Endothelin receptor A and p66Shc regulate spontaneous Ca²⁺ oscillations in smooth muscle cells controlling renal arterial spontaneous motion

Abstract

Adaptor protein p66Shc is overexpressed in smooth muscle cells of renal resistance vessels of hypertensive salt-sensitive rats and is involved in the regulation of renal vascular tone. We applied 2-photon laser scanning fluorescence microscopy to analyze spontaneous dynamic fluctuations in intracellular calcium concentrations ([Ca²⁺]_i) in smooth muscle cells embedded in the walls of freshly isolated renal resistance arteries. The amplitude, number of events, and frequency of spontaneous [Ca²⁺]_i oscillations triggered by endogenously released endothelin-1 were recorded in smooth muscle cells of the renal arteries. Endothelin receptor A antagonist BQ123 dramatically reduced the amplitude and frequency of spontaneous Ca²⁺ events, producing marked inhibition of renal vessels spontaneous motion. Spontaneous Ca²⁺ fluctuations in smooth muscle cells of p66Shc knockout (p66ShcKO) rats had significantly higher amplitude than in control rats. The frequency of spontaneous [Ca²⁺]_i oscillations did not change in p66ShcKO rats, suggesting that p66Shc expression did not affect endothelin-1 release from resident endothelial cells. Acute application of endothelin-1 revealed significantly elevated production of the total [Ca²⁺]_i in p66ShcKO rats. Spontaneous cytosolic Ca²⁺ oscillations in smooth muscle cells of renal vessels mediate their spontaneous motion via the endothelin-1/endothelin receptor A pathway. p66Shc decreases the amplitude of individual changes in [Ca²⁺]_i, which mitigates the spontaneous motion of renal vessels.—Palygin, O., Miller, B. S., Nishijima, Y., Zhang, D. X., Staruschenko, A., Sorokin, A. Endothelin receptor A and p66Shc regulate spontaneous Ca²⁺ oscillations in smooth muscle cells controlling renal arterial spontaneous motion.

Impaired renal autoregulation, resulting in a loss of preglomerular vascular resistance to changes in renal blood flow, contributes to the progression of renal damage in hypertension, diabetes, and other intrinsic renal diseases (1). Endothelial cells are known to release vasoactive signaling molecules, which establish the myogenic tone of vascular smooth muscle cells (SMCs) and regulate renal blood flow (2–4). Endothelin (ET)-1 is constitutively released by vascular endothelium and regulates vascular tone either through the calcium-mediated activation of SMCs or through stimulation of eNOS (2). ET-1 is one of major modulators of renal autoregulation, and its vasoactive action is mediated by endothelin receptor (ETR)A (constriction) and ETRB (vasodilation) of vascular smooth muscle and endothelial cells (1). Excessive ET-1 production is implicated in a variety of renal pathologies and particularly in diseases of elderly individuals (3–6).

The task of preglomerular afferent arterioles is to maintain steady glomerular capillary pressure regardless of dynamic changes in systemic blood pressure (7). The factors mediating the myogenic response and the signal transduction molecules involved in the myogenic control of renal vascular tone remain uncertain (1, 7). However, it is established that calcium influx is necessary for vasomotor responses in rat afferent arteriole (8–10). Furthermore, we have previously identified adaptor protein p66Shc as a major regulator of the myogenic response of afferent arterioles in hypertensive rats and demonstrated that p66Shc controls calcium flux by inhibiting Ca²⁺ influx via transient receptor potential cation (TRPC) channels (11). We have previously shown that p66Shc in renal mesangium is essential for the formation of multiunit signaling complexes and regulates a variety of signaling cascades triggered by ET-1 (6, 12–14).

We have established a novel technique that allows the detailed ex vivo analysis of intracellular Ca²⁺ concentration ([Ca²⁺]_i) in freshly isolated vessels (15). This approach was applied to determine assemblage of individual Ca²⁺ events in SMCs of rat renal resistance arteries in either wild-type or genetically modified rats with the mutated Shc1 gene encoding p66Shc. Previously, we demonstrated increased mobilization of [Ca²⁺]_i in primary SMCs isolated from renal microvessels of p66Shc knockout (p66ShcKO) rats (11). Here we showed a significant increase in [Ca²⁺]_i in SMCs embedded in the vascular wall of renal vessels of p66ShcKO rats when compared with wild-type Dahl salt-sensitive (SS) rats. Effective sensitivity of 2-photon microscopy allowed us to monitor Ca²⁺ oscillations in SMCs embedded in the wall of renal resistance vessels. We tested the hypothesis that ET-1 signaling, modulated by adaptor protein p66Shc, is the molecular mechanism underlying changes in spontaneous Ca²⁺ oscillations and, presumably, the spontaneous motion of renal resistance arteries.

MATERIALS AND METHODS

Animals

Rats with targeted modifications of the Shc1 gene were generated on the genetic background of Dahl SS rats using engineered zinc finger nucleases (16) as previously described (11). Water was provided ad libitum, and male rats were maintained on a high-salt (1.0% NaCl, Purina 5001) diet after weaning for a total of 11–18 wk (14–21 wk of age). Male rats were used exclusively throughout the study. Animal use and welfare procedures adhered to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD, USA) following protocols reviewed and approved by the Medical College of Wisconsin Institutional Animal Care and Use Committee.

Two-photon imaging of [Ca²⁺]_i

Ex vivo imaging of [Ca²⁺]_i in SMCs of isolated renal interlobar arteries was carried out as previously described (15). Dissected arteries (150–200 µm inner diameter) were loaded with low-calcium (0.1 mM Ca²⁺) physiologic saline solution (PSS) supplemented with 5 μM Fluo-4 AM and 0.05% pluronic acid and stored for 1 h on a rotating shaker at room temperature in the dark. After incubation, arteries were washed 2–3 times with PSS (2 mM Ca²⁺) to remove extracellular dye, and all further procedures were done in PSS. Arteries were transferred to a silicon-coated plate, and the plate was then transferred to the upright Olympus Fluoview FV1000 microscope equipped with Ti:sapphire lasers tuned to 820 nm and imaged with a ×25 (N.A. 1.05 and working distance 2 mm) water-immersion objective lens (XLPL25XWMP; Olympus, Tokyo, Japan). The fluorescent layer of SMCs was identified using 2-photon microscopy. The fluorescent signal was observed within the loaded cells and measured as arbitrary units of fluorescence intensity.

Vascular response analyses (vasomotion)

Freshly isolated renal microvessels (150–250 µm) were cannulated with 2 glass micropipettes for continuous measurement of internal diameters with a video system as previously described (17–19). Briefly, vessels were pressurized to 60 mmHg and equilibrated for 1 h at 37°C in Krebs-PSS gassed with a mixture of 21% O₂–5% CO₂–74% N₂ to maintain pH 7.4. Measurements of the internal diameter were continuously recorded by a pressure myograph system equipped with dimension analysis software (DMT 114P with MyoView.4; Danish Myo Technology A/S, Aarhus, Denmark). The obtained signal was sampled with a fast Fourier transform (FFT) algorithm over a period of 200 s and divided into frequency components (OriginPro 9.0; OriginLab, Northampton, MA, USA). Vasomotion of the vessel mediated by the ETRA signaling component was calculated as a ratio between the sum of the FFT amplitudes in the baseline to the corresponding sum of distinct frequencies during BQ123 application for the same time domain.

Western blot analysis and immunohistochemistry

For protein expression analysis, animals were anesthetized by isoflurane inhalation and euthanized by a bilateral pneumothorax. The right kidney was extracted and decapsulated, and the inner and outer medullae were separated from cortex tissue. In separate animals, after the removal of the medulla, renal interlobar arteries were dissected from both renal cortexes and washed in low-calcium PSS. Arteries were then digested in PSS using 240 U/ml collagenase II and 0.1 mg/ml soybean trypsin protease inhibitor (Worthington Biochemicals, Lakewood, NJ, USA) at 37°C for 30 min. After 2 washes in PSS, arteries were flash frozen and stored at −80°C until homogenization. Isolated tissues were homogenized on ice in RIPA buffer [50 mM Tris (pH 8), 150 mM NaCl, 1% Igepal-650 (MilliporeSigma, Burlington, MA, USA), 0.5% sodium deoxycholate, 0.1% SDS, 1 mM PMSF, and Roche Complete protease inhibitor cocktail] (MilliporeSigma) with 25 strokes of a Potter-Elvehjem PTFE pestle and glass tube homogenizer (MilliporeSigma) followed by brief sonication. The homogenate was centrifuged at 12,000 g at 4°C to collect the supernate, and 30 μg (cortex and medulla) or 15 μg (microvessels) of protein per sample was analyzed via Western blot using antibodies for Shc (610879; BD Transduction Laboratories, San Jose, CA, USA) and ETRA (AB3260; MilliporeSigma). The Shc signal was detected by ECL chemiluminescent film exposure, and ETRA was detected using near-infrared secondary antibodies (925-32210; Li-Cor Biosciences, Lincoln, NE, USA). To detect p66Shc expression in vivo, immunohistochemistry was done as previously described (11, 12). Briefly, for immunohistochemistry, specific anti-p66Shc antibodies (DiagnoCure, Quebec City, QC, Canada) were applied to formalin-fixed, paraffin-embedded kidney sections for 1 h prior to using the mouse-on-rat polymer detection system conjugated with alkaline phosphatase (Biocare Medical, Concord, CA, USA) and blue alkaline phosphatase substrate as chromagen and counterstained with Nuclear Fast Red (Vector Laboratories, Burlingame, CA, USA).

Statistical analysis

Data are presented as means ± sem. Data were tested for normality (Shapiro-Wilk) and equal variance. Statistical analysis consisted of Student’s t test and 1-way ANOVA with Tukey’s post hoc test, and P < 0.05 was considered significant (SigmaPlot 12.5, Systat, San Jose, CA, USA).

RESULTS

Ca²⁺ oscillations in SMCs embedded in the wall of renal resistance vessels are linked with spontaneous vascular motion

The isolated intact vascular tree was prepared for 2-photon imaging microscopy as previously described (15). Renal microvessels (diameter, 100–150 µm; identified as interlobular arteries) were loaded with Fluo4 AM calcium indicator to monitor fluctuations in [Ca²⁺]_i. The regions of interest were defined for living SMCs as dynamically fluorescent cells lying within the tunica media and perpendicular to the vascular lumen (Fig. 1A). The resolution of 2-photon microscopy can avoid nonspecific autofluorescence of collagen and elastin fibers as well as Fluo4-loaded fibroblasts, adipocytes, and nerve cells of the tunica externa (15). Effective sensitivity of 2-photon microscopy allowed us to observe and quantitate Ca²⁺ oscillations in SMCs (Fig. 1B). The amplitude, the number of events, and the period of spontaneous Ca²⁺ oscillations in SMCs were recorded in parallel with the renal arteries spontaneous motion (Supplemental Video S1A–F). This motion and Ca²⁺ oscillations likely depend on the influx of extracellular Ca²⁺ and are modulated by the release of vasoactive agents by endothelial cells (20). Because renal vascular endothelium releases ET-1, a strong potent vasoconstrictor with an ability to modulate renal vascular tone by acting on SMCs of renal vessels (21), we tested the involvement of ET-1 signaling and specific ET-1 receptors in spontaneous Ca²⁺ oscillations and motion of renal vessels.

Figure 1 .

Analyses of spontaneous Ca²⁺ oscillations in the freshly isolated interlobular renal microvessels. A) Microphotograph of the renal microvessel obtained with 2-photon microscopy. Shown are a transmitted light image and the corresponding fluorescent signal from Fluo4 AM Ca²⁺ indicator. B) Magnified fragment of the vessel at A and an example of Ca²⁺ spontaneous events analyses from the single SMC [single region of interest (ROI) shown by blue box]. Scale bars, 40 μm. C) Amplitude, number of events, and time between events analyzed for the ROI at B for 166 s with an imaging frequency of 0.6 Hz. Changes in spontaneous characteristics obtained during the control period (black), pharmacological application of BQ123 (400 nM; red), and the addition of ET-1 (100 nM) in the recording chamber (blue) (n ≥ 3 vessels; n ≥ 3 rats). The time between each 166-s period was 3 min.

ETRA signaling modulates Ca²⁺ oscillations and spontaneous vascular motion

ET-1, a powerful vasoconstriction peptide, is expressed constitutively by many cell types, including endothelial cells of resistance vessels (22, 23). ET-1 is known to activate Ca²⁺ influx and to induce Ca²⁺ mobilization pathways (24). We tested the hypothesis that spontaneous Ca²⁺ oscillations are modulated by the endogenous release of ET-1 and corresponding activation of ET-1 receptors in SMCs. ET-1 activates downstream signaling pathways through GPCR ETRA and ETRB, with ETRA being predominant on SMCs (21). The highly selective peptide antagonists BQ123 (for ETRA) and BQ788 (for ETRB) have been routinely used to define functions of ET-1 receptors (6, 21). In the first set of experiments, the amplitude, the number of events, and the period of spontaneous Ca²⁺ oscillations in SMCs were recorded in the presence of BQ123 (400 nM) (Fig. 1C). Treatment with BQ123 in concentration, which completely blocks ETRA, dramatically decreased the amplitude of Ca²⁺ oscillations and the frequency of events, increasing the period between oscillations up to the maximum time exposed. As a consequence, we observed radical inhibition of renal vessel vascular motion (Supplemental Video S1A–F). The effectiveness of ETRA receptor blockade was further tested by the addition of exogenous ET-1 to isolated vessels pretreated with BQ123. In these conditions, ET-1 (100 nM) failed to restore Ca²⁺ oscillations in SMCs embedded in the vascular wall of renal resistance vessels and did not reinstate the spontaneous vascular motion activity of renal vessels.

The mean amplitude and number of Ca²⁺ oscillations dropped dramatically in BQ123-treated vessels (Fig. 1). To verify the viability of vessels treated with BQ123, we measured Ca²⁺ oscillations in the same vessels after 3 min washout of BQ123 and the consequent addition of exogenous ET-1 (100 nM). Ca²⁺ oscillations were observed, albeit with less amplitude than the prior to BQ123 treatment, which is consistent with the difficulties of removing BQ123 from cells and tissues (Fig. 2). In addition to the decrease of amplitude, the frequency of obtained oscillation was restored after ET-1 application (Fig. 2B). To detect vasomotion in freshly isolated renal microvessels, we continuously recorded changes in the internal diameter by a pressure myograph system (Fig. 3A). Similar to the observation of fluorescent Ca²⁺ oscillations in SMCs, the application of BQ123 (100 nM) significantly attenuated vasomotion of microvessel after washout and activation by the ET-1 application. Changes in internal diameter over time were further represented as a sum of sine waves obtained by FFT (Fig. 3B). In control experiments, the presence or absence in microvessels vasomotion can be visually detected and strongly depends on the presence of extracellular Ca²⁺ ions (Supplemental Video S2).

Figure 2 .

The blockade of ETRA receptor dramatically decreased Ca²⁺ oscillations in small resistant renal vessels of p66ShcKO rats. A) Example of single-cell activity profile during the control, ETRA blockade (BQ123; 400 nM), and activation by exogenous application of ET-1 (100 nM). The washout period for BQ123 was 3 min. B) ETRA blockade significantly reduces the amplitude of spontaneous Ca²⁺ events, which was partially restored after washout. The frequency and period of events were also blocked by ETRA antagonist and entirely restored after washout and application of ET-1 (n ≥ 3 vessels; n ≥ 3 rats).

Figure 3 .

The detection of spontaneous vasomotion in freshly isolated renal microvessels. A) Example of microvessel activity profile, estimated as a fluctuation of internal vessel diameter, during the control, ETRA blockade (BQ123; 100 nM), washout, and activation by exogenous application of ET-1 (100 nM). B) The obtained signals (A) were sampled with an FFT algorithm and divided into single sinusoidal oscillations at distinct frequencies.

Similar studies were carried out with BQ788, a specific antagonist of ETRB. A concentration of BQ788 of 400 nM blocks 100% of ETRB, with minimal effect on ETRA (25). BQ788 failed to affect spontaneous Ca²⁺ oscillations in renal SMCs (Fig. 4 and Supplemental Video S3A–F).

Figure 4 .

The blockade of the ETRB receptor did not change the behavior of spontaneous Ca²⁺ oscillations in small resistant renal vessels. A) Example of single-cell activity profile during the control, ETRB blockade (BQ788; 400 nM), and activation by exogenous application of ET-1 in the presence of BQ788 (n ≥ 3 vessels; n ≥ 3 rats). B) ETRB blockade did not produce significant changes in amplitude, frequency, or period of spontaneous Ca²⁺ events. The increase in amplitude in response to ET-1 was mediated by the presence of a significant amount of exogenous ETRA agonist.

As is the case with spontaneous Ca²⁺ oscillations, exogenous ET-1 acts via ETRA receptors to cause [Ca²⁺]_i mobilization in SMCs embedded in renal vascular walls. We carried out a comparison of antagonists of ETRA and ETRB on the effect of ET-1 in the setup where the effect of ET-1 is expected to be at its maximum (Fig. 5). Pretreatment with BQ788 did not affect ET-1–induced Ca²⁺ influx, whereas BQ123 significantly reduced ET-1–mediated changes in [Ca²⁺]_i.

Figure 5 .

The exogenous ET-1 acts via ETRA receptors to cause [Ca²⁺]_i mobilization in SMCs embedded in renal vascular walls. A) Two-photon microphotograph of the renal microvessel before and after ET-1 application in the presence of ETRB antagonist (BQ788). Shown are fluorescence and the corresponding transmitted light image. Changes in the vessel diameter are visible in transmitted light during the application of ET-1 and the corresponding mobilization of [Ca²⁺]_i in SMCs. Scale bar, 40 µm. B) Summary of the response to the ET-1 application in vessels treated by ETRA (BQ123) or ETRB (BQ788) antagonists. Total [Ca²⁺]_i, calculated as integral of ET-1–mediated transient, indicates a significant attenuation of the calcium response during the ETRA blockade (n ≥ 4 vessels; n ≥ 4 rats for each group). *P < 0.05 (ANOVA).

If during the exposed time (2.8 min) we did not observe spontaneous activity in SMCs, we considered it to represent a period when no spontaneous oscillation occurs. The period was calculated as the distance between events, and if no events were observed, the period was marked as maximum or 166 s (2.8 min).

The contribution of p66Shc in ET-1–mediated Ca²⁺ oscillations

Impaired renal microvascular responses accompany the SS, hypertension-induced nephropathy. The Dahl SS rat is a widely used model to study mechanisms of vascular dysfunction associated with SS hypertension in humans (11, 26–28). Chronic exposure of SS rats to a 1% salt diet for 17 wk (Fig. 6A) induces hypertension-induced nephropathy accompanied by the impaired vasoconstrictive response of renal afferent arterioles, and adaptor protein p66Shc contributes to the regulation of renal vascular tone (11). Immunoblotting analyses reveal expression of p66Shc in rat kidney medulla and isolated rat renal arteries (Fig. 6B) but not p66ShcKO rats. Therefore, to test the contribution of p66Shc in controlling function of renal resistance vessels, we performed imaging analyses of SMC activity and spontaneous vascular motion in wild-type SS and rats with genetic manipulation of p66Shc maintained on 1% sodium salt diet.

Figure 6 .

Model of SS, hypertension-induced nephropathy and corresponding p66ShcKO mutant in the Dahl SS rat background. A) Dietary protocol for the development of SS hypertension in SS and p66ShcKO rats. B) Immunoblotting analysis of p66Shc expression in rat kidney medulla and isolated rat renal arteries. C) ETRA protein expression was evaluated in renal cortex tissue collected from SS, p66ShcKO, and p66Shc-S36A rats. The signal corresponding to ETRA was detected at ∼46 kDa (upper panel) and was normalized to total protein detected by Ponceau S staining (lower panel). No significant difference in ETRA expression was seen among the strains (n = 3 each strain). D) Immunohistochemistry analysis of p66Shc expression in rat kidney medulla. Scale bar, 40 μm.

Lack of p66Shc causes increased amplitude of Ca²⁺ oscillations

The lack of p66Shc is accompanied by a significant increase of the mean amplitude of spontaneous ETRA-mediated Ca²⁺ oscillations when compared with wild-type SS rats (Fig. 7A, B). Neither the number of oscillations nor the time between oscillations is affected by p66ShcKO. It is likely that the mechanism by which p66Shc attenuates the amplitude of Ca²⁺ oscillations in SMCs observed here is the same by which p66Shc inhibits the increase of [Ca²⁺]_i in cultured SMCs (i.e., through modulation of TRPC channels). The rat strain that expresses endogenous p66Shc with the single amino acid substitution of Serine 36 (Ser36) is termed p66Shc-S36A as previously described (11). Mutation of regulatory phosphorylation site Ser36 resulted in a p66Shc incapable of translocation to mitochondria and thus p66Shc-S36A rats lack p66Shc-dependent mitochondria action (29). We have demonstrated that weakened ET-1 activity in afferent arterioles characterizes rats expressing S36A mutant of p66Shc (11). It is generally accepted that phosphorylation of Ser36 in p66Shc triggers its translocation to mitochondria (30, 31). An additional indication that S36A mutation decreases the amplitude of Ca²⁺ oscillations due to p66Shc cytosolic location is the opposite to the effect of p66ShcKO. SMCs embedded in vascular walls of renal resistance vessels from p66Shc-S36A rats display significantly decreased amplitude of Ca²⁺ oscillations compared with wild-type SS rats (Supplemental Video S4A, B). Again, the number of events and their frequency are not affected (Fig. 7B). In experiments on the miograph system described in Fig. 3, we observed a strong trend to increase in ETRA-mediated vasomotion of the microvessels form p66ShcKO animals (Fig. 7C, D).

Figure 7 .

The lack of p66Shc accompanied by a significant increase of the mean amplitude of spontaneous ETRA-mediated Ca²⁺ oscillations. A) Example of spontaneous [Ca²⁺]_i in SMCs of the wild-type SS, S36A mutant (devoid of p66Shc mitochondrial action), and p66ShcKO rats. B) The amplitude of Ca²⁺ oscillations, number of spontaneous Ca²⁺ events, and their frequency in SS, S36A, and p66ShcKO rats (n ≥ 10 vessels; n ≥ 5 rats for each group). *P < 0.05 (ANOVA). C) Example of the vasomotion in SS and p66ShcKO rats after by the blockade of ETRA by the application of BQ123 (100 nM). D) Changes in ETRA-mediated vasomotion were elevated in p66ShcKO rats and obtained as a ratio between the sum of the FFT amplitudes in the baseline to the corresponding sum of distinct frequencies during BQ123 application for the same time domain (n ≥ 5 vessels; n ≥ 5 rats for each group).

ET-1–induced intracellular Ca²⁺ mobilization in SMCs of renal vessels is inhibited by p66Shc

The demonstrated capability of p66Shc to modulate spontaneous Ca²⁺ oscillations is the rationale to expect p66Shc expression to have a similar effect upon mobilization of [Ca²⁺]_i caused by exogenous ET-1. Vasoactive ET-1 is essential for the cross-talk between glomerular cells as well as cells of the vascular wall (23, 32). Fluorescent Ca²⁺ indicator Fluo-4 AM reveals a significant increase of [Ca²⁺]_i in SMCs of renal vessels after the bath application of ET-1 (100 nM) (15). Comparison of [Ca²⁺]_i mobilization in SMCs of renal vessels freshly isolated from SS and p66ShcKO rats showed a significant enhancement of ET-1–induced changes in [Ca²⁺]_i when p66Shc is lacking (Fig. 8A). Changes in ET-1–induced [Ca²⁺]_i mobilization in individual embedded SMCs of p66Shc-S36A rats was lessened when compared with SMCs of parental SS rats (Fig. 8). These data are in good accordance with the previously reported blunted autoregulation response of p66Shc-S36A afferent arterioles to increased perfused pressure and ATP (11). The decrease of Ca²⁺ influx in p66Shc-S36A cells when compared with wild-type SS SMCs was consistent with the decreased response to ET-1 of cultured SMCs derived from S36A renal vessels.

Figure 8 .

The comparison of [Ca²⁺]_i mobilization in SMCs of renal vessels freshly isolated from SS rats and p66Shc mutant and KO rats. A) Summary of the calcium response to the exogenous ET-1 application in SS, S36A mutant, and p66SchKO SMCs from the freshly isolated renal vessels. B) Summary graphs of changes in the total [Ca²⁺]_i and maximum amplitude in renal microvessels (n ≥ 5 vessels; n ≥ 4 rats for each group). *P < 0.05 (ANOVA).

DISCUSSION

In this study, we combined 2-photon imaging with targeted modification of rat genome to elucidate the role of adaptor protein p66Shc and ET-1 receptor ETRA in modulating spontaneous Ca²⁺ oscillations in SMCs of renal blood vessels, which control oscillations in the tension of renal resistance arteries. This approach allowed us to investigate directly regulatory mechanisms of spontaneous motion in hypertension-induced nephropathy. It is likely that observed spontaneous motion of isolated renal microvessels is a manifestation of changes in vasomotion or the circular changes of vascular tone. Many diseases, including diabetes and hypertension, alter the pattern of vasomotion (33). Vasomotion is necessary for optimal blood flow and blood supply to tissues (33) and has been shown in many studies of isolated arteries to be dependent on Ca²⁺ oscillations (34, 35) and factors released by endothelium (33, 36). Spontaneous changes in the diameter of freshly isolated rat blood vessels, revealed by video microscopy, are the result of ongoing vasomotion, which is initiated and maintained by mechanisms inherent to the vascular wall (20).

We have previously reported that treatment of isolated rat renal resistance arteries with ET-1 causes a rapid increase of [Ca²⁺]_I in smooth muscle vasculature of these vessels. However, in the current study we registered the amplitude, number of events, and frequency of small spontaneous [Ca²⁺]_i oscillations in these cells without treatment with vasoactive agents. We report that these spontaneous [Ca²⁺]_i oscillations are the consequence of endogenous ET-1 release, most probably from endothelial cells of renal resistance vessels. Furthermore, we uncovered the role of p66Shc expression in the regulation of amplitude of these [Ca²⁺]_i oscillations and their link with vasomotion of renal blood vessels.

Studying Ca²⁺ transients using 2-photon imaging in individual SMCs of the blood vessels is essentially different from measurements of Ca²⁺ transients in the whole vessel, which combines signals from fibroblasts, endothelium, and SMCs. The impact of fibroblast response is not discussed in confocal measurements however, not taking it into consideration could result in misinterpretation of the data.

In this study, we identified p66Shc expression as one of the causes of limited changes in [Ca²⁺]_i of SMCs of renal vessels in response to exogenous vasoactive peptide ET-1, which reflects pathophysiological changes in the renal vascular cells. Pathophysiological changes in the vasculature are linked with age-related diseases such as hypertension, which contributes to the development of chronic kidney disease and hemorrhagic stroke (37–39). Hypertension-induced nephropathy is associated with impaired renal vascular responsiveness and structural changes (40, 41). Thus, uncovering the molecular mechanisms of vascular dysfunction holds possibilities for developing new therapeutic strategies to combat diseases that contribute to the escalation of health care costs (42).

Our measurements of spontaneous calcium oscillations were carried out in interlobular arteries, which may differ from afferent arterioles regarding regulation of spontaneous vascular motion. Nevertheless, the similarities in vasoconstrictor activity, such as conducted vasomotor response, between renal afferent and interlobular arteries have been reported (43). Still, afferent arterioles are unique renal resistance arteries that express components of the fast, smooth muscle gene program important for the myogenic contractile response (44). Analysis of spontaneous calcium oscillations in renal afferent arterioles is hindered by their small size and relative inaccessibility.

Calcium oscillation could play a role in intercellular synchronization in the vascular wall, which is necessary for vasomotion. Accordingly, p66Shc expression by decreasing the amplitude of Ca²⁺ oscillations can disrupt intercellular synchronization and cause inefficient spontaneous oscillation in the tone of blood vessel walls. Because the S36A mutant of p66Shc, which lacks potential for translocation to mitochondria, has an even more deleterious effect on Ca²⁺ oscillations, it is probable that p66Shc inhibitory action is linked to its cytoplasmic location. The mechanism of p66Shc action is likely based on inhibition of TRPC channels and the consequent decrease of amplitudes of [Ca²⁺]_i changes. Accordingly, p66Shc affects spontaneous Ca²⁺ oscillations without preventing activation of ETRA by ET-1. However, because cytoplasmic p66Shc promotes the production of reactive oxidation species (ROS) through regulating the activity of the plasma membrane–bound NADPH oxidase (45–47), a p66Shc-mediated ROS-dependent mechanism of inhibition of vascular motion cannot be excluded. It has been reported that ROS activate Ca²⁺/calmodulin-dependent protein kinase II, which regulates several proteins involved in Ca²⁺ handling (48). Mitochondria also take up Ca²⁺ via the mitochondrial Ca²⁺ uniporter and release it via the Na⁺/Ca²⁺ Li⁺ permeable exchanger (48).

It is generally accepted that vasomotion of the renal vessel results from the interconnected action of multiple intracellular and intercellular systems, thus inhibition of 1 oscillatory component in the vascular wall does not necessarily eliminate vasomotion (33). It seems, however, that addition of antagonist of ETRA stops spontaneous motion of renal resistance arteries. Thus, our data suggest that ET-1–maintained Ca²⁺ oscillations, presumably maintained by ET-1 released by endothelial cells of renal vessels, play a principal role in the spontaneous motion of renal vessels. Removal of endothelium has been shown to prevent vasomotion (20, 33, 49).

This study demonstrates that p66Shc inhibits the amplitude of Ca²⁺ oscillations maintained by ETRA signaling. ET-1 signaling is linked to cardiorenal pathologies associated with old age, such as glomerulosclerosis, hypertension, and atherosclerosis (3, 50), whereas p66Shc is generally considered to act as a promotor of age-related dysfunction (51, 52). Whether these [Ca²⁺]_i changes lead to a decrease of vasomotion of renal resistance blood vessels, thus contributing to renal damage at hypertension-induced nephropathy and old age, remains to be established. Considering that p66Shc causes a lack of autoregulation activity of preglomerular afferent arterioles, efforts to come up with efficient inhibitors of p66Shc signaling and clinical studies evaluating the effect of such inhibitors seem to be warranted.

Supplementary Material

This article includes supplemental data. Please visit http://www.fasebj.org to obtain this information.

Glossary

ET

endothelin

ETR

endothelin receptor

FFT

fast Fourier transform

p66ShcKO

p66Shc knockout

PSS

physiological saline solution

ROS

reactive oxidation species

SMC

smooth muscle cell

SS

salt sensitive

TRPC

transient receptor potential cation

AUTHOR CONTRIBUTIONS

O. Palygin, D. X. Zhang, A. Staruschenko, and A. Sorokin conceptualized the study and designed research; O. Palygin, B. S. Miller, Y. Nishijima, D. X. Zhang, A. Staruschenko, and A. Sorokin analyzed data; O. Palygin, B. S. Miller, Y. Nishijima, and D. X. Zhang performed research; A. Sorokin wrote the original draft; O. Palygin, B. S. Miller, D. X. Zhang, A. Staruschenko, and A. Sorokin edited the manuscript to the final version; and A. Sorokin supervised the study.