‘No Time to be Lost!’

Severe Traumatic Brain Injury (TBI) remains a major cause of death and disability afflicting mostly young adult males and elderly people, resulting in high economic costs to society. Therapeutic approaches focus on reducing the risk on secondary brain injury. Specific ethical issues pertaining in clinical testing of pharmacological neuroprotective agents in TBI include the emergency nature of the research, the incapacity of the patients to informed consent before inclusion, short therapeutic time windows, and a risk-benefit ratio based on concept that in relation to the severity of the trauma, significant adverse side effects may be acceptable for possible beneficial treatments. Randomized controlled phase III trials investigating the safety and efficacy of agents in TBI with promising benefit, conducted in acute emergency situations with short therapeutic time windows, should allow randomization under deferred consent or waiver of consent. Making progress in knowledge of treatment in acute neurological and other intensive care conditions is only possible if national regulations and legislations allow waiver of consent or deferred consent for clinical trials.
Introduction
Severe Traumatic Brain Injury (TBI) remains a major cause of death and disability afflicting mostly young adult males and elderly people, resulting in high economic costs to society [1]. Road traffic accidents, domestic and work-related falls and assaults are the main causes of TBI in Europe. The fatality rate for severe TBI is about 30% and a significant disability in 35–40% in unselected series. The primary injury initiates a complex sequence of events resulting in secondary brain damage, which can be exacerbated by systemic insults, such as hypotension and hypoxia. Therapeutic approaches focus on reducing the risk on secondary brain injury. Pharmacological neuroprotective agents aim to limit secondary brain damage after the primary acute injury and aim to improve overall outcome. Various neuroprotective agents, mainly targeting specific pathophysiologic mechanisms, have been tested in TBI, but convincing benefit has not been shown [2]. These data signify an ethical imperative to develop and test new therapeutic strategies and neuroprotective pharmacological agents in the field of TBI.
The most important ethical issues pertaining to clinical pharmacological trials in severe TBI are: emergency nature of the researchincapacity of the patients to consentshort therapeutic time windowsrisk/benefit ratio based on the concept that in relation to the seriousness of the injury, significant adverse side effects may be acceptable for treatments with possible benefit.
The importance of implications of these issues is not fully recognized outside, and even within, the expert field of treatment of severe TBI.
Therapeutic trials to evaluate the efficacy and safety of pharmacological agents are subject to the ethical and juridical principles of Good Clinical Practice, national legislation and European and international regulations. The guiding ethical principles underlying these investigations of treatment are respect for autonomy of the subjects, protection against discomfort, harm, risk and exploitation and the prospect of benefit. The prospect of benefit is almost always complicated by the equipoise underpinning the statistical null hypothesis of pharmacological trials: the hope that an individual patient will benefit, but that this is not more certain than the chance of non benefit.
Countries in the European Union are amending legislation to comply with the European Union Directive 2001/20/EC [3]. In this European legislation, emergency research under deferred or waiver of consent is however not permitted. This will impede or even obviate emergency research phase III trials in TBI in the European countries [4–6].
The European Clinical trial Directive 2001/20/EC was originally aimed as a European-wide harmonization of the provisions concerning clinical pharmacological trials, with a focus at the facilitation of multi-national clinical research. Since the publication in 2001, several articles drew attention to the serious threat to the development of evidence-based critical care and emergency research within the European Union (EU) posed by the Directive 2001/20/EC which requires prior informed written consent before subjects can be recruited to clinical trials of medicinal products [7–19].
The Directive made no direct exception for emergency and critical care situations, and therefore threatened to prevent all emergency trials involving patients with acute catastrophic illness causing loss of decision-making capacity and facing (very) short therapeutic time windows, such as severe shock, circulatory arrest, acute myocardial infarction, severe stroke and other acute neurological conditions, and moderate and severe traumatic brain injury.
Implementation by all EU countries was required by May 2004. The wording of the Directive permitted some flexibility so that variations were expected that might impact on emergency research [20]. Lemaire et al. [9] have described the variations in national legislative responses to the Directive within Europe; they called on legislators to permit waivers of informed consent for emergency and critical care research, to clarify terms and definitions, and to remove the artificial distinction between interventional and observational research. Concerning practice in The Netherlands, the requirements as described in the Directive have been transposed into the revision of the Medical Research in Human Subjects Act (WMO) and Medicine law (WOG) [21]. The amended WMO will change the rules governing drugs studies in The Netherlands. There will be little, if any, change to non-drug research. The Dutch Parliament has accepted the amended WMO for the implementation (amended WMO) at November 22, 2005 and the revised Act became effective in The Netherlands on March 1, 2006.
The Directive was conceived in part to ensure that participants enrolled in research projects are given adequate information about the nature of the trials and the associated risks. Legislation to protect the interests of patients was necessary and timely. The research community welcomed most of the Articles in the Directive; they offer guidance and will help to maintain confidence in the probity of medical research. Unfortunately, however, neither those responsible for the Directive, nor many who drafted enabling legislation within Member States, considered the special problems relating to research in emergency nor critical care situations, where consent cannot be obtained from subjects and where the need for emergency treatment does not allow time for contact with relatives or other legal representatives. Moreover, in the United States, in 1996, the FDA had published a waiver of informed consent for certain types of emergency and critical care research after earlier strict provisions had brought to a halt important progress in some critical clinical situations.
This shortcoming and the variable response within European Member States to the requirements of the Directive, prompted an expert meeting to be convened in Vienna, Austria on 30 May 2005 (‘Vienna Initiative to save European Research’ [VISEAR]). A final report was presented in December 2005 and full reports appeared in the Wiener Klinische Wochenschrift in 2006. The initiative to the meeting was supported by the Department for Ethics in Medical Research of the Vienna Medical University, in cooperation with the European Forum for Good Clinical Practice (EFGCP), the European Clinical Research Infrastructures Network (ECRIN), and the Vienna School of Clinical Research. One of the six working groups aimed at ‘clinical trials including patients who are not able to consent; the concept of individual direct benefit from research and informed consent in case of the temporarily incapacitated patient’ Their recommendations were published in 2006 [6].
Consent Procedures
Informed consent in TBI victims can, due to the severity of the brain injury, never be obtained from the patients. Proxies or an independent physician must give consent for inclusion in research, or consent must be deferred or waived. Most ethical committees in European countries consider consent by legal representatives (proxy consent) valid. The moral basis for proxy consent is restricted to the substituted judgment about the inclusion into the trial. The proxy is supposed to act as the patient, if competent, would have decided. The question remains if the patient wants to be represented by relatives for inclusion in a trial. Roupie et al. [22] found that only 40.6% of 1,089 patients would want their spouse/partner to be their surrogate, 28% want to be represented by the physician in charge of their care.
Coppolino and Ackerson [23] concluded that surrogate decision makers for critical care research resulted in false-positive consent rates in up to 20%. In the study by Sulmasy et al. [24], agreement between patients and proxies varied between 57% and 81%, depending on whether previous discussions had taken place on similar situations. It is very unlikely that such existential discussions occur frequently in the target population prone to TBI (young adult males), resulting in lack of evidence as to what their relative would have wanted in case of severe TBI. Most proxies seem to make decisions in emergency situations based on what they hope that will happen (survival of the patient), rather than what is likely to happen (possible death or [severe] disability); this will bias decision-making towards possible therapeutic benefit, however small the chance will be [12].
In some European countries consent for randomization may be given by an independent physician. Different perceptives on consent by a physician are reflected in conflicting reports. In one study, 84% of patients with myocardial infarction felt that the physician could independently decide on inclusion, if the patient was unable to consent for himself [25]. In the field of neonatology only 11% of parents believe that physicians should decide regarding research participation [26].
With deferred (proxy) consent patients are included into the research without prior consent. After inclusion, the patient (deferred consent) or his/her representatives (deferred proxy consent) should be informed as soon as possible and subsequent informed consent should be requested.
With waiver of consent, all consent is waived. Emergency research without prior consent (deferred consent or waiver of consent) can morally be accepted on the principles of fairness, justice and beneficence [27].
As severe TBI mostly occur outside the domestic situation (road traffic accidents), proxies are rarely available during the first hours after TBI [28]. This prompted investigators to use deferred (proxy) consent and waiver of consent in emergency research facing very short therapeutic time windows. In the National Acute Brain Injury Study: Hypothermia (NABIS-H) resulted the adoption of waiver of consent in a higher enrolment and reduced the time between injury and treatment by approximately 45 min [27]. In this study, relatives of only 11 out of 113 patients arrived within 6 h of the injury. In a septic shock trial the investigators could not contact the proxies within the inclusion time in 74% of the cases, and these were included under waiver of consent [19]. In the CRASH trial, mean time to randomization was significantly longer in those hospitals where consent was required compared with those it was not (4.4 h [SE = 0.21] vs. 3.2 h [SE = 0.16]), the difference in the mean time to randomization was 1.2 h [95% CI 0.7 to 1.8 h] [29]. In our series in the dexanabinol trial only 174 out of 6,303 (2.7%) were excluded for reason that proxy consent could not be obtained within 6 h after injury [30].
Even when proxies are available, many do not know what the patient’s wishes are [31]. Surrogate decision makers for critical-care research resulted in false-positive consent rates of 16–20.3% [23]. The emotional nature of an emergency situation limited the reliability of proxy consent for clinical research [26, 31, 32]. Only 48% of 79 representatives of European Brain Injury Consortium (EBIC) associated neuro-trauma centers in 19 European countries feel that relatives can make a balanced decision in an emergency situation, 72% believed that a consent procedure forms a significant factor causing decrease in enrolment rate in a TBI study, and 83% believed that prior consent is a significant factor causing delay in initiation of study treatment [12]. Under emergency circumstances, proxy consent does not seem to secure proper patient/subject protection. To our experience the validity of informed consent and proxy consent given in an emergency situation is at least troubling. When consent for clinical research is sought during an emergency situation, comprehension is generally less than optimal [33–35]. A small minority realized that pharmacological trials are designed to assess not only efficacy but safety as well [36]. One study searching for public views on emergency exception to informed consent found that most (88%) of 530 people believed that research subjects should be informed prior to being enrolled, while 49% believed enrolling patients without prior consent in an emergency situation would be acceptable and 70% (369) would not object to being entered into such a study without providing prospective informed consent [37]. In another study 11 of 12 stroke patients stated that, if the patient of family was not able to consent, then the treating physician should make the decision for inclusion in an emergency trial [38].
The requirement for all patients to give written informed (proxy) consent before enrolment can result in major selection biases, such that registry patients were not representative of the typical patient [39].
The Emergency Nature of Research in TBI
Traumatic Brain Injury is by definition an acute condition. The emergency nature of pharmacological research in TBI is reflected by the fact that experimental and clinical studies have shown that patho-physiological cascades are initiated within minutes to hours following primary injury. Time windows for treatment modalities are therefore considered to be short. Experimental studies have shown the efficacy of many neuroprotective agents, if these were administered before, or within 15 min after injury; others have shown a window of efficacy of 3–6 h.
In the most recent international pharmacological trial in TBI, a phase III randomized, placebo-controlled clinical trial investigating the efficacy and safety of a single dose Dexanabinol [40], the experimental data have consistently shown better protection the sooner the agent is administered after TBI [41]. In the animal model for TBI, this agent given up to 3 h after TBI was protective against breakdown of the blood–brain barrier and reduced formation of edema and resulted in less severe neurological symptoms [42]. Administered between 4 h and 6 h after injury, no significant reduction of cerebral edema was noticed, nevertheless neurological symptoms improved. Based on these findings, it may be concluded that in the experimental model the patho-physiologic endpoint can be determined at 3 h. If this time window also form the clinical therapeutic border in patients with severe TBI remains however uncertain. Time windows as applied to clinical trials in TBI have rarely been based on experimental evidence, but were rather determined by organizational and logistical considerations as to the time window within which investigators expected that a considerable number of patients could be enrolled. This was also the case in the recent Dexanabinol trial. One of the inclusion criteria in this trial was ‘sustained TBI within the past 6 h [40] Informed consent could, seen the severity of the brain injury, not be obtained from the patients. Proxy consent was accepted in all participating countries. Deferred patient or proxy consent was only allowed in Australia, Austria, Finland, France and Germany and consent by an independent physician was allowed in Israel, Italy, Spain and the United Kingdom. In all cases of deferred consent, subsequent written assent by patient or proxy was obtained.
As coordinating quality control and assurance center for this trial, we had the opportunity to study time windows in more detail [43]. We defined for this analysis four different time windows: the time between injury and admission in a neuro-trauma center;the time between admission in a neuro-trauma center and first head CT scan;the time between the first head CT scan and proxy consent for inclusion in the trial;the time between proxy consent and study drug administration.
For analysis of these four time windows we selected 631 patients. The only selection criterion was that the study drug was administered after written proxy consent. Patients included in the trial under deferred consent were excluded from our analysis. Furthermore, we only included patients from Europe and Israel, excluding patients from Australia and the United States for other reasons [44]. The time between injury and admission at the neuro-trauma center was for all selected patients between 1 h, 16 h and 2.35 h (Table 1, Fig. 1). 501 (79.4%) patients were directly admitted to the neuro-trauma center, 130 cases (20.6%) concerns secondary referrals. In all patients the window between admission and the first diagnostic CT scan remains within 1 h. With exception of France, in all countries the median time between injury and completion of the CT scan remained within the 3 h (Table 1). The longest time window was found between the first diagnostic CT scan and obtaining the required proxy consent (between 1.71 h and 2.74 h). The median time between injury and obtained proxy consent was between 3.75 h and 5.00 h (IQR 2.75–5.38 h) (Table 1). After proxy consent was given, almost all patients subsequently received the study drug within one hour (Table 1, Fig. 1). In 85.3% of all cases the time between injury and study drug administration was longer than 4 h, in 60% of the cases even longer than 5 h.
Table 1Time windows per country (median + IQR)Country (N)Hours between injury and admission NTC median (IQR)Hours between injury and CT scan median (IQR)Hours between injury and obtained consent median (IQR)Hours between injury and SDA median (IQR) Belgium (23)0.93 (0.65–1.27)1.80 (1.28–2.27)3.75 (2.75–4.75)4.60 (3.98–5.42)Netherlands (73)1.00 (0.75–1.33)1.65 (1.32–2.00)4.53 (3.95–5.05)5.53 (5.07–5.75)Israel (116)0.93 (0.72–1.40)1.91 (1.58–2.47) 4.01 (3.20–4.83)4.67 (4.00–5.33)Spain (75)1.33 (0.97–1.67) 2.07 (1.65–2.53)4.17 (3.33–5.00)5.17 (4.30–5.58)Germany (109)1.20 (0.88–2.00)1.65 (1.30–2.13)4.08 (3.42–4.98)5.25 (4.25–5.67)Italy (146)1.25 (0.83–2.60)1.77 (1.40–2.35)4.92 (4.08–5.28)5.50 (4.98–5.75)France (34)2.17 (1.42–3.00)3.08 (1.97–3.53)5.00 (4.50–5.38)5.75 (5.17–5.83)Other countries* (55) 1.47 (1.00–2.67)1.82 (1.33–2.50)4.00 (3.08–4.75)5.25 (4.33–5.75)*Countries with small patient populations (United Kingdom, Denmark, Austria, Poland, and Turkey) were combinedFig. 1Time between injury and admission neurotrauma center, time between admission and first CT scan, time between first CT scan and informed consent for inclusion in trial and time between consent and start study drug admission (from Ref. 43)
Dexanabinol was one of the promising new pharmaceuticals in the treatment of TBI, but it shown to be safe but not effective in the treatment of severe TBI [40]. Nevertheless, one can conclude that chances of efficacy increase if treatment is provided earlier. Fact is that in almost all of the studied cases the time between injury and completion of the primary diagnostic CT scan remains within 3 h post injury, which is shown to be the therapeutic time window in the animal model. In 60% of the cases the time between injury and study drug administration was however longer than 5 h, and in 85.3% of all cases longer than 4 h (Fig. 1). Our data provide the empirical proof for considering deferred consent or waiver of consent in trials with a very short therapeutic time window.
Risk-Benefit Ratio
To my opinion the balance between risk and benefit should be the guiding principle in emergency research in severe TBI. This also applies to the nature and the type of consent procedures. The ethical principle of respect for the autonomy of the patient underpinning the informed consent procedures is not valid for acutely incapacitated patients as TBI victims. Significant concerns has been raised on the validity and ethics of proxy consent in acute emergency situations, and the required written consent cause a significant delay in treatment initiation, as we have shown with our analysis of the time windows. The possible therapeutic benefit, as has been shown in experimental models, form the moral justification for randomizing patients under deferred consent or waiver of consent within a sufficient period of time. The risks should however be acceptable in relation to the severity of the disease or injury. For trials under deferred consent or waiver of consent in acute emergency situations we would constrain the institution of an independent safety committee, under the auspices of regulatory authorities. The obligation to such a committee is based on the experience of a dramatically harmful outcome in some trials under waiver of consent in other fields of medicine [45, 46].
Conclusions
Specific ethical issues pertaining in clinical testing of pharmacological neuroprotective agents in TBI include the emergency nature of the research, the incapacity of the patients to informed consent before inclusion, short therapeutic time windows, and a risk-benefit ratio based on concept that in relation to the severity of the trauma, significant adverse side effects may be acceptable for treatments with possible benefit.
Time windows as applied to randomized controlled clinical trials in TBI have rarely been based on experimental evidence, but were rather determined by organizational and logistical considerations as to a time window within which investigators expected that a considerable number of patients could be enrolled [12]. The main determinant is now formed by the informed (proxy) consent procedures, as also has been compelled in the new European Union Directive 2001/20/EC [3, 6]. These requirements assume that relatives are available in emergency situations, and that these relatives can be fully informed and given sufficient time to make a balanced decision in a relatively short time period. The conflict between the desire for early initiation of experimental treatment versus the time required for following consent procedures and the conflict between the desire for following consent procedures requiring prior consent and the doubts about the validity of proxy consent in acute situations are the most problematic aspects of emergency research in TBI.
Clinical research in emergency situations without prospective informed or proxy consent is ethically challenging. Severe TBI is without doubt an emergent and life-threatening condition and existing therapy is unsatisfactory seen the high morbidity and mortality in a mostly young group of patients. This should qualify severe TBI for emergency exception form informed consent for randomized clinical controlled trials with pharmacological agents with promising therapeutic benefit facing short therapeutic time windows. Randomized controlled investigations are necessary to determine the safety and effectiveness of new developed agents in these conditions. We have proved that the requirement of previous written proxy consent causes a significant delay till study drug administration in a trial with a neuroprotective agent in TBI. With waiver of consent or deferred (proxy) consent the first dose of the experimental drug can be administered directly after completion of the first diagnostic CT scan, which is very close to the experimental therapeutic time window. Randomized controlled phase III trials investigating the safety and efficacy of agents with promising benefit, conducted in acute emergency situations with short therapeutic time windows, should allow randomization under deferred (proxy) consent or waiver of consent. Making progress in knowledge of treatment in acute neurological and other intensive care conditions is only possible if national regulations and legislations allow waiver of consent or deferred (proxy) consent for clinical trials [15]. As two of us have said before: ‘treat first, ask later’ seems ethically defendable in acute care research [4].

traumatic brain injury
deferred consent
waiver of consent
pharmacological trial
proxy consent
clinical ethics


Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia

Nitric oxide (NO) is an important regulator of renal transport processes. In the present study, we investigated the role of NO, produced by inducible NO synthase (iNOS), in the regulation of renal ATP-binding cassette (ABC) transporters in vivo during endotoxemia. Wistar–Hannover rats were injected with lipopolysaccharide (LPS+) alone or in combination with the iNOS inhibitor, aminoguanidine. Controls received detoxified LPS (LPS−). After LPS+, proximal tubular damage and a reduction in renal function were observed. Furthermore, iNOS mRNA and protein, and the amount of NO metabolites in plasma and urine, increased compared to the LPS− group. Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage. Gene expression of 20 ABC transporters was determined. After LPS+, a clear up-regulation in Abca1, Abcb1/P-glycoprotein (P-gp), Abcb11/bile salt export pump (Bsep), and Abcc2/multidrug resistance protein (Mrp2) was found, whereas Abcc8 was down-regulated. Up-regulation of Abcc2/Mrp2 was accompanied by enhanced calcein excretion. Aminoguanidine attenuated the effects on transporter expression. Our data indicate that NO, produced locally by renal iNOS, regulates the expression of ABC transporters in vivo. Furthermore, we showed, for the first time, expression and subcellular localization of Abcb11/Bsep in rat kidney.
Introduction
The free radical nitric oxide (NO) has been shown to play an important role in various physiological processes in the kidney, including salt and fluid reabsorption, renal hemodynamics, renin secretion, and tubuloglomerular feedback [1]. Endogenous NO is enzymatically produced from conversion of the amino acid l-arginine to l-citrulline, a reaction that is catalyzed by the enzyme NO synthase (NOS). This enzyme exists in three isoforms; the neuronal (nNOS) and endothelial, both of which are Ca2+- and calmodulin-dependent constitutive isoforms, and an inducible (iNOS) isoform [2]. Mediators associated with sepsis, such as endotoxin and the proinflammatory cytokines, interleukin-1β (IL-1β), IL-2, and tumor necrosis factor-α (TNF-α), have been shown to induce iNOS, thereby increasing production of NO. Sustained, high-output generation of NO by iNOS can cause cell death and tissue damage through lipid peroxidation, DNA damage, and proapoptotic effects [3]. This is the result of NO itself and the oxidant peroxynitrite, which is generated after reaction of NO with superoxide anions [4].
In contrast to many other organs, iNOS is constitutively expressed in the kidney, in particular in the medulla and in proximal tubules [5]. Many in vivo animal studies showed the importance of iNOS as a contributor to the pathophysiology of acute kidney injury (AKI). For example, it has been shown that selective chemical inhibitors of iNOS [6, 7], NO scavengers [8], iNOS gene knockout mice [9], or antisense oligodeoxynucleotides to iNOS [10] resulted in less renal tubular injury and improved structural and functional outcome. This renoprotective effect was partly due to the rescue of tubular epithelial cells from injury by NO [3].
Using killifish renal proximal tubules, we found previously that NO has a regulatory role in the transport activity of the ATP-binding cassette (ABC) transporter multidrug resistance protein 2 (Abcc2/Mrp2) via an intracellular signaling pathway in response to the action of several nephrotoxic chemicals in vitro [11]. This pathway involved at least endothelin (ET) release, binding to the basolateral ETB receptor, and activation of NOS, soluble guanylyl cyclase, and protein kinase C [12]. A similar regulatory pathway was found for the ABC transporter P-glycoprotein (Abcb1/P-gp) in rat brain capillaries [13]. Both studies indicate that the activation of NOS is most likely Ca2+-independent and we concluded that the production of NO is a result of the activation of iNOS.
The proximal tubule is responsible for the excretory transport of xenobiotics, xenobiotic metabolites, and waste products of metabolism from blood into urine. Consequently, the proximal tubule is also an important target for toxic effects and is often the first site of damage in AKI [14]. Luminal transport proteins, like the ABC transporters ABCB1/P-gp, ABCC2/MRP2, and ABCC4/MRP4, are important efflux transporters in the kidney [15] and, therefore, play a role in the detoxification of tubule cells. The observed effects of NO on Abcc2/Mrp2 in killifish proximal tubule may be part of the normal progression of cellular events that occur during AKI.
In the present study, we hypothesized that iNOS has a role in the regulation of renal ABC transporters during nephrotoxicity in vivo. An endotoxemia model was used in which rats were exposed to lipopolysaccharide (LPS), and the gene expression of 20 ABC transporters was evaluated. In addition, we examined the effect of the iNOS inhibitor, aminoguanidine, to determine whether the change in expression of the renal efflux transporters is caused directly by NO generated by iNOS.
Materials and methods
Animals and experimental design
Specified pathogen-free male Wistar–Hannover rats (240–295 g; purchased from Harlan, Zeist, The Netherlands) were kept under routine laboratory conditions at the Central Animal Laboratory of the Radboud University Nijmegen Medical Centre. This strain was used because they do not develop hydronephrosis spontaneously like normal Wistar rats, which in turn could have a down-regulating effect on iNOS expression [16]. The local Animal Care Committee approved all experimental procedures.
Different pilot studies have been conducted to determine the route of administration, intravenously or intraperitoneally (IP), concentration of the endotoxin LPS (LPS+, Escherichia coli 0127:B8, Sigma-Aldrich, Zwijndrecht, The Netherlands) and aminoguanidine (Sigma-Aldrich), and the time point of coadministration in this strain. LPS and aminoguanidine were dissolved in sterile Hank’s balanced salt solution (HBSS, Gibco, Paisly, UK) supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES, Gibco), and set to pH 7.4 with NaOH.
Thirty rats were, stratified to body weight, injected IP with 5 mg/kg LPS+ and were killed after 3, 6, 12, 24, or 48 h (n = 6 for each experimental group). Fifteen rats served as controls and received 10 mg/kg detoxified LPS (LPS−, n = 3 per group), in which the biologically active component, lipid A, essential for the activation of NOS, is lacking [17]. Although the control group is small, standard errors appeared to be satisfactory to allow a reduction and refinement of animals in this group. Twelve rats were injected IP with both 5 mg/kg LPS+ and, after 1 h, 100 mg/kg of the iNOS inhibitor aminoguanidine (Sigma-Aldrich), and were killed 6 and 12 h after LPS+ (n = 6 per group). For the isolated perfused kidney experiments, six rats received LPS+, four received LPS−, and four were untreated and served as controls, and were killed after 12 h as described under the “Isolated kidney perfusion” section.
To collect urine before and after the injection, rats were housed in metabolic cages, washed with 2% chlorohexidine (Genfarma, Zaandam, The Netherlands) to prevent bacterial growth. Urine volumes were recorded and samples were snap-frozen in liquid nitrogen and stored at −80°C until assayed. During the experiment, body weight and body temperature were determined. At indicated time points after LPS injection, the rats were anesthetized with 5% isoflurane and heparin (125 IU/100 g) was injected via the penile vein to minimize blood clotting. The left kidney was removed, divided in four pieces, and snap-frozen in liquid nitrogen for immunohistology or fixed in Bouin’s solution for routine histology. Arterial blood was obtained by aortic puncture and the rats were killed by exsanguination. The right renal vein was punctured and, subsequently, the kidney was perfused with HBSS–HEPES for 2 min at 7.5 ml/min (Minipuls 2, Gilson, Villiers-le-Bel, France). After perfusion, the kidney was removed, divided, and snap-frozen in liquid nitrogen for quantitative real-time polymerase chain reaction (RQ-PCR) and Western blotting. Thrombocyte and leukocyte counts were determined in blood as inflammatory parameters, and the concentrations of creatinine, urea, protein, glucose, and sodium were analyzed in blood and urine by routine clinical chemistry. The activity of alkaline phosphatase, a proximal tubule injury marker, was determined in 26-fold diluted urine samples, as described before [18].
Determination of mRNA expression
RNA was isolated using a Mikro-dismembrator U (Sartorius B. Braun Biotech, Melsungen, Germany). To prevent RNA degradation, we washed the metal cylinders with 0.5 M NaOH. Perfused kidneys were pulverized (2,000 rpm, 30 s) and transferred in ice-cold TRIzol reagent (Invitrogen, Breda, The Netherlands), and then RNA was isolated as described previously [19]. RQ-PCR on cDNA was performed according to the TaqMan® protocol in optical tubes using either the ABI PRISM 7700 single reporter Sequence Detection System (n = 6, Applied Biosystems, Zwijndrecht, The Netherlands) or the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card Sequence Detection System (three pooled samples from nine different rats, Applied Biosystems) according to the manufacturer’s instructions. All experiments were performed in triplicate. Different rat genes were amplified with a predeveloped Gene Expression Assay, provided by Applied Biosystems.
Western blotting
Total membrane and cytosolic fractions from perfused kidneys were isolated using a Mikro-dismembrator U (Sartorius BBI Systems GmbH, Melsungen, Germany; 2,000 rpm for 30 s) and subjected to Western blotting as described previously [20]. Protein concentration was determined with the Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA) using bovine serum albumin (BSA) as standard. Samples were separated on a 6 or 12% sodium dodecylsulfate polyacrylamide gel and transferred to Hybond-C pure nitrocellulose membrane (Amersham, Buckinghamshire, UK). The membrane was incubated overnight at 4°C with antibodies against NOSII/iNOS (1:1,000, according to Vos et al. [21]), ABCB1/P-gp (1:200, C219, DakoCytomation, Dako, Denmark), Abcc2/Mrp2 (1:250, M2III-6, Alexis Biochemicals, San Diego, CA, USA), ABCC4/MRP4 (1:1,000, as described by van Aubel et al. [22]), Abcb11/bile salt export pump (Bsep) (1:1,000, kindly provided by Dr. Bruno Stieger, University Hospital Zürich, Switzerland), or β-actin (1:10,000, Sigma) in Tris-buffered saline supplemented with 0.1% Tween-20 and 1% nonfat dried milk.
Nitrate and nitrite determination
The total amount of the stable NO metabolites (NOx), nitrate and nitrite, were determined as a measure of NO radicals production, using the Griess reaction, according to Moshage et al. [23]. Heparinized plasma was diluted 1:4 and urine samples were diluted 1:40 or 1:80 with distilled water. Each sample was determined in triplicate. Calibration curves were made with sodium nitrite and sodium nitrate in distilled water. The nitrate recovery was 90–100% over the entire range tested (0–50 μmol/l). The coefficient of variation between the different experiments was less than 5%.
Histology and immunohistology
Kidneys fixed in Bouin’s solution were dehydrated, embedded in paraplast (Amsterstad, Amsterdam, The Netherlands), and cut into 3-μm sections, which were stained with periodic acid-Schiff for routine histology. For immunohistologic studies, serial tissue sections of 1.5 μm were cut from frozen kidneys using a cryostat as described previously [24]. The slices were incubated for 60 min at room temperature with antibodies against either NOSII/iNOS (1:200, according to Vos et al. [21]) or ABCB1/P-gp (1:50, C219, DakoCytomation) in PBS supplemented with 1% BSA. For the antibodies peroxynitrite (1:500, Sigma) and Abcb11/Bsep (1:1,000, as described by Huls et al. [24]), cryostat slices of 4 μm were used, fixed in acetone for 5 min. Endogenous peroxidase was blocked by incubation with 5% H2O2 for 5 min, and nonspecific protein binding was blocked with 10% normal goat serum (Vector, Burlingame, CA, USA) in PBS for 10 min at room temperature. Slices were incubated with the primary antibody in PBS 1% BSA overnight at 4°C. Primary antibody was detected using the Vectastain Elite peroxidase ABC kit (Vector) with 3,3′-diaminobenzidine as substrate. Finally, slices were counterstained with hematoxylin, dehydrated, and mounted in mounting medium. Sections were examined with a confocal laser-scanning microscope (Leica lasertechnik GmbH, Heidelberg, Germany).
Isolated kidney perfusion
Twelve hours after the administration of LPS+ (n = 6) or LPS− (n = 4), the transport activity of Abcc2/Mrp2 was determined by monitoring the urinary excretion of calcein in an isolated perfused rat kidney, as described in detail previously [25]. Briefly, rats were anesthetized IP with pentobarbital (6 mg/100 g), and furosemide was injected IP (1 mg/100 g) to prevent deterioration of the distal nephron. Heparin (125 IU/100 g) was injected in the spleen. The kidney was excised and placed in a fluid bath with a constant temperature of 37.5°C and rats were killed by exsanguination. Vitamin B12 was used for the determination of the glomerular filtration rate (GFR) and calcein-acetoxymethylester was used as a source for the fluorescent substrate calcein [25].
Statistical analysis
Values are given as mean ± SE. Differences between the experimental groups were tested using one-way ANOVA with Bonferroni’s correction or two-way ANOVA corrected for repeated measurements. A two-sided P value < 0.05 was considered significant.
Results
Signs of inflammation
A few hours after LPS+ administration, inflammatory symptoms appeared, like diarrhea and piloerection. Body temperature was elevated after 24 h and the loss in body weight increased from −3% (t = 3 h), due to normal circadian rhythm, to 12% (t = 48 h), compared to the controls (Table a in the supplementary material 1). Furthermore, administration of LPS+ resulted in the expected leucopenia (t = 3 h) followed by leukocytosis (t = 48 h) and thrombocytopenia, with a maximum at 48 h. Coadministration of LPS+ with aminoguanidine did not affect the inflammatory parameters, except for a greater loss in body weight 12 h after LPS administration.
iNOS induction after LPS treatment
RQ-PCR was used to determine the iNOS mRNA levels in kidney samples. The relative expression of iNOS in LPS− rats was normalized for the average cycle threshold (CT) value for the housekeeping gene, GAPDH (CT = 16.06 ± 0.04, n = 15), and set to 1. GAPDH expression remained stable during endotoxemia (CT = 16.22 ± 0.09, n = 42). Furthermore, no circadian rhythm for the different genes was detected in control animals.
Analysis of three separate experiments revealed a more than 40,000 times induction in iNOS mRNA 3 h after LPS+ compared to controls (Fig. 1a), which decreased with time, but was still 100 times higher than control values 48 h after LPS+. Treatment with both LPS+ and aminoguanidine slightly, but not significantly, reversed iNOS mRNA induction compared to LPS+ alone (Fig. 1a). Maximal iNOS protein expression was observed in cytosolic fractions 6 h after LPS+, and returned to LPS− level at 48 h (Fig. 1b). In contrast to its effect on iNOS mRNA expression, aminoguanidine reversed the induction in protein expression (Fig. 1c).
Fig. 1iNOS mRNA and protein. a iNOS mRNA expression in rat kidney at different time points after LPS+ was determined with RQ-PCR without (closed bars, n = 6) or with coadministration of aminoguanidine (gray bars, n = 6). The iNOS mRNA expression was normalized for the GAPDH CT value (16.06 ± 0.04, n = 15), and ΔCT values for the LPS− were set to 1. Data are expressed as mean±SE. Significantly different compared to the LPS− (double asterisksP < 0.01, triple asterisksP < 0.001, quadruple asterisksP < 0.0001). Cytosolic fractions of rat kidney were isolated and expression of iNOS was determined by Western blotting. Representative images (n = 4) show maximal iNOS protein expression 6 h after LPS+ administration (b), and coadministration with aminoguanidine reversed this induction (c)
NOx levels in plasma and urine were measured using the Griess assay. An increase in NOx concentration in plasma was already visible 3 h after LPS+, with a peak concentration at 12 h. Coadministration with aminoguanidine reversed the increase in NOx significantly at both 6 and 12 h (Fig. 2a). The urinary excretion of NOx increased 12 h after LPS+, and was still elevated 48 h later. Aminoguanidine returned this increase to baseline levels at both time points (Fig. 2b).
Fig. 2NOx in plasma and urine. NOx levels in plasma and urine were measured at various times after LPS− (open bars, n = 3) or LPS+ (closed bars, n = 6) treatment or LPS+ together with aminoguanidine (gray bars, n = 6). a Blood samples show a peak concentration at 12 h and coadministration with aminoguanidine reversed this increase at both 6 and 12 h. b The urinary excretion of NOx increased 12 h after LPS+ treatment, and aminoguanidine returned this increase to baseline levels. Data are expressed as mean±SE. Significantly different compared to the LPS− (single asterisksP < 0.05, double asterisksP < 0.01, triple asterisksP < 0.001) or LPS+ (double sharp signsP < 0.01, triple sharp signsP < 0.001)
Renal localization of iNOS and formation of 3-nitrotyrosine-protein adducts, a reliable biomarker of peroxynitrite generation [26], were determined by immunohistology. There was no iNOS staining visible in kidney sections of rats treated with LPS−. Positive iNOS staining was seen in both experimental groups after LPS+, in inflammatory cells, and in proximal tubule cells. Forty-eight hours after LPS+, iNOS staining was no longer detectable. Figure 3 shows representative immunohistochemical images with iNOS expression in all segments of the proximal tubule (3A+C). After colocalization with Abcb1/P-gp (3B), iNOS expression in the vicinity of the apical membrane was visible (3A+C). 3-Nitrotyrosine-protein adducts were predominantly present in the cortex in peritubulary capillaries and proximal tubules after LPS+ (Fig. 3d,e). Coadministration with aminoguanidine attenuated the formation of adducts in the cortex (Fig. 3f,g).
Fig. 3Immunofluorescence microscopic analysis of iNOS and peroxynitrite in the kidney. a–c Representative immunohistochemical images show that iNOS (green staining) is expressed in the vicinity of the apical membrane (P-gp counterstaining, red) in all segments of the proximal tubule 12 h after LPS+ treatment. d, e Formation of 3-nitrotyrosine-protein adducts, a reliable biomarker of peroxynitrite generation, were visible in the cortex in peritubulary capillaries and proximal tubules 12 h after LPS+ treatment. f, g Coadministration with aminoguanidine resulted in attenuated formation of 3-nitrotyrosine-protein adducts at 12 h. G glomerulus, asterisks peritubular staining, P proximal tubule, D distal tubule. Original magnification d, f ×200 and a–c, e, g ×400
Aminoguanidine restores the slightly impaired kidney function and loss of brush border membrane
Kidney function was slightly impaired after LPS+ as indicated by increased blood urea nitrogen (BUN), an increase in plasma creatinine, and an increased urinary glucose excretion (Table 1). Furthermore, the urine/plasma creatinine ratio was decreased (data not shown) as was the creatinine clearance, although not significantly.
Table 1Renal functional parametersKidney functionTime (h)Controls (LPS−)LPS+LPS+ and aminoguanidineTotal urine volume (ml)31.6 ± 0.241.7 ± 0.2762.4 ± 0.654.45 ± 0.987.3 ± 0.76123.2 ± 0.5710.0 ± 0.70a9.4 ± 0.65a2411.5 ± 0.2513.7 ± 2.114823.5 ± 3.4718.0 ± 1.57BUN (mmol/l)36.6 ± 0.209.9 ± 0.7464.6 ± 0.7214.0 ± 1.48b12.1 ± 0.82b126.3 ± 0.2412.3 ± 1.38a15.4 ± 1.27b247.0 ± 0.5810.6 ± 0.58486.5 ± 0.109.1 ± 0.38Blood creatinine (mmol/l)30.035 ± 0.0020.049 ± 0.002b60.039 ± 0.0020.053 ± 0.002b0.043 ± 0.019d120.037 ± 0.0020.048 ± 0.002c0.036 ± 0.032e240.038 ± 0.0010.051 ± 0.001a480.042 ± 0.0010.052 ± 0.001Glucose excretion (μmol/h)30.290 ± 0.0450.481 ± 0.34160.333 ± 0.0860.420 ± 0.1000.478 ± 0.051120.425 ± 0.1060.537 ± 0.0540.332 ± 0.080240.254 ± 0.0060.984 ± 0.039c480.325 ± 0.0370.606 ± 0.128Creatinine clearance (ml/min)31.33 ± 0.120.78 ± 0.1961.06 ± 0.150.87 ± 0.090.93 ± 0.35121.01 ± 0.050.90 ± 0.031.01 ± 0.13241.19 ± 0.020.91 ± 0.20481.00 ± 0.071.08 ± 0.37FENa (%)30.816 ± 0.0040.365 ± 0.07361.046 ± 0.1450.358 ± 0.094b1.423 ± 0.310e121.094 ± 0.0970.458 ± 0.075a1.610 ± 0.282e241.070 ± 0.0240.429 ± 0.065c481.033 ± 0.0770.121 ± 0.024aData are expressed as mean±SE, n = 6 for each time point for both LPS+ and LPS+ +aminoguanidine and n = 3 for LPS− FENa fractional sodium excretionaSignificantly different compared to the control group; P < 0.01bSignificantly different compared to the control group; P < 0.001cSignificantly different compared to the control group; P < 0.05dSignificantly different compared to the LPS+ group; P < 0.01eSignificantly different compared to the LPS+ group; P < 0.001
Histological examination after LPS+ revealed damage to the proximal tubules, compared to LPS− (Fig. 4a,b). Loss of brush border membranes and formation of vacuoles were observed. Aminoguanidine reduced LPS+-induced damage to the proximal tubule (Fig. 4c). The activity of alkaline phosphatase in urine samples was assayed to get an indication of proximal tubule injury. Twelve hours after LPS+, the activity of alkaline phosphatase was increased in urine samples and was still significantly increased after 48 h (Fig. 4d). Furthermore, the expression of kidney injury molecule 1 (Kim-1 [27]) was strongly up-regulated, 400 times more than control values, 6 and 12 h after LPS+ (Table b in the supplementary material 1). Except for BUN, aminoguanidine improved all parameters of renal function, as shown in Table 1 and Fig. 4. No differences in urinary protein excretion were found and, remarkably, the fractional excretion of filtered sodium was decreased (Table 1).
Fig. 4Proximal tubular damage during endotoxemia. Histological examination after LPS+ revealed damage to the proximal tubules compared to LPS−. a LPS−-treated rat with intact brush border membrane in proximal tubules. b Twelve hours after LPS+, damage to the proximal tubules is observed with loss of brush border membranes and formation of vacuoles. c Treatment with both LPS+ and aminoguanidine resulted in less damage to the proximal tubule. Kidneys of LPS+-treated rats killed after 24 or 48 h also showed signs of proximal tubule damage (data not shown). Proximal tubules are indicated by arrows. Original magnifications (a–c) ×400. d Histological data are supported by an increase in the activity of alkaline phosphatase, marker for proximal tubule damage, in urine samples 12 h after LPS+ administration (closed bars, n = 6), compared to controls (open bars, n = 3). Coadministration with aminoguanidine (gray bars, n = 6) reduced this proximal tubule damage. Data are expressed as mean±SE. Significantly different compared to the LPS− (double asterisksP < 0.01) or LPS+ (sharp signP < 0.05)
Furthermore, the mRNA expression of different biotransformation enzymes [28], enzymes involved in the NO pathway [2] or known to be activated during oxidative stress [29, 30] was determined. Twelve and 24 h after LPS+ treatment, expression of the metabolizing enzymes glutathione S-transferases (GST-α2 and GST-α5) and uridine diphosphate-glucuronosyltransferases (UDPGT1a6) was down-regulated (Table b in the supplementary material 1, at least a 2 times lower expression). The arginine metabolic enzymes, argininosuccinate synthetase and arginase 2, were not changed after LPS+. In contrast, arginase 1 mRNA expression was reduced. The gene expression of the ETB receptor, which gets activated as part of the NO-dependent signaling pathway regulating Abcb1/P-gp and Abcc2/Mrp2 [13, 31], did not change during endotoxemia. Three and 6 h after LPS+ the mRNA expressions of super oxide dismutase 2, heam-oxygenase 1, and nuclear factor κB (NF-κB) were up-regulated (i.e., at least a 2 times higher expression). Coadministration of aminoguanidine had no effect on the gene expression of all these proteins. In contrast, aminoguanidine reversed the down-regulation of nNOS gene expression during endotoxemia (Table b in the supplementary material 1).
LPS+ treatment affects ABC drug transporter expression in the kidney
The mRNA expression of 20 renal ABC transporters was profiled. Abcb1a/P-gp, Abcb1b/P-gp, Abcc2/Mrp2 and Abcc4/Mrp4 expression was determined with the ABI PRISM 7700 single reporter sequence detection system. In rodents, Abcb1/P-gp exists as two functional isoforms, which are encoded by two different genes: Abcb1a and Abcb1b [32]. The other ABC transporters were assessed with the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card (Table 2). Their expression was normalized for the average CT value for GAPDH, as described for iNOS. Other housekeeping genes, like glucuronidase and hypoxanthine guanine phosphoribosyl transferase 1, gave comparable values to GAPDH (data not shown). Abcb1b/Pg-p and Abcc2/Mrp2 were clearly up-regulated 3 h after LPS+ and were still elevated 12 and 24 h later (Fig. 5b,c). Furthermore, Abcb1a/P-gp and Abcc4/Mrp4 were slightly, but not significantly, increased 12 h after LPS+ treatment (Fig. 5a,d). Table 2 presents a list of the expression of other transporter genes during endotoxemia. Abca1 showed a slight up-regulation 3 h after LPS+, but returned to basal levels within 12 h. Furthermore, Abcb11 was up-regulated, but only significantly after 48 h. A down-regulation was observed for Abcb4 and Abcc8 (at least a 2 times lower expression). The expression of the Abcg genes was not affected by endotoxemia.
Table 2Gene expression of efflux transporters during endotoxemiaGene symbolProtein synonymAssay IDsDelta CT valuesaLPS− (controls)LPS+t = 3 hLPS+t = 6 hLPS+t = 12 hLPS+t = 24 hLPS+t = 48 hLPS+ + amino t = 6 hLPS+ + amino t = 12 hAbca1Abca 1Rn00710172_m17.1 ± 0.315.1 ± 0.165.8 ± 0.227.3 ± 0.557.3 ± 0.356.8 ± 0.236.1 ± 0.238.0 ± 0.12Abca2Abca 2Rn00577821_m13.5 ± 0.403.5 ± 0.354.1 ± 0.154.5 ± 0.384.3 ± 0.353.6 ± 0.064.4 ± 0.384.4 ± 0.12Abca5Abca 5Rn00597824_m19.8 ± 0.069.8 ± 0.359.8 ± 0.109.5 ± 0.1210.0 ± 0.2310.2 ± 0.219.5 ± 0.259.7 ± 0.35Abcb1aP_gpRn00591394_m18.4 ± 0.01Fig. 5aAbcb1bP_gpRn00561753_m110.8 ± 0.04Fig. 5bAbcb4Abcb 4Rn00562185_m110.7 ± 0.6212.3 ± 0.1612.5 ± 0.2112.8 ± 0.1510.9 ± 0.157.3 ± 0.4213.2 ± 0.4612.4 ± 0.24Abcb6Abcb 6Rn00589801_m15.5 ± 0.355.9 ± 0.266.4 ± 0.256.5 ± 0.525.9 ± 0.385.4 ± 0.146.4 ± 0.256.2 ± 0.32Abcb9Abcb 9Rn00573757_m18.1 ± 0.208.5 ± 0.389.3 ± 0.298.8 ± 0.178.6 ± 0.108.7 ± 0.179.2 ± 0.428.9 ± 0.15Abcb11BsepRn00582179_m110.5 ± 0.408.6 ± 0.3710.3 ± 0.2411.9 ± 0.218.7 ± 0.175.4 ± 0.499.9 ± 0.219.7 ± 0.21Abcc1Mrp1Rn00574093_m15.9 ± 0.256.2 ± 0.266.1 ± 0.256.1 ± 0.256.3 ± 0.215.9 ± 0.236.4 ± 0.406.2 ± 0.15Abcc2Mrp2Rn00563231_m16.7 ± 0.31Fig. 5cAbcc3Mrp3Rn00589786_m18.4 ± 0.158.7 ± 0.128.8 ± 0.5088.6 ± 0.068.0 ± 0.358.2 ± 0.128.8 ± 0.328.7 ± 0.23Abcc4Mrp4Rn01465699_q16.6 ± 0.06Fig. 5dAbcc5Mrp5Rn00588341_m17.1 ± 0.017.9 ± 0.037.6 ± 0.156.4 ± 0.066.5 ± 0.506.5 ± 0.387.6 ± 0.456.4 ± 0.36Abcc6Mrp6Rn00578778_m17.0 ± 0.147.0 ± 0.018.4 ± 0.218.4 ± 0.127.8 ± 0.056.8 ± 0.358.6 ± 0.078.3 ± 0.14Abcc8Sur1Rn00564778_m111.5 ± 0.4011.4 ± 0.2111.4 ± 0.2613.4 ± 0.1514.8 ± 0.1513.2 ± 0.2511.7 ± 0.2313.7 ± 0.31Abcc9Sur2Rn00564842_m16.8 ± 0.017.7 ± 0.268.4 ± 0.318.1 ± 0.157.2 ± 0.326.8 ± 0.408.0 ± 0.358.2 ± 0.32Abcg1Abcg1Rn00585262_m16.8 ± 0.157.8 ± 0.256.8 ± 0.257.1 ± 0.406.8 ± 0.316.7 ± 0.216.4 ± 0.317.3 ± 0.36Abcg2Abcg2Rn00710585_m14.1 ± 0.244.5 ± 0.074.6 ± 0.215.0 ± 0.064.5 ± 0.144.5 ± 0.074.9 ± 0.425.2 ± 0.07Abcg5Abcg5Rn00587092_m118.8 ± 0.29baData of nine pooled samples, measured in triplicate, are expressed as mean ± SDbLow expressed transport protein: CT value is not considered as validFig. 5mRNA expression of different renal ABC transporters. Gene expression of Abcb1a/P-gp (a) and Abcb1b/P-gp (b), Abcc2/Mrp2 (c), and Abcc4/Mrp4 (d) were determined in rat kidney at different time points after LPS− (open bars, n = 3) or LPS+ (closed bars, n = 6) treatment or LPS+ together with aminoguanidine (gray bars, n = 6). ΔCT values for the LPS−, given in Table 2, were set to 1. Data are expressed as mean±SE. Significantly different compared to the LPS− (single asterisks P < 0.05, double asterisks P < 0.01, triple asterisks P < 0.001) or LPS+ (sharp signs P < 0.05)
Coadministration with aminoguanidine blocked the induction in mRNA expression of both Abcb1b/P-gp and Abcc2/Mrp2 at 6 and 12 h, respectively (Fig. 5b,c). Furthermore, aminoguanidine reduced the increased protein expression of both transporters 12 h after LPS+ treatment (Fig. 6b,d), indicating a regulatory role for NO in the expression of these efflux transporters. Abcc4/Mrp4 protein levels, on the other hand, decreased even more after treatment with both LPS+ and aminoguanidine. Gene expression levels of other transporters were not affected by aminoguanidine (Table 2).
Fig. 6Protein expression of different renal ABC transporters. Total membrane fractions of rat kidney were isolated and expression of Abcb1/P-gp (a, b), Abcb11/Bsep (c), Abcc2/Mrp2 (d, e), and Abcc4/Mrp4 (f, g) was determined by Western blotting. Representative images (n = 4) show maximal Abcb1/P-gp protein expression 12 and 24 h after LPS+ (a), and coadministration with aminoguanidine reversed this induction (b). The protein expression of Abcb11/Bsep was up-regulated 12 h after LPS+ treatment, which was reversed by aminoguanidine (c). Maximal Abcc2/Mrp2 protein expression was seen 12 h after LPS+ (d) and aminoguanidine reversed this induction (e). Abcc4/Mrp4 protein expression was down-regulated during endotoxemia (f), which was intensified by aminoguanidine (g)
The expression patterns of Abcb1/P-gp, Abcb11, Abcc2/Mrp2, and Abcc4/Mrp4 were investigated further. Using total membrane fractions, we found an up-regulation of Abcb1/P-gp 6 h after LPS+, with maximal protein expression 12 and 24 h after LPS+ (Fig. 6a). Abcb11 is the major liver canalicular bile salt export pump (Bsep), but surprisingly, this transporter is also expressed at the apical membrane of mice renal proximal tubule, as we discovered recently [24]. In agreement with an increased mRNA expression, Abcb11/Bsep protein was up-regulated after LPS+ (Fig. 6c). Although we are not completely certain whether the C219 antibody against Abcb1/P-gp also detects Abcb11/Bsep [33], our Western blot results showed expression of Abcb1/P-gp, with a molecular weight of 130 kDa, and it does not seem to cross-react with Bsep at 160 kDa.
By using immunohistochemistry, the expression and localization of Abcb11/Bsep was investigated in endotoxemic rat kidneys. In agreement with mouse kidney, positive staining was observed in the apical membranes of the proximal tubule 12 h after LPS+ treatment (Fig. 7). In addition, we observed staining in rat glomerular endothelium and peritubular capillaries.
Fig. 7Distribution of Abcb11/Bsep in rat kidney cortex. a, b Representative immunohistochemical images show apical staining in proximal tubules (indicated by arrows). In addition, glomerular and peritubular staining of Bsep in the cortex was found. G glomerulus, asterisks peritubular staining, P proximal tubule, D distal tubule. Original magnification in a ×200, and in b ×400
Furthermore, a clear increase in Abcc2/Mrp2 protein was observed after LPS+ (Fig. 6d), but a down-regulation in Abcc4/Mrp4 was found (Fig. 6f). We measured the function of Abcc2/Mrp2 by monitoring the urinary excretion of calcein in an isolated perfused rat kidney 12 h after LPS+ or LPS−. A 16 ± 3% increase in calcein excretion was observed after LPS+, as shown in Fig. 8. We do not expect calcein to be a Bsep substrate because Mrp2-deficient (TR−) rats clearly showed that calcein excretion was highly reduced [25], despite the presence of Bsep in these strains (unpublished data). Twenty-four hours after LPS+, Abcc2/Mrp2 activity was still elevated, although not significantly. In accordance, endotoxemia affected renal function as observed by diminished GFR and fractional reabsorption of water in isolated perfused rat kidneys (Table 3).
Fig. 8Renal calcein excretion in isolated perfused rat kidneys. Secretion of the fluorescent calcein into the urine was measured after 12 h in untreated (closed squares control; n = 4), LPS− (open squaresn = 4), or LPS+ (trianglesn = 6) rats. Urinary excretion was corrected for GFR. Significantly different compared to the LPS− (asterisk P < 0.05)Table 3Functional parameters of isolated perfused rat kidneys in control rats (no additional treatment) and after exposure to LPS− or LPS+ for 12 hParameterControls (n = 4)LPS− (n = 4)LPS+ (n = 6)GFR (μl/min)350 ± 25380 ± 17290 ± 20aDiuresis (μl/min)18.6 ± 1.821.1 ± 1.317.4 ± 1.0FRwater (%)94.7 ± 0.294.5 ± 0.392.7 ± 0.6aRPP (mmHg)b88.4 ± 1.584.5 ± 1.788.7 ± 1.0Data are expressed as mean ± SE of four to six perfused kidney experiments over the period 25-115 min. Untreated rats served as controls for this model. Kidneys were perfused for 150 min as described in the “Materials and methods” sectionaSignificantly different from perfused control kidney (P < 0.05)bConstant flow during the experiments was 18 ± 0.5 ml/minFR fractional reabsorption, RPP renal perfusion pressure
Discussion
In the present study, we investigated the role of NO, produced by iNOS, in the differential expression of renal ABC transporters in rats in vivo. A clear up-regulation of Abca1, Abcb1/P-gp, Abcb11/Bsep, and Abcc2/Mrp2 expression was found after LPS+. In addition, we observed a functional up-regulation of Abcc2/Mrp2 in perfused kidneys. This supports a detoxifying role of the efflux pump, which is in accordance with the up-regulation of Abcc2/Mrp2 protein in killifish renal tubules [34, 35], in rat kidney, and in MDCKII cells overexpressing human ABCC2/MRP2 after long-term exposure to the nephrotoxicant gentamicin [20], and of abcb1/P-gp in mouse kidneys after ischemic reperfusion injury [24]. Indeed, blocking Abcb1/P-gp during endotoxemia leads to further renal damage (Heemskerk, Wouterse, Russel, and Masereeuw, preliminary findings). The renal drug transporters are likely to be under direct influence of NO produced by iNOS, as coadministration of the iNOS inhibitor, aminoguanidine, attenuated the LPS-induced effects on transporter expression. Aminoguanidine treatment results in decreased NO production and, subsequently, reduced oxidative stress by decelerating the formation of peroxynitrite. Under these conditions, less toxic metabolites may be produced, explaining the restoration in transporter expression and reversal of proximal tubular damage.
In contrast to our findings, Cherrington et al. [36] reported that coadministration of aminoguanidine failed to change the expression of different hepatic drug transporters during endotoxemia. This discrepancy might be due to organ differences and the fact that Cherrington et al. pretreated the animals with the NOS inhibitor, whereas we gave aminoguanidine 1 h after LPS+. The lack of an effect of aminoguanidine on the expression of different biotransformation enzymes, enzymes involved in the iNOS pathway and during oxidative stress, supports a direct function of NO itself in ABC transporter regulation.
The role of two up-regulated efflux pumps, Abca1 and Abcb11/Bsep, in the kidney is unknown. In humans, ABCA1 is widely expressed and found to be the causative gene in Tangier disease, a disorder of cholesterol transport between tissues and the liver, mediated by binding of the cholesterol onto high-density lipoprotein particles [37]. ABCA1 controls the extrusion of membrane phospholipid and cholesterol toward specific extracellular acceptors; however, the exact role of the protein in this process is not known. Moreover, its expression and function in the kidney requires further investigation. The presence of Abcb11/Bsep, the major canalicular bile salt export pump, was recently demonstrated by us in mouse kidney [24], and here we show a clear apical expression in rat kidney proximal tubules for the first time as well. Hence, the function of Abcb11/Bsep in the kidney still needs to be determined. In addition to their up-regulation in the kidney, we found a down-regulation of Abcb1/P-gp, Abcc2/Mrp2, and Abcb11/Bsep in the liver (data not shown). This is in agreement with previous findings for the hepatic expression of the transporters during inflammation [36, 38–40], obstructive jaundice [41], and cholestasis [42]. An induction of the transporters in the kidney during liver failure may function as a compensatory elimination mechanism to reduce enhanced levels of circulating bile salts caused by endotoxin-induced cholestasis [39] and/or to protect proximal tubular cells against the higher exposure via primary urine.
A differential regulation was found for Abcc4. Although this transporter is expressed in the same nephron segments as Abcb1/P-gp and Abcc2/Mrp2, the apically expressed efflux pump clearly has a distinct regulation. In killifish, we previously found renal proximal tubules exposed to nephrotoxicants or ET-1 that Abcc2/Mrp2-mediated transport and Mrp2 expression in the luminal membrane were increased after 24 h [34], whereas the expression of Abcc4/Mrp4 remained unchanged [35]. Furthermore, Abcc4/Mrp4 protein levels in rat kidney were decreased during cholestasis in contrast to the Abcc2/Mrp2 levels [43]. No changes in Abcc4/Mrp4 mRNA were found, suggesting a posttranscriptional regulation of transporter expression, including phosphorylation and protein routing. A down-regulation of ABC transporters, as observed for Abcc8/Sur1 and initially also for Abcb4, may be explained by the different functions of these ABC members in the kidney.
LPS itself may affect transporter expression after binding to the Toll-like receptor 4 and a second, not yet identified, factor through activation of NOS-dependent pathways, as was found for Abcb1/P-gp in rat brain capillaries [44]. Furthermore, systemic TNF-α, released during endotoxemia, may regulate efflux pumps, including ABC transporters [45, 46]. This cytokine might act through NOS activation [44], but also through a pathway that does not involve iNOS-derived NO during endotoxin-associated renal failure [47]. Furthermore, regulation of iNOS after exposure to LPS occurs at the level of gene transcription and may require activation of the NF-κB pathway [48]. It has been shown previously that an up-regulation of Abcb1/P-gp in renal failure, i.e., after ischemic–reperfusion damage [24] or exposure to cadmium [49], might be signaled through an NF-κB-dependent pathway. Consistent with these findings, we observed a clear induction (sevenfold) of NF-κB expression 3 h after LPS+ as well.
Selective inhibition of renal iNOS has important implications for the treatment of septic acute kidney disease. Changes in the expression levels of renal drug transporters alters the pharmacokinetics of many clinically important drugs [50] and has implications for the prediction of overall drug disposition during sepsis. For example, glucocorticoids are substrates for Abcb1/Pg-p and are often prescribed for their anti-inflammatory properties in septic patients. An up-regulation of Abcb1/Pg-p, as observed in our study, might have consequences for therapeutic levels of these substrates [51].
In conclusion, after LPS+, a clear up-regulation in Abca1, Abcb1/P-gp, Abcb11/Bsep, and Abcc2/Mrp2 was found, whereas Abcc8/Sur1 was down-regulated. Abcb4 and Abcc4/Mrp4 show a differential pattern during the course of endotoxemia. The renal drug transporters are likely to be under direct influence of NO produced by iNOS, as coadministration of the iNOS inhibitor, aminoguanidine, attenuated the LPS-induced effects on their expression. Apparently, an up-regulation of important efflux pumps diminishes the renal accumulation of toxic compounds and attenuates further proximal tubular damage during endotoxemia.
Electronic supplementary material
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inos
lipopolysaccharide
aminoguanidine
nephrotoxicity
renal drug transport


Accumulation of fibronectin in the heart after myocardial infarction: a putative stimulator of adhesion and proliferation of adipose-derived stem cells

Stem cell therapy is a promising treatment after myocardial infarction (MI). A major problem in stem cell therapy, however, is that only a small proportion of stem cells applied to the heart can survive and differentiate into cardiomyocytes. We hypothesized that fibronectin in the heart after MI might positively affect stem cell adhesion and proliferation at the site of injury. Therefore, we investigated the kinetics of attachment and proliferation of adipose-tissue-derived stem cells (ASC) on fibronectin and analysed the time frame and localization of fibronectin accumulation in the human heart after MI. ASCs were seeded onto fibronectin-coated and uncoated culture wells. The numbers of adhering ASC were quantified after various incubation periods (5–30 min) by using DNA quantification assays. The proliferation of ASC was quantified after culturing ASC for various periods (0–9 days) by using DNA assays. Fibronectin accumulation after MI was quantified by immunohistochemical staining of heart sections from 35 patients, after different infarction periods (0–14 days old). We found that ASC attachment and proliferation on fibronectin-coated culture wells was significantly higher than on uncoated wells. Fibronectin deposition was significantly increased from 12 h to 14 days post-infarction, both in the infarction area and in the border-zone, compared with the uninfarcted heart. Our results suggest that a positive effect of fibronectin on stem cells in the heart can only be achieved when stem cell therapy is applied at least 12 h after MI, when the accumulation of fibronectin occurs in the infarcted heart.
Introduction
Cardiovascular diseases are a leading cause of morbidity and mortality in the western world (Wang and Sjoquist 2006), with heart failure attributable to myocardial infarction (MI) being the most common aetiology (Lu et al. 2004). Heart failure partly is caused by the loss of cardiomyocytes and the limited capacity of spontaneous regeneration of the heart. This results in the replacement of injured myocardium by scar tissue, which adversely affects cardiac function (Smits et al. 2005; Wang and Sjoquist 2006).
To reduce heart failure after MI, current therapeutic strategies focus on the restoration of the blood flow to the myocardium and on reducing cardiac remodelling by using drug therapy (inhibitors of angiotensin-converting enzyme and beta blockers; Fraser et al. 2004). Although these strategies significantly reduce mortality (Khalil et al. 2001; Lange and Hillis 2002), they do not replace the lost cardiomyocytes.
A promising tool to restore contractile function after myocardial infarction is stem cell therapy. Stem cells not only restore neovascularization, but also replace lost cardiomyocytes, since they have the capacity to differentiate into cardiomyocytes (Orlic et al. 2001; Strauer et al. 2002; Wang and Sjoquist 2006). However, to restore contractile function of the heart effectively after MI, sufficient stem cells need to attach to the infarcted area and subsequently proliferate and differentiate into cardiomyocytes, thereby replacing a substantial fraction of the jeopardized cardiomyocytes. Unfortunately, recent studies suggest that only a small proportion of stem cells applied to the heart invade the infarcted area and finally differentiate into cardiomyocytes. Furthermore, most of the applied stem cells die within the first week after transplantation (Wang and Sjoquist 2006; Zhang et al. 2001).
This high stem cell death rate is probably caused by the inferior environment that is found in the heart after myocardial infarction and into which the cells are transplanted; this poor environment is caused by the presence of inflammatory mediators and ischemia (Azarnoush et al. 2005; Wang and Sjoquist 2006; Zhang et al. 2001). Therefore, stem cell therapy has to be applied at that moment after infarction when the environment is most favourable for stem cell adhesion and cardiomyocyte formation. However, to the best of our knowledge, this ideal time frame has yet not been determined (Mishra 2005). In most animal studies, investigators apply stem cells during the same operational procedure as the infarct induction, whereas recent studies have shown that later time points might be more favourable for the survival of the stem cells (Bermejo et al. 2006; Lu et al. 2004; Wang and Sjoquist 2006).
Homing, growth and differentiation of stem cells after myocardial infarction is known to depend on several environmental factors, including the presence of adhesion factors at the site of injury (Chastain et al. 2006; Lu et al. 2004; Malek et al. 2006; Salasznyk et al. 2004; Wang and Sjoquist 2006). Fibronectin has been demonstrated to enhance the attachment of bone-marrow-derived stem cells in vitro, compared with several other extracellular matrix (ECM) molecules (Cool and Nurcombe 2005; Ogura et al. 2004). Fibronectin is a large molecular weight glycoprotein, present at low levels in the ECM of the healthy heart (Heng et al. 2004; Willems et al. 1996). In addition to the influence of fibronectin on cell attachment, the presence of fibronectin can also affect stem cell behaviour such as migration and proliferation, both in vitro and in vivo (Tate et al. 2002; van der Loo et al. 1998; Voermans et al. 1999; Wijelath et al. 2004). However, the effect of fibronectin on adipose-tissue-derived stem cells (ASC) has as yet not been investigated. Therefore, we have examined whether fibronectin stimulates the attachment of ASC and have compared this with the attachment of ASC to other ECM proteins that are normally expressed in the heart and that are upregulated after acute MI (AMI), namely collagen type 1 and laminin (Deten et al. 2001; Morishita et al. 1996). We have also investigated the effect of fibronectin on the proliferation of ASC in vitro.
In vivo, fibronectin is strongly upregulated in the heart after MI (Froen and Larsen 1995; Knowlton et al. 1992; Willems et al. 1996). We have hypothesized that fibronectin serves as an attractant for ASCs, thereby improving stem cell homing, attachment and proliferation. Therefore, we have studied the kinetics and the localization of fibronectin accumulation in the human heart after MI in detail, in heart sections from 35 patients, representing various infarction periods.
Materials and methods
Adipose tissue donors
Human subcutaneous adipose tissue samples were obtained as waste material after elective surgery and donated upon informed consent of the patients from various clinics in Amsterdam, The Netherlands. Adipose tissue was harvested from the abdomen or hip and thigh region by using either resection or tumescent liposuction. Five donors (age-range: 27–49 years) were included in this study.
Isolation of the stromal vascular fraction of adipose tissue
Adipose tissue was stored in sterile phosphate-buffered saline (PBS) at 4°C and processed within 24 h after surgery as described previously (Oedayrajsingh-Varma et al. 2006). In brief, resected material was minced by using a surgical scalp before processing. After extensive washes with PBS, the ECM was enzymatically digested with 0.1% collagenase A (Roche Diagnostics, Mannheim, Germany) in PBS containing 1% bovine serum albumin (BSA; Roche Diagnostics) under intermittent shaking for 45 min at 37°C. Then the material was washed with Dulbecco’s modified Eagle’s medium (DMEM-glucose; BioWhittaker, Cambrex, Verviers, Belgium) containing 10% fetal bovine serum (FBS) and centrifuged for 10 min at 600g. The cell pellet was resuspended in PBS and passed through a 200-μm mesh (Braun/Beldico, Marche-en-Famenne, Belgium) to obtain a single cell suspension. To remove contaminating erythrocytes, the cells were subjected to Ficoll density centrifugation (lymphoprep, ρ=1.077 g/ml, osmolarity: 280±15 mOsm; Axis-Shield, Oslo, Norway). The cell-containing interface was harvested and washed with DMEM containing 10% FBS. Cells were frozen under “controlled rate” conditions and stored in liquid nitrogen until needed in experiments.
Cell culture
Cells from the stromal vascular fraction were seeded at 1×105 cells/cm2 and cultured for several passages in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine (all from Gibco, Invitrogen, Calif., USA; normal culture medium), in a humidified atmosphere of 5% CO2 at 37°C. Media were changed twice a week. When reaching 80%–90% confluency, cells were detached with 0.5 mM EDTA/0.05% trypsin (Gibco, Invitrogen) for 5 min at 37°C and replated.
Attachment of ASC
To investigate attachment of ASC to different coatings, culture-expanded cells (passages 3–6; p3-6) were seeded in a 96-well culture plate at a density of 25,000 cells per well. Wells were either uncoated or coated with fibronectin (5 or 0.5 μg/cm2; Roche Diagnostics), laminin (0.12 μg/cm2; Roche Diagnostics) or collagen (2.5 μg/cm2, rat tail collagen type I; BD Biosciences, Bedford, Mass., USA). Cells were then allowed to attach undisturbed in a humidified incubator (5% CO2, 37°C) for different incubation times (5 min, 10 min, 30 min), after which unattached cells were removed by washing with PBS. To standardize the conditions, all the conditions at the same timepoint were added simultaneously to one microplate. The number of attached cells was quantified by assaying for DNA with a CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s protocol.
To achieve an in vitro situation more comparable to the in vivo situation after AMI, attachment of ASC to cardiac muscle cells and to fibronectin-coated cardiac muscle cells was investigated. Cardiomyoblasts (H9c2, p18; ATCC, Manassas, Va., USA) were seeded into 24-well culture dishes, at a density of 10,000 cells per well, and when wells were 70% confluent, cells were either coated with fibronectin (0.5 or 5.0 μg/cm2) or remained uncoated. Then culture-expanded cells (p3–p7) from four donors were seeded into the culture dishes, at a density of 40,000 cells per well. Cells were allowed to attach undisturbed in a humidified incubator (5% CO2, 37°C) for various incubation times (5 min, 10 min, 30 min), after which the unattached cells were removed by washing with PBS. The cells were subsequently trypsinized, washed, centrifuged at 600g for 5 min, resuspended in 50 μl PBS and incubated with anti-CD90 phycoerythrin-labelled antibody (1:20; BD Biosciences Pharmingen, San Diego, Calif., USA) for 30 min on ice. The percentage of CD90-positive cells (ASC) was quantified by using fluorescence-activated cell sorting (FACS) analysis (FACScaliber, Becton Dickinson, San Jose, Calif., USA).
Proliferation of ASC
To assess ASC proliferation on fibronectin-coated culture dishes, culture-expanded cells (p3–p6) from three donors were plated into 6-well culture plates, at a density of 25,000 cells per well, either coated with 5 μg/cm2 fibronectin or uncoated, in normal culture medium. After a 1-day incubation in a humidified incubator (5% CO2, 37°C), unattached cells were removed by washing with PBS. Cells were then allowed to proliferate for various incubation times (0 days, 3 days, 6 days, 9 days). Cells were washed with PBS to remove dead cells. The amount of living cells removed by this washing was negligible (<0.5%). The number of attached cells was quantified by assaying for DNA with a CyQUANT Cell Proliferation Assay Kit (Invitrogen) according to the manufacturer’s protocol.
Myocardial tissue from infarction
Myocardial tissue from 27 autopsied patients who had died from AMI and from eight autopsied patients with no cardiac lesions of any kind was obtained from the Department of Pathology for authopsy, with approval of the ethics committee of the VU Medical Centre, Amsterdam. The use of left-over material after the pathological examination is completed, is part of the standard patient contract in our hospital. Heart tissue was sampled as soon as possible, within 24 h of death.
Characterization of infarction phase
Recent AMI was defined by using macroscopic (lactate dehydrogenase decolouration) and microscopic criteria to estimate infarct duration and the viability of cardiomyocytes in all myocardial tissue specimens (paraffin-wax-embedded sections stained with haematoxylin and eosin). Clinical data with respect to the duration of the infarction corresponded to the time intervals of the various morphological phases of infarction. Jeopardized myocardium was characterized by the intensity of eosinophilic staining of involved myofibres, cell condensation, loss of nuclei and cross striation and was visualized by complement (C3d) staining. We defined heart tissue sections without microscopic changes but with macroscopic lactate dehydrogenase decolourization as a phase 1 infarct (3– to 12-h-old AMI), infiltration of polymorphonuclear leucocytes as a phase 2 infarct (12-h to 5-day-old AMI) and infiltration of lymphocytes and macrophages and fibrosis as a phase 3 infarct (5– to 15-day-old AMI; Krijnen et al. 2003). Table 1 shows the distribution of the various infarct phases among the different patients.
Table 1Characterization of the different infarct phases used in the immunohistochemical study (LDH lactate dehydrogenase)InfarctionphaseNumberof patientsInfarct durationDefinition08No infarctionDoes not apply1113–12 hLDH decolourization,without extravascularneutrophilic granulocytes.2912 h–5 daysExtravascular neutrophilicgranulocytes.375–14 daysGranulation tissueformation
Immunohistochemistry
Myocardial tissue was fixed with 4% buffered formaldehyde and embedded in paraffin. Paraffin sections (3–4 μm) were mounted on glass slides, dried, deparaffinized, rehydrated, and then used for immunohistochemistry. Sections were stained for fibronectin. Serial sections were stained for complement factor C3d to define the microscopical infarction area (Krijnen et al. 2003). All antibodies used were from DakoCytomation (Glostrup, Denmark).
For C3d staining, antigen retrieval was performed by boiling sections in 10 mM sodium citrate buffer, pH 6.0, for 10 min in a microwave oven. Sections were pre-incubated with normal swine serum (1:10; DakoCytomation) for 10 min at room temperature, followed by an incubation with rabbit antibodies against human C3d (1:1,000) for 1 h at room temperature. The sections were then washed with PBS and incubated with biotin-conjugated swine-anti-rabbit antibodies (1:300) for 30 min, washed in PBS and incubated with streptavidin-biotin complex/horseradish peroxidase (1:200) for 1 h. Staining was visualized by using 3,3′-diaminobenzidine (DAB; 0.1 mg/ml, 0.02% H2O2).
For fibronectin staining, antigen retrieval was performed by incubating the sections with 0.1% pepsine-HCl for 30 min at 37°C. Sections were incubated with rabbit antibodies against human fibronectin (1:18,000) for 1 h at room temperature, followed by incubation with 100 μl envision (ready for use kit, anti-Mouse IgG and anti-Rabbit IgG) for 30 min at room temperature. Staining was visualized with envision DAB (DakoCytomation). Finally, the sections were counterstained with haematoxylin, dehydrated and cover-slipped. Control sections were incubated with PBS instead of the primary antibody.
All slides were judged and scored for infarct age and anatomical localization of fibronectin and C3d, as visualized by immunohistochemical staining. The border-zone of the infarct was defined as the area surrounding the microscopic infarction area, as characterized by C3d staining. Since only the fibronectin on the plasma membrane and in the ECM are available for ASC attachment, fibronectin deposition was scored separately for the ECM, plasma membrane and intracellular components (nucleus and cytoplasm). The extent of the fibronectin deposits was determined by quantifying the mean surface area occupied by cardiomyocytes positive for fibronectin and expressed as a percentage of the total surface area of the sections in the infarcted region, border-zone and non-infarcted areas. Areas were scored as negative (0), 1%–5% of the cells positive (1), 6%–25% positive (2), 26%–50% positive (3), 51%–75% positive (4) or 76%–100% positive (5). Since positivity of fibronectin in the ECM is difficult to quantify, fibronectin staining in ECM was related to its intensity score as follows: negative (0), minor positive (1), intermediate positive (2) or strongly positive (3).
Statistics
Statistics were performed with the SPSS statistics program (Windows version 9.0) and Graphpad Prism (version 4). Attachment and proliferation data were analysed by using Repeated Measures. Fibronectin accumulation was analysed by the Kruskal Wallis and the Dunn’s Multiple Comparison Test. A P-value of less than 0.05 was considered to be significant.
Results
Attachment and proliferation of ASCs on fibronectin
To investigate the attachment of ASC to various ECM molecules, the percentage of ASCs adhering to laminin, fibronectin, collagen-1 and uncoated culture wells was analysed. We monitored the number of ASCs in the culture wells and compared this with the numbers of cells in uncoated culture wells. As shown in Fig. 1, after 5 and 10 min of incubation, significantly higher numbers of ASC were detected on fibronectin-coated culture wells as compared with the uncoated culture wells (P<0.05). This effect was dose-dependent, since a lower concentration of fibronectin (0.5 μg/cm2 instead of 5.0 μg/cm2) did not increase ASC attachment. Laminin and collagen-1 did not significantly affect ASC attachment, although collagen-1 showed a trend towards increased attachment of ASC. After 30 min, a plateau level seemed to be reached for all conditions. This suggested that ASC adhered more rapidly to fibronectin than to laminin, collagen or uncoated culture wells.
Fig. 1ASC adherence to laminin-coated, collagen-1 (col-1)-coated and fibronectin (fibro)-coated (5 and 0.5 μg/cm2) and uncoated culture wells monitored with time. Numbers of adhered ASCs were quantified after 5 (a), 10 (b) and 30 (c) min of attachment. Results are expressed as the percentage of applied cells. Error bars represent means±SEM (n=4, *P<0.05, Repeated measures)
To investigate the kinetics of ASC attachment in a setting more comparable to the in vivo situation after AMI, the percentage of ASCs adhering to fibronectin-coated and uncoated cardiac muscle cells was monitored with time. Numbers of adhered ASCs were quantified after 5, 10 and 30 min of attachment and are shown as the percentage of total cells applied in Fig. 2. ASC rapidly adhered to the fibronectin-coated cardiac muscle cells. This rapid attachment was most prominent after 5 min of incubation, when 5 times more ASC attached to the fibronectin-coated cells than to the uncoated cells (P<0.05). This effect was independent of the dose of fibronectin, since the ASC also attached rapidly to the cardiac muscle cells coated with a lower concentration of fibronectin (0.5 μg/cm2 instead of 5.0 μg/cm2). On the uncoated cardiac muscle cells, however, attachment was limited when compared with the fibronectin-coated cells and increased only gradually the first 10 min. For all conditions, a plateau level was reached after 30 min of attachment. These findings suggested that ASC attached more rapidly on fibronectin-coated cardiac muscle cells than to uncoated cardiac muscle cells.
Fig. 2Kinetics of ASC adherence to fibronectin-coated (5 and 0.5 μg/cm2) and uncoated cardiac muscle cells monitored with time. Numbers of adhered ASCs were quantified after 5 (a), 10 (b) and 30 (c) min of attachment. Results are expressed as the percentage of applied cells. Error bars represent means±SEM (n=4, *P<0.05, Repeated measures)
To study the proliferation of ASCs on fibronectin, ASCs were seeded in fibronectin-coated culture wells. Unattached cells were washed away after 24 h (day 0). We monitored the number of ASCs in the culture wells with time (days 0, 3, 6, 9) and compared this with the numbers of cells in uncoated culture wells. Significantly higher numbers of ASC were detected on fibronectin-coated culture wells compared with the uncoated culture wells during the 9-day culture period (P=0.012, Repeated measures, n=3; Fig. 3).
Fig. 3Kinetics of ASC proliferation on fibronectin-coated (5 μg/cm2) and uncoated culture wells. Numbers of adhered ASCs were quantified after 0, 3, 6 and 9 days of culture by using the CyQUANT cell proliferation assay. Results are expressed as percentage of cells, compared to day 0, both for the fibronectin-coated and uncoated wells. Error bars represent means ± SEM. (n = 3, p = 0,012, Repeated measures)
Fibronectin accumulation after infarction
Fibronectin accumulation after myocardial infarction was investigated in heart sections from 35 patients, representing various infarction periods (no infarction, infarction of 3–12 h old, 12 h–5 days old, and 5–14 days old). To define jeopardized cardiomyocytes and thus the microscopical infarction area, we stained serial sections for complement factor C3d (Fig. 4c). Fibronectin was found not only in cardiomyocytes (plasma membrane, cytoplasm and nucleus; Fig. 4a), but also in the ECM (Fig. 4b). Fibronectin co-localized with complement factor C3d but fibronectin deposition was also found in the border-zone adjacent to the infarction area (complement-negative). To analyse fibronectin depositions in the human heart after myocardial infarction, we scored all sections for the extent and the anatomical localization of fibronectin depositions. Notably, in AMI phase 1, the border-zone and infarcted area could not be analysed as such with the slides from the macroscopical infarction area, since it was not possible to identify a microscopical infarction area clearly at this early phase, as complement was then negative. As shown, the intensity of fibronectin depositions in the ECM (Fig. 5) was significantly increased at the site of myocardial infarction of AMI phases 2 (P<0.01) and 3 (P<0.05) compared with the uninfarcted heart. The extent of fibronectin deposition was also increased in the border-zone of phases 2 and 3; however, this increase was not statistically significant. In phase 1 infarction and in the uninfarcted areas, no increase in ECM fibronectin intensity score was found, when compared with uninfarcted hearts (not shown).
Fig. 4Localization of fibronectin in myocardial infarction of the human heart. Fibronectin accumulation after myocardial infarction was investigated in heart sections representing different infarction periods. a High power view of fibronectin deposition in infarct phase 2. Fibronectin depositions were found on the plasma membrane (arrow I), in the nucleus (arrow II) and in the cytoplasm (arrow III) of jeopardized cardiomyocytes. b Fibronectin depositions in infarct phase 3 in the extracellular matrix (ECM; arrow IV). c C3d deposition in infarct phase 2 (+ jeopardized cardiomyocytes, i.e. complement-positive cells, − complement-negative cells). d Fibronectin colocalization within C3d-positive areas in a serial section from the same patient as in c, in infarct phase 2. As a control, AMI sections were incubated with PBS instead of mAb and gave no positive staining (not shown)Fig. 5Fibronectin deposition in the ECM of the human heart after myocardial infarction. Intensity of the fibronectin deposition in the ECM was scored and compared with that of the non-infarcted heart (stadium 0). Areas were scored negative (0), minor positive (1), intermediate positive (2) or strongly positive (3). Error bars represent means±SEM (*P<0.05, **P<0.01)
We subsequently scored fibronectin positivity in the cardiomyocytes. The extent of fibronectin deposition was significantly increased in the infarcted area of AMI phase 2, compared with controls, at the plasma membrane (P<0.05), in the cytoplasm (P<0.001) and in the nucleus (P<0.05). Fibronectin depositions were increased not only in the infarcted area, but also in the border-zone of phase 2; however, this increase was only statistically significant in the nucleus when compared with the non-infarcted heart. In AMI phase 3, no cardiomyocytes in the infarction area could be analysed since this area no longer contains cardiomyocytes in this stadium, but only granulation tissue. The extent of fibronectin deposition was still significantly increased in the border-zone of AMI phase 3 in the nucleus and cytoplasm but was not positive on the plasma membrane (Fig. 6a). Neither in AMI phase 1 (Fig. 6) nor in non-infarcted areas (not shown) was an increase in fibronectin positivity found in the cardiomyocytes when compared with controls (Fig. 6).
Fig. 6Fibronectin deposition in cardiomyocytes in the human heart after myocardial infarction (IH immunohistochemical). a Area of fibronectin deposition in the plasma membrane. b Area of fibronectin deposition in the cytoplasm. c Area of fibronectin deposition in the nucleus. Areas are scored from 0 for no fibronectin-positive cells up until 5 for 75%–100% fibronectin-positive cells. Error bars represent means±SE (*P<0.05, **P<0.01) (Phase 0: n=8, phase 1: n=11, phase 2 border: n=6, phase 2 infarct: n=9, phase 3 border: n=7)
Discussion
We hypothesized that accumulation of fibronectin in the heart after MI might improve the fate of ASC after transplantation in the heart. Therefore, we have studied the effect of this protein on the attachment and proliferation of ASC in vitro. In addition, we have investigated the time course and the localization of the accumulation of fibronectin after myocardial infarction in the human infarcted heart. We have found that ASC attachment and proliferation on fibronectin-coated plates is significantly increased compared with that on uncoated plates (early attachment: P<0.05, proliferation: P=0.009, Repeated measures, n=4) and that attachment of ASC to cardiac muscle cells is significantly increased when cells are coated with fibronectin (0.5 μg/cm2 and 5.0 μg/cm2), after 5 min of attachment. In the ECM of the human heart, fibronectin depositions are significantly increased at AMI phase 2 and phase 3 in the infarction area, whereas fibronectin deposition in the cytoplasm, at the plasma membrane and in the nucleus of cardiomyocytes is significantly increased at AMI phase 2, but not in AMI phase 3, except for depositions in the nucleus.
When culture wells are coated with fibronectin, we have found an increased attachment of ASC when compared with uncoated plates, even though ASC are mesenchymal stem cells that are defined by their strong attachment to plastic culture wells (Wagner et al. 2005). We have also found that neither laminin nor collagen-1 significantly increases the attachment of ASC, unlike fibronectin, which does cause an increase. These findings are comparable with studies of Ogura et al. (2004) and Cool and Nurcombe (2005), in which an increased attachment of human bone-marrow-derived MSC to fibronectin-coated culture plates has been found when compared with albumin-coated or other ECM-coated culture plates. Ogura et al. (2004) have also reported that attachment to fibronectin is most prominent within the first 20 min of incubation. We now have shown that fibronectin also improves the attachment of ASC.
In an experiment mimicking the in vivo situation after a myocardial infarction, we have demonstrated that fibronectin also increases the attachment of ASC to cardiac muscle cells, even when a relatively low concentration of fibronectin is used. These results are in agreement with our hypothesis that fibronectin depositions will also increase stem cell attachment in vivo, independent of fibronectin concentration, underlining the importance of fibronectin in stem cell transplantation.
In this study, we have additionally demonstrated a significant increase in the proliferation of ASC cultured in fibronectin-coated wells, compared with proliferation in uncoated culture wells (P=0.012). This effect is most prominent during the first 5 days of culture; thereafter, the cells grow confluently and the effect diminishes. Cool and Nurcombe (2005) have found no significant effect of fibronectin on proliferation, although a trend is visible; this might be caused by the timepoint of their study, since they have only measured the difference in cell numbers at day 7, or could be related to the source of stem cells.
Since the presence of fibronectin promotes ASC attachment and proliferation in vitro in our study, we have hypothesized that the presence of fibronectin in the heart after myocardial infarction might positively affect stem cell adhesion and proliferation at the site of injury. As such, stem cell therapy would be most efficient when fibronectin depositions are high. We have found an increase in the extent of fibronectin depositions in cardiomyocytes during AMI phase 2, whereas fibronectin deposition in the ECM is significantly increased in the infarction area during AMI phase 2 and phase 3, when compared with the uninfarcted heart.
These results are comparable with the findings of several animal studies, in which increased fibronectin mRNA expression has been demonstrated, together with increased fibronectin deposition in the infarcted area as early as the first day after infarct induction (Carlyle et al. 1997; Froen and Larsen 1995; Knowlton et al. 1992; Shekhonin et al. 1990; Ulrich et al. 1997). Moreover, several studies have shown increased deposition of fibronectin in the infarction area after MI in human (Hu et al. 1996; Ishikawa et al. 2000, 2003; Ortmann et al. 2000; Willems et al. 1996). However, none of these studies have scored the fibronectin depositions as accurately as we have during the different infarction phases, i.e. by using complement to define the microscopic infarction area, by differentiating between infarction area and border-zone and by separately scoring the ECM and the various anatomical locations in the cells.
We have studied the various anatomical locations of fibronectin depositions separately, since theoretically only fibronectin on the plasma membrane and in the ECM should play a direct role in stem cell attachment. Although fibronectin has been detected on the ECM and the plasma membrane of cardiomyocytes, depositions in the cytoplasm and the nuclei are more widespread than on the plasma membrane. These strong depositions in the nucleus and cytoplasm have also been reported by Ishikawa et al. (2000); however, they have only described these nuclear depositions and not scored them.
Notably, we have found that the extent of fibronectin depositions in the heart is only increased in the infarcted area and the border-zone, and not in the non-infarcted areas. This is an important observation, since stem cell attachment and differentiation is only desirable in the infarcted area and the area surrounding it for stem cell therapy. In accordance with this finding, Tran et al. (2006) have indeed shown that rat bone marrow MSC are retained better in the infarcted area, when compared with the intact myocardium.
Several other studies have also investigated the time frame after MI with respect to when best to apply stem cell therapy, both in animal models and human stem cell transplantation studies, by investigating molecular events and stem cell retention at the site of infarction (Bermejo et al. 2006; Lu et al. 2004; Ma et al. 2005). These studies suggest that stem cell therapy should neither be applied during the acute inflammation phase (within 1 day after infarction), nor after a period longer than 2 weeks when scar tissue has been formed. In agreement with this suggestion, our results indicate that a putative effect of fibronectin on stem cell therapy can only be achieved when stem cell therapy is applied more than 12 h but within 14 days after infarction.
In conclusion, we have shown, for the first time, that fibronectin improves ASC attachment and proliferation. To achieve a positive effect of fibronectin on stem cell therapy after AMI in the heart, this therapy should be applied when fibronectin depositions in the heart are high. We have accurately scored fibronectin deposition in the human heart after AMI and found that the fibronectin intensity score significantly increases from 12 h after MI in both the infarction area and the border-zone.

heart
infarction
adhesion
stem cell
human
adipose tissue