*2.1. Uremic Inflammation*

An impaired renal function leads to the accumulation of nitrogenous substances in the blood that would normally be excreted in the urine. At progressively increasing concentration, these substances exert toxic e ffects, which eventually become apparent as symptoms of *uremia*. The altering effects of the uremic milieu on the immune system has been described as *uremic inflammation* [10,11], and include mechanisms of both immunoactivation and immunosuppression. Uremic inflammation resembles the premature ageing phenotype in many ways. On the one hand, it is characterized by an abnormal activation of the innate immune system, especially monocytes [6,12]. This immunoactivation contributes to systemic inflammation via increased synthesis of pro-inflammatory cytokines, such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF) [12], and is similar to the chronic low-grade state of systemic inflammation that is associated with an ageing immune system and has been coined "inflammageing" [13]. Furthermore, inflammation is also a major component of other diseases that are independent risk factors for CKD, such as obesity [14]. Importantly, an increased synthesis of pro-inflammatory cytokines and chemokines by senescent cells is one of the main features of cellular senescence [15,16], and has been coined senescence-associated secretory phenotype (SASP). The SASP suggests a further bi-directional link between inflammation and ageing in CKD. On the other hand, a downregulation and reduced function of the adaptive immune system, particularly of T and B lymphocytes, during uremic inflammation parallels "immunosenescence" in the premature ageing phenotype [6,10,12].

The causes of uremic inflammation are multifactorial. Exogenous factors, such as catheterization, exposition to microbial contaminants, or biocompatibility issues during dialysis treatment [10] may play an obvious role in the activation of the immune system and are avoidable using good clinical practice. Possible exposure to bacterial endotoxin, which activates the immune system and contributes to systemic inflammation, can furthermore result from comorbidities, such as gingivitis and periodontitis [17]. Patients with CKD may also show signs of intestinal dysbiosis and increased gu<sup>t</sup> permeability [10], which lead to the presence of bacterial DNA and elevated endotoxin levels, as well as elevated plasma levels of the macrophage-derived cluster of di fferentiation (CD)14 [10], a co-receptor in the recognition of bacterial endotoxin [18].

Conversely, endogenous factors that provoke uremic inflammation in CKD are linked to metabolic deviations from normal physiology and can be categorized as (i) changes in the mineral metabolism, especially in the levels of phosphate and sodium concentrations; (ii) regulation of oxidative stress; and (iii) increased nonenzymatic glycation. However, these categories are interconnected and may influence each other.

The endocrine fibroblast growth factor-23 (FGF-23)–klotho pathway (Figures 1 and 2) is important for the resorption of phosphate in the kidney and is dysregulated in CKD. Decreased renal clearance produces a relative overload of inorganic phosphate (Pi), which results in hyperphosphatemia and contributes to systemic inflammation and vascular calcification/early vascular ageing (EVA) [19] (Figure 2). Hyperphosphatemia promotes endothelial dysfunction and trans-di fferentiation of vascular smooth muscle cells (VSMC) into osteoblast-like cells [20]. In bovine aortic smooth muscle cells, high Pi promotes an osteogenic phenotype via an inflammatory mechanism involving nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling, which increases the generation of reactive oxygen species (ROS). This phenotype can be prevented with the Pi binder lanthanum carbonate [21]. Similarly, another Pi binder, sevelamer, increases levels of fetuin A, an inhibitor of extracellular matrix mineralization, in patients with CKD [22]. As a negative acute phase protein, low levels of fetuin A indicate systemic inflammation and may shorten telomeres in leukocytes [23]. Furthermore, high Pi induces the expression of the pro-inflammatory transcription factor NF-κB in human aortic VSMC [24].

**Figure 1.** The phosphate-fibroblast growth factor-23 (FGF-23)–klotho endocrine axis. Main effectors of phosphate homeostasis with potential effects on ageing components (simplified overview). Phosphate (Pi) is taken up by the intestine and accumulates in the circulation of patients with advanced chronic kidney disease (CKD). In the circulation, fetuin A (blue circles)-bound calcium Pi in calciprotein particles (CPP) prevents precipitation of calcium Pi in the circulation. Increased Pi is further regulated by parathyroid hormone (PTH) secreted from the four parathyroid glands (orange circles) at the back of thyroid gland by increasing intestinal Pi resorption but also inducing phosphaturia. The 1,25(OH)2 vitamin D3 metabolite is activated in the kidneys and increases intestinal Pi resorption. Furthermore, bone-secreted fibroblast growth factor-23 (FGF-23) also exerts phosphaturic effects through its mandatory co-receptor klotho in the kidneys.

**Figure 2.** The phosphate-FGF-23–klotho endocrine axis and premature ageing in CKD. Association of the different effectors of the phosphate homeostasis with ageing components (light blue arrows: inducers; red arrows: inhibitors). In advanced CKD, calciprotein particles (CPP) can induce early vascular ageing (EVA) and inflammation. However, contradictory findings also sugges<sup>t</sup> a beneficial role of CPP on EVA. Circulating phosphate (Pi) promotes EVA, cellular senescence, and oxidative stress by different mechanisms. Vitamin D inhibits inflammation and oxidative stress directly or indirectly. The anti-ageing-associated klotho counteracts EVA, cellular senescence, inflammation, and oxidative stress. Data for fibroblast growth factor-23 (FGF-23) are limited and further studies need to investigate whether FGF-23 directly induces different ageing components or whether the clinical associations to ageing are mediated by other factors, such as Pi.

Although Pi levels increase in the circulation of CKD patients, sodium accumulates in the tissue under uremic conditions and may lead to chronic systemic inflammation via activation of the p38 mitogen-activated protein kinase (MAPK) pathway and induction of IL-17-producing CD4+ T helper cells [6,25].

The transcription factor NRF2 plays an important and ancient role in the anti-oxidant response. Following oxidative stress, NRF2 dissociates from its repressor kelch-like erythroid cell-derived protein with CNC homology [ECH]-associated protein 1 (KEAP1) in the cytosol and subsequently translocates to the nucleus, where it binds to the anti-oxidant response element of numerous promoter regions of over 300 genes encoding anti-oxidant and detoxifying molecules [26]. In peripheral blood mononuclear cells from CKD patients, NRF2 is down-regulated, whereas NF-κB is in turn up-regulated [27]. NF-κB plays a key role in regulating the inflammatory response and is stimulated by ROS [10]. This suggests that uremia induces an impaired NRF2 system in CKD and hemodialysis (HD) patients, which contributes to the pathogenesis of oxidative stress and inflammation [27]. Importantly, increased oxidative stress and its sequalae are major contributors to premature atherosclerosis and calcification [28], resulting in increased cardiovascular (CV) morbidity and mortality in CKD [29,30]. Retained uremic toxins may further both become substrates for oxidative injury and increase the burden of oxidative stress [29,30].

Advanced glycation end-products (AGE) are formed from reducing sugars and biomolecules such as proteins, lipids, and nucleic acids by a sequence of nonenzymatic glycation reactions, collectively known as the Maillard reaction [26,31]. Exogenous sources of AGE originate from diet [32] and tobacco smoking [33]. Endogenously, AGE formation is promoted by hyperglycemia, but also by high levels of oxidative stress [26]. In fact, oxidative stress and AGE formation mutually stimulate each other [34]. AGE increase the levels of ROS via activation of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase through interaction with the advanced glycation end product-specific receptor (RAGE), as well as through receptor-independent pathways, whereas ROS decrease the levels of glyoxalase I (Glo-1), an enzyme that detoxifies AGE precursor molecules [26]. In CKD, AGE accumulate as a result from both a decreased renal clearance and increased formation [26]. AGE–RAGE interaction activates the NF-κB pathway and thus perpetuates uremic inflammation via release of pro-inflammatory cytokines, such as IL-1, IL-6, and TNF [35].

### *2.2. Consequences of Inflammation on Premature Ageing in CKD*

Persistent uremic inflammation directly promotes premature ageing and further increases inflammatory e ffects through a vicious circle. For instance, NF-κB promotes cellular senescence and accelerated ageing but it is also activated in senescent cells [36–38]. In more detail, senescent cells promote inflammation through the transcription factor GATA4, which increases NF-κB to initiate the SASP [39]. Furthermore, cell replication is amplified in acute and chronic inflammation, resulting in increased telomere attrition, which is directly linked to cellular ageing [40]. Importantly, the association between telomere length and inflammation also seems to be reciprocally. Thus, mice deficient in the telomerase genes telomerase RNA component (*TERC*) and telomerase reverse transcriptase *(TERT)* show a higher amount of mRNA expression of various pro-inflammatory cytokines [41]. In a cross-sectional study, the negative association of leukocyte telomere length with circulating inflammatory markers was also confirmed in prevalent HD patients [23]. Moreover, increased oxidative stress in CKD and adverse lifestyle factors (e.g., smoking and diet), as well as psychological stress, may further promote telomere shortening through inflammation [40].
