David A. Vesey

Effect of luminal growth factor preservation on intestinal growth

Intestinal atrophy contributes to the clinical difficulties of patients who cannot eat normally. Atrophy is prevented by luminal food proteins but not by the equivalent aminoacids. This observation is not explained by current theories of intestinal physiology. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) are secreted into the gut lumen. We speculated that these are digested by pancreatic enzymes in fasting juice, but preserved when food proteins block the active sites of these enzymes. Studies based on molecular size and bioactivity confirmed that fasting human jejunal juice destroys EGF and TGF alpha. EGF, but not TGF alpha, was preserved when the milk protein casein or an enzyme inhibitor were present; elemental diets were ineffective. Diversion of pancreatic juice to the mid point of the small intestine in rats significantly increased luminal EGF-like bioactivity and all variables of growth in the proximal enzyme-free segment. Our findings support a novel mechanism of control of intestinal growth, which has important clinical implications. The addition of enzyme-inhibiting proteins such as casein to elemental diets may preserve intestinal integrity and function.

Transport pathways for cadmium in the intestine and kidney proximal tubule: focus on the interaction with essential metals.

Cadmium (Cd) is a toxic metal with a propensity to accumulate in the proximal tubules cells (PTC) of the kidney where it can lead to tubular dysfunction and eventually renal failure. Although Cd(2+)-induced nephrotoxicity has been well described there is still uncertainty about how this metal gains entry into these cells to induce toxicity. As a non-essential metal, specific transport proteins for Cd are unlikely to exist. Rather transport proteins/channels used by essential metals (iron, zinc, calcium) are thought to be responsible. When these dietary essential metals are in short supply and deficiencies develop, Cd absorption and toxicity are enhanced. This is primarily due to increased expression of essential metal transport proteins such as divalent metal transporter 1 (DMT1) which can transport Cd in the intestine and enhance toxicity in the kidney. The zinc/bicarbonate sympoters ZIP8 and 14 are expressed at the apical membrane of enterocytes and PTC, and can transport Cd into cells. TRPV5 and 6 are major transporters for calcium in intestine and kidney and may be involved in Cd transport in these locations. Cd in the circulation is bound to proteins such as metallothioneins (MT) which are readily filtered. Two multiligand receptors, megalin and cubulin, reabsorb filtered proteins including albumin and MT by the process of receptor-mediated endocytosis. This review summarises the transport pathways for Cd in the intestine and kidney proximal tubule focusing in particular at how Cd uses essential metal transport processes to gain entry to the circulation and the kidney.

An antagonist of human protease activated receptor-2 attenuates PAR2 signaling, macrophage activation, mast cell degranulation, and collagen-induced arthritis in rats

Multiple serine proteases exert proinflammatory actions by signaling through protease‐activated receptor‐2 (PAR2) on the cell surface. Although inhibitors of individual proteases are anti‐inflammatory, we sought to discover whether the first potent antagonist of their common target PAR2 might be beneficial in treating chronic arthritis‐like inflammatory disease. Using a fluorescence assay, a novel compound, GB88, was shown to antagonize PAR2‐induced intracellular Ca2+ release in human monocyte‐derived macrophages, being 1000 times more potent than a control compound, ENMD‐1068 (IC50 1.6±0.5 μM vs. 1.2±0.4 mM, respectively). In Wistar rats, GB88 was orally bioavailable (F=55%, Tmax 4 h, Cmax 1.7 μM, 10 mg/kg). GB88 inhibited the acute paw edema induced in Wistar rats by intraplantar λ‐carrageenan or PAR2 agonists 2‐furoyl‐LIGRLO‐NH2 or mast cell β‐tryptase, without inhibiting proteolytic activity of tryptase in vitro. In the chronic collagen‐induced model of arthritis in rats, GB88 (10 mg/kg) was disease modifying and ameliorated pathological and histopathological changes (edema, pannus formation, synovial hyperplasia, collagen degradation, macrophage invasion, mast cell degranulation) compared to untreated arthritic controls. The results suggest that an orally active PAR2 antagonist is effective in treating chronic arthritis in rats through inhibiting macrophage infiltration, mast cell degranulation, and β‐tryptase‐PAR2 signaling in joint inflammation.—Lohman, R.‐J., Cotterell, A. J., Barry, G. D., Liu, L., Suen, J. Y., Vesey, D. A., Fairlie, D. P. An antagonist of human protease activated receptor‐2 attenuates PAR2 signaling, macrophage activation, mast cell degranulation, and collagen‐induced arthritis in rats. FASEB J. 26, 2877–2887 (2012). www.fasebj.org

Antagonism of protease-activated receptor 2 protects against experimental colitis.

Many trypsin-like serine proteases such as β-tryptase are involved in the pathogenesis of colitis and inflammatory bowel diseases. Inhibitors of individual proteases show limited efficacy in treating such conditions, but also probably disrupt digestive and defensive functions of proteases. Here, we investigate whether masking their common target, protease-activated receptor 2 (PAR2), is an effective therapeutic strategy for treating acute and chronic experimental colitis in rats. A novel PAR2 antagonist (5-isoxazoyl-Cha-Ile-spiro[indene-1,4′-piperidine]; GB88) was evaluated for the blockade of intracellular calcium release in colonocytes and anti-inflammatory activity in acute (PAR2 agonist-induced) versus chronic [2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced] models of colitis in Wistar rats. Disease progression (disease activity index, weight loss, and mortality) and postmortem colonic histopathology (inflammation, bowel wall thickness, and myeloperoxidase) were measured. PAR2 and tryptase colocalization were investigated by using immunohistochemistry. GB88 was a more potent antagonist of PAR2 activation in colonocytes than another reported compound, N1-3-methylbutyryl-N4-6-aminohexanoyl-piperazine (ENMD-1068) (IC50 8 μM versus 5 mM). Acute colonic inflammation induced in rats by the PAR2 agonist SLIGRL-NH2 was inhibited by oral administration of GB88 (10 mg/kg) with markedly reduced edema, mucin depletion, PAR2 receptor internalization, and mastocytosis. Chronic TNBS-induced colitis in rats was ameliorated by GB88 (10 mg/kg/day p.o.), which reduced mortality and pathology (including colon obstruction, ulceration, wall thickness, and myeloperoxidase release) more effectively than the clinically used drug sulfasalazine (100 mg/kg/day p.o.). These disease-modifying properties for the PAR2 antagonist in both acute and chronic experimental colitis strongly support a pathogenic role for PAR2 and PAR2-activating proteases and therapeutic potential for PAR2 antagonism in inflammatory diseases of the colon.

Protein-bound uremic toxins, inflammation and oxidative stress: A cross-sectional study in stage 3-4 chronic kidney disease

BACKGROUND AND AIMS Indoxyl sulfate (IS) and p-cresyl sulfate (PCS) are nephro- and cardiovascular toxins, produced solely by the gut microbiota, which have pro-inflammatory and pro-oxidative properties in vitro. We undertook this study to investigate the associations between IS and PCS and both inflammation and oxidative stress in the chronic kidney disease (CKD) population. METHODS In this cross-sectional observational cohort study, participants with stage 3-4 CKD who enrolled in a randomized controlled trial of cardiovascular risk modification underwent baseline measurements of serum total and free IS and PCS (measured by ultraperformance liquid chromotography), inflammatory markers (interferon gamma [IFN-γ], interleukin-6 [IL-6] and tumor necrosis factor-alpha [TNF-α]), antioxidant and oxidative stress markers (plasma glutathione peroxidase [GPx] activity, total antioxidant capacity [TAC] and F2-isoprostanes) and pulse wave velocity (PWV), a marker of arterial stiffness. RESULTS There were 149 CKD patients (59% male; age 60 ± 10 years; 44% diabetic) with a mean eGFR of 40 ± 9 mL/min/1.73 m(2) (range 25-59). Serum free and total IS were independently associated with serum IL-6, TNF-α and IFN-γ, whereas serum free and total PCS were independently associated with serum IL-6 and PWV. Free IS and PCS were additionally independently associated with serum GPx but not with TAC or F2-isoprostanes. CONCLUSIONS IS and PCS were associated with elevated levels of selected inflammatory markers and an antioxidant in CKD patients. PCS was also associated with increased arterial stiffness. Inflammation and oxidative stress may contribute to the nephro- and cardiovascular toxicities of IS and PCS. Intervention studies targeting production of IS and PCS by dietary manipulation and the subsequent effect on cardiovascular-related outcomes are warranted in the CKD population.

Novel renoprotective actions of erythropoietin: new uses for an old hormone.

SUMMARY:  Erythropoietin (EPO) has been used widely for the treatment of anaemia associated with chronic kidney disease and cancer chemotherapy for nearly 20 years. More recently, EPO has been found to interact with its receptor (EPO‐R) expressed in a large variety of non‐haematopoietic tissues to induce a range of cytoprotective cellular responses, including mitogenesis, angiogenesis, inhibition of apoptosis and promotion of vascular repair through mobilization of endothelial progenitor cells from the bone marrow. Administration of EPO or its analogue, darbepoetin, promotes impressive renoprotection in experimental ischaemic and toxic acute renal failure, as evidenced by suppressed tubular epithelial apoptosis, enhanced tubular epithelial proliferation and hastened functional recovery. This effect is still apparent when administration is delayed up to 6 h after the onset of injury and can be dissociated from its haematological effects. Based on these highly encouraging results, at least one large randomized controlled trial of EPO therapy in ischaemic acute renal failure is currently underway. Preliminary experimental and clinical evidence also indicates that EPO may be renoprotective in chronic kidney disease. The purpose of the present article is to review the renoprotective benefits of different protocols of EPO therapy in the settings of acute and chronic kidney failure and the potential mechanisms underpinning these renoprotective actions. Gaining further insight into the pleiotropic actions of EPO will hopefully eventuate in much‐needed, novel therapeutic strategies for patients with kidney disease.

Health Risk Assessment of Dietary Cadmium Intake: Do Current Guidelines Indicate How Much is Safe?

Background: Cadmium (Cd), a food-chain contaminant, is a significant health hazard. The kidney is one of the primary sites of injury after chronic Cd exposure. Kidney-based risk assessment establishes the urinary Cd threshold at 5.24 μg/g creatinine, and tolerable dietary intake of Cd at 62 μg/day per 70-kg person. However, cohort studies show that dietary Cd intake below a threshold limit and that tolerable levels may increase the risk of death from cancer, cardiovascular disease, and Alzheimer’s disease. Objective: We evaluated if the current tolerable dietary Cd intake guideline and urinary Cd threshold limit provide sufficient health protection. Discussion: Staple foods constitute 40–60% of total dietary Cd intake by average consumers. Diets high in shellfish, crustaceans, mollusks, spinach, and offal add to dietary Cd sources. Modeling studies predict the current tolerable dietary intake corresponding to urinary Cd of 0.70–1.85 μg/g creatinine in men and 0.95–3.07 μg/g creatinine in women. Urinary Cd levels of < 1 μg/g creatinine were associated with progressive kidney dysfunction and peripheral vascular disease. A urinary Cd of 0.37 μg/g creatinine was associated with breast cancer, whereas dietary Cd of 16–31.5 μg/day was associated with 25–94% increase in risk of estrogen receptor–positive breast cancer. Conclusion: Modeling shows that dietary intake levels for Cd exceed the levels associated with kidney damage and many other adverse outcomes. Thus, the threshold level of urinary Cd should be re-evaluated. A more restrictive dietary intake guideline would afford enhanced health protection from this pervasive toxic metal. Citation: Satarug S, Vesey DA, Gobe GC. 2017. Health risk assessment of dietary cadmium intake: do current guidelines indicate how much is safe? Environ Health Perspect 125:284–288; http://dx.doi.org/10.1289/EHP108

Pathway-selective antagonism of proteinase activated receptor 2

Proteinase activated receptor 2 (PAR2) is a GPCR associated with inflammation, metabolism and disease. Clues to understanding how to block PAR2 signalling associated with disease without inhibiting PAR2 activation in normal physiology could be provided by studies of biased signalling.

Potential physiological and pathophysiological roles for protease-activated receptor-2 in the kidney

SUMMARY:  The protease‐activated receptor‐2 (PAR‐2), the second of four members of a unique subfamily of G‐protein coupled receptors, is abundantly expressed in the kidney. In a similar manner to other PAR cleavage of its extracellular N‐terminus exposes a tethered ligand, SLIGKV in humans, which acts as an intramolecular ligand to activate itself. In the kidney, PAR‐2 expression has been variably reported in collecting duct cells, mesangial cells, interstitial fibroblasts, vascular endothelial cells, vascular smooth muscle cells and proximal tubular cells. Despite this renal expression data, the function of PAR‐2 in the kidney remains unknown. More than 15 different mammalian serine proteases have been shown to activate PAR‐2 in an in vitro setting, but it is still unclear which of these are physiologically relevant activators of PAR‐2 in specific tissues. Their identification could provide novel therapeutic targets. PAR‐2 activates a number of down‐stream signalling molecules that include protein kinase C, extracellular signal regulated kinase and nuclear factor kappa‐B. Proteases that can activate PAR‐2 are generated and released from cells during injury, inflammation and malignancy and can thus signal to cells under these conditions. Potential physiological and pathophysiological roles for PAR‐2 in the kidney include the regulation of inflammation, blood flow, and ion transport and tissue protection, repair and fibrosis. In this review the potential roles of PAR‐2 in the kidney are highlighted and discussed.

Erythropoietin reduces cisplatin-induced apoptosis in renal carcinoma cells via a PKC dependent pathway

Anaemia which develops as a consequence of malignancies is often treated using recombinant human erythropoietin (rhEpo). Epo is now known as an anti-apoptotic factor for a wide range of cell types that express Epo receptors (EpoRs) and its co-use with cancer therapies can act detrimentally to diminish therapy-induced apoptosis. This had not been analyzed for renal cell carcinomas (RCCs). We examined the influence of rhEPO on the ability of cisplatin to induce apoptosis in RCCs. Two RCC cell lines (SN12K1 and ACHN) were compared with a non-RCC renal epithelial cell line (HK2). Cells were treated with 50μM cisplatin with and without 200IU/mL rhEpo and were compared for apoptosis, mitosis and protein expression of EpoR, nuclear factor-κB (NFκB), protein kinase C (PKC), Bcl-2, Bax and cyclin-D1. Experiments were repeated with PKC promotion (PMA, 20nM) or inhibition (H7, 10μM). rhEpo reduced cisplatin-induced apoptosis in RCCs (p