The guanylate cyclase C agonist linaclotide ameliorates the gut-cardio-renal axis in an adenine-induced mouse model of chronic kidney disease.

BACKGROUND: Cardiorenal syndrome is a major cause of mortality in patients with chronic kidney disease (CKD). However, the involvement of detrimental humoral mediators in the pathogenesis of cardiorenal syndrome is still controversial. Trimethylamine-N-oxide (TMAO), a hepatic metabolic product of trimethylamine generated from dietary phosphatidylcholine or carnitine derived by the gut microbiota, has been linked directly with progression of cardiovascular disease and renal dysfunction. Thus, targeting TMAO may be a novel strategy for the prevention of cardiovascular disease and chronic kidney disease. METHODS: Linaclotide, a guanylate cyclase C agonist, was administered to adenine-induced renal failure (RF) mice and changes in renal function and levels of gut-derived uremic toxins, as well as the gut microbiota community, were analyzed using metabolomic and metagenomic methods to reveal its cardiorenal effect. RESULTS: Linaclotide decreased the plasma levels of TMAO at a clinically used low dose of 10 µg/kg in the adenine-induced RF mouse model. At a high concentration of 100 µg/kg, linaclotide clearly improved renal function and reduced the levels of various uremic toxins. A reduction in TMAO levels following linaclotide treatment was also observed in a choline-fed pro-atherosclerotic model. Linaclotide ameliorated renal inflammation and fibrosis and cardiac fibrosis, as well as decreased the expression of collagen I, transforming growth factor-ß, galectin-3 (Gal-3) and ST2 genes. Plasma levels of Gal-3 and ST2 were also reduced. Because exposure of cardiomyocytes to TMAO increased fibronectin expression, these data suggest that linaclotide reduced the levels of TMAO and various uremic toxins and may result in not only renal, but also cardiac, fibrosis. F4/80-positive macrophages were abundant in small intestinal crypts in RF mice, and this increased expression was decreased by linaclotide. Reduced colonic claudin-1 levels were also restored by linaclotide, suggesting that linaclotide ameliorated the 'leaky gut' in RF mice. Metagenomic analysis revealed that the microbial order Clostridiales could be responsible for the change in TMAO levels. CONCLUSION: Linaclotide reduced TMAO and uremic toxin levels and could be a powerful tool for the prevention and control of the cardiorenal syndrome by modification of the gut-cardio-renal axis.

p-Cresyl glucuronide is a major metabolite of p-cresol in mouse: in contrast to p-cresyl sulphate, p-cresyl glucuronide fails to promote insulin resistance.

BACKGROUND: The role of uraemic toxins in insulin resistance associated with chronic kidney disease (CKD) is gaining interest. p-Cresol has been defined as the intestinally generated precursor of the prototype protein-bound uraemic toxins p-cresyl sulphate (p-CS) as the main metabolite and, at a markedly lower concentration in humans, p-cresyl glucuronide (p-CG). The objective of the present study was to evaluate the metabolism of p-cresol in mice and to decipher the potential role of both conjugates of p-cresol on glucose metabolism. METHODS: p-CS and p-CG were measured by high performance liquid chromatography-fluorescence in serum from control, 5/6 nephrectomized mice and mice injected intraperitoneously with either p-cresol or p-CG. The insulin sensitivity in vivo was estimated by insulin tolerance test. The insulin pathway in the presence of p-cresol, p-CG and/or p-CS was further evaluated in vitro on C2C12 muscle cells by measuring insulin-stimulated glucose uptake and the insulin signalling pathway (protein kinase B, PKB/Akt) by western blot. RESULTS: In contrast to in humans, where p-CS is the main metabolite of p-cresol, in CKD mice both conjugates accumulated, and after chronic p-cresol administration with equivalent concentrations but a substantial difference in protein binding (96% for p-CS and <6% for p-CG). p-CG exhibited no effect on insulin sensitivity in vivo or in vitro and no synergistic inhibiting effect in combination with p-CS. CONCLUSIONS: The relative proportion of the two p-cresol conjugates, i.e. p-CS and p-CG, is similar in mouse, in contrast to humans, pinpointing major inter-species differences in endogenous metabolism. Biologically, the sulpho- (i.e. p-CS) but not the glucuro- (i.e. p-CG) conjugate promotes insulin resistance in CKD.

Comparative palatability of five supplements designed for cats suffering from chronic renal disease.

BACKGROUND: Intestinal phosphate binders, uremic toxin binders and some other types of supplements are an integral part of the management of chronic kidney disease (CKD) in various species, including cats. This pathology in domestic carnivores requires life-long nutritional and medical management. In this context, the compliance of owners and patients cannot be achieved without an adequate level of palatability for oral medication or supplementation. Knowing that hyporexia and anorexia are among the most commonly seen clinical signs in cats suffering from CKD this is already, in itself, a serious obstacle to acceptable compliance in sick animals. The aim of the present study was to investigate the palatability of four commercially available products designed for cats suffering from CKD: Ipakitine® (Vetoquinol, France), Azodyl® (Vetoquinol, USA), Renalzin® (Bayer, France), Rubenal® (Vetoquinol, France) and an additional recently developed product: Pronefra® (Virbac, France). The study was performed with a group of previously-characterised cats, all living in an enriched and well-being securing environment of an independent centre housing panels of pets expert in palatability measurement. In total 172 monadic testings were performed. The palatability of each product was assessed by measuring their rates of prehension and consumption, and the consumption proportions were also analysed. RESULTS: The most palatable presentation (based on useful consumption) was Pronefra®, which was significantly higher than Azodyl® (p = 0.046), Ipakitine® (p < 0.0001), Renalzin® (p < 0.0001) and Rubenal® (p < 0.0001). The product with the highest rate of prehension was also Pronefra®, which was significantly higher than Azodyl® (p = 0.0019), Ipakitine® (p = 0.0023), Renalzin® (p = 0.0008) and Rubenal® (p < 0.0001). CONCLUSION: Pronefra® was the most palatable presentation tested, meaning it may be useful for improving ease of supplementation in CKD cats.

The G protein-coupled receptor ligand apelin-13 ameliorates skeletal muscle atrophy induced by chronic kidney disease.

BACKGROUND: Targeting of the apelin-apelin receptor (Apj) system may serve as a useful therapeutic intervention for the management of chronic kidney disease (CKD)-induced skeletal muscle atrophy. We investigated the roles and efficacy of the apelin-Apj system in CKD-induced skeletal muscle atrophy. METHODS: The 5/6-nephrectomized mice were used as CKD models. AST-120, a charcoal adsorbent of uraemic toxins (8 w/w% in diet), or apelin (1 µmol/kg) was administered to CKD mice to investigate the mechanism and therapeutic potential of apelin on CKD-induced skeletal muscle atrophy. The effect of indoxyl sulfate, a uraemic toxin, or apelin on skeletal muscle atrophy was evaluated using mouse myoblast cells (C2C12 cells) in vitro. RESULTS: Skeletal muscle atrophy developed over time following nephrectomy at 12 weeks, as confirmed by a significant increase of atrogin-1 and myostatin mRNA expression in the gastrocnemius (GA) muscle and a decrease of lower limb skeletal muscle weight (P < 0.05, 0.01 and 0.05, respectively). Apelin expression in GA muscle was significantly decreased (P < 0.05) and elabela, another Apj endogenous ligand, tended to show a non-significant decrease at 12 weeks after nephrectomy. Administration of AST-120 inhibited the decline of muscle weight and increase of atrogin-1 and myostatin expression. Apelin and elabela expression was slightly improved by AST-120 administration but Apj expression was not, suggesting the involvement of uraemic toxins in endogenous Apj ligand expression. The administration of apelin at 1.0 µmol/kg for 4 weeks to CKD mice suppressed the increase of atrogin-1 and myostatin, increased apelin and Apj mRNA expression at 30 min after apelin administration and significantly ameliorated weight loss and a decrease of the cross-sectional area of hindlimb skeletal muscle. CONCLUSIONS: This study demonstrated for the first time the association of the Apj endogenous ligand-uraemic toxin axis with skeletal muscle atrophy in CKD and the utility of therapeutic targeting of the apelin-Apj system.

Time-averaged concentration estimation of uraemic toxins with different removal kinetics: a novel approach based on intradialytic spent dialysate measurements.

Background: Kt/Vurea is the most used marker to estimate dialysis adequacy; however, it does not reflect the removal of many other uraemic toxins, and a new approach is needed. We have assessed the feasibility of estimating intradialytic serum time-averaged concentration (TAC) of various uraemic toxins from their spent dialysate concentrations that can be estimated non-invasively online with optical methods. Methods: Serum and spent dialysate levels and total removed solute (TRS) of urea, uric acid (UA), indoxyl sulphate (IS) and ß2-microglobulin (ß2M) were evaluated with laboratory methods during 312 haemodialysis sessions in 78 patients with four different dialysis treatment settings. TAC was calculated from serum concentrations and evaluated from TRS and logarithmic mean concentrations of spent dialysate (MlnD). Results: Mean (± standard deviation) intradialytic serum TAC values of urea, UA, ß2M and IS were 10.4 ± 3.8 mmol/L, 191.6 ± 48.1 µmol/L, 13.3 ± 4.3 mg/L and 82.9 ± 43.3 µmol/L, respectively. These serum TAC values were similar and highly correlated with those estimated from TRS [10.5 ± 3.6 mmol/L (R 2 = 0.92), 191.5 ± 42.8 µmol/L (R 2 = 0.79), 13.0 ± 3.2 mg/L (R 2 = 0.59) and 82.7 ± 40.0 µmol/L (R 2 = 0.85)] and from MlnD [10.7 ± 3.7 mmol/L (R 2 = 0.92), 191.6 ± 43.8 µmol/L (R 2 = 0.80), 12.9 ± 3.2 mg/L (R 2 = 0.63) and 82.2 ± 38.6 µmol/L (R 2 = 0.84)], respectively. Conclusions: Intradialytic serum TAC of different uraemic toxins can be estimated non-invasively from their concentration in spent dialysate. This sets the stage for TAC estimation from online optical monitoring of spent dialysate concentrations of diverse solutes and for further optimization of estimation models for each uraemic toxin.

Sponge-like Chitosan Based Porous Monolith for Uraemic Toxins Sorption.

More than three million patients are treated for kidney failure world-wide. Haemodialysis, the most commonly used treatment, requires large amounts of water and generates mountains of non-recyclable plastic waste. To improve the environmental footprint, dialysis treatments need to develop absorbents to regenerate the waste dialysate. Whereas conventional dialysis clears water-soluble toxins, it is not so effective in clearing protein-bound uraemic toxins (PBUTs), such as indoxyl sulfate (IS). Thus, developing absorption devices to remove both water-soluble toxins and PBUTs would be advantageous. Vapour induced phase separation (VIPS) has been used in this work to produce polycaprolactone/chitosan (PCL/CS) composite symmetric porous monoliths with extra porous carbon additives to increase creatinine and albumin-bound IS absorption. Moreover, these easy-to-fabricate porous monoliths can be formed into the required geometry. The PCL/CS porous monoliths absorbed 436 µg/g of albumin-bound IS and 2865 µg/g of creatinine in a single-pass perfusion model within 1 h. This porous PCL/CS monolith could potentially be used to absorb uraemic toxins, including PBUTs, and thus allow the regeneration of waste dialysate and the development of a new generation of environmentally sustainable dialysis treatments, including wearable devices.

Investigation of hallmarks of carbonyl stress and formation of end products in feline chronic kidney disease as markers of uraemic toxins.

OBJECTIVES: Cats are commonly affected by chronic kidney disease (CKD). Many reactive carbonyl intermediates and end products originating from the oxidative stress pathways are recognised as uraemic toxins and may play a role in CKD progression. The aim of the present study is to confirm whether carbonyl end-product formation is higher in cats affected by CKD and to assess whether an angiotensin-converting enzyme inhibitor (ACEi) might affect these hallmarks. METHODS: Twenty-two cats were divided into three groups: a control group (CG), cats with CKD and cats with CKD treated with an ACEi. Serum levels of pentosidine, carboxymethyllysine, advanced oxidation protein products, malondialdehyde, methylglyoxal and hexanoyl-lysine were measured. In addition, biochemical parameters and systolic blood pressure were evaluated. After checking for normality, comparisons between groups were performed followed by multiple comparison tests. P values ⩽0.05 were considered significant. Correlations between concentrations of the considered biomarkers and of the other metabolic parameters were investigated. RESULTS: Advanced oxidation protein products, malondialdehyde and hexanoyl-lysine concentrations were significantly higher in CKD and ACEi-treated groups compared with the CG ( P <0.05). Carboxymethyllysine increased in the ACEi-treated group when compared with the CG, whereas intermediate values of these biomarkers were found in the CKD group ( P <0.05). The ACEi-treated group showed the highest values of carboxymethyllysine, advanced oxidation protein products and hexanoyl-lysine. By contrast, the CKD group had the highest concentration of malondialdehyde. No statistically significant difference was found in the levels of pentosidine or methylglyoxal. End products correlated with creatinine and urea and with each other. CONCLUSIONS AND RELEVANCE: Significantly high concentrations of both intermediate and end products of carbonyl/oxidative stress were detected in CKD cats. This is the first study to have concurrently taken into account several uraemic toxins and biochemical parameters in cats affected by CKD.

Modulation of multidrug resistance-associated proteins function in erythrocytes in glycerol-induced acute renal failure rats.

OBJECTIVES: Evaluation of the function of multidrug resistance-associated proteins (MRPs) expressed in erythrocytes and screening of endogenous MRPs modulator(s) in glycerol-induced acute renal failure (ARF) rats. METHODS: Concentrations of 2,4-dinitrophenyl-S-glutathione (DNP-SG), a substrate for MRPs, in erythrocytes after administration of 1-chloro-2,4-dintrobenzene (CDNB), a precursor of DNP-SG, were determined in control and ARF rats. The screening of endogenous MRPs modulator(s) was performed using washed erythrocytes and inside-out erythrocyte membrane vesicles (IOVs) in vitro. KEY FINDINGS: Accumulation of DNP-SG in erythrocytes was observed in ARF rats. Uraemic plasma components exhibited a greater inhibitory effect on DNP-SG uptake by IOVs than control plasma components and increased the DNP-SG accumulation significantly in washed erythrocytes. Several protein-bound uraemic toxins at clinically observed concentrations and bilirubin significantly inhibited DNP-SG uptake by IOVs. In washed erythrocytes, bilirubin (10 µm) and l-kynurenine (100 µm), a precursor of kynurenic acid being MRPs inhibitor, increased DNP-SG accumulation significantly. CONCLUSIONS: Glycerol-induced ARF rats contain various MRPs inhibitors in plasma, and membrane-permeable MRP substrates/inhibitors including their precursors inhibit the MRPs function in erythrocytes cooperatively.

Detection of indoxyl sulfate levels in dogs and cats suffering from naturally occurring kidney diseases.

Indoxyl sulfate (IS), a protein-bound uraemic toxin, has been found to accumulate in the serum of people with renal diseases and is associated with free radical induction, nephrotoxicity cardiovascular toxicity, and osteoblast cytotoxicity. Although IS has been studied in humans and in experimental models, the role of IS in dogs and cats with kidney disease has not been investigated. A high performance liquid chromatography system was applied to detect plasma IS concentrations in non-azotaemic animals (63 dogs, 16 cats) and in animals with renal azotaemia (66 dogs, 69 cats). The IS levels of azotaemic animals were significantly higher (P <0.01) than those of non-azotaemic animals (median [IQR] 20.4 (9.5) mg/L vs. 7.2 (8.8) mg/L for dogs; median [IQR] 21 (18.9) mg/L vs. 14.8 (12.3) mg/L for cats). The IS level was significantly correlated with blood urea nitrogen, serum creatinine and phosphate concentrations. Dogs with acute kidney injury had significantly higher IS levels (P <0.01) than those with chronic kidney diseases (CKD) (median [IQR] 57.7 (40.8) mg/L vs. 17.7 (25.1) mg/L). When CKD was graded using the International Renal Interest Society (IRIS) staging system, IS levels were correlated with CKD severity in both dogs and cats. The IS concentration is directly related to loss of renal function. Further studies are necessary to determine whether measurement of IS provides any additional diagnostic or prognostic information in dogs and cats with kidney disease.