasingly used as alternatives to traditional one-point clot time assays employed in clinical laboratories to detect thrombotic disorders. The parameters derived from these analyses are analogous to thromboelastography viz. time, rate and maximum extent of clot formation. The derived parameters, based on the whole course of the clotting reaction are more robust, informative and quantitative than single-point clot time assays. However, the parameters themselves are usually obtained arbitrarily by crude graphical analysis of subjectively selected points of progress curves. The current work aimed to investigate the sensitivity and reproducibility of an MTP clotting assay and examine its suitability for measuring tissue factor (TF) levels in cell culture medium and patient urine. The results demonstrate that progress curves can be analysed by fitting a logistic equation, derived from a simplified autocatalytic clot formation model. The parameters, maximum amplitude (Fm), rate constant (k), time to half-maximum amplitude (tm) and maximum rate of clot formation (vm), fit a power curve showing limiting effects with increasing TF concentration. Log/log plots of tm and k against TF concentration provide standard curves for assessment of unknowns.