Intact Microtubules Are Required for Natriuretic Responses to Nitric Oxide and Increased Renal Perfusion Pressure
Extracellular renal interstitial (RI) cGMP modulates NO- and pressure-induced natriuresis in vivo in the rat. The present study objective was to test the hypothesis that an intact microtubulin network is required for transport of cGMP from intracellular sites into the extracellular compartment in vivo and that this transport is required for natriuresis induced by NO and increased renal perfusion pressure. After a 1-hour control period, uninephrectomized rats received an RI infusion of NO donor S-nitroso-N-acetylpenicillamine (SNAP), SNAP+microtubule inhibitor nocodazole (NOC), SNAP+NOC+cGMP, or NOC alone for 2 consecutive 1-hour collection periods. SNAP alone increased RI cGMP (P<0.05 during both experimental periods) and urinary sodium excretion (P<0.05 at 1 hour and P<0.005 at 2 hours). In contrast, when SNAP+NOC were coinfused, there was no increase in either RI cGMP or urinary sodium excretion. However, when cGMP was coinfused with SNAP+NOC, the natriuretic response to SNAP was fully restored. Similarly, NOC abolished SNAP-induced increases in the fractional excretion of Na+ and Li+. NOC also prevented the increase in both RI cGMP and natriuresis engendered by raising renal perfusion pressure in uninephrectomized rats, and pressure-natriuresis was re-established by coadministration of RI cGMP. As demonstrated by confocal microscopy after in vivo renal perfusion fixation, β-tubulin was disrupted in renal cortical nephrons of kidneys infused intrarenally with NOC. These observations indicate that a functioning microtubulin network is required for the transport of cGMP into the extracellular space to modulate NO- and pressure-induced natriuresis.
Microtubules are protein polymers that have diverse roles in eukaryotic cells. Through their continuous state of disequilibrium, also known as dynamic instability, they support the cytoskeleton, motility, intracellular transport, and mitosis.1,2 The role of microtubules in renal intracellular transport processes has been demonstrated in vitro in LLC-PK1 cells by Kruse et al,3 wherein functional microtubules were necessary for agonist-induced recruitment of renal dopamine D1 receptors from intracellular sites to the cell surface. When the microtubulin network was depolymerized with the benzimidazole derivative nocodazole (NOC), D1 receptors were not translocated to the cell membrane in response to a D1-like receptor agonist.3 Other studies have shown that microtubules are required for perinuclear positioning of aquaporin-2 after endocytic retrieval in renal principal cells.4 These observations suggested that microtubules may play an important role in intracellular trafficking within the nephron.
cGMP is a second messenger that modulates renal Na+ excretion and blood pressure.5 cGMP is formed from GTP when NO activates soluble guanylyl cyclase, which is present in proximal tubule cells. Lahera et al6 demonstrated that competitively blocking NO leads to antinatriuresis in rats. Previous studies from our laboratory have shown that cGMP is formed intracellularly but must be transported into the renal interstitial (RI) compartment via a probenecid-sensitive organic anion transporter to induce natriuresis in the rat kidney.7,8 Chevalier et al,9 using LLC-PK1 cells in vitro, demonstrated that cGMP may be transported across the plasma membrane into the extracellular environment by a probenecid-sensitive organic anion transporter and that an intact microtubule network is required for this process.
Pressure-natriuresis is defined as a physiological mechanism whereby an acute rise in renal perfusion pressure (RPP) leads to an increase in renal Na+ excretion.10 Recent studies have demonstrated that extracellular cGMP modulates both NO- and pressure-induced natriuresis in vivo.11 Administration of an NO donor or a rise in RPP increased RI cGMP and urine Na+ excretion, both of which were prevented by intrarenal administration of probenecid and restored with exogenous RI cGMP.11
The aforementioned studies demonstrated the importance of RI cGMP in mediating NO- and pressure-induced natriuresis. In the present study, we explored the hypothesis that NO- and pressure-induced natriuresis require an intact microtubulin network in vivo in the rat kidney.
The experiments were approved by the Animal Care and Use Committee of the University of Virginia and were conducted on 12-week–old 225- to 250-g female Sprague-Dawley rats. Rats were housed in a vivarium under controlled conditions (temperature: 21±1°C; humidity: 60±10%; light: 8:00 am to 8:00 pm) and received a normal 0.28% NaCl diet. Experiments were performed at similar times each day to prevent any diurnal variation in blood pressure measurements.
On the day of the experiment, the rats were anesthetized with an IP injection of pentobarbital (5 mg/100 g) or ketamine (60 mg/kg) and xylazine (4 mg/kg; protocols 2 and 3). A catheter (polyethylene 50) was placed in the carotid artery for measurement of mean arterial pressure. A venous catheter (polyethylene 10) was placed in the internal jugular vein for IV infusion of 5% dextrose solution (D5W), inulin, and lithium chloride at 20 μL/min. After a midline incision and unilateral nephrectomy, the remaining ureter was cannulated (polyethylene 10) for urine collection.
Renal Cortical Interstitial Infusion
RI infusions were conducted using an open-bore microinfusion catheter (polyethylene 10) that was inserted underneath the capsule in the renal cortex of the remaining kidney. Vetabond tissue adhesive (3 mol/L, Animal Care products) was added to the site of insertion to prevent the accidental removal of the catheter and interstitial pressure loss in the kidney. The infusion microcatheters were connected to a Harvard pump (model 55-222), and substances were infused directly in the RI space at 2.5 μL/min.
RI Fluid Microdialysis Technique
For determination of RI fluid concentrations of cGMP, we used an RI fluid microdialysis technique that has been developed and successfully used in our laboratory.8,11,12
Measurement of Renal Function
Glomerular filtration rate (GFR) was measured by inulin clearance as published previously.13 Tubular Na+ reabsorption was quantified in selected experiments by fractional excretion of Na+ (FENa), and renal proximal tubule (RPT) Na+ reabsorption was estimated by the fractional excretion of Li+ (FELi).
The standard pressure-natriuresis model of Roman and Cowley14 was used in Sprague-Dawley rats.
cGMP was measured with an enzyme immunoassay (Cayman Chemical), which has been used previously in our laboratory.7,8,11,12
Plasma and Urinary Na+ and Li+ Concentrations
Plasma and urinary Na+ and Li+ concentrations were measured using an Instrumentation Laboratory-943 flame photometer.
Effects of Intrarenal NO Donor Administration With and Without NOC or NOC+cGMP on RI cGMP and Renal Function
Uninephrectomized rats (n=48) were studied over a 4-hour period in a 1-hour control period with vehicle (V) infusion, 2 consecutive 1-hour experimental periods, and a 1-hour postcontrol period with V infusion. Five groups were evaluated: (1) time control where rats (n=8) received V infusion throughout the entire 4-hour length of the study; (2) S-nitroso-N-acetylpenicillamine (SNAP) alone, where rats (n=12) received V infusion during the control period followed by SNAP (0.12 nmol/kg per minute) alone during the 2 consecutive experimental periods; (3) SNAP+NOC, where rats (n=11) received NOC (3 μg/kg per minute) during the 1-hour control period and both SNAP and NOC during the 2 experimental periods; (4) NOC alone, where rats (n=8) received an infusion of NOC alone during all 4 hours of the experiment; and (5) SNAP+NOC+cGMP, where rats (n=9) received NOC in the control period and then an infusion of cGMP (18 μg/kg per minute) together with SNAP and NOC during the 2 experimental periods. All of the RI infusions were at 2.5 μL/min. RI cGMP, urinary sodium excretion (UNaV), GFR, FENa, FELi, and mean arterial pressure were measured during each control and/or experimental period.
Effects of Increased RPP on RI cGMP and UNaV in the Presence and Absence of Intrarenal Administration of NOC or NOC+cGMP
The purpose of this study was to determine whether inhibiting cGMP transport into the extracellular compartment with NOC would reduce RI cGMP and abolish pressure-natriuresis. Rats (n=27) were uninephrectomized and studied using the pressure-natriuresis protocol, which has previously been used in our laboratory.11,12 A catheter was placed in the carotid artery to transduce systolic blood pressures. Renal infusion microcatheters were placed into the renal cortex for infusion of V, NOC, or NOC+cGMP. A renal cortical interstitial microdialysis catheter was placed to sample RI cGMP. High RPP was achieved by adjusting 2 ultra–micro-Blalock clamps on the aorta, one above the superior mesenteric artery and the other below the left renal artery, as published previously. Urine was collected by placing an indwelling ureteral catheter to measure UNaV. Pressure-natriuresis was studied with (n=6) and without RI infusions of NOC (n=14) or NOC+cGMP (n=7).
In Vivo Renal Perfusion Fixation
At the end of the second experimental period, while NOC alone or V was still interstitially infused, the rat heart left ventricular cavity was cannulated, and the animal was perfused with 40 mL of 4% sucrose in Dulbecco’s PBS with calcium and magnesium followed by perfusion with 40 mL of 4% paraformaldehyde in Dulbecco’s PBS. The kidney was removed, bisected, and postfixed by immersion in fresh 4% paraformaldehyde for an additional 2 hours at room temperature. Cortex slices were rinsed in several changes of Dulbecco’s PBS, immersed in 100 mmol/L of Tris for 30 minutes, and then stored in 30% sucrose in Dulbecco’s PBS overnight at 4°C. Sections were embedded in Tissue Tek OCT Compound freezing medium in Cryomold vinyl specimen molds, frozen on liquid nitrogen, and stored at −80°C. Cryostat thin sections (6 to 8 μm) were placed on Probe On Plus positively charged microscope slides (Fisher Scientific). Immediately before staining, an ImmEdge pen (Vector Laboratories) was used to draw a hydrophobic ring around the sections.
Confocal Immunofluorescence Microscopy
After thin sections of cortex had been spotted onto slides and washed with triphosphate-buffered saline (TBS), they were permeabilized with 0.2% Triton x-100 in triphosphate-buffered saline for 5 minutes. The sections were then washed several times with triphosphate-buffered saline with 0.02% Tween 20 (TBST), and then blocked in 1% milk in TBST for 1 hour. The kidney sections were incubated with anti–β-tubulin primary antibody (Oncogene Research Products, Incclone DM1B) at 1:2000 dilution in 1% milk made in TBST overnight at 4°C. After washing with TBST, ALEXA 488 conjugated goat anti-mouse secondary antibody diluted at 1:500 was added for 90 minutes at room temperature. The slides were then washed with TBST. To identify RPTs, the preparation was stained with Texas Red–labeled phalloidin (Invitrogen), which labels actin-containing structures, including proximal tubule cell apical plasma membranes, before being covered with a glass coverslip in fluoromount G. Stained tubules were photographed with a 14-bit Hamamatsu electron multiplying cooled charge-coupled device (C9100-02) on an Olympus IX81 spinning disk confocal microscope using either a 20× UApo water immersion NA 0.70 or a 60× UPlanSApo NA 1.2 water immersion objective. Six-micron z-axis image stacks were captured every 0.3 μm and deconvolved using Slidebook 4.1 software and its integrated deconvolution module.
Results are expressed as mean±SE. Data were analyzed by paired Student’s t test. ANOVA with a repeated-measures term was used for multiple comparisons, and P<0.05 was considered significant.
Intrarenal Disruption of β-Tubulin by NOC
As illustrated in Figure 1A, a low power (×200) photomicrograph demonstrates β-tubulin expression in both proximal and distal tubules of V-infused kidneys. Figure 1B, also at ×200, demonstrates a reduction, but not complete elimination, of the β-tubulin signal predominantly in RPT cells after 3 hours of RI NOC infusion. Figure 1C, a high-power (×600) view of RPT cells from a control kidney, demonstrates a prominent dense network of microtubules within the intracellular compartment. Figure 1D (×600), from an NOC-treated kidney, shows partial disruption of the microtubulin network.
Effects of Intrarenal SNAP With and Without NOC, NOC Alone, or SNAP+NOC+cGMP on RI cGMP and UNaV
As illustrated in Figure 2, SNAP increased RI cGMP from control values of 5.1±0.9 to 8.4±1.7 fmol/min (P<0.05) in the first experimental period and to 10.5±2.3 fmol/min (P<0.05) during the second experimental period, after which RI cGMP returned to baseline levels during the postcontrol period. In contrast, NOC abolished the SNAP-induced increase in RI cGMP (P<0.01 from SNAP alone in the second experimental period). The time control or administration of NOC alone did not influence RI cGMP. RI cGMP coinfusion with SNAP+NOC restored RI cGMP levels to those observed with SNAP alone (P<0.001 from cGMP+SNAP+ NOC control).
As depicted in Figure 3, RI SNAP infusion increased UNaV from 0.046±0.009 to 0.087±0.022 μmol/min (P<0.05) after 1 hour and to 0.105±0.018 μmol/min (P<0.01) at 2 hours of SNAP infusion, followed by a reduction to near-baseline levels (P value not significant from control) during the postcontrol period. In contrast, SNAP failed to increase UNaV (P value not significant) when coinfused with NOC during either experimental period. However, the natriuretic response to SNAP was re-established when cGMP was coinfused with SNAP and NOC (P<0.05). Neither the time control nor NOC alone had any effect on UNaV.
Figure 4 demonstrates that mean arterial pressure was not significantly influenced by any of the RI infusions.
As shown in Figure 5A, GFR was not influenced by RI infusion of SNAP alone, SNAP+NOC, V, or NOC alone. In Figure 5B, SNAP increased FENa from 1.2±0.3% in the control period to 2.9±0.9% (P<0.05) in experimental period 2. NOC alone did not alter FENa but did abolish the SNAP-induced increase in FENa in experimental period 2 (P<0.05 from SNAP). As illustrated in Figure 5C, SNAP increased FELi from 27±3% to 42±7% (P<0.05) in experimental period 1 and to 47±6% (P<0.01) in experimental period 2, followed by a return to baseline levels in the postcontrol period. NOC abolished this response during both experimental periods (P<0.05). V or NOC alone did not affect FELi.
Effects of Increased RPP on RI cGMP and UNaV in the Presence and Absence of Exogenous Intrarenal NOC With or Without cGMP
As shown in Figure 6A and 6C, an increase in RPP from 102.4±2.20 to 156.69±4.40 mm Hg (P<0.00001) increased RI cGMP from 4.57±1.21 to 11.01±1.95 fmol/min (P<0.0001 from control). Increasing RPP from 97.14±3.51 to 155.56±5.27 mm Hg (P<0.00001) during RI infusion of NOC did not lead to a rise in RI cGMP during the high RPP (P value not significant). However, coinfusion of cGMP with NOC led to the rise of cGMP during the high RPP (P<0.0001 from control).
As illustrated in Figure 6B and 6C, the increase in RPP from 97±3.5 to 155±5.3 mm Hg increased the UNaV from 0.047±0.06 to 0.49±0.16 μmol/min (P<0.01). The pressure-natriuresis was completely abolished by intrarenal administration of NOC (P<0.01 from RPP 155±5.3 mm Hg in the absence of NOC). However, coinfusion of cGMP+NOC rescued the natriuretic response to increased RPP (P<0.05 from RPP 97±3.5 mm Hg). As demonstrated in Figure 6C, RI administration of NOC or NOC+cGMP did not alter the increase in RPP as compared with that during V administration.
Extracellular RI cGMP is an important modulator of NO- and pressure-induced natriuresis in the rat kidney in vivo via a direct tubular effect not associated with hemodynamic change.8,11,12 In LLC-PK1 cells, cGMP extrusion into the extracellular environment is inhibited by the organic anion transporter inhibitor probenecid and the microtubule inhibitor NOC.9 In human RPT cells, Na+ transport is inhibited by increasing extracellular cGMP, and this response is abolished when cGMP extrusion is prevented by probenecid.7 In intact animals, both NO- and pressure-induced natriuresis are abolished when cGMP is prevented from being transported into the RI space with probenecid.11 The present study addresses the hypothesis that microtubules participate in cGMP transport from intracellular sites to the region of the basolateral membrane of renal tubule cells, from which organic anion transporters translocate the cyclic nucleotide into the RI compartment modulating natriuresis in vivo.
The major results of this study are as follows: (1) RI infusion of NOC disrupts the microtubulin network of RPT cells; (2) depolymerization of microtubules with NOC inhibits both NO- and pressure-induced increases in RI cGMP and Na+ excretion in parallel; (3) the natriuresis abolished with NOC can be restored by exogenous RI infusion of cGMP; and (4) NOC infused alone does not alter basal Na+ excretion. These results suggest that an intact microtubulin network is required for the movement of cGMP into the interstitial compartment after increased intracellular cGMP formation, where it acts as a modulator of renal Na+ excretion.
The precise mechanism by which cGMP moves through the cell and is transported into the extracellular environment is unknown. Probenecid-sensitive organic anion transporters play an important role in extruding cGMP from the cells where it is formed. However, after intracellular synthesis, whether cGMP binds/interacts directly with microtubules or is packaged in transport vesicles and shunted along microtubules is currently unknown. Other studies have evaluated the role of microtubules in intracellular protein trafficking. Toomre et al15 reported that microtubules are important in protein trafficking from the trans-Golgi network to the cell surface. Kruse et al3 studied intracellular trafficking of dopamine D1 receptors to the cell surface. The receptors were stored in vesicles that were transported to the cell surface.3 One alternative mechanism is that cGMP diffuses along microtubules down a concentration gradient into the extracellular compartment.
NOC is a benzimidazole derivative that was originally developed as a fungicide and antihelminthic drug.16 However, NOC has been used experimentally as a chemotherapeutic agent because of its action on microtubules.16 Benzimidazoles have a similar mechanism of action to that of colchicine; both inhibit microtubules by binding to a major component of microtubules, tubulin. In the present study, we demonstrated that β-tubulin was disrupted, whereas actin filaments were preserved, in RPT cells from NOC-infused kidneys. Although the transport of many molecules that may affect renal Na+ excretion theoretically may have been interrupted by microtubule disruption, the present in vivo studies demonstrating reconstitution of the natriuretic response by exogenous cGMP in the presence of NOC and the failure of NOC alone to alter basal Na+ excretion render this possibility unlikely. The precise manner by which microtubules mediate intracellular trafficking of cGMP has yet to be determined. However, from the present in vivo experiments, an intact microtubulin network seems to be important in transporting cGMP from the intracellular space into the RI compartment.
Similar to other in vivo studies,11 the present study demonstrates that NO-induced natriuresis is accompanied by increases in FENa and FELi, with no change in GFR. Because FELi is associated with a maximal 4% error rate in predicting proximal tubule Na+ transport in volume-replete animals,17 these findings implicate the proximal tubule as the target site for the natriuretic response to NO. However, micropuncture studies will have to be used to validate the proximal tubule as the major site of alterations in Na+ transport in response to NO in our model.
The cellular mechanism whereby extracellular RI cGMP modulates natriuresis is currently unknown. However, a large quantity of the cyclic nucleotide is transported within minutes into the RI compartment in response to NO donor administration or angiotensin type 2 receptor activation.8,18 cGMP extrusion from cells occurs against a concentration gradient, is nonsaturable, and is unidirectional, because extracellular cGMP does not re-enter the cell.7,14,19 On the basis of this information, we speculate that RI cGMP may bind to a tubule basolateral plasma membrane constituent that transmits the signal for inhibition of Na+ transport by as-yet-undiscovered mechanisms.
Our studies suggest that extracellular RI cGMP regulates Na+ excretion in response to NO and increased RPP in the rat. The mechanism whereby cGMP is exported from the intracellular to the extracellular environment depends on an intact microtubule network. We also show that the RPT seems to be a target cell for NO- and pressure-induced natriuresis. The exact mechanisms by which cGMP is exported and regulates Na+ excretion are currently being investigated.
Source of Funding
This work was supported by National Institutes of Health grant RO1-HL-081891 to R.M.C.
- Received October 11, 2007.
- Revision received November 7, 2007.
- Accepted November 20, 2007.
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