Causes and therapy of microinflammation in renal failure

Prof. Dr. Ralf Schindler

Dept. of Nephrology and Intensive Care Medicine,
Charité-Virchow-Klinikum, Humboldt Universität zu
Berlin. Germany

ralf.schindler@charite.de

Microinflammaton in renal failure has been the subject of numerous contributions to the literature. It has been suggested that the inflammatory process may not merely be an epiphenomenon but rather a pathogenetic factor in the genesis of atherosclerosis [1]. Cardiovascular mortality is greatly increased in hemodialysis patients [2]. On the other hand, plasma levels of CRP and IL-6 are strong predictors for mortality in subsequent years [3, 4]. The causes for the inflammatory response in patients with renal failure are not obvious. There are several potential causes and possible therapies (table 1) that will be discussed subsequently.

Uremia

The dialysis procedure itself may only be partially responsible for inflammation in renal disease because even patients with renal insufficiency not yet on dialysis have elevated levels of CRP that further rise after starting regular hemodialysis treatment [5]. Stenvinkel summarized the prevalence of elevated CRP-levels from several studies and concluded that app. 35% of patients with renal failure not yet on dialysis showed elevated levels of CRP but more than 50% of HD patients [6]. Thus, in the uremic patient inflammation starts long before renal replacement therapy. Accumulation of mediators (pro-inflammatory cytokines, advanced glycation end products, AGEs) in renal failure may contribute to inflammation. Reduced elimination in renal failure leads to accumulation of factor D, the rate-limiting step in complement activation and to subsequent amplification of C3 activation [7].

The kidney is the major site of elimination of many cytokines, as evidenced in studies of IL-1 and TNF clearance in nephrectomized rats [8] as well as by pharmacokinetic studies of injected recombinant IL-10 in humans with different degrees of renal dysfunction [9]. Descamps-Latscha demonstrated that plasma levels of IL-1 receptor antagonist were significantly increased from the earliest stage of renal failure. Plasma levels of TNF- and soluble TNF-receptors rise with the severity of renal failure and correlate with GFR [10].

Naturally, both pro- and anti-inflammatory cytokines and mediators accumulate in renal failure and it has been questioned whether the result is inflammation or rather a balance between pro-inflammatory mediators and their inhibitors. However, when investigating cytokine production in whole blood (best reflecting the in vivo situation), we observed that spontaneous and LPS-induced production of IL-1 and IL-6 in whole blood from HD patients is almost doubled compared to normal subjects (figure 1, [11]). Thus, the net effect of accumulation of mediators appears to be pro-inflammation. Among the numerous other products that accumulate in renal failure possibly contributing to inflammation are AGEs [12] and advanced oxidation protein products [13].

Acidosis is a well-known complication of uremia and might also contribute to inflammation. An established consequence of acidosis in chronic renal failure is protein catabolism and loss of lean body mass [14]. In animal models, feeding of NH4Cl suppresses the growth of normal rats and increases their excretion of urea nitrogen [15]. Correction of acidosis on the other hand has beneficial effects on body weight and muscle mass in patients on peritoneal dialysis [16]. Pickering et al. recently reported similar results in 8 CAPD patients and observed a decrease in muscle ubiquitin mRNA as well as of tumor-necrosis-factor (TNF) plasma levels after correction of acidosis [17]. An involvement of pro-inflammatory cytokines in protein catabolism is likely since TNF injection into rats is associated with muscle protein and branch-chain amino acid degradation [18]. We recently observed that acidosis augments the production of IL-6 and Rantes from smooth muscle cells in vitro [19] indicating that acidosis may contribute to the inflammatory state in uremia.

Heart failure and volume overload

Overhydration is common in patients with renal failure and may contribute to inflammation. Niebauer et al. [20] reported elevated endotoxin plasma levels in oedematous patients with chronic heart failure than in stable patients with chronic heart failure. Oedematous patients had the highest concentrations of several cytokines. After diuretic treatment, endotoxin concentrations decreased significantly, suggesting that overhydration and/or heart failure leads to increased endotoxin levels possibly triggering immune activation.

There are also reports on negative correlations between excess in extracellular fluid volume and serum albumin in dialysis patients [21]. The greater the fluid excess, the lower the albumin concentration. Thus, overhydration may lead to increases in endotoxin and cytokine levels reduce albumin synthesis.

Upregulation of cytokines has also been reported in heart failure as evidenced by elevated levels of IL-6 and TNF in patients with heart failure [22]. TNF is not expressed in normal myocardium but myocardial cells are capable to express TNF mRNA and to produce TNF in response to increased left ventricular pressure or volume overload [23]. Elevated sympathetic activity in heart failure may contribute to enhanced cytokine response because chronic beta-adrenergic stimulation induces myocardial expression of TNF and IL-6 [24].

In addition, treatment with beta-blockers leads to reduction in TNF plasma levels [25]. Thus, every effort should be undertaken to continuously search for the correct "dry weight" of hemodialysis patients to avoid overhydration and subsequent heart failure that are additional causes of inflammation.

Oxidative stress

Neutrophils and monocytes produce reactive oxygen species (ROS) in the course of host defense against microorganisms. Neutrophils obtained from hemodialysis patients exhibit a higher spontaneous rate of production of ROS than neutrophils from healthy subjects [26]. Furthermore, neutrophils from HD patients are primed for an enhanced respiratory burst following additional stimuli. The enhanced phagocytosis-stimulated H2O2 production is conferred by uremic plasma because it is observed not only in neutrophils from uremic patients but also in neutrophils from normal subjects incubated with uremic plasma [27].

In addition, a single high-flux hemodialysis session leads to a decrease and almost normalization in H2O2 production by neutrophils regardless of the membrane used [28]. Consistent with an increased production of ROS, proteins and lipids from HD patients exhibit higher levels of oxidation as evidenced by an increase in protein carbonyl groups and advanced protein oxidation products [29]. These oxidated proteins are capable to stimulate monocytes and exert an pro-inflammatory effects themselves [13]. When renal failure proceeds, more and more uremic solutes accumulate that may serve as targets for increased oxidation. Thus, some authors concluded that the primary stimulus for oxidative stress in renal failure patients is uremia per se and not the dialysis procedure [30].

An additional factor contributing to oxidative stress in uremia may be therapy with intravenous iron. Intravenous iron can release free iron that may react with hydrogen peroxide to produce the strong oxidant hydroxyl radical. Drueke at al reported that in HD patients, AOPP levels correlate with serum-ferritin and the dose of intravenous iron [31].

Furthermore, early signs of atherosclerosis (wall-to-lumen ratio) were associated with plasma AOPP, serum ferritin, and the annual intravenous iron dose administered [31]. Thus, iron overload should be avoided.

Bioincompatibility

According to PubMed, there have been 610 publications on biocompatibility and hemodialysis within the last 20 years. The term biocompatibility involves coagulation, thrombocytes, leukocytes, complement activation, cytokine and bradykinin production, each of which may affect inflammation. Craddock described the activation of the alternative complement pathway by cuprophan in 1977 [32]. Activated complement factors such as C3a and C5a rise during HD and reach maximal levels after 15 to 30 minutes. At the same time, leukopenia is maximal. There are large differences between membranes in complement activation.

Usually, cellulosic membranes in which polar OH-groups are substituted with acetyl- or DEAE-groups or synthetic membranes activate less complement. Complement factors C3a and C5a activate granulocytes but also monocytes that subsequently become "primed" to produce cytokines. Cells exiting a cuprophan dialyzer express large amounts of mRNA for IL-1 and IL-6 while HD with non-complement-activating membranes express much less mRNA for these cytokines [33].

When subsequently stimulated with endotoxin, these mRNA-expressing cells are sensitised to produce much more cytokines than non-activated cells. During the course of granulocyte activation, these cells release their granular products (elastase, myeloperoxidase, lactoferrin) and express surface markers such as CD11b, Mac-1 or CD66b [34]. While the latter process is also mainly dependent on complement activation, degranulation is not complement-dependent but is also observed with the use of reused cuprophan membranes that activate little complement.

In vivo studies underline the clinical relevance of these observations and support membrane differences regarding induction of inflammatory processes. For instance, Tayeb et al switched hemodialysis patients from cuprammonium membranes to polysulfone [35]. Serum albumin levels increased significantly both in patients with and without diabetes. Memoli et al. dialyzed the same patients with cuprophan, synthetically modified cellulosic membranes and cellulose diacetate [36]. The authors observed significant differences in plasma levels of CRP, IL-6 and albumin between membranes. During dialysis with cuprophan higher levels of CRP and IL-6 and lower levels of albumin were observed.

In a randomized cross-over study, 18 hemodialysis patients were subsequently treated with dialyzers containing polyamide, polycarbonate or cuprophan for 8 weeks on each dialyzer [11]. CRP levels were lower when patients were dialyzed with polyamide compared to the levels when the same patients were dialyzed with cuprophan. The whole blood content of IL-1Ra was higher when patients were dialyzed with cuprophan compared to the same patients on polyamide or on polycarbonate. Thus, the degree of inflammation in hemodialysis patients is affected by the choice of the dialyzer.

It should be noted that not only the type of membrane but also its flux and the extent of convective transport may influence inflammation. Cytokine induction on the blood side is the product of complement activation, the permeation of bacterial products from the dialysate and direct blood-membrane interactions (figure 2).

With high-flux membranes and convective therapies one should expect that more pro-inflammatory products (C3a, C5a, AGEs, advanced protein oxidation products, OPP) be removed while the passage of bacterial products is hindered resulting in less inflammation on the blood side. However, clinical data on the effect of convective therapies on inflammation are scarce.

Dialysate contamination

Since the introduction of hemodialysis into clinical practice, there has been concern about the transfer of bacterial cytokine-inducing substances (CIS) from the dialysate into the blood compartment and subsequent deleterious effects on the patient. A number of bacterial products (figure) such as lipopolysaccharides (LPS), exotoxins and peptidoglycans share the ability to induce cytokines and are known activators of immune functions. A number of in vitro and in vivo studies have been performed investigating the permeation of these substances through dialysis membranes.

In many studies, the biological test of cytokine-induction in peripheral blood mononuclear cells has been used to detect these substances. By this test, all biological relevant bacterial substances are detected whereas the Limulus-test (LAL) detects only LPS-derived substances. Moreover, only LPS-fragments above app. 8 kDa are reactive in the LAL-test but may still be pyrogenic [37]. The exact chemical nature of bacterial CIS is not completely understood, and most likely CIS consist of a mixture of bacterial products. LPS is not the only product in dialysate that induce cytokines. This is supported by the observation that the cytokine-inducing activity of pseudomonas products that appear on the blood side of dialyzers cannot entirely be blocked by polymyxin B [38] and are negative in the LAL test [39].

New pyrogenic candidates that may pass dialyzer membranes are bacterial-derived short DNA fragments. It has been reported that PBMC ingest bacterial DNA [40]. The structural requirements for immunostimulation by ODN were defined to be a cytosine-guanosine (CG) core that had to be unmethylated to stimulate mammalian cells [41].

Mammalian DNA shows extensive suppression of CG sequences and they are commonly methylated; only in bacterial DNA unmethylated CG-motifs can be found. This unmethylated CG-motif (CpG) distinguishes bacterial from mammalian DNA and allows phagocytic cells to recognize and to be activated by bacterial DNA. CpG ODN of 15 to 20 base pairs (bp) are able to induce natural killer cell activity and induce IFN-, TNF- and IL-6 from PBMC [42, 43]. When injected intraperitoneally, CpG ODN induce IL-6, TNF and IL-12 in mice and may even lead to septic shock [44]. The signaling pathways by which CpG ODN activate cells are currently being characterized and involve Toll-like receptor 9 (TLR9).

Macrophages and dendritic cells from TLR9-deficient mice do not respond to ODN [44]. Cytokine-inducing ODN are of sufficient small size (10 bp ˜ 2500 Da) to pass through dialyzer membranes (Schindler, manuscript submitted). Thus, bacterial ODN may be a factor contributing to cytokine induction during hemodialysis that are not easily removed by conventional ultrafiters.

Several studies investigated the permeability of low- and high-flux dialyzer membranes for CIS. Most of these studies demonstrated prompt transfer of CIS through low-flux cellulosic membranes but no or less transfer through high-flux polysulfone or polyamide membranes [45-47]. It was concluded that the sponge-like structure of these high-flux membranes adsorbs bacterial products; this feature even enabled the use of polysulfone and polyamide as ultrafilters to efficiently remove CIS from aqueous solutions [48]. High-flux membranes were considered to be a safe barrier against possible bacterial products in dialysate. However, this may not be true for all high-flux membranes. We observed recently that there are large differences between high-flux membranes regarding their permeability for cytokine-inducing substances from E. coli as well as for LPS derived from E. coli and Sten. Maltophilia [49].

Ultrapure dialysate is not yet the standard of dialysate quality in most dialysis centres. Although the consequences of inflammation in dialysis patients are not fully understood, preventing the penetration of bacterial products from the dialysate seems prudent. When using high-flux membranes that are possibly permeable for bacterial CIS, the use of CIS-free dialysate is essential. To completely remove all CIS including bacterial DNA from dialysate, supplementary measures in addition to ultrafiltration may be required.

Access infection

Access infection is a frequent and often overlooked cause for inflammation. Especially the types of access involving foreign materials may become infected and be a source for bacteremia. Ayus reported a series of infected, old non-functioning grafts in hemodialysis patients [50]. Not always are there physical signs of graft infection and detection of infectivity requires a high index of suspicion.

Venous catheters are associated with increased rates of infection, including bacteremia, osteomyelitis and endocarditis compared to other forms of vascular access [51]. Recently, Pastan et al. [52] reported that medium-term all-cause mortality and mortality due to infection is correlated wit the use of venous catheters (cuffed or non-cuffed).

Possible drug therapy for microinflammation

Several drugs have been reported to reduce inflammation assessed by CRP and cytokine levels. ACE-inhibitors reduce the synthesis of IL-1 and TNF in vitro [53] and IL-6 levels in patients with heart failure in vivo [54]. In HD patients, the use of ACE-inhibitors is associated with lower levels of CRP and TNF [55]. Angiotensin receptor blockers (candesartan) also lower levels of IL6 and TNF in patients with heart failure [56]. Whether this effect is direct or due to amelioration of heart failure is not known.

The CRP-lowering effect of statins is well described for the general population [57, 58] as well as for patients with renal failure [59]. All statins including atorvastatin, simvastatin and cerivastatin seem to lower CRP. This effect appears to be dose-dependent [57] but CRP-lowering does not correlate with the decrease in cholesterol [58].

The effect of aspirin and anti-oxidants such as vitamin E on inflammation is more controversial. One study reported reductions in both CRP and IL-6 with aspirin in patients with coronary artery disease [60] but this effect may be dose-dependent and may not be observed with the usual 100 mg daily dose of aspirin [61]. Devaraj reported that supplementation with -tocopherol lowers CRP levels and IL-6 release from monocytes in 47 diabetic patients and in normal controls [62].

In contrast, Bruunsgaard failed to observe an effect of -tocopherol and vitamin C on CRP levels in 55 healthy subjects [63]. The authors concluded that long-term combined supplementations with -tocopherol and vitamin C in reasonable doses have no detectable systemic anti-inflammatory effects in healthy men. The effect of vitamin E and aspirin on inflammation in dialysis patients needs to be clarified in further trials.

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