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.

A cost-effective three-dimensional culture platform functionally mimics the adipose tissue microenvironment surrounding the kidney.

There is an increasing interest in studying the crosstalk between tumor-associated adipose tissue and tumor progression. In proximity to the primary site of kidney tumors, perinephric adipose tissue has direct contact with cancer cells when kidney cancer becomes invasive. To mimic the perinephric adipose tissue microenvironment, we applied the liquid overlay-based technique, which cost-effectively generated functional adipocyte spheroids using mesenchymal stem cells isolated from human perinephric adipose tissue. Thereafter, we co-cultured adipocyte spheroids with unpolarized macrophages and discovered an M2 phenotype skew in macrophages. Moreover, we discovered that, in the presence of adipocyte spheroids, M2 macrophages exhibited stronger invasive capacity than M1 macrophages. We further showed that the perinephric adipose tissue sampled from metastatic kidney cancer exhibited high expression of M2 macrophages. In conclusion, the liquid overlay-based technique can generate a novel three-dimensional platform enabling investigation of the interactions of adipocytes and other types of cells in a tumor microenvironment.

The inverse association of glomerular function and urinary ß2-MG excretion and its implications for cadmium health risk assessment.

Urinary ß2-microgroblin (ß2-MG) excretion levels above 300 µg/g creatinine are used to indicate defective tubular reabsorption. Arguably, increased urinary ß2-MG excretion could also reflect glomerular filtration rate decline. Thus, we investigated an association between urinary ß2-MG and estimated glomerular filtration rate (eGFR). We studied 527 subjects, aged 30-87 years (mean 51.2), who lived in a rural area of Thailand polluted with cadmium (Cd). Of this cohort, 10.3% had urinary Cd levels <2 µg/g creatinine and 53.5% had urinary Cd levels ≥5 µg/g creatinine. Half (53.1%) of the participants had urinary ß2-MG levels ≥ 300 µg/g creatinine, and 11.6% had low GFR, defined as eGFR <60 mL/min/1.73 m2. Lower eGFR values were associated with older age (ß = -0.568, P < 0.001), higher urinary ß2-MG (ß = -0.170, P < 0.001), higher urinary Cd (ß = -0.103, P = 0.005) and diabetes (ß = 0.074, P = 0.032). An inverse association between eGFR and urinary ß2-MG was evident in subjects with low GFR (ß = -0.332, P = 0.033), but not in those with GFR >90 mL/min/1.73 m2 (ß = -0.008, P = 0.896). These findings suggested Cd-induced nephron loss and reduced tubular reabsorption in low eGFR subjects. Urinary ß2-MG levels <300 µg/g creatinine were associated with 4.66 (95% CI: 1.92, 11.32) fold increase in the POR for low GFR, compared with urinary ß2-MG levels <100 µg/g creatinine. Findings in the present study cast doubt on a cut-off value for urinary ß2-MG, while lending support to the notion that elevated urinary ß2-MG excretion could indicate a fall of GFR.

Optimising assessment of kidney function when managing localised renal masses.

Increased early and incidental detection, improved surgical techniques and technological advancement mean that the management of renal mass lesions is constantly evolving. The treatment of choice for renal mass lesions has historically been radical nephrectomy. Partial nephrectomy is now recommended for localised renal masses, owing to favourable renal functional outcomes. Ablative renal surgery confers a significant risk of chronic kidney disease. There are few studies assessing long term outcomes of nephrectomy on renal outcomes, and virtually no studies assessing long term outcomes for less invasive therapies such as ablation. Unless a renal mass is clearly benign on imaging, management decisions will be made with an assumption of malignancy. The content of this review applies to both benign and malignant renal mass lesions. We advocate for improved strategies for kidney function assessment and risk stratification, early targeted referral, and regular screening for chronic kidney disease for all patients after surgery.

Current health risk assessment practice for dietary cadmium: Data from different countries.

Cadmium (Cd) is an environmental toxicant with high rates of soil-to-plant transference. This makes exposure to Cd through the food-chain contamination a public health concern. Cd accumulates in kidneys, and the most frequently reported adverse effect of long-term Cd intake is injury to kidneys. The FAO/WHO Joint Expert Committee on Food Additives established a tolerable dietary intake level and a threshold to safeguard population health. The FAO/WHO tolerable intake was set at 25 µg per kg body weight per month (58 µg per day for a 70-kg person) with urinary Cd threshold at 5.24 µg/g creatinine. Worldwide population data indicate that urinary Cd excretion reflects cumulative Cd exposure or body burden more accurately than estimated Cd intake, derived from total diet study (TDS). For the adult population, TDS estimated Cd intake of 8-25 µg/day, while urinary Cd levels suggest higher intake levels (>30 µg/day). These Cd intake estimates are below the FAO/WHO intake guideline, but they exceed the levels that are associated with distinct pathologies in many organ systems. A wide diversity of Cd toxicity targets and Cd toxicity levels argue for a more restrictive dietary Cd intake guideline and the measures that minimize Cd levels in foodstuffs.

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.

Angiotensin II facilitates autoregulation in the perfused mouse kidney: An optimized in vitro model for assessment of renal vascular and tubular function.

BACKGROUND AND AIMS: We have optimized the isolated perfused mouse kidney (IPMK) model for studying renal vascular and tubular function in vitro using 24-28 g C57BL6J mice; the wild type controls for many transgenic mice. METHODS AND RESULTS: Buffer composition was optimized for bovine serum albumin concentration (BSA). The effect of adding erythrocytes on renal function and morphology was assessed. Autoregulation was investigated during stepped increases in perfusion pressure. Perfusion for 60 min at 90-110 mmHg with Krebs bicarbonate buffer containing 5.5% BSA, and amino acids produced functional parameters within the in vivo range. Erythrocytes increased renal vascular resistance (3.8 +/- 0.2 vs 2.4 +/- 0.1 mL/min.mmHg, P < 0.05), enhanced sodium reabsorption (FE(Na) = 0.3 +/- 0.08 vs 1.5 +/- 0.7%, P < 0.05), produced equivalent glomerular filtration rates (GFR; 364 +/- 38 vs 400 +/- 9 microL/min per gkw) and reduced distal tubular cell injury in the inner stripe (5.8 +/- 1.7 vs 23.7 +/- 3.1%, P < 0.001) compared to cell free perfusion. The IPMK was responsive to vasoconstrictor (angiotensin II, EC50 100 pM) and vasodilator (methacholine, EC50 75 nM) mediators and showed partial autoregulation of perfusate flow under control conditions over 65-85 mmHg; autoregulatory index (ARI) of 0.66 +/- 0.11. Angiotensin II (100 pM) extended this range (to 65-120 mmHg) and enhanced efficiency (ARI 0.21 +/- 0.02, P < 0.05). Angiotensin II facilitation was antagonized by methacholine (ARI 0.76 +/- 0.08) and papaverine (ARI 0.91 +/- 0.13). CONCLUSION: The IPMK model is useful for studying renal physiology and pathophysiology without systemic neurohormonal influences.