The Dual Roles of Protein-Bound Solutes as Toxins and Signaling Molecules in Uremia

In patients with severe kidney disease, renal clearance is compromised, resulting in the accumulation of a plethora of endogenous waste molecules that cannot be removed by current dialysis techniques, the most often applied treatment. These uremic retention solutes, also named ure-mic toxins, are a heterogeneous group of organic compounds of which many are too large to be filtered and/or are protein-bound. Their renal excretion depends largely on renal tubular secretion, by which the binding is shifted towards the free fraction that can be eliminated. To facilitate this process, kidney proximal tubule cells are equipped with a range of transport proteins that cooperate in cellular uptake and urinary excretion. In recent years, innovations in dialysis techniques to advance uremic toxin removal, as well as treatments with drugs and/or dietary supplements that limit uremic toxin production, have provided some clinical improvements or are still in progress. This review gives an overview of these developments. Furthermore, the role protein-bound uremic toxins play in inter-organ communication, in particular between the gut (the side where toxins are produced) and the kidney (the side of their removal), is discussed

Protein-Bound Uremic Toxins in Senescence and Kidney Fibrosis

Chronic kidney disease (CKD) is a progressive condition of kidney dysfunction due to diverse causes of injury. In healthy kidneys, protein-bound uremic toxins (PBUTs) are cleared from the systemic circulation by proximal tubule cells through the concerted action of plasma membrane transporters that facilitate their urinary excretion, but the endogenous metabolites are hardly removed with kidney dysfunction and may contribute to CKD progression. Accumulating evidence suggests that senescence of kidney tubule cells influences kidney fibrosis, the common endpoint for CKD with an excessive accumulation of extracellular matrix (ECM). Senescence is a special state of cells characterized by permanent cell cycle arrest and limitation of proliferation, which promotes fibrosis by releasing senescence-associated secretory phenotype (SASP) factors. The accumulation of PBUTs in CKD causes oxidative stress and increases the production of inflammatory (SASP) factors that could trigger fibrosis. Recent studies gave some clues that PBUTs may also promote senescence in kidney tubular cells. This review provides an overview on how senescence contributes to CKD, the involvement of PBUTs in this process, and how kidney senescence can be studied. Finally, some suggestions for future therapeutic options for CKD while targeting senescence are given

Protein-Bound Uremic Toxins in Senescence and Kidney Fibrosis

Chronic kidney disease (CKD) is a progressive condition of kidney dysfunction due to diverse causes of injury. In healthy kidneys, protein-bound uremic toxins (PBUTs) are cleared from the systemic circulation by proximal tubule cells through the concerted action of plasma membrane transporters that facilitate their urinary excretion, but the endogenous metabolites are hardly removed with kidney dysfunction and may contribute to CKD progression. Accumulating evidence suggests that senescence of kidney tubule cells influences kidney fibrosis, the common endpoint for CKD with an excessive accumulation of extracellular matrix (ECM). Senescence is a special state of cells characterized by permanent cell cycle arrest and limitation of proliferation, which promotes fibrosis by releasing senescence-associated secretory phenotype (SASP) factors. The accumulation of PBUTs in CKD causes oxidative stress and increases the production of inflammatory (SASP) factors that could trigger fibrosis. Recent studies provide some clues that PBUTs may also promote senescence in kidney tubular cells. This review provides an overview on how senescence contributes to CKD and the involvement of PBUTs in this process, and how kidney senescence can be studied, Finally, some suggestions for future therapeutic options for CKD while targeting senescence are given

Selective transport of protein-bound uremic toxins in erythrocytes

To better understand the kinetics of protein-bound uremic toxins (PBUTs) during hemodialysis (HD), we investigated the distribution of hippuric acid (HA), indole-3-acetic acid (IAA), indoxyl sulfate (IS), and p-cresyl sulfate (pCS) in erythrocytes of HD patients. Their transport across the erythrocyte membrane was explored in the absence of plasma proteins in vitro in a series of loading and unloading experiments of erythrocytes from healthy subjects and HD patients, respectively. Furthermore, the impact of three inhibitors of active transport proteins in erythrocytes was studied. The four PBUTs accumulated in erythrocytes from HD patients. From loading and unloading experiments, it was found that (i) the rate of transport was dependent on the studied PBUT and increased in the following sequence: HA < IS < pCS < IAA and (ii) the solute partition of intra-to extra-cellular concentrations was uneven at equilibrium. Finally, inhibiting especially Band 3 proteins a ff ected the transport of HA (both in loading and unloading), and of IS and pCS (loading). By exploring erythrocyte transmembrane transport of PBUTs, their kinetics can be better understood, and new strategies to improve their dialytic removal can be developed

The Uremic Toxin Indoxyl Sulfate Accelerates Senescence in Kidney Proximal Tubule Cells

Kidney fibrosis is the common final pathway of nearly all chronic and progressive nephropathies. One cause may be the accumulation of senescent cells that secrete factors (senescence associated secretory phenotype, SASP) promoting fibrosis and inflammation. It has been suggested that uremic toxins, such as indoxyl sulfate (IS), play a role in this. Here, we investigated whether IS accelerates senescence in conditionally immortalized proximal tubule epithelial cells overexpressing the organic anion transporter 1 (ciPTEC-OAT1), thereby promoting kidney fibrosis. Cell viability results suggested that the tolerance of ciPTEC-OAT1 against IS increased in a time-dependent manner at the same dose of IS. This was accompanied by SA-β-gal staining, confirming the accumulation of senescent cells, as well as an upregulation of p21 and downregulation of laminB1 at different time points, accompanied by an upregulation in the SASP factors IL-1β, IL-6 and IL-8. RNA-sequencing and transcriptome analysis revealed that IS accelerates senescence, and that cell cycle appears to be the most relevant factor during the process. IS accelerates senescence via TNF-α and NF-ĸB signalling early on, and the epithelial-mesenchymal transition process at later time points. In conclusion, our results suggest that IS accelerates cellular senescence in proximal tubule epithelial cells

Effects of an SGLT Inhibitor on the Production, Toxicity, and Elimination of Gut-Derived Uremic Toxins: A Call for Additional Evidence

Sodium-glucose cotransporter (SGLT) inhibitors are a class of oral hypoglycemic agents, which, in recent years, have been shown to improve renal and cardiovascular outcomes in patients with diabetic and non-diabetic chronic kidney disease. There remains considerable debate regarding the potential glucose-independent mechanisms by which these benefits are conferred. SGLT inhibitors, to a variable extent, impair small intestinal glucose absorption, facilitating the delivery of glucose into the colon. This suppresses protein fermentation, and thus the generation of uremic toxins such as phenols and indoles. It is acknowledged that such a shift in gut microbial metabolism yields health benefits for the host. SGLT inhibition, in addition, may be hypothesized to foster the renal clearance of protein-bound uremic toxins. Altered generation and elimination of uremic toxins may be in the causal pathway between SGLT inhibition and improved cardiometabolic health. Present review calls for additional research

Cholangiocyte organoids to study drug-induced injury

Background: Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. Methods: Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. Results: CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. Conclusion: These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.</p

Cholangiocyte organoids to study drug-induced injury

Background: Drug induced bile duct injury is a frequently observed clinical problem leading to a wide range of pathological features. During the past decades, several agents have been identified with various postulated mechanisms of bile duct damage, however, mostly still poorly understood. Methods: Here, we investigated the mechanisms of chlorpromazine (CPZ) induced bile duct injury using advanced in vitro cholangiocyte cultures. Intrahepatic cholangiocyte organoids (ICOs) were driven into mature cholangiocyte like cells (CLCs), which were exposed to CPZ under cholestatic or non-cholestatic conditions through the addition of a bile acid cocktail. Results: CPZ caused loss of monolayer integrity by reducing expression levels of tight junction protein 1 (TJP1), E-cadherin 1 (CDH1) and lysyl oxidase homolog 2 (LOXL2). Loss of zonula occuludens-1 (ZO-1) and E-cadherin was confirmed by immunostaining after exposure to CPZ and rhodamine-123 leakage further confirmed disruption of the cholangiocyte barrier function. Furthermore, oxidative stress seemed to play a major role in the early damage response by CPZ. The drug also decreased expression of three main basolateral bile acid transporters, ABCC3 (ATP binding cassette subfamily C member 3), SLC51A/B (solute carrier family 51 subunit alpha/beta) and multidrug resistance transporter ABCB1 (ATP binding cassette subfamily B member 1), thereby contributing to bile acid accumulation. CPZ did not induce an inflammatory response by itself, but addition of TNFα revealed a synergistic effect. Conclusion: These results show that ICOs present a model to identify toxic drugs affecting the bile ducts while providing mechanistic insights into hepatotoxicity.</p

Regulation of P-Glycoprotein in Renal Proximal Tubule Epithelial Cells by LPS and TNF-α

During endotoxemia, the ATP-dependent drug efflux pump P-glycoprotein (Abcb1/P-gp) is upregulated in kidney proximal tubule epithelial cells. The signaling pathway through which lipopolysaccharide (LPS) or tumor necrosis factor-α (TNF-α) regulates P-gp expression and activity was investigated further in the present study. Exposure of rat kidney proximal tubule cells to TNF-α alone or TNF-α and LPS increased P-gp gene and protein expression levels and efflux activity, suggesting de novo P-gp synthesis. Upon exposure to TNF-α in combination with LPS, P-gp activity in renal proximal tubule cells is increased under influence of nitric oxide (NO) produced by inducible NO synthase. Upon exposure to TNF-α alone, P-gp upregulation seems to involve TLR4 activation and nuclear factor kappaB (NF-κB) translocation, a pathway that is likely independent of NO. These findings indicate that at least two pathways regulate P-gp expression in the kidney during endotoxemia