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Myosin heavy chain expression in renal afferent and efferent arterioles: relationship to contractile kinetics and function¹

Smooth Muscle Research Group, University of Calgary, Calgary, Alberta T2N 4N1, Canada

²Correspondence: University of Calgary Faculty of Medicine, Smooth Muscle Research Group, 3330 Hospital Dr. NW, Calgary, Alberta, Canada, T2N 4N1. E-mail:rloutzen{at}ucalgary.ca

SPECIFIC AIMS

Vascular smooth muscle expresses four myosin heavy chain (MHC) isoforms (1A, 1B, 2A, 2B) derived by alternative splicing from a single gene. MHC-1 and MHC-2 differ in the length and amino acid sequence of the carboxyl terminus but do not exhibit clearly discernible differences in function. In contrast, MHC-A and MHC-B, which differ in the absence (MHC-A) or presence (MHC-B) of a 7 amino acid insert in the ATP binding pocket of the motor domain of the myosin head, exhibit markedly different actin-activated Mg ATPase activities and cross bridge cycling rates and have been associated with differing smooth muscle contractile kinetics. The physiologic significance of this smooth muscle MHC diversity is not fully understood, especially at the level of the vasculature.

We sought to determine MHC isoform expression patterns in the renal afferent and efferent arterioles and to determine whether MHC isoform expression correlated with function, specifically with regard to contractile kinetics. Renal afferent and efferent arterioles play distinctly different roles in regulating renal function and the smooth muscles comprising these two vessels exhibit striking differences in activation characteristics. The preglomerular afferent arteriole regulates glomerular inflow resistance and must respond quickly to changes in blood pressure in order to prevent the transmission of elevated systolic pressure to downstream glomerular capillaries. This arteriole has evolved a robust and rapid myogenic vasoconstrictor response. The postglomerular efferent arteriole is not exposed to rapid changes in perfusion pressure and does not exhibit myogenic reactivity. Rather, in settings of reduced renal perfusion, a tonic maintenance of efferent arteriolar vasoconstriction increases glomerular outflow resistance to maintain an adequate filtration pressure within the upstream glomerular capillaries and preserve renal function. The extremely small size (10–20 µm in diameter, 1/10th the size of an eyelash) and inaccessibility of the terminal renal arterioles have hampered characterization of their biochemical and functional attributes.

PRINCIPAL FINDINGS

1. The preglomerular afferent arteriole exhibits threefold faster contractile kinetics than the postglomerular efferent arteriole in response to angiotensin II and norepinephrine
The time courses for angiotensin II- and norepinephrine-induced vasoconstriction of renal afferent and efferent arterioles were examined using the in vitro perfused hydronephrotic rat kidney model. Changes in diameter were measured by online image processing. Upon administration of 0.1 nM angiotensin II, afferent arteriolar diameters were reduced from 16.8 ± 1.0 to 4.9 ± 0.7 µ (n=5) and efferent arteriolar diameters from 13.0 ± 1.4 to 5.0 ± 0.6 µ (n=5). The time to half-maximal response (t_1/2) was 35 ± 5 s in afferent arterioles, threefold faster than that for efferent arterioles (99±12 s, P=0.0013). As shown in Fig. 1 , norepinephrine produced similar steady-state reductions in afferent (from 15.6±0.7 to 5.0±0.7 µ, n=7) and efferent (from 14.2±0.5 to 6.0±0.6 µ, n=7) arteriolar diameters, but elicited much faster responses in both vessels. These data are plotted as the percent of maximal response in Fig. 1C . As depicted in Fig. 1D , the time course for the afferent response (t_1/2=9.3±1.6 s) was three- to fourfold faster than that of the efferent arteriole (35.0±7.3 s).

View larger version (32K): [in this window] [in a new window]   Figure 1. Time courses of norepinephrine (0.1 µM)-induced vasoconstriction in afferent and efferent arterioles. Data expressed as actual diameters (A, B), % of maximal response (100% representing the maximal decrease in diameter, C), and time to half-maximal constriction (t1/2, D).

2. The faster afferent arteriole expresses the more rapidly cycling MHC-B isoform; the efferent arteriole expresses only MHC-A
The MHC isoform expression patterns in isolated afferent and efferent arterioles were determined using RT-PCR, Western analysis, and immunohistochemistry. RT-PCR revealed mRNAs for MHC-A and MHC-B in afferent and efferent arterioles. Using the same primers, MHC-B mRNA was clearly predominant in the afferent arteriole whereas message for MHC-A was predominant in the efferent arteriole (Fig. 2 A). By Western blot analysis we were able to demonstrate marked differences in MHC protein expression in afferent and efferent arterioles. Because of the small amount of protein present in the isolated arterioles it was necessary to increase the sensitivity of the assay. To achieve this, membranes were first treated with rabbit pan-MHC antibody (which detects all MHC isoforms) or anti-MHC-B antibody, then incubated with biotin-conjugated goat anti-rabbit IgG. This was followed by application of streptavidin-conjugated horseradish peroxidase. Proteins were then visualized using enhanced chemiluminescence (SuperSignal West Femt substrate, Pierce). As shown in Fig. 2B , MHC-B protein was detected in afferent arteriole and tail artery but not in the efferent arteriole or aorta. Reprobing with a pan-MHC antibody indicated that the inability to detect MHC-B in the efferent arteriole did not reflect lower MHC loading, as the total efferent MHC was 84 ± 10% of afferent MHC. This preferential expression of MHC-B in the afferent arteriole was confirmed by immunofluorescence microscopy.

View larger version (65K): [in this window] [in a new window]   Figure 2. Expression of MHC-A and MHC-B. A) Primers for the amino-terminal region containing the MHC-A/B alternative splice site were used for RT-PCR. Bands at 240 and 261 bp correspond to MHC-A and MHC-B, respectively. B) Western blot analysis of MHC-B (upper panel) and total MHC (lower panel) expression in rat tail artery, aorta, and afferent and efferent arteriole using anti-MHC-B and anti-MHC, respectively.

3. Expression patterns for MHC-1 and MHC-2 isoforms were similar in afferent and efferent arterioles
RT-PCR and Western analysis was also used to examine the expression patterns of MHC-1 and MHC-2 in afferent and efferent arterioles. RT-PCR revealed the presence of mRNA for MHC-1 and MHC-2 in both vessels. Western analysis demonstrated that expression levels of MHC-1 and MHC-2 protein in afferent and efferent arterioles were not different and that the patterns were similar to that seen in the rat aorta and tail artery. The proportions of MHC-1 and MHC-2 were 77 ± 3 and 23 ± 3% in tail artery and 73 ± 2 and 27 ± 2% in the aorta. Similarly, the afferent arteriole contained 80 ± 3% MHC-1 and 20 ± 3% MHC-2, values not significantly different from those of the efferent arteriole (77±8% and 23±8%).

CONCLUSIONS AND SIGNIFICANCE

The present study is the first to compare the contractile kinetics of the afferent and efferent arterioles and to relate these properties to MHC isoform expression patterns. We found the afferent arteriole predominantly expressed MHC-B and exhibited a more rapid response to angiotensin II and norepinephrine. In contrast, the efferent arteriole expressed only the MHC-A isoform and exhibited much slower contractile kinetics. These findings are consistent with previous studies linking differences in MHC-A/MHC-B isoform expression to smooth muscle contractile kinetics. Phasic smooth muscle types, such as bladder, tend to express MHC-B whereas tonic smooth muscles, such as aorta, are reported to express the MHC-A isoform. The MHC-B isoform is also reported to be expressed at higher levels in terminal resistance vessels vs. proximal conduit vessels, but expression patterns vary with blood vessel type. Our finding that terminal resistance arterioles of the kidney express different MHC isoforms has important implications for the physiologic function of these two vessel segments, as the afferent and efferent arteriole play distinctly differing roles in regulating renal function. This interesting example of MHC isoform diversity may provide insight into the physiologic relevance of variations in vascular smooth muscle myosin expression patterns in the circulatory system in general.

The afferent and efferent arterioles regulate the inflow and outflow resistances of the renal glomerulus (Fig. 3 ). The afferent arteriole, which originates at the interlobular artery and terminates at the glomerular capillaries, must respond rapidly to prevent sudden changes in systemic blood pressure from being transmitted to the glomerulus. The hydrostatic pressure within the glomerular capillaries (PGC) is a primary determinant of the glomerular filtration rate (GFR) and must be maintained within precise limits to regulate renal function and prevent glomerular injury. A disruption of PGC regulation and the ensuing glomerular capillary hypertension are considered to be primary events linking pathophysiologic states such as diabetes and hypertension to kidney disease and renal failure. Accordingly, the preferential expression of MHC-B in the afferent arteriole represents an important adaptation that would allow this vessel to rapidly modulate tone and insulate the glomerular capillaries from rapid fluctuations in systemic blood pressure, thereby maintaining GFR within precise limits and protecting the glomerular capillaries from elevations in PGC.

View larger version (42K): [in this window] [in a new window]   Figure 3. Schematic diagram illustrating the anatomical relationship of the afferent arteriole, glomerulus, and the efferent arteriole. The afferent arteriole must respond rapidly to changes in blood pressure to prevent pressure elevations from being transmitted to downstream glomerular capillaries. This vessel predominantly expresses the faster cycling MHC-B isoform. The efferent arteriole regulates glomerular outflow resistance and must be able to efficiently maintain a tonic increase in tone when renal perfusion pressure is compromised. This vessel predominantly expresses MHC-A, which has a slower cross bridge cycling rate and lower ATPase activity.

The postglomerular efferent arteriole plays a distinctly different role. The efferent arteriole originates at the glomerular capillaries and terminates in the peritubular capillaries or vasa recta (in cortical and juxtamedullary nephrons, respectively). This vessel is not exposed to the same fluctuations in pressure as the afferent arteriole and is not myogenic. The physiologic importance of the efferent arteriole is seen in conditions in which renal perfusion pressure is compromised, such as congestive heart failure or renal arterial stenosis. In such settings, efferent arteriolar vasoconstriction elevates glomerular outflow resistance, thereby maintaining an adequate filtration pressure. This important mechanism preserves GFR when blood pressure or renal perfusion pressure is reduced. This function does not involve rapid adjustments in tone, but rather requires an ability of the efferent arteriole to maintain a tonic level of vasoconstriction for extended periods. We found that the efferent arteriole expresses only the slower cycling MHC-A isoform. This isoform has an intrinsically lower ATPase activity and would provide an efficient mechanism for maintaining a tonic level of vasoconstriction. Thus, MHC-A expression in the efferent arteriole represents a functional adaptation that would allow this vessel to efficiently maintain an elevation in glomerular outflow resistance and preserve GFR under conditions of reduced renal perfusion pressure.

In summary, we found that afferent and efferent arterioles differ in their expression of MHC-A and MHC-B isoforms and that this biochemical difference in myosin isoform expression corresponded to functional differences in contractile kinetics. We suggest that the differing MHC expression patterns and kinetic characteristics of these two vessels would, in each case, facilitate their distinct physiologic roles in regulating glomerular capillary pressure. This striking heterogeneity in smooth muscle MHC expression within the renal microvasculature represents a unique and important example of how smooth muscle myosin diversity is related to physiologic function.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0096fje; doi: 10.1096/fj.03-0096fje

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