Bioengineered Kidney Tubules Efficiently Clear Uremic Toxins in Experimental Dialysis Conditions

Patients with end-stage kidney disease (ESKD) suffer from high levels of protein-bound uremic toxins (PBUTs) that contribute to various comorbidities. Conventional dialysis methods are ineffective in removing these PBUTs. A potential solution could be offered by a bioartificial kidney (BAK) composed of porous membranes covered by proximal tubule epithelial cells (PTECs) that actively secrete PBUTs. However, BAK development is currently being hampered by a lack of knowledge regarding the cytocompatibility of the dialysis fluid (DF) that comes in contact with the PTECs. Here, we conducted a comprehensive functional assessment of the DF on human conditionally immortalized PTECs (ciPTECs) cultured as monolayers in well plates, on Transwell® inserts, or on hollow fiber membranes (HFMs) that form functional units of a BAK. We evaluated cell viability markers, monolayer integrity, and PBUT clearance. Our results show that exposure to DF did not affect ciPTECs’ viability, membrane integrity, or function. Seven anionic PBUTs were efficiently cleared from the perfusion fluid containing a PBUTs cocktail or uremic plasma, an effect which was enhanced in the presence of albumin. Overall, our findings support that the DF is cytocompatible and does not compromise ciPTECs function, paving the way for further advancements in BAK development and its potential clinical application.</p

Improving home haemodialysis: Stability evaluation of routine clinical chemistry analytes in blood samples of haemodialysis patients

Introduction: A growing number of dialysis patients is treated with home haemodialysis. Our current pre-analytical protocols require patients to centrifuge the blood sample and transfer the plasma into a new tube at home. This procedure is prone to errors and precludes accurate bicarbonate measurement, required for determining dialysate bicarbonate concentration and maintaining acid-base status. We therefore evaluated whether cooled overnight storage of gel separated plasma is an acceptable alternative. Materials and methods: Venous blood of 34 haemodialysis patients was collected in 2 lithium heparin blood collection tubes with gel separator (LH PSTTM II, REF 367374; Becton Dickinson, New Jersey, USA). One tube was analysed directly for measurement of bicarbonate, potassium, calcium, phosphate, glucose, urea, lactate, aspartate aminotransferase (AST), and lactate dehydrogenase (LD); whereas the other was centrifuged and stored unopened at 4 °C and analysed 24 h later. To measure analyte stability after 24 h of storage, the mean difference was calculated and compared to the total allowable error (TEa) which was used as acceptance limit. Results: Potassium (Z = - 4.28, P < 0.001), phosphate (Z = - 3.26, P = 0.001), lactate (Z = - 5.11, P < 0.001) and AST (Z = - 2.71, P = 0.007) concentrations were higher, whereas glucose (Z = 4.00, P < 0.001) and LD (Z = 3.13, P = 0.002) showed a reduction. All mean differences were smaller than the TEa and thus not clinically relevant. Bicarbonate (Z = 0.69, P = 0.491), calcium (Z = - 0.23, P = 0.815) and urea (Z = 0.81, P =0.415) concentrations were stable. Conclusions: Our less complex, user-friendly pre-analytical procedure resulted in at least 24 h stability of analytes relevant for monitoring haemodialysis, including bicarbonate. This allows shipment and analysis the next day

A Uremic Pig Model for Peritoneal Dialysis

With increasing interest in home dialysis, there is a need for a translational uremic large animal model to evaluate technical innovations in peritoneal dialysis (PD). To this end, we developed a porcine model with kidney failure. Stable chronic kidney injury was induced by bilateral subtotal renal artery embolization. Before applying PD, temporary aggravation of uremia was induced by administration of gentamicin (10 mg/kg i.v. twice daily for 7 days), to obtain uremic solute levels within the range of those of dialysis patients. Peritoneal transport was assessed using a standard peritoneal permeability assessment (SPA). After embolization, urea and creatinine concentrations transiently increased from 1.6 ± 0.3 to 7.5 ± 1.2 mM and from 103 ± 14 to 338 ± 67 µM, respectively, followed by stabilization within 1-2 weeks to 2.5 ± 1.1 mM and 174 ± 28 µM, respectively. Gentamicin induced temporary acute-on-chronic kidney injury with peak urea and creatinine concentrations of 16.7 ± 5.3 mM and 932 ± 470 µM respectively. PD was successfully applied, although frequently complicated by peritonitis. SPA showed a low transport status (D/P creatinine at 4 h of 0.41 (0.36-0.53)) with a mass transfer area coefficient of 9.6 ± 3.1, 4.6 ± 2.6, 3.4 ± 2.3 mL/min for urea, creatinine, and phosphate respectively. In conclusion, this porcine model with on-demand aggravation of uremia is suitable for PD albeit with peritoneal transport characterized by a low transport status

Diabetic proximal tubulopathy: Can we mimic the disease for in vitro screening of SGLT inhibitors?

Diabetic kidney disease (DKD) is the foremost cause of renal failure. While the glomeruli are severely affected in the course of the disease, the main determinant for disease progression is the tubulointerstitial compartment. DKD does not develop in the absence of hyperglycemia. Since the proximal tubule is the major player in glucose reabsorption, it has been widely studied as a therapeutic target for the development of new therapies. Currently, there are several proximal tubule cell lines available, being the human kidney-2 (HK-2) and human kidney clone-8 (HKC-8) cell lines the ones widely used for studying mechanisms of DKD. Studies in these models have pushed forward the understanding on how DKD unravels, however, these cell culture models possess limitations that hamper research, including lack of transporters and dedifferentiation. The sodium-glucose cotransporters (SGLT) are identified as key players in glucose reabsorption and pharmacological inhibitors have shown to be beneficial for the long-term clinical outcome in DKD. However, their mechanism of action has, as of yet, not been fully elucidated. To comprehend the protective effects of SGLT inhibitors, it is essential to understand the complete functional, structural, and molecular features of the disease, which until now have been difficult to recapitulate. This review addresses the molecular events of diabetic proximal tubulopathy. In addition, we evaluate the protective role of SGLT inhibitors in cardiovascular and renal outcomes, and provide an overview of various in vitro models mimicking diabetic proximal tubulopathy used so far. Finally, new insights on advanced in vitro systems to surpass past limitations are postulated

Diabetic proximal tubulopathy: Can we mimic the disease for in vitro screening of SGLT inhibitors?

Diabetic kidney disease (DKD) is the foremost cause of renal failure. While the glomeruli are severely affected in the course of the disease, the main determinant for disease progression is the tubulointerstitial compartment. DKD does not develop in the absence of hyperglycemia. Since the proximal tubule is the major player in glucose reabsorption, it has been widely studied as a therapeutic target for the development of new therapies. Currently, there are several proximal tubule cell lines available, being the human kidney-2 (HK-2) and human kidney clone-8 (HKC-8) cell lines the ones widely used for studying mechanisms of DKD. Studies in these models have pushed forward the understanding on how DKD unravels, however, these cell culture models possess limitations that hamper research, including lack of transporters and dedifferentiation. The sodium-glucose cotransporters (SGLT) are identified as key players in glucose reabsorption and pharmacological inhibitors have shown to be beneficial for the long-term clinical outcome in DKD. However, their mechanism of action has, as of yet, not been fully elucidated. To comprehend the protective effects of SGLT inhibitors, it is essential to understand the complete functional, structural, and molecular features of the disease, which until now have been difficult to recapitulate. This review addresses the molecular events of diabetic proximal tubulopathy. In addition, we evaluate the protective role of SGLT inhibitors in cardiovascular and renal outcomes, and provide an overview of various in vitro models mimicking diabetic proximal tubulopathy used so far. Finally, new insights on advanced in vitro systems to surpass past limitations are postulated

New mixed matrix membrane for the removal of urea from dialysate solution

Urea removal is one of the biggest challenges in dialysate regeneration in Wearable Artificial Kidney (WAK) devices. In this work, a new Mixed Matrix Membrane (MMM) is developed for urea removal in WAK applications. The MMM consists of polystyrene-based ninhydrin particles within a polyethersulfone/polyvinylpyrrolidone polymer blend matrix. The MMM is prepared via dry-wet spinning technique and characterized in terms of its morphology via electron microscopy and clean water permeance. Urea removal is studied both in static and in dynamic conditions. Thanks to the good dispersion of small size ninhydrin particles (size < 63 µm), the MMM removed under static conditions, at 70 °C, 2.1 ± 0.1 mmol of urea per grams of particles at 24 h, while urea removal by the particles in suspension reached 1.7 ± 0.1 mmol/g under the same conditions. Importantly, in continuous recirculation experiments, performed at 70 °C using a laboratory scale module, the MMM removed 3.4 ± 0.3 mmol of urea per grams of particles, in 4 h, due to the high particle accessibility by urea within the membrane. Based on these results it is estimated that only 215 g of MMM are needed for removing the daily produced urea from spent dialysate (400 mmol) making MMM suitable for application to WAK, where miniaturization and lightweight are required

New mixed matrix membrane for the removal of urea from dialysate solution

Urea removal is one of the biggest challenges in dialysate regeneration in Wearable Artificial Kidney (WAK) devices. In this work, a new Mixed Matrix Membrane (MMM) is developed for urea removal in WAK applications. The MMM consists of polystyrene-based ninhydrin particles within a polyethersulfone/polyvinylpyrrolidone polymer blend matrix. The MMM is prepared via dry-wet spinning technique and characterized in terms of its morphology via electron microscopy and clean water permeance. Urea removal is studied both in static and in dynamic conditions. Thanks to the good dispersion of small size ninhydrin particles (size < 63 µm), the MMM removed under static conditions, at 70 °C, 2.1 ± 0.1 mmol of urea per grams of particles at 24 h, while urea removal by the particles in suspension reached 1.7 ± 0.1 mmol/g under the same conditions. Importantly, in continuous recirculation experiments, performed at 70 °C using a laboratory scale module, the MMM removed 3.4 ± 0.3 mmol of urea per grams of particles, in 4 h, due to the high particle accessibility by urea within the membrane. Based on these results it is estimated that only 215 g of MMM are needed for removing the daily produced urea from spent dialysate (400 mmol) making MMM suitable for application to WAK, where miniaturization and lightweight are required

De draagbare kunstnier: hoe staan de zaken?

Draagbare dialyseapparaten geven dialysepatiënten de mogelijkheid om thuis te dialyseren en vergroten daarmee hun mobiliteit en autonomie. Centraal in de ontwikkeling van deze apparaten staat het ontwikkelen van een strategie om dialysaat te hergebruiken. Momenteel wordt in Nederland toegewerkt naar een klinische trial met een draagbaar hemodialyseapparaat van circa 10 kg en naar een klinische trial met een draagbaar systeem van circa 10 kg voor peritoneale dialyse met continue flow

Drugs commonly applied to kidney patients may compromise renal tubular uremic toxins excretion

In chronic kidney disease (CKD), the secretion of uremic toxins is compromised leading to their accumulation in blood, which contributes to uremic complications, in particular cardiovascular disease. Organic anion transporters (OATs) are involved in the tubular secretion of protein-bound uremic toxins (PBUTs). However, OATs also handle a wide range of drugs, including those used for treatment of cardiovascular complications and their interaction with PBUTs is unknown. The aim of this study was to investigate the interaction between commonly prescribed drugs in CKD and endogenous PBUTs with respect to OAT1-mediated uptake.We exposed a unique conditionally immortalized proximal tubule cell line (ciPTEC) equippedwithOAT1 to a panel of selected drugs, including angiotensin-converting enzyme inhibitors (ACEIs: captopril, enalaprilate, lisinopril), angiotensin receptor blockers (ARBs: losartan and valsartan), furosemide and statins (pravastatin and simvastatin), and evaluated the drug-interactions using an OAT1-mediated fluorescein assay.We show that selected ARBs and furosemide significantly reduced fluorescein uptake,with the highest potency forARBs. Thiswas exaggerated in presence of some PBUTs. Selected ACEIs and statins had either no or a slight effect at supratherapeutic concentrations on OAT1-mediated fluorescein uptake. In conclusion, we demonstrate that PBUTs may compete with co-administrated drugs commonly used in CKD management for renal OAT1 mediated secretion, thus potentially compromising the residual renal function

Effect of substituents on the reactivity of ninhydrin with urea

Ninhydrin, i. e. the stable hydrate of the reactive species indanetrione, is a well-known compound used for the quantification of ammonia and amino acids. However, substituent effects on the reactivity of ninhydrin with nucleophiles are not described. In this work, the kinetics of the reaction of C4-and C5-substituted ninhydrins with urea was studied and monitored by 13C-NMR. Surprisingly, the obtained results show that electron donating groups (EDGs) as well as electron withdrawing groups (EWGs) decrease the rate of the reaction. EDGs decrease the electrophilicity of indanetrione, resulting in slower overall kinetics than unsubstituted ninhydrin. The calculated Gibbs free energy differences for the dehydration of unsubstituted and substituted ninhydrins and the subsequent reaction with urea showed that the dehydration of the compounds is more sensitive to electronic effects than the subsequent reaction with urea. Therefore, although EWGs increase the electrophilicity of indanetrione, this is more than counterbalanced by an adverse shift of the hydration equilibrium towards the unreactive hydrate (i. e. ninhydrin), resulting in slower kinetics as well