In Vivo Readout of CFTR Function: Ratiometric Measurement of CFTR-Dependent Secretion by Individual, Identifiable Human Sweat Glands

Abstract

To assess CFTR function in vivo, we developed a bioassay that monitors and compares CFTR-dependent and CFTR-independent sweat secretion in parallel for multiple (∼50) individual, identified glands in each subject. Sweating was stimulated by intradermally injected agonists and quantified by optically measuring spherical sweat bubbles in an oil-layer that contained dispersed, water soluble dye particles that partitioned into the sweat bubbles, making them highly visible. CFTR-independent secretion (M-sweat) was stimulated with methacholine, which binds to muscarinic receptors and elevates cytosolic calcium. CFTR-dependent secretion (C-sweat) was stimulated with a β-adrenergic cocktail that elevates cytosolic cAMP while blocking muscarinic receptors. A C-sweat/M-sweat ratio was determined on a gland-by-gland basis to compensate for differences unrelated to CFTR function, such as gland size. The average ratio provides an approximately linear readout of CFTR function: the heterozygote ratio is ∼0.5 the control ratio and for CF subjects the ratio is zero. During assay development, we measured C/M ratios in 6 healthy controls, 4 CF heterozygotes, 18 CF subjects and 4 subjects with ‘CFTR-related’ conditions. The assay discriminated all groups clearly. It also revealed consistent differences in the C/M ratio among subjects within groups. We hypothesize that these differences reflect, at least in part, levels of CFTR expression, which are known to vary widely. When C-sweat rates become very low the C/M ratio also tended to decrease; we hypothesize that this nonlinearity reflects ductal fluid absorption. We also discovered that M-sweating potentiates the subsequent C-sweat response. We then used potentiation as a surrogate for drugs that can increase CFTR-dependent secretion. This bioassay provides an additional method for assessing CFTR function in vivo, and is well suited for within-subject tests of systemic, CFTR-directed therapeutics.

A Little CFTR Goes a Long Way: CFTR-Dependent Sweat Secretion from G551D and R117H-5T Cystic Fibrosis Subjects Taking Ivacaftor

“… Fig. 2C also shows that the C/M ratio was not constant across the glands, but increased significantly as a function of the M-sweat rate (r = 0.31, p<0.02), see also [10]. The positive relationship between C/M and M, which was significant for all but one subject, was used to arrive at better estimates of the% of CFTR function restored by ivacaftor (see discussion).…”

“…Paired t-tests on log transformed data, Pearson r, ANOVA, and mixed models regression with lmer() from the lme4 package in R (see methods, ref [10]) were used as appropriate.…”

Low Beta-Adrenergic Sweat Responses in Cystic Fibrosis and Cystic Fibrosis Transmembrane Conductance Regulator-Related Metabolic Syndrome Children

“…5,6 The β response was seen in 16–21% of sweat glands of medicated patients and in no glands in patients not in therapy, indicating that the single-gland method is a more sensitive measure of sweat response and therefore more cabable of detecting residual CFTR function. 6 Unfortunately, monitoring the sweat secretion from individual glands requires complete subject cooperation to accomodate relatively long periods of inactivity and is therefore probably not feasible in young children or infants.…”

The magnitude of ivacaftor effects on fluid secretion via R117H-CFTR channels: Human in vivo measurements

“…Heavy mineral oil was from EMD Chemicals, Gibbstown, NJ, and was water-saturated before use as previously described [17]. Erioglaucine disodium salt (CAS No.…”

“…The goal of the present study was to quantify effects of ivacaftor on R117H function in vivo using a new sweat rate bioassay for CFTR function in individually identified sweat glands [14, 17]. The standard sweat chloride assay induces sweating via pilocarpine iontophoresis and then measures the concentration of chloride in the collected sweat [18].…”