Fluorescence-Based Transport Assays Revisited in a Human Renal Proximal Tubule Cell Line.

Apical transport is key in renal function, and the activity of efflux transporters and receptor-mediated endocytosis is pivotal in this process. The conditionally immortalized proximal tubule epithelial cell line (ciPTEC) endogenously expresses these systems. Here, we used ciPTEC to investigate the activity of three major efflux transporters, viz., breast cancer resistance protein (BCRP), multidrug resistance protein 4 (MRP4), and P-glycoprotein (P-gp), as well as protein uptake through receptor-mediated endocytosis, using a fluorescence-based setup for transport assays. To this end, cells were exposed to Hoechst33342, chloromethylfluorescein-diacetate (CMFDA), and calcein-AM in the presence or absence of model inhibitors for BCRP (KO143), P-gp (PSC833), or MRPs (MK571). Overexpression cell lines MDCKII-BCRP and MDCKII-P-gp were used as positive controls, and membrane vesicles overexpressing one transporter were used to determine substrate and inhibitor specificities. Receptor-mediated endocytosis was investigated by determining the intracellular accumulation of fluorescently labeled receptor-associated protein (RAP-GST). In ciPTEC, BCRP and P-gp showed similar expressions and activities, whereas MRP4 was more abundantly expressed. Hoechst33342, GS-MF, and calcein are retained in the presence of KO143, MK571, and PSC833, showing clearly redundancy between the transporters. Noteworthy is the fact that both KO143 and MK571 can block BCRP, P-gp, and MRPs, whereas PSC833 appears to be a potent inhibitor for BCRP and P-gp but not the MRPs. Furthermore, ciPTEC accumulates RAP-GST in intracellular vesicles in a dose- and time-dependent manner, which was reduced in megalin-deficient cells. In conclusion, fluorescent-probe-based assays are fast and reproducible in determining apical transport mechanisms, in vitro. We demonstrate that typical substrates and inhibitors are not specific for the designated transporters, reflecting the complex interactions that can take place in vivo. The set of tools we describe are also compatible with innovative kidney culture models and allows studying transport mechanisms that are central to drug absorption, disposition, and detoxification.

Proximal tubular efflux transporters involved in renal excretion of p-cresyl sulfate and p-cresyl glucuronide: Implications for chronic kidney disease pathophysiology.

The uremic solutes p-cresyl sulfate (pCS) and p-cresyl glucuronide (pCG) accumulate in patients with chronic kidney disease (CKD), and might contribute to disease progression. Moreover, retention of these solutes may directly be related to renal tubular function. Here, we investigated the role of the efflux transporters Multidrug Resistance Protein 4 (MRP4) and Breast Cancer Resistance Protein (BCRP) in pCS and pCG excretion, and studied the impact of both solutes on the phenotype of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC). Our results show that p-cresol metabolites accumulate during CKD, with a shift from sulfation to glucuronidation upon progression. Moreover, pCS inhibited the activity of MRP4 by 40% and BCRP by 25%, whereas pCG only reduced MRP4 activity by 75%. Moreover, BCRP-mediated transport of both solutes was demonstrated. Exposure of ciPTEC to pCG caused epithelial-to-mesenchymal transition, indicated by increased expression of vimentin and Bcl-2, and diminished E-cadherin. This was associated with altered expression of key tubular transporters. In conclusion, BCRP is likely involved in the renal excretion of both solutes, and pCG promotes phenotypical changes in ciPTEC, supporting the notion that uremic toxins may be involved in CKD progression by negatively affecting renal tubule cell phenotype and functionality.

Optimized metabolomic approach to identify uremic solutes in plasma of stage 3-4 chronic kidney disease patients.

BACKGROUND: Chronic kidney disease (CKD) is characterized by the progressive accumulation of various potential toxic solutes. Furthermore, uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes. METHODS: In this study, we applied (1)H-nuclear magnetic resonance (NMR) spectroscopy, following three distinct deproteinization strategies, to determine differences in the plasma metabolic status of stage 3-4 CKD patients and healthy controls. Moreover, the human renal proximal tubule cell line (ciPTEC) was used to study the influence of newly indentified uremic solutes on renal phenotype and functionality. RESULTS: Protein removal via ultrafiltration and acetonitrile precipitation are complementary techniques and both are required to obtain a clear metabolome profile. This new approach, revealed that a total of 14 metabolites were elevated in uremic plasma. In addition to confirming the retention of several previously identified uremic toxins, including p-cresyl sulphate, two novel uremic retentions solutes were detected, namely dimethyl sulphone (DMSO2) and 2-hydroxyisobutyric acid (2-HIBA). Our results show that these metabolites accumulate in non-dialysis CKD patients from 9±7 µM (control) to 51±29 µM and from 7 (0-9) µM (control) to 32±15 µM, respectively. Furthermore, exposure of ciPTEC to clinically relevant concentrations of both solutes resulted in an increased protein expression of the mesenchymal marker vimentin with more than 10% (p<0.05). Moreover, the loss of epithelial characteristics significantly correlated with a loss of glucuronidation activity (Pearson r = -0.63; p<0.05). In addition, both solutes did not affect cell viability nor mitochondrial activity. CONCLUSIONS: This study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using (1)H-NMR spectroscopy, and provide insight into the negative impact of DMSO2 and 2-HIBA on ciPTEC, which could aid in understanding the progressive nature of renal disease.

Mesenchymal stem cell-conditioned medium accelerates regeneration of human renal proximal tubule epithelial cells after gentamicin toxicity.

Bone marrow-derived mesenchymal stem cells (MSCs) have the capacity to regenerate renal tubule epithelia and repair renal function without fusing with resident tubular cells. The goal of the present project was to investigate the role of MSCs secreted cytokines on tubule cell viability and regeneration after a toxic insult, using a conditionally immortalized human proximal tubule epithelial cell (ciPTEC) line. Gentamicin was used to induce nephrotoxicity, and cell viability and migration were studied in absence and presence of human MSC-conditioned medium (hMSC-CM) i.e. medium containing soluble factors produced and secreted by MSCs. Exposure of ciPTEC to 0-3000 µg/ml gentamicin for 24 h caused a significant dose-dependent increase in cell death. We further demonstrated that the nephrotoxic effect of 2000 µg/ml gentamicin was recovered partially by exposing cells to hMSC-CM. Moreover, exposure of ciPTEC to gentamicin (1500-3000 µg/ml) for 7 days completely attenuated the migratory capacity of the cells. In addition, following scrape-wounding, cell migration of both untreated and gentamicin-exposed cells was increased in the presence of hMSC-CM, as compared to exposures to normal medium, indicating improved cell recovery. Our data suggest that cytokines secreted by MSCs stimulate renal tubule cell regeneration after nephrotoxicity.

FXYD2 and Na,K-ATPase expression in isolated human proximal tubular cells: disturbed upregulation on renal hypomagnesemia?

Autosomal dominant renal hypomagnesemia (OMIM 154020), associated with hypocalciuria, has been linked to a 121G to A mutation in the FXYD2 gene. To gain insight into the molecular mechanisms linking this mutation to the clinical phenotype, we studied isolated proximal tubular cells from urine of a patient and a healthy subject. Cells were immortalized and used to assess the effects of hypertonicity-induced overexpression of FXYD2 on amount, activity and apparent affinities for Na(+), K(+) and ATP of Na,K-ATPase. Both cell lines expressed mRNA for FXYD2a and FXYD2b, and patient cells contained both the wild-type and mutated codons. FXYD2 protein expression was lower in patient cells and could be increased in both cell lines upon culturing in hyperosmotic medium but to a lesser extent in patient cells. Similarly, hyperosmotic culturing increased Na,K-ATPase protein expression and ATP hydrolyzing activity but, again, to a lesser extent in patient cells. Apparent affinities of Na,K-ATPase for Na(+), K(+) and ATP did not differ between patient and control cells or after hyperosmotic induction. We conclude that human proximal tubular cells respond to a hyperosmotic challenge with an increase in FXYD2 and Na,K-ATPase protein expression, though to a smaller absolute extent in patient cells.

Decreased intracellular ATP content and intact mitochondrial energy generating capacity in human cystinotic fibroblasts.

Cystinosis is an autosomal recessive lysosomal storage disorder caused by a defect in the lysosomal cystine carrier cystinosin. Cystinosis is the most common cause of inherited Fanconi syndrome leading to renal failure, in which the pathogenesis is still enigmatic. Based on studies of proximal tubules loaded with cystine dimethyl ester (CDME), altered mitochondrial adenosine triphosphate (ATP) production was proposed to be an underlying pathologic mechanism. Thus far, however, experimental evidence supporting this hypothesis in humans is lacking. In this study, energy metabolism was extensively investigated in primary fibroblasts derived from eight healthy subjects and eight patients with cystinosis. Patient's fibroblasts accumulated marked amounts of cystine and displayed a significant decrease in intracellular ATP content. Remarkably, overall energy-generating capacity, activity of respiratory chain complexes, ouabain-dependent rubidium uptake reflecting Na,K-ATPase activity, and bradykinin-stimulated mitochondrial ATP production were all normal in these cells. In conclusion, the data presented demonstrate that mitochondrial energy-generating capacity and Na,K-ATPase activity are intact in cultured cystinotic fibroblasts, thus questioning the idea of altered mitochondrial ATP synthesis as a keystone for the pathogenesis of cystinosis.

Strict cysteamine dose regimen is required to prevent nocturnal cystine accumulation in cystinosis.

Cystinosis is an autosomal recessive disorder, caused by mutations in the lysosomal cystine carrier cystinosin, encoded by the CTNS gene. The disease generally manifests with Fanconi syndrome during the first year of life and progresses towards end stage renal disease before the age of 10 years. Cysteamine depletes intralysosomal cystine content, postpones the deterioration of renal function and the occurrence of extra-renal organ damage. Based on the pharmacokinetic data, patients with cystinosis are advised to use cysteamine every 6 h. The aim of this study was (1) to evaluate the cysteamine dose regimen in Dutch patients with cystinosis and (2) to determine morning polymorphonuclear (PMN) leukocyte cystine content 6 h vs 9 h after the last evening cysteamine dose. Only 5/22 of Dutch cystinosis patients ingested cysteamine every 6 h. Morning (8 a.m.) PMN cystine content in 11 examined patients was elevated 9 h after 12.5-15 mg/kg evening cysteamine dose compared to the value 6 h after the ingestion of the same dose (0.73+/-0.81 nmol vs 0.44+/-0.52 nmol cystine/mg protein, p =0.02). In conclusion, only the minority of Dutch cystinosis patients follows the recommended strict cysteamine dose regimen. We provide evidence that cysteamine has to be administered every 6 h, including the night, as it has much better effect for maintaining low PMN cystine levels.

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells.

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.