Pathogenesis of Interstitial Renal Damage Leading to Renal Failure in Cystinosis
Pathogenesis of interstitial renal damage leading to renal failure in cystinosis1st Progress Report Cystinosis Research Foundation
6 months
E.N. Levtchenko
Department of Pediatrics
UMC St. Radboud
P.O. 9101
6500 HB Nijmegen
The Netherlands
Tel. +31 24 3616872
FAX +31 24 3619348
Summary of the project
Nephropathic cystinosis is characterized by the lysosomal accumulation of cystine and is generally leading to Fanconi syndrome in the first year of life. This autosomal recessive disorder frequently progresses to end stage renal disease, which is the leading cause of morbidity in patients with cystinosis.
In the current project funded by the Cystinosis Research Foundation we have addressed two key objectives:
1) Study of cytokines/chemokines and endothelin 1 production in mature proximal tubular cells from patients with cystinosis compared to healthy controls at different stages of cystine accumulation and after albumin application
The development of a conditionally immortalized proximal tubular cell (ci-PTC) model of cystinosis phenotype by our group allows us to investigate the cytokine/chemokine production in vitro. In cells cultured on Transwell chambers, the polarity of secretion can be evaluated and provides information about the cause of the progressive development of tubulointerstitial lesions observed in cystinosis patients.
2) Study of intracellular glutathione status, transport and generation of reactive oxygen species (ROS) in a conditionally immortalized proximal tubular cell line
The altered status of intracellular glutathione observed in cystinosis tissue, points to the role of oxidative stress in the pathogenesis of cystinosis. The in vitro cell model mentioned above allows us to investigate the glutathione status and ROS production. Furthermore, we can evaluate the effects of anti-oxidants and cystine depleting agent cysteamine. This might lead to improvements in the therapy of patients with cystinosis.
In this first progress report we describe the preliminary data that we have obtained for both key objectives in the first six months. Preliminary ResultsKey objective 1)
Study of cytokines/chemokines and endothelin 1 production in mature proximal tubular cells from patients with cystinosis compared to healthy controls at different stages of cystine accumulation and after albumin application
A. Mature ci-PTC’s will be cultured until confluent monolayers are obtained
We had already succeeded in culturing ci-PTC’s of 3 healthy controls and 3 cystinotic patients, by culturing urine sediment and transfection using SV40T antigen, containing both tsA58 and U19 mutations, and hTERT, containing the essential catalytic subunit of human telomerase. This resulted in cells that proliferate at 33°C and mature at 37°C. To obtain a homogenous cell line, we now have subcloned cell lines of 2 healthy donors and 3 cystinotic patients.
In previous studies we have shown the cobblestone morphology, presence of aminopeptidase N and alkaline phosphatase activity, all characteristic for proximal tubular cells. To underscore this proximal phenotype in the subclones, we have performed additional characterization studies, indicating the presence of dipeptidyl-peptidase IV (dpp-IV, CD26), aquaporin-1 (AQP1), p-glycoprotein (pGP) and organic cation transporter 2 (OCT2) by immunoblot analysis after culturing at 33°C and after 10 days maturation at 37°C (figure 1). Furthermore, the expression of SV40T disappeared after 10 days maturation. By immunohistochemistry, the presence of zona occludens 1 (ZO-1) tight junction protein was detected in cells, indicating their epithelial origin (figure 2). The presence of tight junctions in the ci-PTC contributes to an impermeable cell monolayer, which is important for the transport experiments using Transwell chambers.
According to this additional characterization of the proximal tubular phenotype of the subcloned cell lines, we have selected one clone of each donor for further experiments.
To study the cytokine production, one control and one cystinotic ci-PTC subclone was cultured in triplo in 24 well plates and after 7 days maturation at 37°C, medium was changed to serum free medium. At day 8 of maturation, cells were stimulated with IL1a for 48hr and supernatant tissue culture medium was harvested at day 10. For this preliminary report, we have measured IL-8 in the supernatants in the presence or absence of IL1a using DuoSet ELISA development system (R&D systems, USA). The results show an increase in IL1a-stimulated IL-8 production in both control and cystinotic cells. To perform statistical analysis, we will perform this test in all selected ci-PTC subclones in triplo.
B. Examination of polarity of secretion
The polarity of secretion will be examined in a Transwell chamber system. To study the polarity of ci-PTC cultured on Transwells, we have performed electron-microscopy (EM). Cells were seeded on Transwells and after 16hr incubation at 33°C to allow the cells to attach, Transwells were transferred to 37°C for 10 days. Subsequently, cells were fixed in situ and further processed suitable for EM (fig. 4). The image shows clearly some apical microvilli indicating polarization of the ci-PTC. Secondly, the EM pictures show some clathrin coated pits, indicating that the endocytic apparatus is functioning.
When we have analyzed more cytokines, we can evaluate the results and select an appropriate cytokine antibody array. By doing this, we can highly sensitively detect a broad range of cytokines excreted by cystinosis ci-PTC and compare the results with control ci-PTC.
D. Identifying intracellular signaling mechanism
To investigate whether albumin is reabsorbed in proximal tubules via endocytosis in cystinosis we examined the expression of megalin/cubilin in cystinotic renal tissue. These receptors are cooperating in the proximal reabsorption of proteins such as albumin in clathrin-coated pits via the endocytic pathway. The results show megalin and cubilin expression at the brush border of proximal tubule cells in cystinotic tissue (figure 5). Presence of albumin in endocytic vesicles indicates that albumin is reabsorbed in cystinotic proximal tubule cells. This finding might be of importance in studying the effects of albumin on cytokine production and the development of interstitial fibrosis.
A detailed mechanism involved in the progressive development of tubulointerstitial lesion found in cystinosis patients might be found after the evaluation of the cytokine antibody array.
A. Study of proximal tubular GSH transport in conditionally immortalized PTC
So far, we do not have conclusive data on GSH transport in ci-PTC.
B. Study of intracellular glutathione status
The investigation of total and oxidized GSH in ci-PTC provides us with information about the oxidative status in cystinosis.
For this purpose, cells were cultured for 14 days at 37°C and harvested at different stages of maturation. Subsequently, levels of total GSH, oxidized GSSH and intracellular cystine levels were determined using HPLC methods. The data are presented as means of two control and two cystinosis ci-PTC (figure 6). These data confirm our published data in fibroblasts, polymorphonuclear granulocytes and immortalized PTC using HPV E6/E7 gene, in which we have shown increased oxidized GSSH in cystinotic cells (Levtchenko et al, NDT (20), 2005; Wilmer et al, BBRC (337), 2005).
C. Study of intracellular ROS production, ROS-induced cellular damage and effects of the anti-oxidants.
So far, we have not studied this part of the project.
Ongoing researchKey objective 1)
Study of cytokines/chemokines and endothelin 1 production in mature proximal tubular cells from patients with cystinosis compared to healthy controls at different stages of cystine accumulation and after albumin application
Cytokine IL-8 productions in confluent ci-PTC monolayers have been studied so far in one control and one cystinotic cell line. These studies will be extended in 2 control and 3 cystinotic subclones and will be performed in triplo to obtain statistically relevant data. Additionally, the production of MCP1, TGF-b, RANTES and ET-1 will be studied in these cell lines.
In addition, we want to investigate the influence of cystine depleting agent cysteamine cytokine production. This part might be scheduled for the second year, if CRF decides to approve this part of the grant.
B. Examination of polarity of secretionWe have successfully investigated the culture of ci-PTC on Transwell chambers. We want to extend these experiments in the next 6 months in addition of albumin at the apical side of the cells and serum free medium at the basal compartment. Subsequently, we can measure cytokine production in both compartments.
C. Application of a new techniqueWhen we evaluate the production of selected cytokines mentioned above, we have planned to extend this research in the second year of the grant using a cytokine array (RayBio human Cytokine Antibody Array). This will provide us with detailed information on cytokine production in cystinotic PTC.
D. Identifying intracellular signaling mechanismAccording to the results mentioned under (C), we can extend our research in this field, such as the activation of the transcription factor NFk-B. This has become of more importance, since we have shown albumin reabsorption in cystinotic kidney tissue.
Key objective 2 Study of intracellular glutathione status, transport and generation of reactive oxygen species (ROS) in a conditionally immortalized proximal tubular cell lineA. Study of proximal tubular GSH transport in conditionally immortalized PTC
At this point, we are investigating the presence of GSH transporters (OAT1/3 and SDCT2) on ci-PTC by immunoblotting. When we have established these transporters in the next six months, we will perform transport experiments to compare GSH influx and efflux in cystinotic cells with control cells.
B. Study of intracellular glutathione status
We have shown increase of oxidized GSSH in cystinotic ci-PTC. We will confirm these results by a second approach using a colorimetric assay (Cayman Chemical).
C. Study of intracellular ROS production, ROS-induced cellular damage and effects of the anti-oxidants.
In the second part of this study, we will investigate the effect of cysteamine exposure on GSH, GSSH and ROS levels using fluorescent reporter molecules and video-imaging microscopy.
