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NDT Advance Access published online on August 26, 2008
Nephrology Dialysis Transplantation, doi:10.1093/ndt/gfn483
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© The Author [2008]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Renal endothelial function and blood flow predict the individual susceptibility to adriamycin-induced renal damage

Peter Ochodnicky*, Robert H. Henning, Hendrik Buikema, Alex C. A. Kluppel, Marjolein van Wattum, Dick de Zeeuw and Richard P. E. van Dokkum

Department of Clinical Pharmacology, University Medical Center Groningen (UMCG) and Graduate School for Drug Exploration (GUIDE), University of Groningen, The Netherlands

Correspondence and offprint requests to: Peter Ochodnicky, Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University Bratislava, Slovak Republic, Odbojarov 10, 832 32 Bratislava, Slovak Republic. Tel: +421-2-50117-376; Fax: +421-2-50117-100; E-mail: ochodnicky{at}fpharm.uniba.sk



   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Background. Susceptibility to renal injury varies among individuals. Previously, we found that individual endothelial function of healthy renal arteries in vitro predicted severity of renal damage after 5/6 nephrectomy. Here we hypothesized that individual differences in endothelial function in vitro and renal perfusion in vivo predict the severity of renal damage in a model of adriamycin-induced nephropathy.

Methods. In three separate studies, the following baseline parameters were measured in healthy male Wistar rats: (1) acetylcholine (ACh)-induced relaxation in small renal arteries in vitro (n = 16) and the contribution of prostaglandins, nitric oxide (NO) and endothelium-dependent hyperpolarizing factor (EDHF) to the relaxation; (2) glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) in spontaneously voiding rats in vivo (n = 16) and (3) the acute effect of the NO-synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, n = 12) on renal blood flow (RBF) as compared to vehicle infusion (n = 9). Following these measurements, adriamycin (1.75 mg/kg i.v.) was injected and subsequent renal damage after 6 weeks was related to the baseline parameters.

Results. Total ACh-induced (r = 0.51, P < 0.05) and EDHF-mediated relaxation (r = 0.68, P < 0.05), as well as ERPF (r = 0.66, P < 0.01), positively correlated with the severity of proteinuria 6 weeks after injection. In contrast, pronounced baseline NO-mediated dilation was associated with lower proteinuria (r = 0.71, P < 0.01). Nevertheless, an acute L-NAME infusion, strongly reducing RBF by 22 ± 8%, during adriamycin administration provided protection against the development of proteinuria.

Conclusions. Individual animals with pronounced baseline endothelial dilatory ability measured in vitro and high ERPF in vivo are vulnerable to renal damage after the adriamycin injection. Acute inhibition of NO during adriamycin administration, resulting in a decrease of RBF, protects against renal injury, probably by limiting the delivery of the drug to the kidney. Therefore, interindividual variability in renal haemodynamics might be crucially involved in susceptibility to nephrotoxic renal damage.

Keywords: adriamycin nephrosis; endothelial function; nitric oxide; predictive value proteinuria; renal blood flow



   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
The development and progression of chronic renal damage is largely variable among individuals both in experimental and clinical settings. Environmental systemic factors, such as severity of diabetes or hypertension, cannot fully explain this variation, suggesting that some individuals might be intrinsically predisposed to develop renal impairment [1]. Several specific animal strains spontaneously develop renal function loss [2,3], indicating that predisposition of an individual to renal damage involves genetically conditioned factors [4]. However, variable susceptibility to renal damage could also be observed among individuals within a given animal strain. For instance, after a standardized nephrotoxic challenge, such as 5/6 nephrectomy (5/6Nx), outbred Wistar rats develop renal impairment of highly variable severity [5]. Seeking for the factors responsible for this variability, we previously observed that in vitro measured endothelium-dependent dilatory capacity of small renal arteries in healthy Wistar rats predicts the severity of subsequent renal damage inflicted by 5/6Nx [6]. This indicates that intrinsic variability in renal vascular function might be responsible for variable susceptibility to renal injury. However, at present it is unclear whether this finding is specific for haemodynamically induced renal impairment, such as that seen in 5/6Nx, or whether variability in renal vascular function might also be involved in other types of renal injury.

Therefore, in the present study, we investigated whether the concept of the predictive value of renal endothelial function is valid in a model of nephropathy induced by the nephrotoxic drug adriamycin. In adriamycin nephropathy, a single injection of cytostatic agent adriamycin leads to progressive renal damage with proteinuria, tubulointerstitial damage and glomerulosclerosis [7,8]. Remarkably, this relatively uniform challenge (standard adriamycin injection) results in largely variable renal damage among individuals [9,10], indicating that some individual rats are more susceptible to adriamycin challenge than others. To elucidate the factors responsible for this variability, we measured total endothelium-mediated and specific endothelial mediators (e.g. nitric oxide: NO, endothelium-dependent hyperpolarizing factor: EDHF, prostaglandins: PGs)-dependent relaxation of small intrarenal arteries prior to the administration of adriamycin and related this in vitro vascular reactivity to the severity of subsequent renal damage. Endothelial mediators, such as endothelial nitric oxide synthase (eNOS)-derived NO, might not only modulate renal disease by direct control of renal vascular resistance but also modulate other nonhaemodynamic mechanisms including anti-inflammatory, antithrombotic, antiproliferative or antioxidant activity [11,12] that could contribute to its renal effect. Moreover, nonhaemodynamic effects of NO produced in large amounts by inducible NOS may participate in the development of adriamycin nephropathy [13].

To study the contribution of haemodynamic mechanisms to the development of adriamycin nephropathy, we sought to determine whether variability in the level of renal perfusion in vivo (measured as effective renal plasma flow: ERPF and renal blood flow: RBF) in conscious healthy rats predicts the nephrotoxic effect of adriamycin. To address the role of direct NO-mediated haemodynamic mechanisms, we additionally designed an intervention study to examine whether an acute NO inhibition during the adriamycin injection modulates the severity of chronic renal damage. Here, we report that both renal endothelium-dependent reactivity measured in vitro and ERPF measured in vivo in healthy individual rats predict the development of adriamycin-induced nephropathy. Additionally, acute RBF reduction by NO inhibition during the adriamycin injection attenuates the severity of renal damage.



   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Three separate experimental studies were performed using outbred male Wistar rats (12–13 weeks old, Harlan, Zeist, The Netherlands) housed under standardized conditions in animal facilities of the University of Groningen with free access to food and drinking water. All animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Committee for Animal Experiments of the University of Groningen.

Study I: Relation between baseline in vitro endothelial function and the severity of adriamycin-induced nephropathy
To investigate the relationship between individual renal endothelial function in vitro and subsequent renal damage, we performed unilateral nephrectomy (UnX) on rats (n = 16) under 3% isoflurane in N2O/O2 anaesthesia. Small renal arteries isolated from nephrectomized kidney were employed for in vitro measurements of vascular function as described below. Following UnX, a single i.v. injection of adriamycin in tail vein in a dose of 1.75 mg/kg (Pharmachemie BV, Haarlem, The Netherlands) was administered to induce renal damage. Subsequently, systolic blood pressure (SBP) was measured weekly in restrained awake animals by means of the tail-cuff method (IITC Inc., Ithaca, NY, USA) for 6 weeks. Urinary protein excretion was determined weekly by nephelometry (Dade Behring III, Mannheim, Germany) in 24-h urine samples obtained by putting the animals in metabolic cages. At the end of the study, animals were killed under anaesthesia and remaining kidneys and blood samples were harvested. Plasma creatinine was measured by means of a colorimetric assay with the Jaffé method without deproteinization (Chema Diagnostica, Jesi, Italy). The focal glomerulosclerosis (FGS) score was determined according to standard procedures in kidneys subjected to fixation and embedding in paraffin. Sections of 3 µm were stained with periodic acid Schiff (PAS) and microscopically evaluated for the incidence of FGS as previously described [14].

Measurements of baseline renal endothelial function in vitro.
Small renal (interlobar) arteries (250–350 µm) were isolated from the nephrectomized kidney and transferred to an arteriograph system for pressurized arteries (Living System Instrumentation, Burlington, VT, USA) as previously described [6]. Artery segments were cannulated on glass micropipettes, and intraluminal pressure was held constant at 70 mmHg. The vessel chamber was continuously recirculated with a warmed (37°C) and oxygenated (5% CO2 in O2) Krebs solution with a pH of 7.4 (120.4 NaCl, 5.9 KCl, 2.5 CaCl2, 1.2 MgSO4, 25.0 NaHCO3, 1.2 NaH2PO4, 11.5 mmol glucose). An inverted light microscope attached to a video camera and video dimension analyser was used to continuously register lumen diameter.

Following the 40-min equilibration period, arteries were pre-constricted submaximally with phenylephrine (PE, 3 x 10–7–10–6 mol/l) and studied for endothelium-dependent and endothelium-independent relaxation by administering cumulative doses of acetylcholine (ACh; 3 x 10–8–3 x 10–5 mol/l) and sodium nitroprusside (SNP, 10–9–10–4 mol/l) to the recirculating bath, respectively. ACh-induced relaxation was also studied in the same artery in the presence of either indomethacin (10–5 mol/l, to inhibit PGs), indomethacin and N{omega}-monomethyl-L-arginine (L-NMMA, 10–4 mol/l, to additionally inhibit NO) or indomethacin plus L-NMMA and a combination of charybdotoxin (chtx, 10–7 mol/l) and apamin (apa, 5 x 10–7 mol/l) applied into the lumen of the artery as well as to the superfusion medium (to additionally inhibit EDHF). ACh and SNP concentration–response curves were successfully obtained in all 16 animals, whereas curves in the presence of all inhibitors were obtained in 13 rats. Previously, we established that endothelial dilatory function did not differ between renal arteries within the used diameter range isolated from the same kidney, and therefore ACh-induced relaxation of one artery can be considered representative [6].

Study II: Relation between baseline in vivo renal haemodynamic function and proteinuria in adriamycin-induced nephropathy
To investigate whether in vivo determinants of renal haemodynamics in the healthy rats predict subsequent renal damage, measurements of renal function were performed prior to the injection of adriamycin (1.75 mg/kg) in another group of rats (n = 16). Following the injection, SBP and urinary protein excretion were measured weekly in the same way as in the first experimental study.

Measurements of baseline renal haemodynamic function in vivo.
Glomerular filtration rate (GFR) and ERPF were measured by clearance of simultaneously infused iothalamate and para-amino hippuric acid (PAH), respectively, in freely moving and spontaneously voiding rats as previously described [15]. Briefly, the rats instrumented with a jugular and carotid catheter were infused with a bolus of iothalamate (9 mg/kg) and PAH (12 mg/kg) followed by continuous intra-arterial infusion of these markers (iothalamate 0.9 mg/h and PAH 4.5 mg/h). Following an initial equilibration period of 2 h, the clearance period was determined by the urine collection depending on spontaneous voiding of the rats and a blood sample was drawn via the jugular catheter after each urine collection. Plasma and urine levels of iothalamate and PAH in the samples were determined by HPLC [16]. ERPF and GFR were calculated as a plasma clearance of PAH and urinary clearance of iothalamate, respectively, according to the method described by de Vries et al. [15]. Mean arterial pressure (MAP) was measured continuously during the experiment by connecting the carotid catheter to a pressure transducer (Baxter Healthcare, Irvine, CA, USA). Renal vascular resistance was calculated as the ratio of MAP and ERPF.

Study III: The effect of acute intervention in the renal NO system on the severity of adriamycin-induced nephropathy
To determine whether an acute intervention in the NO system modulates the severity of renal damage, in two additional experimental groups of rats, the adriamycin (1.75 mg/kg) was administered under concomitant infusion of either vehicle (n = 9) or NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, n = 12). Following the injection, urinary protein excretion was measured weekly and FGS was determined at the end of the study.

In vivo acute inhibition of NO during the adriamycin injection.
Under 3% isoflurane in N2O/O2 anaesthesia, the left kidney was exposed by abdominal midline incision and flowprobe (Transonic Systems Europe Inc., Maastricht, the Netherlands) was placed around the left renal artery to monitor the RBF. The rats instrumented with a jugular and carotid catheter were intravenously infused with either vehicle (n = 9) or L-NAME (10 to 100 µg/kg/min, n = 12), and MAP was measured continuously by connecting the carotid catheter to a pressure transducer. The dose of L-NAME was adjusted to achieve a minimum reduction of 15% in RBF. After 30 min of equilibration, adriamycin was injected (1.75 mg/kg) in tail vein and vehicle or L-NAME infusion was continued for additional 15 min, since it has been previously shown that adriamycin nephrotoxicity can be completely prevented by clipping the renal artery for 15 min following the injection [10].

Statistical analysis
Data are expressed as mean ± standard deviation (SD), unless stated otherwise. Concentration–response curves to ACh or SNP were expressed in percentage of pre-constriction to PE. The concentrations of drugs causing half-maximal responses were expressed as the negative logarithm of the molar concentration (pD2 values). The area under each individual ACh curve (AUC) was determined (Sigma Plot, SPSS Inc., Chicago, IL, USA) and expressed in arbitrary units. The contribution of PGs, NO and EDHF to endothelial relaxation was calculated as a difference between corresponding AUCs (Figure 1A). The characteristics of the concentration–response curves were compared by one-way ANOVA or ANOVA for repeated measures when appropriate. Statistical comparisons between parameters at baseline and at the end of the study were performed by Student's paired t-test, whereas potential relationships were tested by Pearson's parametric or Spearman's non-parametric correlation test (SPSS), where appropriate. Multiple linear regression analyses were performed in studies II and III to identify significant predictors of renal damage (proteinuria, FGS as dependent variables) among baseline haemodynamic parameters (ERPF, RBF, MAP, RVR, GFR as independent variables) (SPSS).


Figure 1
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  		Fig. 1 (A) Concentration-response curves to endothelium-dependent vasodilator acetylcholine (ACh) in small renal arteries isolated before the administration of adriamycin. The curves were constructed in absence of any inhibitor (total), in presence of indomethacin (indo, 10–5 mol/l), in additional presence of L-NMMA (10–4 mol/l) and in additional presence of charybdotoxin (chtx, 10–7 mol/l) and apamin (apa, 5 x 10–7 mol/l). Data are given as mean ± SEM. (B) Variability in total endothelium-dependent relaxation, prostaglandins (PGs)-, nitric oxide (NO)- and EDHF-mediated relaxation of small renal arteries isolated from healthy rats before the administration of adriamycin. AUC-Area Under Curve expressed in arbitrary units; box whisker plot: central box encloses middle 50% of all the data, horizontal line inside the box represents median and the whiskers encompass 5 to 95 percentiles.

 


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Markers of renal damage in adriamycin nephropathy
Functional characteristics of the rats used in the first two studies are presented in Table 1. Six weeks after the injection of adriamycin, rats in the first study developed overt nephropathy, characterized by elevated proteinuria, FGS and increased plasma creatinine when compared to values measured in healthy animals before the injection. SBP remained stable over the entire experimental period. Interestingly, despite the standardized injection of adriamycin, proteinuria varied considerably among individual rats, ranging from 145 to 883 mg/24 h in the first and comparably from 124 to 869 mg/24 h in the second experimental group. Both plasma creatinine and FGS score positively correlated with proteinuria (r = 0.56, P < 0.01 and r = 0.63, p = 0.01 respectively), suggesting that proteinuria adequately reflects renal damage in this model.


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  		Table 1 Functional parameters of animals in both studies measured prior to the administration of adriamycin (baseline) and 6 weeks thereafter and in vivo measurements of renal haemodynamics at baseline in study II

 
Study I: Endothelial function predicts renal damage in adriamycin nephropathy
Variability of renal endothelial function in healthy rat.
ACh induced concentration-dependent relaxation of small renal arteries. The average group response and curve characteristics are presented in Figure 1A and Table 2. Relaxation to ACh was highly variable in these—at that time—healthy animals (Figure 1B) and was independent of the level of PE-induced pre-constriction or endothelium-independent relaxation to SNP. Blockade of PGs by indomethacin resulted in variable small changes of the ACh curve in individual rats, however, not being significantly different on average (Figure 1A, Table 2). Additional administration of the NO inhibitor consistently decreased endothelium-dependent relaxation (Figure 1A, Table 2), however, to a highly variable extent in individual animals (Figure 1B). As a result, the remaining EDHF-mediated relaxation also displayed considerable variability (Figure 1B). This relaxation was completely blocked by the combination of indomethacin, L-NMMA and chtx+apa (Figure 1A, Table 2).


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  		Table 2 Baseline characteristics of the concentration–response curves to endothelium-dependent vasodilator acetylcholine and endothelium-independent vasodilator sodium-nitroprusside (SNP) in small renal arteries isolated from healthy rats prior to the administration of adriamycin

 
Correlation analysis.
Correlation analysis was performed to investigate the relation between baseline endothelial relaxation and renal damage 6 weeks after the adriamycin injection. ACh-induced relaxation (expressed as AUC) positively correlated with the level of proteinuria (Figure 2A, r = 0.51, P = 0.04). Endothelium-dependent relaxation also predicted plasma creatinine levels and FGS score (r = 0.68, P < 0.01 and r = 0.56, P < 0.05, respectively). Thus, the rats with a pronounced baseline endothelial relaxation developed more severe renal damage after the adriamycin injection. There was no correlation between the individual level of PE pre-constriction or endothelium-independent relaxation by SNP on the one hand and renal damage on the other.


Figure 2
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  		Fig. 2 (A) Correlation between individual total endothelium-dependent relaxation of small renal arteries measured prior to the administration of adriamycin and proteinuria determined 6 weeks after the administration of adriamycin (n = 16). (B) Correlation between individual relative effective renal plasma flow (ERPF) measured prior to the administration of adriamycin and proteinuria determined 6 weeks after adriamycin injection (n = 16). AUC-Area Under Curve expressed in arbitrary units, BW-body weight.

 
Additionally, we studied the relation between the endothelial dilatory mediators and proteinuria. PGs-mediated relaxation tended to correlate positively with proteinuria, but this was of marginal statistical significance (Figure 3C). A positive correlation was also found between the individual EDHF-mediated relaxation and proteinuria (Figure 3B). In contrast, individual NO-mediated relaxation was inversely correlated with the level of proteinuria (Figure 3A) as well as FGS (r = –0.69, P = 0.01), suggesting that individuals with a large NO-mediated relaxation are protected from the development of renal damage in adriamycin nephropathy.


Figure 3
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  		Fig. 3 Correlation between individual nitric oxide (A)-, EDHF (B)- and prostaglandins (C)-mediated endothelium-dependent relaxation of small renal arteries measured prior to the administration of adriamycin and proteinuria determined 6 weeks after administration of adriamycin (n = 13 each). AUC- Area Under Curve expressed in arbitrary units.

 
Study II: RBF predict proteinuria in adriamycin nephropathy
Baseline values of renal functional parameters have been included in Table 1. GFR, ERPF and RVR displayed considerable variability in healthy animals prior to the adriamycin injection.

Neither average value nor variability in individual values of proteinuria at Week 6 in study II was different from study I, suggesting that potential minor changes in RBF following UnX performed in study I did not have any impact on the renal outcome.

Individual ERPF markedly correlated with the proteinuria 6 weeks after the induction of the disease (Figure 2B, r = 0.66, P = 0.005), indicating that animals with highly perfused kidneys at the time of adriamycin administration developed more renal damage. Additionally, a negative correlation was found between individual RVR and proteinuria 6 weeks after the adriamycin injection (r = –0.65, P = 0.007). In contrast, no correlation was found between individual baseline GFR and proteinuria at Week 6 (r = 0.24, P = NS). Multiple regression analysis identified ERPF as the only independent predictor for proteinuria (best fitting model: proteinuria = –37.1 + 179.6 x ERPF), when haemodynamic variables (MAP, GFR, ERPF, RVR) were included as independent factors.

Study III: Acute NO inhibition protects against adriamycin-induced nephropathy
In the third intervention study, the acute administration of vehicle or L-NAME directly modulated haemodynamic parameters (Table 3). Thirty-minute infusion of L-NAME resulted in a 22 ± 8% decline of RBF, an 18 ± 10% increase of MAP and consequently a 53 ± 18% elevation of RVR. The median dose of L-NAME used to achieve these values was 50 µg/kg/min. No significant changes in any of these parameters were recorded after the infusion of vehicle. A subsequent injection of adriamycin had no effect on the haemodynamic parameters maintained by concomitant infusion of either vehicle or L-NAME up to 15 min thereafter.


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  		Table 3 Haemodynamic effects of acute vehicle or L-NAME administration in the interventional study III. The parameters were measured in anaesthetized rats at the start of infusion (0 min) and after 30 min prior to the administration of adriamycin

 
Concomitant inhibition of NO during the adriamycin injection markedly attenuated the severity of renal damage when compared to vehicle infusion as evidenced by significant reduction in proteinuria during 4–6 weeks (Figure 4A, P = 0.02) and the lower FGS score at the end of the study (21 ± 5% versus 15 ± 7% in vehicle and L-NAME-treated rats, respectively). This protective effect seemed to be mediated by the RBF-limiting effect of L-NAME, since both RBF and RVR measured prior to the adriamycin injection significantly predicted proteinuria (Figure 4B; r = 0.44, P = 0.049 and r = –0.51, P = 0.02 for RBF and RVR, respectively) or FGS (r = 0.52, P = 0.02 and r = –0.55, P = 0.01 for RBF and RVR, respectively) at Week 6. In contrast, no correlation was found between individual MAP and the severity of renal damage. Similarly, multiple regression analysis, with both vehicle and L-NAME-injected animals included, identified RBF as the best independent predictor of proteinuria (best fitting model: proteinuria = 132.3 + 252.6 x RBF) and FGS (best fitting model: FGS = 8.4 + 11.9 x RBF) among haemodynamic variables (MAP, RBF, RVR).


Figure 4
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  		Fig. 4 (A) The effect of acute NO blockade by L-NAME infusion during adriamycin injection (n = 12) on the development of proteinuria 6 weeks thereafter as compared to acute vehicle infusion during adriamycin administration (n = 9). Data are given as mean ± SEM. (B) Correlation between individual renal blood flow (RBF) acutely manipulated either by vehicle or L-NAME (n = 21) during the administration of adriamycin and proteinuria determined 6 weeks after adriamycin injection.

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In the present study, we found that the individual level of renal endothelial function of healthy rats measured in vitro predicts their susceptibility to renal damage after the injection of adriamycin. The baseline level of renal perfusion measured in vivo was related to renal damage as well. Moreover, the acute reduction in RBF by in vivo NO inhibition during the adriamycin injection protects against renal injury. Collectively, these data indicate that the state of renal vasculature measured both in vitro and in vivo might predispose certain healthy individual animals to a more severe course of toxic renal damage.

Endothelium-dependent relaxation of intrarenal arteries varies considerably among healthy animals within an outbred rat strain, which allows us to test for its predictive value in the development of renal damage. The animals with pronounced baseline relaxation developed higher proteinuria after the adriamycin injection. This finding might seem surprising since ACh-induced relaxation is considered a measure of protective abilities of vascular endothelium [17,18]. Furthermore, in contrast to our present data, we previously observed that animals with large renal endothelial relaxation were protected against renal injury after 5/6Nx [6]. In this remnant kidney model, intact endothelial function might provide protection against haemodynamic and hypertrophic adaptations following renal mass reduction, such as hyperfiltration and compensatory growth of the remaining nephrons, which maintain GFR acutely, but prove deleterious in the long term [5,19]. On the other hand, in adriamycin nephropathy, these specific adaptations are largely absent. Rather, a particular role of haemodynamics in the initiation of adriamycin-related injury might explain the discrepancy of predictive endothelial findings as compared to 5/6Nx. In the present study, we found that enhanced ERPF and reduced RVR, but not GFR, measured in conscious rats prior to the injection were associated with higher proteinuria. Since endothelium crucially participates in the regulation of renal haemodynamics [20,21], the rats with pronounced endothelial function (potentially those with high ERPF) may be exposed to a larger amount of toxic agent leading to an adverse renal outcome. Since adriamycin is not only filtered but also actively secreted and reabsorbed in the kidney [22], ERPF, rather than GFR, may represent a marker of adriamycin delivery to the sites of toxicity, including podocytes and tubular cells. After the adriamycin injection, a transient clipping of the renal artery for only several minutes completely prevents subsequent renal damage [10], indicating that acute delivery of the drug within this time scale may govern the long-term renal impairment. Moreover, in our intervention study, the acute inhibition of NO by L-NAME during the adriamycin injection reduced proteinuria. Though acute NO blockade can interfere with adriamycin nephropathy in several ways, including direct limitation of adriamycin toxic metabolite formation [23], renoprotection was correlated with the blood flow-limiting effect of L-NAME, suggesting that the acute restriction of the drug inflow is probably responsible for the attenuation of nephrotoxicity. Although UnX was performed in the present study to allow for the isolation of small renal arteries, this intervention, potentially associated with a minor acute RBF increase, likely did not have an impact either on the renal outcome or its variability and thus the potential predictive value, as suggested by the comparison between adriamycin-injected animals with and without nephrectomy. Collectively, the predictive value of endothelial function seems to be crucially dependent on the type and aetiology of renal damage, reflecting acute endothelium-mediated RBF regulation after the adriamycin injection as opposed to vasculoprotective endothelial properties against haemodynamic adaptations after 5/6Nx.

ACh-induced relaxation measured in this study reflects the sum of functional activity of NO, EDHF and PGs, as evidenced by the complete blockade of endothelial response by the combination of indomethacin, L-NMMA and chtx+apa. Interestingly, when addressing in vitro NO-mediated relaxation specifically, the rats with a large response were protected from proteinuria, in contrast to the in vivo observation that acute inhibition of NO limits renal damage. Moreover, we found a similar relation between in vitro NO-mediated relaxation and proteinuria in rats subjected to 5/6Nx [6]. Therefore, it seems that NO-mediated vasodilation measured in vitro reflects a NO renoprotective capacity of an individual against the development of renal injury with various aetiologies. Indeed, eNOS-derived NO, responsible for ACh-induced relaxation, may provide protection by suppressing inflammation, proliferation, leucocyte adhesion and other processes involved in the progression of renal damage [24] regardless of its effects on renal haemodynamics. On the other hand, during short-term in vivo L-NAME administration, the acute NO-mediated flow-limiting effect seems to prevail. Furthermore, L-NAME as being a non-selective NOS inhibitor may additionally inhibit neuronal NOS involved in the regulation of tubuloglomerular feedback [25] and thus modulate in vivo kidney perfusion differentially from in vitro vascular preparation. The third inducible NOS isoform (iNOS) is only induced during the course of renal damage and probably does not contribute to the effect of acute L-NAME blockade in the healthy animals. Collectively, it appears that although the severity of adriamycin toxicity is mainly driven by the acute haemodynamic status of the kidney, individual NO-mediated vasodilation measured in vitro might provide additional predictive power on the susceptibility to renal damage.

The contribution of additional endothelial mediators, PGs and EDHF predicted proteinuria in agreement with total ACh-induced relaxation and RBF. Both vasodilatory PGs and EDHF are directly involved in RBF regulation [26,27] and thus their increased contribution might be associated with higher RBF during the adriamycin injection and therefore worsen the renal outcome. EDHF, which exerts its effects by hyperpolarization of underlying smooth muscle cells, although its identity remains elusive [28], might also serve as a backup dilatory mechanism under circumstances when NO production is decreased [29,30]. In the present study, individual NO-mediated relaxation inversely correlated with that of EDHF, possibly reflecting the lack of NO-mediated protection. Overall, these data suggest that measurement of endothelium-dependent relaxation attributed to specific mediators, such as NO, EDHF and PGs, might provide important information on the interindividual susceptibility to renal damage.

Additional data are needed to investigate whether the predictive value of renal vascular function might identify individuals with increased risk for adverse renal outcomes following the injection of other nephrotoxic agents. Additionally, findings in a model of adriamycin nephropathy may be important for adriamycin-induced cardiomyopathy that displays similar variability in outcomes [31]. It remains to be elucidated whether and how these results can be transferred to a human clinical situation. In conclusion, this study showed that both baseline endothelial function of isolated renal vessels and the level of renal perfusion measured in conscious animals predict the severity of renal damage imposed by administration of a nephrotoxic drug. The acute manipulation of RBF prevents the development of drug-induced nephropathy. Further investigation into the nature of renal vascular variability may help to characterize better the mechanisms of progressive renal disease.



   Acknowledgments
 
P.O. is a recipient of an Ubbo Emmius Fellowship from the Graduate School for Drug Exploration (GUIDE). Authors acknowledge technical assistance of J. J. Duker and A. Wagenaar. This work was in part presented at World Congress of Nephrology, Berlin, Germany, 8–12 June 2003.

Conflict of interest statement. None declared.



   Notes
 
* Present address: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Comenius University Bratislava, Slovak Republic. Back



   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received for publication: 30. 7.08
Accepted in revised form: 4. 8.08


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