Proceedings of the Fourth Genoa Meeting on Hypertension, Diabetes, and Renal Diseases
Inflammation as a Mediator of the Link between Mild to Moderate Renal Insufficiency and Endothelial Dysfunction in Essential Hypertension
Carmine Zoccali*,
Raffaele Maio,
Giovanni Tripepi*,
Francesca Mallamaci* and
Francesco Perticone
* Consiglio Nazionale delle Ricerche–Istituto di Bio-Medicina, National Research Council–Institute of Biomedicine, Clinical Epidemiology and Physiopathology of Renal Diseases and Hypertension, Reggio Calabria, and Internal Medicine and Cardiovascular Diseases Unit, Department of Experimental and Clinical Medicine G. Salvatore, University Magna Graecia of Catanzaro, Catanzaro, Italy
Address correspondence to: Prof. Carmine Zoccali, CNR-IBIM, Istituto di Biomedicina, Epidemiologia Clinica e Fisiopatologia, delle Malattie Renali e dell’Ipertensione Arteriosa, c/o Divisione di Nefrologia, Ospedali Riuniti, Via Vallone Petrara, 89124 Reggio Calabria, Italy. Phone: +39-965-397010; Fax: 0039-0965-397000; E-mail: carmine.zoccali{at}tin.it
The relationship among inflammation (plasma high-sensitivityC-reactive protein [CRP]), endothelial function (hemodynamicresponse to acetylcholine [ACh] in the forearm), and renal function(serum creatinine and GFR [Modification of Diet in Renal Diseaseformula]) was investigated in 264 never-treated individualswith uncomplicated essential hypertension and serum creatininewithin the normal range. Multiple regression models of renalfunction (creatinine) were constructed in sequence includingFramingham risk factors as well the hemodynamic response toACh and plasma CRP. The inclusion of endothelial function intoa model based on Framingham risk factors added highly significant(P < 0.001) power to this model (+5%). Of note, in an alternativemodel that included CRP (instead of endothelial function), thecreatinine variance explained by this factor was two times higher(+10%) than that associated with endothelial function in thefirst model. In the full model that included both endothelialfunction and CRP, CRP maintained a much stronger independentlink with the outcome measure than endothelial function. Inindividuals with untreated, uncomplicated essential hypertension,multivariate modeling indicated that inflammation is a crucialmechanism mediating the endothelial–renal function link.The proatherogenic potential of inflammation associated withsubtle impairment in renal function may contribute to the cardiovascularrisk of essential hypertension.
Mild to moderate renal insufficiency now has emerged fully asa major public health problem. Population-based studies (1)and secondary analyses of intervention studies in hypertensivepatients (2) and in selected patients at high cardiovascularrisk (3) have shown coherently that classical risk factors suchas hypertension, diabetes, hyperlipidemia, smoking, and overweight/obesityrepresent major correlates of renal dysfunction. Although itsimportance for prevention is still debated, convincing evidencewas provided recently that low-grade inflammation, as measuredby highly-sensitivity C-reactive protein (CRP), is an earlymarker of renal dysfunction and of cardiovascular events inthe general population (4) and a risk factor for cardiovascularcomplications and progressive renal insufficiency in patientswith chronic kidney disease (CKD) (5). We recently reportedan association between mild to moderate renal impairment andthe hemodynamic response to acetylcholine (ACh), an establishedtest of endothelial function, in a large group of never-treatedindividuals with uncomplicated essential hypertension (6). Acorollary finding in this study was that renal function alsowas associated with serum CRP, suggesting that inflammationmay be a critical factor mediating endothelial dysfunction andrenal insufficiency in individuals with essential hypertension.Although merely associative, these observations in a carefullyselected population of hypertensive individuals are importantin that they may be hypothesis generating for understandingthe high risk associated with mild to moderate renal insufficiency.
In this study, we therefore performed a secondary analysis inthe same database to test the influence of inflammation, asmeasured by serum CRP, on the association between altered hemodynamicresponse to ACh and mild to moderate renal insufficiency inindividuals with essential hypertension. This analysis, basedon multivariate modeling, suggests that inflammation is a criticalfactor in the chain of events that lead to renal dysfunctionin hypertensive individuals.
The local ethics committee approved the study, and all participantsgave written, informed consent for all procedures.
Patients
A total of 246 never-treated individuals who had uncomplicatedessential hypertension (Table 1) and had simultaneous measurementsof high-sensitivity CRP and forearm blood flow studies wereselected from a population of approximately 3500 individualswho were referred to the hypertension clinic of the UniversityHospital of Catanzaro University between September 1994 andJanuary 2003. To be selected, patients had to have newly diagnosedessential hypertension, a serum creatinine 1.5 mg/dl, and absenceof proteinuria on the dipstick test and had to have never receivedantihypertensive medications. None of the patients had a historyor clinical evidence of angina, myocardial infarction, valvularheart disease, diabetes, serum cholesterol exceeding 280 mg/dl,peripheral vascular disease, coagulopathy, or any disease predisposingto vasculitis or Raynaud’s phenomenon. Causes of secondaryhypertension were excluded by appropriate investigations, includingmeasurement of plasma renin activity and aldosterone, Dopplerstudies of the renal arteries, and/or renal scintigraphy orrenal angiography. The following risk factors for atherosclerosiswere assessed at the time of the first evaluation: Arterialpressure (measured three times at 2- to 5-min interval witha mercury sphygmomanometer), glucose, lipids, smoking, and CRP.
Table 1. Main demographic, somatometric, clinical, and biochemical data of patients divided into GFR quartilesa
Hemodynamic and Laboratory Studies
These studies were started at 9 a.m. after overnight fasting,with the patients lying supine in a quiet, air-conditioned room(22 to 24°C). For the measurement of the vasodilatory responseto ACh, we adopted the protocol by Panza et al. (7), and thedetailed description of these tests was described elsewhere(6). For our study, the maximal response to ACh was consideredfor statistical analysis.
Serum creatinine was measured in the routine laboratory by anautomated technique based on the measurement of Jafféchromogen and implemented in an autoanalyzer. The GFR was estimatedby the Modification of Diet in Renal Disease equation developedby Levey et al. (8). CRP was measured by a high-sensitivityturbidimetric immunoassay (Behring, Marburg, Germany).
Statistical Analyses
Data are expressed as mean ± SD, median and interquartilerange, or percentage of frequency, as appropriate, and comparisonsamong groups were made by P for trend. Relationships betweenpaired parameters (continuous variables) were analyzed by Pearsonproduct moment correlation coefficient. Variables with positivelyskewed distribution were log transformed before the correlationstudy. To test the independent relationship between renal function(as estimated by serum creatinine or the GFR) and inflammation(CRP) and endothelial function (hemodynamic response to ACh),we constructed a multiple regression model (basic model) thatincluded a series of traditional risk factors (age, gender,smoking, body mass index, systolic and pulse pressure, heartrate, total cholesterol, LDL cholesterol, HDL cholesterol, triglycerides,and serum glucose). Multiple regression models of serum creatinineand GFR (including all variables of the "basic" model and eitherthe GFR or creatinine) were confronted by comparing the correspondingR2 and associated P values. Data are expressed as standardizedregression coefficient (), partial correlation coefficient,and P value. All calculations were made using a standard statisticalpackage (SPSS for Windows Version 9.0.1, Chicago, IL).
In Table 1, we categorized hypertensive individuals on the basisof the GFR into four quartiles. As expected, the GFR was stronglyassociated (in an inverse manner) with age and male gender.Weaker associations existed with systolic pressure and fastingplasma glucose (P = 0.12 and 0.08 for trend, respectively).These associations were substantially confirmed by linear correlationanalyses, which again identified age (r = –0.27) and gender(r = 0.46) as fundamental correlates of the GFR. Furthermore,the link between renal function and these risk factors heldtrue also when plasma creatinine was used as the outcome measureof renal function (creatinine versus age: r = 0.14, P = 0.02;creatinine versus gender: r = 0.12, P = 0.06). Remarkably, bothon categorical (Table 1) and linear correlation analyses (Figure 1),plasma CRP and the vasodilatory response to ACh correlated withthe GFR, CRP being progressively higher as the GFR declinedand the response to ACh being consistently higher in individualswith relatively higher GFR values. These correlations againwere confirmed fully when plasma creatinine was used as an indicatorof renal function (r = 0.32 and r = –0.28, P < 0.001).
Figure 1. Relationship between maximal vasodilatory response to acetyl choline (ACh) with the GFR (left) and with plasma C reactive protein (CRP; right).
Multivariate Analyses
To test the influence of inflammation (as measured by CRP) inthe statistical association between endothelial and renal function(serum creatinine), we constructed hierarchical multivariatemodels based on Framingham risk factors as well as the hemodynamicresponse to ACh. In multivariate modeling, we elected to usecreatinine rather than the GFR as an outcome measure to avoidthe analytical distortion that is derived by the use of GFR(i.e., a variable calculated on the basis of age and gender,which also are independent covariates in the same models). Abasic model that included these risk factors (age, gender, BP,LDL cholesterol and triglycerides, body mass index, and smoking)explained 8% of the creatinine variance. As shown in Figure 2,the addition of the forearm blood flow response to ACh addedsignificant information to the model in that the explained creatininevariance rose to 13% (P < 0.001). CRP seemed to be a strongerindependent correlate of creatinine than the response to AChbecause a model that included this variable (instead of theresponse to ACh) explained an even higher proportion of serumcreatinine variance (18%; P < 0.001). Of note, in a modelthat included both covariates (the response to ACh and CRP),the independent association between CRP with creatinine lostmodest strength (partial r = 0.27, P < 0.001), whereas theendothelial function–creatinine link, although still significant,lost substantial strength (partial r = –0.15, P = 0.02)so that CRP remained a much stronger correlate of serum creatininethan endothelial function (P < 0.001). Notwithstanding thepossible analytical distortion that is derived by the use ofthe GFR as an outcome measure, the results of GFR modeling didnot materially differ from the previously described creatininemodeling (data not shown).
Figure 2. Explained creatinine variance in statistical models that included classical risk factor (basic model) and CRP or endothelial function or both factors.
This study shows that endothelial dysfunction and inflammationare associated with renal insufficiency in cardiovascular events–freeindividuals with essential hypertension. Furthermore, modelingof renal function by multiple regression analysis suggests thatthese two risk factors are in the same causal pathway conduciveto renal damage in primary hypertension.
In the past decade, convincing evidence has been accrued thateven minor degrees of renal insufficiency as estimated on thebasis of plasma creatinine predict adverse cardiovascular outcomesin uncomplicated essential hypertension (9,10) and in the generalpopulation (11). The strength of this association is such thatguidelines that are promoted by major scientific societies nowrecommend GFR estimates that are based on serum creatinine forrefining risk stratification in essential hypertension and inCKD as well. Renal endothelial dysfunction as an expressionof systemic endothelial dysfunction is suspected to be a contributoryfactor to kidney disease in primary hypertension (12). Thishypothesis has experimental support in animal models in thatin the rat, the nitric oxide inhibitor NG-nitro-l-arginine methylester induces an arteriolopathy very similar to nephroangiosclerosisin humans (13).
Endothelial dysfunction that is measured by the hemodynamicresponse to ACh in the forearm is a very consistent findingin essential hypertension (14). This alteration is only in minorpart accounted for by the severity of hypertension and otherrisk factors, such as overweight, smoking, and hypercholesterolemia(15). The systemic nature of this disturbance (15) is highlightedby the fact that it affects disparate vascular beds, includingperipheral vessels, the coronary circulation (16), and the kidney(17). In line with a primary analysis reported elsewhere (6),this secondary analysis confirms that the forearm blood flowresponse to ACh is strongly associated with renal function inessential hypertensives.
Inflammation as measured by serum or plasma CRP concentrationis associated with BP levels in seemingly healthy individuals(4). CRP levels are considered a risk marker for cardiovasculardisease in the general population (18) as well as in patientswith cardiac ischemia (3). The importance of this risk factoris highlighted by the fact that in intervention studies, changesin CRP concentration are paralleled by coherent changes in therate of cardiovascular events (19). Notably, CRP was associatedwith the brachial artery flow–mediated dilation in a community-basedstudy (20). Such an association suggests that a microinflammatorystate, perhaps triggered by the synthesis of inflammatory cytokinesin abdominal and visceral fat cells (21), may alter the hemodynamiccontrol of the circulation by the endothelium. Renal insufficiency,a condition whereby endothelial dysfunction is pervasive (22),very frequently is accompanied by raised CRP. CRP was foundrecently to be associated with a subtle reduction in creatinineclearance in the Prevention of Renal and Vascular Endstage Disease(PREVEND) study (23). In keeping with these observations, wereported that CRP was related inversely both with the vasodilatoryresponse to ACh and with renal function. These coherent findingsare compatible with the hypothesis that endothelial dysfunctionmay be an intermediate mechanism mediating the effect of inflammationon renal function. To explore this hypothesis in our study,we modeled renal function by sequentially including endothelialfunction and CRP in multiple regression analyses. This techniquecan be used for investigating causal inferences in mechanistichypotheses (24). It is intriguing that this analysis indicatedthat CRP is a stronger correlate of renal function and showedthat endothelial function loses considerable explanatory powerfor the creatinine variance when tested in a model that includesboth factors, CRP retaining a much stronger association withthe outcome measure. Such statistical observation suggests thatCRP and endothelial dysfunction are in the same causal pathwaythat leads to renal insufficiency in hypertensive individualsand that CRP may be a causative factor for endothelial dysfunctionin the pathogenetic sequence that causes renal damage.
Our observations are cross-sectional and therefore need to beconfirmed in prospective and in interventional studies. In thisregard, it is worth noting that secondary analyses of clinicaltrials (25) demonstrated that interventions that are known tomodify CRP, such as statins, also reduce the risk for progressionof CKD in patients with hypercholesterolemia. Such a favorableeffect may not depend entirely on amelioration of hypercholesterolemiabecause, independent of changes in serum cholesterol, reductionsin CRP that are induced by statins predict a reduction in adversecardiovascular outcomes (19).
CRP and endothelial dysfunction are markers of renal functionloss in patients with essential hypertension. Inflammation signaledby high serum CRP may be the trigger of systemic endothelialdysfunction and renal insufficiency in these patients. Interventionstudies aimed at modifying inflammation are needed to confirmwhether this risk factor is a crucial element in the perversecardiovascular–renal connection that is conducive to cardiovascularcomplications in hypertension.
Acknowledgments
This study was supported by grants of University Magna Graeciaof Catanzaro and of CNR-IBIM National Research Council of Italy.
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Top
Abstract
Introduction
1.5 mg/dl, and absence of proteinuria on the dipstick test and had to have never received antihypertensive medications. None of the patients had a history or clinical evidence of angina, myocardial infarction, valvular heart disease, diabetes, serum cholesterol exceeding 280 mg/dl, peripheral vascular disease, coagulopathy, or any disease predisposing to vasculitis or Raynaud’s phenomenon. Causes of secondary hypertension were excluded by appropriate investigations, including measurement of plasma renin activity and aldosterone, Doppler studies of the renal arteries, and/or renal scintigraphy or renal angiography. The following risk factors for atherosclerosis were assessed at the time of the first evaluation: Arterial pressure (measured three times at 2- to 5-min interval with a mercury sphygmomanometer), glucose, lipids, smoking, and CRP. 
), partial correlation coefficient, and P value. All calculations were made using a standard statistical package (SPSS for Windows Version 9.0.1, Chicago, IL). 
