Atrial natriuretic peptide modulation of albumin clearance and contrast agent permeability in mouse skeletal muscle and skin: role in regulation of plasma volume

Abstract

Atrial natriuretic peptide (ANP) via its guanylyl cyclase-A (GC-A) receptor participates in regulation of arterial blood pressure and vascular volume. Previous studies demonstrated that concerted renal diuretic/natriuretic and endothelial permeability effects of ANP cooperate in intravascular volume regulation. We show that the microvascular endothelial contribution to the hypovolaemic action of ANP can be measured by the magnitude of the ANP-induced increase in blood-to-tissue albumin transport, measured as plasma albumin clearance corrected for intravascular volume change, relative to the corresponding increase in ANP-induced renal water excretion. We used a two-tracer method with isotopically labelled albumin to measure clearances in skin and skeletal muscle of: (i) C57BL6 mice; (ii) mice with endothelium-restricted deletion of GC-A (floxed GC-A x tie2-Cre: endothelial cell (EC) GC-A knockout (KO)); and (iii) control littermates (floxed GC-A mice with normal GC-A expression levels). Comparison of albumin clearances in hypervolaemic EC GC-A KO mice with normovolaemic littermates demonstrated that skeletal muscle albumin clearance with ANP treatment accounts for at most 30% of whole body clearance required for ANP to regulate plasma volume. Skin microcirculation responded to ANP similarly. Measurements of permeability to a high molecular mass contrast agent (35 kD Gadomer) by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) enabled repeated measures in individual animals and confirmed small increases in muscle and skin microvascular permeability after ANP. These quantitative methods will enable further evaluation of the contribution of ANP-dependent microvascular beds (such as gastro-intestinal tract) to plasma volume regulation.

Figure 1. Albumin clearance and plasma volume measured in C57BL6 mice with and without ANP

A, the clearance of albumin in skin, muscle and trachea in wild type C57BL6 mice with no ANP (control; open bars) and C57BL6 mice with continuous infusion of ANP (0.5 ng min⁻¹ (g body weight)⁻¹; heavy hatched bars). The 30 min clearances were measured as the difference between the plasma equivalent volume containing the amount of albumin labelled with ¹²⁵I in the tissue (vascular plus extravascular space) after 35 min and the plasma equivalent volume of albumin labelled with ¹³¹I measured after 5 min (mainly vascular space, and also used as a measure of tissue plasma volume. B, the figure shows that the amount of albumin in the extravascular space after 30 min may be less than or equal to the amount of tracer in vascular space. There was a tendency for ANP to increase albumin clearances in all tissues but only back and tail skin and hamstring muscle showed significant increases (*P < 0.05) relative to vehicle control. There was no significant effect of ANP on measured plasma volumes (light hatched bars in B). All values are expressed per gram of tissue dry weight.

Figure 2. Clearances (30 min) in wild type C57BL6 mice with (hatched bars) and without ANP (open bars) were normalized to the measured plasma volume in each tissue (data fromFig. 1)

The normalization reduces some of the variability in the data (*P < 0.05). Furthermore, the significant increases in the normalized clearance (in back and tail skin and hamstring muscle) are consistent with a real increase in vascular permeability, not just an increase in surface area for exchange. The alternating background stipple separates different tissue samples. Normalized clearances falling below the broken line indicate 30 min clearances smaller than the measured plasma volume in the tissue sample. Note that the units of the 30 min clearance are μl (g dry weight)⁻¹. Thus, a 30 min clearance is a real clearance (expressed as a rate (ml g⁻¹ min⁻¹) multiplied by a measuring time (min).

Figure 3. Comparison of the action of ANP (hatched bars) to increase albumin clearance in mice with endothelial-specific KO of the ANP GC-A receptor (open bars) and their control littermates (dark bars)

The format is the same as used in Fig. 2 for the wild type mice. In KO mice, ANP does not significantly increase normalized albumin clearance relative to vehicle controls. On the other hand, normalized albumin clearance in the presence of ANP is significantly increased in control littermates relative to the vehicle control (*) and relative to ANP-treated KO mice for back skin, tail and hamstring muscle (**). Note that the units of the 30 min clearance are μl (g dry weight)⁻¹. Thus, a 30 min clearance is a real clearance (expressed as a rate (ml g⁻¹ min⁻¹) multiplied by a measuring time (min).

Figure 4. Measurement of 35 kD Gadomer contrast agent apparent permeability coefficient in skin and muscle tissue of C57BL6 control mouse muscle and cheek during vehicle (saline) infusion

A, MR image (axial slice) of mouse head acquired 200 s after contrast agent injection via the tail vein. The ROIs were carefully selected using anatomical references for muscle, skin and vessels. B, shown is the subtracted image (image in A minus baseline image) recording the signal increase in tissue after injection of Gadomer, where cold colours indicate low signal enhancement and warm colours indicate high signal enhancement. Note the high signal intensity in large arteries showing that most of the high molecular weight Gadomer contrast agent is mainly in the vascular space. C, curve showing the signal intensity changes over time in a ROI used to estimate Gadomer permeability coefficient in the skin. As the contrast agent is injected there is a step increase in tracer signal intensity above background as the vascular volume in the ROI is filled with the contrast agent. Tracer continues to accumulate in the ROI as it enters the extravascular space. The initial rate of tracer accumulation is estimated from the slope of the signal intensity over the first 100–150 s. An initial estimate of the vascular permeability is obtained from the magnitude of the initial slope and step. This initial estimate can be corrected for the fall in vascular tracer concentration (as measured from the signal intensity over an adjacent artery; see inset). D, signal intensity over time in ROI over masseter muscle. Muscle permeability is less than in skin. The analysis to estimate vascular permeability is over an ROI containing no vessels larger than 100 μm diameter. Thus, assuming a mean plasma volume to exchange surface area of 4.4 × 10⁻⁴ cm, the vascular permeability coefficients in muscle and skin tissue were 4.6 ± 0.6 × 10⁻⁷ cm s⁻¹ and 26 ± 3 × 10⁻⁷ cm s⁻¹, respectively.

Figure 5. Mean values of 35 kD Gadomer contrast agent apparent permeability coefficients in masseter muscle tissue and skin of control and EC GC-A KO mice before (open bars) and after (hatched bars) infusion of ANP

Using the slope/step method in Fig. 4, paired measurements of Gadomer tracer permeability were made before and after ANP infusion in mice later used for the two-tracer isotope uptake methods summarized in Fig. 3. ANP increased cheek skin permeability coefficient (*P < 0.05) and there was a small increase in muscle. In the presence of ANP there was no significant permeability increase in skin or muscle of EC GC-A KO mice.