Role of Nitric Oxide in the Renal Hemodynamic Response to Unilateral Nephrectomy
David H. Sigmon*,
Edgard Gonzalez-Feldman,
Maria A. Cavasin,
D’Anna L. Potter and
William H. Beierwaltes
*Science Division of Mercy College of Northwest Ohio, Toledo, Ohio; and Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan.
Correspondence to Dr. William H. Beierwaltes, Hypertension and Vascular Research Division, 7121 E & R Building, Henry Ford Hospital, 2799 West Grand Boulevard, Detroit, MI 48202-2689. Phone: 313-916-7494; Fax: 313-916-0524; E-mail: wbeierw1{at}hfhs.org
ABSTRACT. Reduction of renal mass by unilateral nephrectomyresults in an immediate increase in renal blood flow (RBF) tothe remnant kidney, followed by compensatory renal hypertrophy.Whether the increase in RBF after unilateral nephrectomy ismediated by nitric oxide (NO) was tested. It was found thatimmediately after nephrectomy, blood flow to the remaining kidneyincreased by 8% (P < 0.01), and inhibition of NO synthesiswith N-nitro-L-arginine methyl ester (L-NAME) blocked the increasein RBF. In addition, 2 d after nephrectomy, there was a 49%increase in RBF (corrected per gram of kidney weight), a 25%increase at 7 and 14 d, and a 16% increase after 28 d. Acuteinhibition of NO synthesis with L-NAME in uninephrectomizedrats caused a greater decrease in RBF on days 2 and 7 comparedwith controls, whereas by 14 and 28 d, the response to L-NAMEwas similar to controls. Urinary excretion of cyclic guanosinemonophosphate, a marker for renal NO production, increased 2.5-foldby 2 d after uninephrectomy (P < 0.005) and remained at thislevel through 28 d. Pretreating rats chronically with a subpressordose of L-NAME beginning 2 d before nephrectomy blocked theincrease in RBF seen at 2 and 7 d and retarded the renal hypertrophythat should have developed by 7 d. It is concluded that afterunilateral nephrectomy, immediate and sustained increases inRBF are mediated at least in part by NO. The hypertrophic responseto unilateral nephrectomy may be partially initiated by thesignal of hemodynamic changes.
Nitric oxide (NO) is a vasodilator that contributes to the regulationof regional blood flow and BP by tonically lowering vascularresistance (1,2). This effect is mediated through activationof soluble guanylate cyclase, which increases cellular cyclicguanosine monophosphate (cGMP), resulting in vascular relaxationand decreased vascular resistance (3). In the kidney, continuousrelease of NO derived from the vascular endothelium is an importantdeterminant of basal perfusion and resistance. Release of NOcan be stimulated by changes in the hydromechanical forces associatedwith pulsatile blood flow (4–6). Vascular shear stressis a primary stimulus for endogenous NO production from theendothelium (7–10). Miller et al. (7,8) demonstrated invitro that chronic alterations in local blood flow, producedby opening an arteriovenous anastomosis, increased endothelium-dependentrelaxation. Increasing coronary blood flow by chronic cardiacpacing or exercise also enhanced endothelium-dependent dilatation(9,10). Although the mechanism responsible for flow-induceddilatation is not completely understood, there is considerableevidence that it is mediated, in part, by NO (11–15).Removing the endothelium or inhibiting NO synthesis blocks flow-induceddilation (13,15). Studies in dogs have demonstrated that chronicexercise enhances the release of nitrites from the coronaryarteries and microvessels, accompanied by increased blood flow,and this is associated with increased expression of the endothelialisoform of NO synthase (eNOS) (16).
After a reduction in renal mass, the kidney undergoes both hemodynamicand structural changes. The hemodynamic changes involve increasedrenal blood flow (RBF) and decreased renal vascular resistance(RVR) (17,18). The structural adaptation to unilateral nephrectomyis hypertrophy of the remaining kidney. Valdivielso et al. (19)found that 2 d after unilateral nephrectomy, RBF was significantlyhigher in the remaining kidney compared with controls. NOS inhibitionwith N-nitro-L-arginine methyl ester (L-NAME) resulted in agreater decrease in RBF than in controls, although BP increasedsimilarly in both. They suggested that NO may play an importantrole in mediating the hemodynamic changes associated with reducedrenal mass. We investigated the importance of NO in mediatingthe immediate and early changes in renal hemodynamics seen aftera reduction in renal mass by unilateral nephrectomy. We hypothesizedthat after unilateral nephrectomy, NO-mediated flow-inducedvasodilation initiates compensatory dilation in the remainingkidney, which could be part of the signal to initiate contralateralrenal hypertrophy. We propose that NO-mediated vasodilationis an initial adaptive step before the development of renalhypertrophy.
General Methods: Acute Hemodynamic Measurements
Rats fasted for 24 h before the experiments. On the day of theexperiment, they were anesthetized by intraperitoneal injectionof 125 mg/kg body wt thiobutabarbital (Inactin; Research Biochemical,Natick, MA) and placed on a heating pad to maintain constantbody temperature. A tracheotomy was performed using PE-260 tubing(Fisher Scientific, Chicago, IL) for spontaneous breathing ofroom air. Then the femoral artery was catheterized using PE-50tubing to monitor femoral BP directly using a Statham pressuretransducer (Viggo-Spectramed, Oxnard, CA). The femoral veinwas catheterized using PE-50 tubing for an initial supplementaladministration of 1 ml 6% heat-inactivated BSA, a constant maintenanceinfusion of physiologic saline at 40 µl/min, and administrationof drugs. The (remaining) left kidney was exposed through anabdominal incision; the renal artery was separated from therenal vein and fitted with a noncannulating 2.0-mm electromagneticflow probe connected to an electromagnetic flow meter (CarolinaMedical Electronics, King, NC). The probe was calibrated invivo as described previously (20). Absolute zero flow throughthe renal artery was determined by occluding it distal to theprobe using shielded hemostats. The pressure transducer andflow meter were connected to a Gould recorder (Valley View,OH) for simultaneous recording of RBF and BP. Renal vascularresistance was calculated as the ratio of systemic BP to RBF,resulting in units of mmHg/ml per g kidney wt, hereafter referredto as resistance units (RU). Our institutional animal care anduse committee approved the experimental protocols, and experimentswere carried out in accordance with the NIH Guide for the Careand Use of Laboratory Animals. The experiments were dividedinto three groups as described below.
Role of NO in Renal Hemodynamic Responses to Acute Unilateral Nephrectomy.
For assessing the role of NO in mediating the immediate renalhemodynamic response in the left kidney to nephrectomy of theright kidney, 16 male Sprague-Dawley rats that weighed 225 to250 g were prepared as described above. Before the left renalartery was fitted with the electromagnetic flow probe, a 3-0silk ligature was placed around the right renal artery, renalvein, and ureter at the hilus of the right kidney. After theprobe was fitted onto the left renal artery, rats were alloweda 30-min recovery period, during which BP and RBF were recorded.After this period or when BP and RBF had stabilized, a 15-minbaseline value was recorded. Then the ligature around the rightkidney of eight rats was tied to occlude blood flow, and thekidney was decapsulated and removed. Blood flow to the undisturbedleft kidney was monitored continuously for 15 min after nephrectomy.When RBF to the remaining left kidney was stable and hemodynamicmeasurements were taken, the rats were treated with 10 mg/kgL-NAME to block NOS (20–22) and monitored for an additional15 min. We have used this acute renal response to NOS inhibitionas a bioassay to reflect the influence of NO on BP and renalhemodynamics (20–22).
A second group of eight rats were treated with L-NAME 15 minbefore removal of the right kidney. Renal hemodynamics and BPwere monitored in the left kidney for 15 min after nephrectomy.At the conclusion of either protocol, rats were killed and theleft kidney was decapsulated, excised, and weighed.
Role of NO in the Evolving Renal Hemodynamic Response to Unilateral Nephrectomy over 4 Weeks.
For assessing the influence of NO on systemic and renal hemodynamicresponses to unilateral nephrectomy, male Sprague-Dawley ratsthat weighed 225 to 250 g were anesthetized with sodium pentobarbital(Nembutal; Abbott Laboratories, Chicago, IL). Day 0 controls(n = 8) had both kidneys intact, and acute hemodynamic measurementsof the left kidney were taken. In the experimental groups, aright flank incision was made using sterile technique and theright kidney was removed as described above. The wound was closed,and the rats were allowed to recover for 2 (n = 8), 7 (n = 8),14 (n = 7), or 28 d (n = 8) before a second, acute terminalprocedure was run. Rats fasted for 24 h before the acute experimentat each time point. On the day of the experiment, rats wereprepared as described above. After the flow probe was placedon the left renal artery, the rats were allowed a 30-min recoveryperiod, during which BP and RBF were recorded. After this periodor when BP and RBF had stabilized, a 15-min baseline recordingwas obtained. Next, the rats were given a full 10-mg/kg blockingdose of L-NAME. Fifteen minutes later, after BP and RBF werestable, an additional 15-min experimental time point was recorded.At the conclusion of the experiment, rats were killed and theleft kidney was excised and weighed.
A separate group of rats were prepared as described above andplaced in metabolic cages at 0, 2, 7, 14, or 28 d (n = 8 foreach group) after unilateral nephrectomy. A 24-h urine samplewas collected to determine urinary cGMP excretion as an indexof renal NO production (23,24). Each vial contained 1 ml of1 mM isobutylmethylxanthine to prevent cGMP degradation. UrinarycGMP was determined by ELISA using a commercially availablekit (R&D Systems, Minneapolis, MN). cGMP excretion is presentedas both excretion and excretion corrected by kidney weight.
Response to Nephrectomy after Chronic NOS Inhibition.
For determining how chronic NOS inhibition would affect therenal hemodynamic response to unilateral nephrectomy, 13 ratswere pretreated for 4 (n = 6) or 9 d (n = 7) with a subpressorregimen of 1 mg/kg per d L-NAME in drinking water. After 2 don L-NAME, the right kidney was removed as described above andthe rats were allowed to recover for 2 or 7 d, still receivingL-NAME. These periods were chosen because they represented themaximum changes in RBF and hypertrophy (respectively) obtainedfrom the previous protocols. On the day of the acute experiment,rats were prepared as described above. After the flow probewas placed on the left kidney, rats were allowed a 30-min recoveryperiod, during which BP and RBF were recorded. After this period,BP and RBF were monitored for 15 min. Rats were then given acomplete blocking dose of 10 mg/kg L-NAME as a bioassay to determinethe degree of NO synthesis inhibition after chronic subpressorL-NAME treatment. BP and RBF were monitored for 15 min or untilthe parameters had stabilized, and post–L-NAME valueswere recorded. At the conclusion of the protocol, rats werekilled and the left kidney was decapsulated, excised, and weighed.
Evaluation of the Cortical Total Protein:DNA Ratio.
Because the primary site of cortical hypertrophy in responseto unilateral nephrectomy is reported to be the proximal tubules(25), we measured the ratio of total protein to DNA in a preparationof proximal tubules. An increase in this ratio is reportedlyan index of hypertrophy. Proximal tubules were isolated usinga Percoll gradient. The cortex was harvested from right or leftkidneys using a Stadie-Riggs microtome and placed in ice-coldPBS containing the protease inhibitors aprotinin (2 µg/ml),pepstatin A (2 µg/ml), leupeptin (5 µg/ml), andPMSF (0.1 mg/ml). Slices were washed three times with Prep-Media(50:50 mixture of low-glucose DMEM and Ham’s F12; LifeTechnologies BRL), suspended in 10 ml of 95% O2/5% CO2 pre-equilibratedwith Prep-Media containing 1.8 mg/ml collagenase B (BoehringerMannheim) and incubated in a 37% shaking water bath for 30 min.After collagenase digestion, 10 ml of ice-cold Prep-Media wasadded to the tissue suspension to stop the digestion and kepton ice continuously. The digested suspension was filtered througha double layer of gauze, then centrifuged at 800 rpm for 30s and washed twice with PBS. After the final wash, the pelletwas resuspended in 6 ml of cold 45% Percoll (3 M NaCl, 154 MKCl, 200 mM KH2PO4, 155 mM MgSO4, 110 mM CaCl2 pre-equilibratedwith 95% O2/5% CO2) and centrifuged at 14,000 rpm for 40 minat 4°C. The separated bottom band was carefully withdrawnusing a Pasteur pipette, placed in 10 ml of PBS, and centrifugedat 800 rpm for 40 s at 4°C; the supernatant was discarded;and the tubules were rewashed. After the final wash, the pelletwas resuspended in 1 ml of hypotonic lysis buffer (50 mM NaH2PO4[pH 7.4]) and sonicated on ice for 5 s x 4. The resulting suspensionwas used to measure both DNA and protein content.
Total protein was determined by the Bradford method (26). DNAwas quantified using a Pico Green assay kit (Molecular Probes,Eugene, OR) according to published instructions. Briefly, 250µl of the proximal suspension was mixed with 250 µlof TE buffer (Tris-HCl and EDTA [pH 7.5]) and 500 µl ofPico Green reagent in DMSO. The reagents were mixed and incubatedfor 5 min, after which samples were read in a fluorometer (excitation480 nm, emission 520 nm).
Determination of Glomerular and Proximal Tubule Size by Histology.
Histology of the renal cortex was performed on cortical tissuefixed in 10% neutral buffered formalin and sectioned for stainingwith periodic acid-Schiff. Cortical surface area of glomeruliand proximal tubules (in µM2) was measured in the rightkidneys removed on day 0 and the remaining left kidneys 7 dafter uninephrectomy, with or without chronic L-NAME treatment.Outer circumferential measurements were taken of the anatomicalstructures from which total surface area was calculated using"Spot Advanced" imaging software (V3.4.2) from Diagnostic Instruments(Sterling Heights, MI). Glomeruli were identified by structuralcriteria, and proximal tubules were recognized by the presenceof brush borders. For proximal tubules, tubular lumenal circumferenceswere also measured, and these values were subtracted from thetotals to provide measurements of only the wall areas. Analysiswas performed by measuring this cross-sectional surface areaof all complete proximal tubules and glomeruli within 18 sequentialbut nonoverlapping fields from each kidney. Analysis of theright and left kidneys from the same rat was paired. Other thansize, there were no apparent gross histologic differences betweengroups.
Statistical Analyses
Analysis was designed and carried out by the Department of Biostatisticsand Epidemiology at Henry Ford Hospital. Acute procedures weretested using paired t tests. In the chronic procedures, differencesbetween groups were tested using one-way ANOVA, followed bya comparison of all groups beyond day 0, compared back to 0using Dunnett’s procedure. All pair-wise comparisons betweendays 2 and 28 were tested using Hochberg’s method of multiplecomparisons (27) and P values adjusted for the constraints ofmultiple testing.
Role of NO in the Acute Renal Hemodynamic Response to Unilateral Nephrectomy
Rats had a basal BP of 104 ± 5 mmHg, RBF of 8.3 ±0.2 ml/min per g kidney wt, and RVR of 12.7 ± 0.6 RU.Unilateral nephrectomy had no effect on basal BP but increasedRBF by 8% (to 9.0 ± 0.2 ml/min per g kidney wt; P <0.01) and decreased RVR by 8% (to 11.8 ± 0.5 RU; P <0.01; Figure 1). After acute removal of the right kidney, L-NAMEsignificantly increased BP by 24 ± 3 mmHg (to 128 ±6 mmHg; P < 0.001), decreased RBF by 26% (to 6.57 ±0.32 ml/min per g kidney wt; P < 0.001), and increased RVRby 69% (to 19.9 ± 1.0 RU; P < 0.001), similar to theresponses to L-NAME we previously reported in rats with bothkidneys intact (20–22).
Figure 1. Immediate changes in renal blood flow (RBF) and renal vascular resistance in the remaining kidney after unilateral nephrectomy in controls and rats pretreated acutely with 10 mg/kg body wt N-nitro-L-arginine methyl ester (L-NAME). All data are mean ± SEM. *P < 0.01 versus basal.
In rats that were given L-NAME before nephrectomy, basal BPwas 103 ± 4 mmHg, RBF was 8.6 ± 0.3 ml/min perg kidney wt, and RVR was 12.3 ± 0.9 RU. L-NAME significantlyincreased BP by 28 mmHg (to 131 ± 6; P < 0.01), decreasedRBF by 26% (to 6.4 ± 0.3 ml/min per g kidney wt; P <0.01), and increased RVR by 72% (to 21.1 ± 1.7 RU; P< 0.01). Removal of the right kidney in L-NAME–treatedrats produced an additional 7 ± 1 mmHg increase in BP(P < 0.001) but had no significant effect on either RBF orRVR (Figure 1).
Role of NO in the Evolving Renal Hemodynamic Response to Unilateral Nephrectomy over 4 Weeks Table 1 shows basal BP, RBF, and RVR in controls (both kidneysintact) and rats whose right kidney was absent for 2, 7, 14,or 28 d before study. Unilateral nephrectomy did not significantlyalter BP in any group during the 28-d study period. As seenin Figure 2, RBF (normalized by remnant kidney weight) was increasedby 49% (P < 0.002) 2 d after nephrectomy. This increase wasdiminished to 25% above control at 7 and 14 d (P < 0.042),and by 28 d had dropped to control levels. RBF (per g kidneywt) at 28 d was significantly less than at 2 d (P < 0.002).Unilateral nephrectomy decreased RVR by 38% at day 2 (P <0.003), but this decrease dropped to 21% by day 7, 28% at day14 (both P < 0.003), and by day 28 was no different fromcontrol. This suggests that the trend for basal RBF to returnto control levels on days 7, 14, and 28 is related to coincidentrenal hypertrophy, because absolute RBF (not corrected by kidneyweight, in ml/min) was increased at all time points comparedwith control (P < 0.05), although it was similar in all postnephrectomytime periods. The remnant kidney weight was no different fromcontrol at day 2 but significantly greater (28%; P < 0.025)by 7 d (Figure 2, Table 1) and remained proportionately larger(when normalized by body weight) through 28 d.
Figure 2. Absolute RBF (top), (remaining) left kidney weight (middle), and RBF corrected by kidney weight (bottom) in controls and rats whose right kidney was removed 2, 7, 14, or 28 d earlier. All data are mean ± SEM. *Significant difference from controls.
A bioassay of acute NOS inhibition with a bolus blocking doseof L-NAME was used to evaluate the contribution of NO to BPand renal perfusion in each animal at each time point afterthe basal measurements (Figure 3). L-NAME resulted in a similarincrease in BP (26 to 35 mmHg) in controls and all experimentalgroups. Figure 3 also shows the decrease in RBF in responseto acute NOS inhibition at each time point. Post–L-NAMEvasoconstriction in the remaining kidney increased to 166% ofcontrol (P < 0.003) by day 7, then receded to control levelsby day 28. Urinary cGMP excretion, another indicator of renalNO production (23), was 29.3 ± 4.2 nM/24 h in the controls.Two days after nephrectomy, it was 30% higher (to 37.9 ±4.1 nM/24 h). By 7 d, it had increased by 63% (P < 0.04)to 47.8 ± 4.8 nM/24 h and remained near or slightly belowthis level at 14 (45.5 ± 3.2 nM/24 h; P < 0.04) and28 d (43.5 ± 4.9 nM/24 h; P < 0.04). When urinarycGMP was corrected for the variable of changing kidney weight,under control conditions excretion was 9.4 ± 1.3 nM/24h per g kidney wt and after uninephrectomy increased significantlyby 2.5 times after 2 d to 23.7 ± 2.6 nM/24 h per g kidneywt (P < 0.005) and remained at this high level throughoutall subsequent time periods (23.9 ± 2.4, 22.8 ±1.6, and 19.3 ± 2.2 at 7, 14, and 28 d, respectively;P < 0.01 versus control).
Figure 3. Changes in BP and RBF induced by nitric oxide synthase (NOS) inhibition with 10 mg/kg body wt L-NAME in controls and in rats whose right kidney was removed 2, 7, 14, or 28 d earlier. All data are mean ± SEM. *Significant difference from controls.
Response to Nephrectomy after Chronic Subpressor NOS Inhibition
For determining the importance of NO in either the renal vasodilatoror hypertrophic response to unilateral nephrectomy, renal hemodynamicsand kidney weight were determined after 2 and 7 d in rats chronicallytreated with a subpressor dose of L-NAME. At day 2, L-NAME–treatedrats had a BP of 118 ± 2 mmHg, not significantly higherthan untreated controls. RBF at 2 d was 8.2 ± 0.4 ml/minper g kidney wt, similar to controls but only 72% of RBF inuntreated rats 2 d postnephrectomy (11.3 ± 0.9 ml/minper g kidney wt; P < 0.001; Figure 4), whereas RVR was 27%greater than in untreated at 2 d (14.6 ± 0.6 RU; P <0.001). Chronic treatment with low-dose L-NAME did not significantlyalter either RBF or RVR compared with controls (Table 1, Figure 4).The remaining kidney did not grow during chronic L-NAMEtreatment (1.24 ± 0.03 g). In rats previously treatedwith a subpressor dose of L-NAME, a single bolus of 10 mg/kgincreased BP by only 10 mmHg (to 128 ± 2 mmHg), decreasedRBF by only 11%, and increased RVR by 25%. These are only approximatelyone third of the changes that we observed in untreated rats2 d after uninephrectomy.
Figure 4. Effect of chronic subpressor treatment with L-NAME on renal hemodynamics and remaining kidney weight 2 and 7 d after unilateral nephrectomy. All data are mean ± SEM. *Significant difference from controls.
As a further index of hypertrophy in untreated controls, glomerulararea increased by 24% (P < 0.001) and proximal tubular areaincreased by 16% (P < 0.001) over the 7 d after uninephrectomy(Table 2). Ratios of proximal tubule total protein to DNA forright kidneys at day 0 and left kidneys at day 7 were no different(9.46 ± 0.82 at day 0 versus 9.80 ± 0.70 at day7; ratios are divided by a factor of 1000).
Table 2. Area of renal glomeruli and proximal tubules in uninephrectomized right kidneys and remaining left kidneys 7 days after surgery, with or without chronic L-NAME treatment
Seven days after uninephrectomy, rats treated chronically withL-NAME had a BP of only 104 ± 6 mmHg, no different fromday 0 (105 ± 3 mmHg). RBF was 6.42 ± 0.51 ml/minper g kidney wt, which was no different from day 0 but 32% lessthan RBF in untreated rats 7 d after uninephrectomy (P <0.005). RVR was 16.8 ± 1.6 RU, 48% greater than in untreatedrats at 7 d (P < 0.025) but no different from controls. Thus,chronic treatment with low-dose L-NAME did not significantlyalter either RBF or RVR. In addition, the weight (or weightnormalized by body weight) of the remaining kidneys from L-NAME–treatedrats (1.45 ± 0.03 g or 0.42 ± 0.01 g/100 g bodywt) was no different from day 0 controls (Table 1, Figure 4)and significantly less than hypertrophied kidneys from untreatedrats 7 d after uninephrectomy (P < 0.005). In L-NAME–treatedrats, glomerular size remained unchanged over 7 d, whereas proximaltubular area increased by 7% (P < 0.05), less than half thatin the untreated kidneys (Table 2). In proximal tubules isolatedfrom L-NAME–treated rats, there was a slight but significantincrease in the protein/DNA ratio (10.28 ± 0.56 at day0 versus 11.57 ± 0.51 at day 7).
Our results suggest that NO may play an important role in thesequential renal hemodynamic response to unilateral nephrectomy.We found that the immediate increase in flow to the remainingkidney was blocked by acute NOS inhibition, whereas the increasein RBF seen after 2 and 7 d was also eliminated by chronicallytreating rats with a subpressor dose of L-NAME. In addition,data from our bioassay of acute complete NOS inhibition suggestthat the renal response at days 2 and 7 postnephrectomy wasexaggerated but the systemic response was not. These data suggestthat NO plays an important role in mediating the increased flowto the remaining kidney, primarily during the initial week.Finally, we found that the NO-mediated increase in RBF precededmeasurable hypertrophy of the remaining kidney, but this growthcould be greatly attenuated by subpressor administration ofL-NAME. The hypertrophic response to unilateral nephrectomymay be secondary to these hemodynamic changes.
It is well documented that NO plays an important role in regulatingsystemic and renal hemodynamics. Acute inhibition of NO synthesisresults in increased BP and RVR and decreased RBF (20–22).It has been suggested that 2 d after unilateral nephrectomy,the hemodynamic response in the remaining kidney may be mediatedby NO. Valdivielso et al. (19) reported that acute NOS inhibition2 d after unilateral nephrectomy caused a similar systemic pressorresponse compared with controls, but they found an exaggerateddecrease in RBF and increased RVR in the remnant kidney, similarto what we observed at both 2 and 7 d. They also demonstratedan increase in NOS expression and activity, nitrate production,and cGMP in isolated glomeruli from the remaining kidney 2 dafter uninephrectomy, confirming earlier reports that cGMP andguanylate cyclase activity (but not cAMP) in the remaining kidneyincreases 2 to 300% in the first 2 d after unilateral nephrectomy(28). Overall our data, as well as Valdivielso’s, suggestthat NO-mediated renal vasodilation contributes to the adaptationof blood flow to unilateral nephrectomy, beginning immediatelyand becoming even more prominent during the first 7 d afternephrectomy.
Beyond the initial week, absolute RBF remained elevated, butwhen corrected by kidney weight, this difference became similarto prenephrectomy levels. In addition, the acute renal pressorresponse to NOS inhibition returned toward the control response,all suggesting an adaptation of perfusion that is no longerdependent on elevated NO. This is similar to Griffen’sreport that the renal response to NOS inhibition 3 to 4 wk afterunilateral nephrectomy was no different from controls (29).Despite our data suggesting a role for NO mediating renal vasodilationin the adaptation to uninephrectomy, we cannot rule out thatthe vasodilation alone and not specifically NO mediation ofthis phenomenon is the underlying signal for these adaptationsby the kidney. Although it is clear that there are changes inthe renal production of NO, other vasodilator systems mightalso come into play to mediate renal adaptation, especiallyin the long-term regulation of RBF and even hypertrophy. Althoughthis is certainly an interesting alternative explanation ofrenal NO, the ability to test such possibilities is beyond thescope of the current protocols.
In compensatory renal growth, the proximal tubule and collectingtubule both undergo hypertrophy, but the enlargement in theproximal tubule is greater than in any other renal cell type.Thus, the proximal tubule is considered the primary target forthe hypertrophic stimulus (30). As the extent of such growthis limited, renal epithelial cell growth must be regulated;however, the initiating or regulating mechanisms or signalsthat control such growth are unknown. Compensatory growth seemsto be totally independent of the need for an intact nerve supply(31). Although the ratio of proximal tubular total protein toDNA did not increase in our studies, we observed a significantincrease in both proximal tubule and glomerular size in theremaining kidney 7 d after uninephrectomy, consistent with theoverall increase in kidney size. These changes were greatlyattenuated by subpressor partial inhibition of NOS. Preisig(30) has reviewed the process by which hypertrophic growth stopsin the growth phase of the cell cycle and does not progressto DNA duplication and mitosis. Although compensatory enlargementof the residual nephrons is considered to compensate for nephronloss, tubular atrophy, interstitial scarring, and progressivedeterioration of renal function eventually ensue. The progressiveloss of renal function associated with prolonged hypertrophyis related to interstitial expansion as a result of invasionby inflammatory cells or synthesis and expansion of the extracellularmatrix (32,33). However, these are long-term changes and arenot considered a factor in the time frame studied here. Ourdata suggest that compensatory hypertrophy results in a newequilibrium between elevated renal mass and perfusion.
Hypertrophy of the remaining kidney in response to unilateralnephrectomy was not measurable by 2 d and thus seems to followNO-mediated renal vasodilation, reaching its peak by 7 d. Thebiggest change in kidney size came between 2 and 7 d, whereasthe gradual increases from 7 to 28 d after nephrectomy wereconsistent with increasing body weight. We also found significantincreases in both proximal tubule and glomerular size at 7 d.It is not clear why the protein to DNA ratio did not reflectthese patterns of growth as it is reportedly an index of hypertrophy(25). Possibly it is not as good an index in vivo as in culture.Surprising is that we observed that subpressor doses of theNOS inhibitor L-NAME not only eliminated increased RBF at 2and 7 d but also greatly attenuated the renal hypertrophic responseseen in untreated rats at 7 d. It is not obvious from our datahow NOS inhibition may retard the hypertrophic response in theremaining kidney or which isoforms of NOS might be involved(19). This could be related to some signaling pathway initiatedby either the increased RBF or increased NO production associatedwith the 7-d postnephrectomy period or a combination of thesefactors. It does not seem to be related to BP or renal perfusionpressure, as subpressor chronic administration of L-NAME didnot significantly alter BP by 7 d.
The vasodilation induced by NO is produced by activation ofsoluble guanylate cyclase and the resultant increase in cellularcGMP. Elevation of smooth muscle cGMP results in vascular relaxationand decreased vascular resistance. It has been suggested thaturinary cGMP serves as an index of renal NO production (24).The increased role of NO in mediating the hemodynamic changesassociated with unilateral nephrectomy is further supportedby the urinary cGMP values. We found that absolute urinary cGMPexcretion increased from day 0 to day 7, consistent with theobservation that both renal cGMP and guanylate cyclase activitywere increased 2 d after unilateral nephrectomy (28) and withWight et al. (24), who found an increase in urinary cGMP excretionafter unilateral nephrectomy that was blocked by L-NAME. Inaddition, Schlondorff and Weber (34) reported that after unilateralnephrectomy, there was an increase in the (NO-mediated) particulateform of guanylate cyclase. When we corrected the cGMP excretionby the changing kidney weight, we found that cGMP increasedcompared with controls by 2.5-fold at 2 d and remained similarlyelevated at all subsequent time periods. In comparison withthe uncorrected values, these data suggest an increased rolefor NO in the total and sustained adaptation to unilateral nephrectomy.Collectively, these findings suggest that the increased production(and excretion) of cGMP after unilateral nephrectomy is dueto an increase in NO, which is consistent with the exaggeratedrenal response to L-NAME. Although the endothelial isoform ofNOS is largely considered to be the most important in regulationof renal vascular resistance, Valdivielso et al. (19) demonstratedthat in the glomerulus, iNOS rather than eNOS may be upregulatedafter unilateral nephrectomy. Although increased RBF shoulddirectly activate eNOS in the endothelium by increasing shearstress, the relative contribution of the different NOS isoformscannot be addressed by our data.
In conclusion, NO synthesis and NOS expression may be modulatedby a number of factors, including shear stress. After unilateralnephrectomy, there is an increase in renal perfusion and presumablyshear stress, resulting in flow-mediated renal vasodilationthat is seems to be NO dependent. This is supported by our findingthat the increase in RBF after unilateral nephrectomy was blockedby NOS inhibition. In addition, there is a greater renal responseto L-NAME after unilateral nephrectomy up to day 7, which correlateswell with the levels of urinary cGMP. These observations suggestthat the renal hemodynamic changes associated with unilateralnephrectomy are mediated at least in part by NO. The hypertrophicresponse to unilateral nephrectomy seems to be secondary tothe renal hemodynamic changes, but these changes may or maynot be the result of altered NO metabolism.
Acknowledgments
This work was supported in part by grant HL28982 from the NationalInstitutes of Health and also by a grant from the American HeartAssociation, Michigan Affiliate.
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Received for publication July 21, 2001.
Accepted for publication March 3, 2004.
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Abstract
Introduction
-nitro-L-arginine methyl ester (L-NAME) blocked the increase in RBF. In addition, 2 d after nephrectomy, there was a 49% increase in RBF (corrected per gram of kidney weight), a 25% increase at 7 and 14 d, and a 16% increase after 28 d. Acute inhibition of NO synthesis with L-NAME in uninephrectomized rats caused a greater decrease in RBF on days 2 and 7 compared with controls, whereas by 14 and 28 d, the response to L-NAME was similar to controls. Urinary excretion of cyclic guanosine monophosphate, a marker for renal NO production, increased 2.5-fold by 2 d after uninephrectomy (P < 0.005) and remained at this level through 28 d. Pretreating rats chronically with a subpressor dose of L-NAME beginning 2 d before nephrectomy blocked the increase in RBF seen at 2 and 7 d and retarded the renal hypertrophy that should have developed by 7 d. It is concluded that after unilateral nephrectomy, immediate and sustained increases in RBF are mediated at least in part by NO. The hypertrophic response to unilateral nephrectomy may be partially initiated by the signal of hemodynamic changes. 
30% higher (to 37.9 ± 4.1 nM/24 h). By 7 d, it had increased by 63% (P < 0.04) to 47.8 ± 4.8 nM/24 h and remained near or slightly below this level at 14 (45.5 ± 3.2 nM/24 h; P < 0.04) and 28 d (43.5 ± 4.9 nM/24 h; P < 0.04). When urinary cGMP was corrected for the variable of changing kidney weight, under control conditions excretion was 9.4 ± 1.3 nM/24 h per g kidney wt and after uninephrectomy increased significantly by 2.5 times after 2 d to 23.7 ± 2.6 nM/24 h per g kidney wt (P < 0.005) and remained at this high level throughout all subsequent time periods (23.9 ± 2.4, 22.8 ± 1.6, and 19.3 ± 2.2 at 7, 14, and 28 d, respectively; P < 0.01 versus control). 
and its receptors in the pathogenesis of diabetic nephropathy. Kidney Int Suppl 60: S7–S11, 1997[Medline]
