**4.1.1.2 The Interleukin Hypothesis**

100 Progress in Hemodialysis – From Emergent Biotechnology to Clinical Practice

(Liuzzo et al 1994, Lagrand et al., 1999, Badht et al, 2002). C-reactive protein may directly promote the development of atherosclerosis, through complement activation, tissue damage and activation of endothelial cells. Recent studies performed in CKD patients have shown that CRP is a strong predictor of cardiovascular death (Stenvinkel, 2001, Kaysen, 2005). The link between CRP and cardiovascular risk was initially thought to be indirect, reflecting circulating CRP only to the extent of the acute phase reaction in response to nonspecific stimuli such as confounding risk factors, atherosclerosis, vascular injury, ischemia and

Fig. 4. Acute phase response is a defence response which occurs as a consequence of an inflammatory stimulus occurring in the blood or at tissue level. The enhanced production of interleukin-6 (IL-6), the most potent inducer of this reaction at the level of the liver, triggers the synthesis of newly synthesized proteins, e.g., C-reactive protein (which plasma levels may increase up to 50-to 100-fold the normal levels) as well as to the shut-down of the

Stenvinkel et al (1999) first convincingly showed that the malnutrition-inflammation complex syndrome described as MIA syndrome is associated with the highest mortality rates in ESRD. Their results were confirmed and extended (Panichi et al. 2008). As reviewed by Stenvinkel & Barany (2002), there is consensus on a link between CKD and inflammation. A number of studies have highlighted the association between increased inflammatory indexes and a reduced response to Erythropoietin-stimulating agents (ESAs), in particular, high CRP levels were found in HD patients requiring higher ESAs doses (Singh et al., 2007; Bradbury et al. 2009). However, the association between ESAs resistance and increased CRP levels (Barany et al. 1997; Gunnell et al. 1999) is unclear. Plasma IL-6 rather than CRP seem to better predict outcomes in CKD patients (Panichi et al., 2004). Various possible explanations may underline the advantage of IL-6 over CRP as a predictor of ESAs resistance. One possibility is that IL-6, being located upstream in the cascade of events

translation of genes coding for proteins, e.g., albumin.

necrosis. **(Figure 4).** 

Originally introduced as an elegant concept in 1986 (Bingel et al., 1986), the "interleukin hypothesis" was first coined to indicate the production of interleukin-1, the endogenous pyrogen as produced by the result of complement-activated mononuclear cells. Indeed, the interleukin hypothesis explained much more than was initially predictable. Several studies have ever since reported an increased cytokine production secondary to blood interaction with contaminated dialysate. Interleukin-1 (IL-1), tumor necrosis factor-a (TNF-a) and mainly IL-6 are the 3 proinflammatory cytokines that are involved in the pathogenetic aspects of HD-related disease (as reviewed by Lonnemann, 2004, Panichi et al., 2000 (**Figure 5**).

Fig. 5. Here are schematically depicted the mechanisms related to the backdiffusion/backfiltration of bacteria-derived contaminants from the dialysate into the blood. Their interaction with circulating monocytes/macrophages leads to the activation of innate immunity and to the attendant triggering of proinflammatory cytokines (interleukin-1 (IL-1), tumor necrosis factor-. Abbreviations: CIS, cytokine-inducing substances; LAL, *Limulus amoebocyte* lysate, UF, ultrafiltrate.

The Evolution of Biocompatibility: From Microinflammation to Microvesiscles 103

For long time MVs were considered to be inert cellular debris. The frequently observed vesicles by electron microscopy in the interstitial space of tissues or in blood were considered as the consequence of cell damage or the result of dynamic plasma membrane turnover (Siekevitz et al., 1972). As the vesicle population detectable both *in vitro* and *in vivo*  is a mixed population of exosomes and shedding vesicles, we will refer to them collectively as MVs. Released MVs may remain in the extracellular space in proximity of the place of origin or may enter into the biological fluids reaching distant sites. This may explain the presence of MVs in plasma, urine, milk and cerebrospinal fluid. The bulk of MVs present in the circulation is derived from platelets (George, 1982), and in less extent from other blood cells and endothelial cells (Martinez et al., 2005). The MVs derived from platelets are also designed as microparticles while those derived from polymorphonuclear leukocytes are also named ectosomes (Hess et al., 1999). Finally, MVs released during morphogenesis of multicellular organisms are indicated as argosomes (Greco et al., 2001). Besides normal cells, also tumor cells may release MVs and in patients suffering for neoplastic diseases tumorderived MVs may be detected within the biological fluids (Kim et al, 2003, Iero et al., 2008). Therefore, MVs are an assorted population, differing in cellular origin, number, size and antigenic composition (Diamant et al., 2004) shed by various cell types in physiological and pathological conditions. The release of MVs may be constitutive or consequent to cell activation by soluble agonists, by physical or chemical stress such as the oxidative stress and hypoxia, and by shear stress (Ratajczak et al., 2006). Exosomes have an endosome origin and are a rather homogenous population with a size ranging from 30 to 120nm (7). They are stored as intraluminal vesicles within multivesicular bodies of the late-endosome and are released when these multivesicular bodies fuse with the cell membrane. Our knowledge on

Fig. 6.

The proposed mechanisms include blood interaction with endotoxins from the contaminated dialysate through HD membranes. A large number of studies have greatly contributed to increasing our knowledge in the mechanisms of endotoxin transfer across the membrane In fact, when using high permeability membranes, backfiltration and backdiffusion occur and have ebeen extensively described (Fiore & Ronco, 2007, Ronco, 2007). Thus, the transmembrane passage of endotoxins or other cytokine stimulating substances (CIS) occurs during HD (Schindler et al., 2004, Tetta et al., 2006). The reduction of backfiltration of standard dialysate may reduce the plasma concentration of IL-1ra, a sensitive indicator of inflammation in HD patients (*Dinarello personal communication, 2004*), and IL-1 (Panichi et al., 1998). Studies on large groups of patients have shown that highvolume exchange HDF, a treatment in which dialysate backfiltration is minimal, if any, is associated with significantly lower CRP plasma values (Panichi et. 1998). Comparing in a double cross-over study patients treated with high-flux and on line HDF using ultrapure dialysate and infusate, it was shown that a significant reduction of pro-inflammatory CD14+/CD16+ mononuclear subset (Carracedo et al., 2006) occurs in on line HDF. These studies emphasize that the convective component has an additional anti-inflammatory effects (Ramirez et al., 2007).

The new technology of pyrogen-adsorbing, non-complement activating, high-permeability synthetic membrane and dedicated machines (Tetta et al., 2011), as well as the awareness of the deleterious effects derived from contamination of dialysis fluids has reduced the clinical impact to a periodic microinflammatory stimulus. Undoubtedly, the availability of monitors for on-line HDF and its increased popularity have spurred more restrictive measures on safety issues and monitoring. Water quality is a mandatory issue. The safety of online HDF has been shown repeatedly in several monocenter (Canaud et al., 1998, Pizzarelli et al., 1998 and multicenter studies (Canaud et al., 2001,Vaslaki et al., 2000).

Nowadays, the philosophy of ''ultrapure dialysate''is in common practice (Kessler et al., 2002). The clinical, consolidated experience on line HDF warrants well-defined procedures and leaves no space for ''experiments'' in what is now routine (Canaud et al, 2011). The ''hemocompatibility network'' should eventually prevent the periodic microinflammation induction through the implementation of rigid protocols of disinfection and maintenance of water-treatment systems and HD monitors (Cappelli et al., 2006; Kessler et al. 2002).
