Signos distintivos del teatro costumbrista

The glomerular filtration rate (GFR) is regarded as the best indicator for kidney function and is widely used to classify renal failure, however, direct measurement of this index is difficult to achieve [270,271]. Therefore, serum creatinine (SCr) is generally used as an estimate for GFR. Unfortunately, changes in SCr are insensitive to detect injury, since a relative large amount of injury can occur without affecting GFR, especially in patients with a renal reserve or during unilateral renal dysfunction [270,271]. Furthermore, GFR can drop with 50% before SCr levels increase [270,271]. The limitations of SCr as a marker for kidney failure is further supported by the fact that SCr concentrations do not only depend on GFR, but also on tubular secretion and systemic production of creatinine, which is for instance affected by muscle wasting [270]. Thus, SCr is subjected to high interindividual variability and is not suitable for early and site-specific detection of kidney injury. Reasoning from the notion that (1) CKD is synonymous with uremic retention solutes and (2) a number of studies, including the research described in Chapters 2-4, have demonstrated that plasma levels of multiple uremic toxins, e.g. indole-3-acetic acid, hippuric acid and indoxyl sulfate, are significantly elevated in non-dialysis CKD patients [96,272], we investigated in a preliminary study whether uremic toxins levels may predict the rate of eGFR decrease in CKD patients. To this end, we determined baseline concentrations of several uremic toxins, using HPLC and LC-MS/MS, in serum samples of patients that participated in the MASTERPLAN trial [273]. Cases were rapid progressors, i.e. patients with decline of eGFR > -4.5 ml/min/1.73m2_{/year, and as controls we selected patients with a}

lower rate of progression matched for baseline eGFR. In total, 27 cases and 65 controls were included, of whom 43 were slow progressors (decline 4.5 to 1.0 ml/min/1.73m2_/year)

and 22 stable patients (decline < 1 ml/min/1.73m2_{/year). Mean age in the selected patients}

was 59 ± 13 years and 67% was male. Most patients had moderate CKD with a mean eGFR of 37 ± 12 ml/min/1.73m2 _{and received blood pressure lowering drugs (96%), and ACE}

Figure 2

A multiple adjusted linear regression analysis between uremic toxin concentration and change in eGFR was created. The model included baseline eGFR, proteinuria, blood pressure, age and gender. Dots represent regression coefficient (i.e. eGFR decline per unit of the solute) with the 95% confidence interval. HA, hippuric acid; I3A, indole-3-acetic acid; IS, indoxyl sulfate; Kyn, kynurenine; KynA, kynurenic acid; pCG, p-cresyl glucuronide; pCS, p-cresyl sulfate.

128 129 General Discussion

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reduce uremic retention solute levels. AST-120, an oral charcoal adsorbent, has been demonstrated to reduce plasma levels of uremic solutes, including p-cresyl sufate, indoxyl sulfate and hippuric acid [275], and is suggested to reduce a number of comorbidities associated with CKD such as atherosclerosis [276-279]. However, the beneficial impact of AST-120 still needs to be studied in large clinical trials. Other potential suppressive therapies include dietary and probiotic treatments. Several small intervention studies (n<30) have demonstrated that preparations with lactic acid bacilli and Bifidobacterium

longum reduce serum levels of indoxyl sulfate with 9-30% [93,280-282]. Yet, studies on the

impact of probiotics on hard clinical end points (e.g. mortality) in CKD patients have not been performed to date.

Finally, there is much attention for the risk that CKD possess for cardiovascular mortality and morbidity [283], yet little effort is made to elucidate the kidney-brain axis [284]. Several known uremic toxins, e.g. kynurenic acid and quinolinic acid, have neuroactive properties and can modulate the activity of important receptors in the brain, including the α7- nicotinic-acetylcholine (α7nAch)- and the N-methyl-d-aspartate (NMDA) receptor.

Recently, we demonstrated that kynurenic acid concentration-dependently increased glucose uptake by malignant neuroblastoma (N2a) cells, and we unveiled a link between BCRP and kynurenic acid transport with brain energy metabolism, indicating that the efflux pump is an interesting therapeutic target to modulate cerebral glucose and kynurenic acid levels (Dankers et al. unpublished data). This exciting observation warrants further investigation into uremic toxin-induced neuronal dysfunction as well as CKD-associated cognitive impairment and neuropathy.

The results within this thesis expanded the currently known universe of uremic solutes by implementing and improving both targeted and untargeted analytical techniques. Furthermore, a multitude of uremic solutes, irrespective of the source of origin, were demonstrated to negatively influence numerous physiological processes and phenotypic characteristics that define the healthy renal proximal tubule cell. Moreover, we shed a light on the essential role of efflux transporters in the renal extrusion of uremic solutes, laying the foundation for future studies aiming to utilize these pumps to suppress or halt CKD progression and associated pathologies.

renal handling of uremic solutes, and we demonstrated that inactivity of these ABC-trans- porters directly results in accumulation of several potential toxic metabolites. In addition, the nephrotoxic actions of multiple uremic retention solutes were elucidated using a unique proximal tubular cell model, identifying diverse targets affected by these compounds, including mitochondria, drug metabolism enzymes and transporters. Since dialysis is inapt in removing uremic solutes from the circulation, harnessing the ability of transporters to clear these metabolites might prove to be the long coveted therapeutic modality needed to improve clinical outcomes for CKD patients. Recently, Toyohara and coworkers demonstrated that statins could be used to increase the expression of OATP4C1 and improve renal uremic toxin clearance [60]. However, ‘therapeutic manipulation’ of transporters, while intriguing, should be approached with caution. It is conceivable that upregulation of uptake transporter expression might ensue improved uremic toxin clearance. Yet, it could also result in increased renal proximal tubule cell death from toxic exposure. Therefore, it is key to simultaneously target both basolateral and apically expressed transporters in order to maintain the perfect, and delicate, balance between cellular uptake and efflux. An alternative might be employing “living dialysis membranes” consisting of bioreactors containing stem cell derived, and possibly patient specific, trans- porter-expressing cells to better mimic normal renal function and improve therapy efficacy. In our lab, first steps have been undertaken to realize a bio-artificial kidney (BioKid) device using a unique and stable proximal tubule cell model, i.e. ciPTEC. This cell line has been established from cells exfoliated in the urine from a healthy volunteer. ciPTEC have been demonstrated to maintain proximal tubular characteristics, including expression of important in- and efflux transporters as well as megalin-mediated albumin uptake and sodium-dependent phosphate transport, over a prolonged period of cell culturing [98]. Moreover, ciPTEC can be used to study nephrotoxicity as shown by their sensitivity to gentamicin [101], and OCT-mediated organic cation transport [78]. Moreover, using a similar immortalization approach, our group was able to create ciPTEC from renal tissue (ciPTEC-T; Jansen et al., unpublished data). This novel model showed an analogous expression and activity profile as compared to ciPTEC. Of note, ciPTEC-T appeared to have retained the ability to create their own extracellular matrix, illustrated by increased gene expression levels of collagen I and –IV α1. Unfortunately, both ciPTEC and ciPTEC-T lack the important influx transporters OAT1 and OAT3 on gene, protein and functional level. Clearly, there is still much ground for the transporter field to cover, but the very fact that BioKid concepts are being discussed and practically achievable [274], foreshadows some exciting times to come.

Progress can also be made in reducing the exogenous intake of uremic toxins as well as the diminishing the production of uremic solutes by the intestinal flora. Aronov et al. recently reported that in plasma of dialysis patients without a colon several uremic toxins were absent or present at lower concentrations compared to patients with a colon [92]. This finding underlines the notion that the gut is an important therapeutic target to

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