Dottorato in Fisiopatologia Medica

Glomerulus on a chip

Chronic kidney disease (CKD) is increasingly recognized as a global public health problem. According to the World Health Organization’s Global Burden of Disease (GBD) project, kidney and urinary tract disease is the 12th most common cause of death and the 17th most common cause of disability(1).This phenomenon requires constant evolution in research methods, but only recently significant progresses have been made in field of pre-clinical cell models in nephrology.
“Organ-on-a-chip” is a 3-D microfluidic cell culture device able to mimic the activities, mechanics and response of entire organs, in a physiologically most relevant way than cell cultured in dishes(2). In the last few years, different parts of nephron, the functional unit of kidney, have been simulated by microfluidic chip.
Glomerulus exerts its function of blood filtration through the exact organization of glomerular endothelial cells (GEC), podocytes (POD) and mesangial cells (MES), that communicate with each other supported by a specialized glomerular basement membrane (GBM)(3).
In this contest, I would focus my work on a bioartificial device that replicates the function of glomerulus. The starting point of my project is an interesting reviews published in August 2016, showing that not only it is possible to realize a glomerular 3D co-culture on microfluidic chip, but that it can be possible to mimic a pathological condition, in this case glomerular hypertension(4). GENERAL AIM To develop an in vitro 3D model of glomerular filtration: a three-layers structure composed by two specialized and interdependent cell populations, h-GEC and h-POD-SV 40 cell lines, and the basement membrane in between coated with a matrix of Collagen type IV. Once designed this device, it could be possible to examine filtration in different pathological conditions, such us hypertension, proteinuria or Alport Syndrome, reproduced through specific protocols. PRELIMINARY RESULTS The work done so far has mainly had the goal to standardize and make reproducible the process and timings of co-culture in static condition. We adapted the protocol Zhou by using human and not murine cells and shorter times of incubation. The day after coating with collagen type IV (0.25mg/ml), we seed h-GEC (150.000) on the underside of an insert membrane; the following day are sown podocytes, in the same quantities, on the upper side of the insert, each cell line in its medium, DMEM High Glucose for podocytes and EndoGRO for GEC . After 2 days of cell interactions, the two cell lines are subjected to filtration test: putting INULIN-FITC (0.05 mg/ml) and BSA-FITC (1mg/ml) in each well of plate, it is possible calculate the intensity of fluorescence. This parameter indicates the amount of filtered molecule. The amount of BSA or INULIN filtered through the co-culture is smaller than cells in monoculture or controls with only collagen IV, this experimental data suggest that we set up a functional model of glomerular filtration. We are also performing tests in the microflow system, obtaining a minor filtration from the co-coltured chip than chip without cells. However, in this case, many attempts need to be done before achieving an optimal parameters setting. We are also
setting the protocol to induce cell damage on both GEC and podocytes, in order to reproduce a pathological injury. ROADMAP After examining the data obtained so far the most important steps to define a roadmap are as folllow: 1. SETTING UP A MODEL OF FILTRATION IN STATIC:
As proved by our preliminary results, It is possible to re-create glomerular barrier in multiwell inserts: membrane of insert is alternatively coated on both sides with collagen type IV, Endothelial cells are seeded first on one side of the membrane and covered by medium. Once attached, podocytes were seeded on the other side of the membrane (5).
We can test this barrier system using a sample permeability assay (6): FITC-inulin or FITC-BSA are introduced into each well and the passage of these molecules across the barrier can be determined by measurement of fluorescence intensity.
2. SETTING UP A MODEL OF FILTRATION IN A DYNAMIC SYSTEM: It was reported that cultured cells display a more physiological expression of cell specific biomarker in a dynamic system. We propose to introduce the chip developed in static in a microfluidic system. We will evaluate filtration of the reproduced glomerular barrier, but also biomarkers expression by co-cultured cells in dynamic conditions; for example, we will evaluate through immunofluorescence the expression increase of CD 31 and Von Willebrand Factor in h-GEC, Podocin and Nephrin in Podocytes. Microfluidic chips and the perfusion system were purchased from Micronic Microfluidic and Fluigent respectively. This system is composed by a three-layer chip that allow co-colture and expose cells to different shear stress and flow rates ranging from 1 to 100 μL/min in order to mimic human glomerulus. Our system is composed by a microfluidic flow control platform with two independent flow sensors controlled through a computer program witch can achieve flow rate. These can be connected to the four slot chip platform. The chip will be integrated with microsensors that report on the cultured cells or microenvironmental conditions and that can be modulated by the operator(7). 3. MIMICKING DYSFUNCTIONS AND DISEASES: Organs-on-chips have great potential for the investigation of basic mechanism of organ physiology and disease. Glomerulus-on-a-chip could be the starting point for many possible studies. In particular, I think we could focus on:
-Proteinuria(8): a symptom of glomerular diseases and is due to leakage of proteins from the glomerular filtration barrier. In short, we could induce a damage by adding to the Podocytes medium substances known to alter cell morphology and function, such us doxorubicin hydrochloride. Dose-dependent and time-dependent permeability tests will be used to evaluate the increased passage of albumin. This system could be also used to test the nephrotoxicity of different drugs.
- Glomerular hypertension(9): caused by systemic hypertension, it promotes kidney disease progression and manifests as high perfusion pressure, high filtration pressure, and high transmembrane pressure in glomeruli. We could simulate this symptom by modifying flow parameters in the microfluidic system. For example we could perfuse the culture medium at a
higher flow-rate, about of 10 and 15 μL/min, through the endothelium channel of the GC. Then, we could examine filtration functions in different glomerular hemodynamic conditions by measuring the permeability of the microengineered GFB to FITC-inulin and FITC-BSA added into the endothelium channel.
-Alport Syndrome(10): It is a hereditary glomerulopathy leading to end-stage renal disease (ESRD), frequently during adolescence. It is caused by the absence or abnormal composition of the type IV collagen α3/4/5 network normally present in the glomerular basement membrane (GBM). We could reproduce this conditions using patient podocytes and eliminating ColIV coating from treatment of chip.
4. TESTING MICROVESICLES AS THERAPUTIC MOLECULES:
The natural consequence of creating injury models, could be testing molecules able to reverse that injury. Several studies have demonstrated that mesenchymal stem cells have the capacity to reverse acute and chronic kidney injury through a paracrine mode, in particular through mesenchymal stem cell-derived microvesicles (MVs). MVs are circular fragments of membrane
released from the endosomal compartment as exosomes or shed from the surface membranes of most cell types. They may either transfer transcripts from injured cells to stem cells, or vice versa, realizing a transfer of self repair-factors, transcripts, cycle re-entry factors that could improve tissue functionality. Important researches of G. Camussi group shows that in vivo MVs accelerate the functional and morphological recovery of glycerol induced acute kidney injury in severe combined immunodeficient mice(11). Though a collaboration, we could test potential therapeutic of MVs using the static and dynamic glomerular filtration model.
REFERENCES (1) K. B. Jayasekara, D. M. Dissanayake, R. Sivakanesan, A. Ranasinghe, R. H. Karunarathna, and G. W. Kumara. Epidemiology of Chronic Kidney Disease, With Special Emphasis on Chronic Kidney Disease of Uncertain Etiology, in the North Central Region of Sri Lanka- https://dx.doi.org/10.2188%2Fjea.JE20140074 (2) S. Kim, S.Takayama- kidney Research and Clinical Practice. Organ-on-a-chip and the kidney. http://dx.doi.org/10.1016/j.krcp.2015.08.001 (3) T. DesRochers, E. Palma, and D. L. Kaplan. Tissue-Engineered Kidney Disease Models. Adv Drug Deliv Rev. 2014 Apr 20; 0: 67–80. (4) M. Zhou, X. Zhang, X. Wen, T. Wu, W. Wang, M. Yang, J. Wang, M. Fang, B. Lin & H. Lin-S. Reports. Development of a Functional Glomerulus at the Organ Level on a Chip to Mimic Hypertensive Nephropathy. Scientific Reports 6:31771 DOI: 10.1038/srep31771
(5) M. Li, A. Corbelli, S. Watanabe, S. Armelloni, M. Ikehata, V. Parazzi, C. Pignatari, L. Giardino, D. Mattinzoli, L. Lazzari, A. Puliti, F. Cellesi, C. Zennaro , P. Messa, M. Rastaldi. Three-dimensional podocyte–endothelial cell co-cultures: Assembly,validation, and application to drug testing and intercellular signaling studies. http://dx.doi.org/10.1016/j.ejps.2016.02.013 (6) M. Zhou, X. Zhang, X. Wen, T. Wu, W. Wang, M. Yang, J. Wang, M. Fang, B. Lin & H. Lin-S. Reports. Development of a Functional Glomerulus at the Organ Level on a Chip to Mimic Hypertensive Nephropathy. Scientific Reports 6:31771 DOI: 10.1038/srep31771
(7) S. N Bhatia & D. Ingber. Microfluidic organs-on-chips. doi:10.1038/nbt.2989
(8) M. Li, A. Corbelli, S. Watanabe, S. Armelloni, M. Ikehata, V. Parazzi, C. Pignatari, L. Giardino, D. Mattinzoli, L. Lazzari, A. Puliti, F. Cellesi, C. Zennaro , P. Messa, M. Rastaldi. Three-dimensional podocyte–endothelial cell co-cultures:
Assembly,validation, and application to drug testing and intercellular signaling studies. http://dx.doi.org/10.1016/j.ejps.2016.02.013 (9) M. Zhou, X. Zhang, X. Wen, T. Wu, W. Wang, M. Yang, J. Wang, M. Fang, B. Lin & H. Lin-S. Reports. Development of a Functional Glomerulus at the Organ Level on a Chip to Mimic Hypertensive Nephropathy. Scientific Reports 6:31771 DOI: 10.1038/srep31771.
(10) O. Gross, D. Borza, H. Anders, C. Licht, M Weber, S. Segerer, R. Torra, M. Gubler, L. Heidet, S. Harvey, D. Cosgrove, G. Lees, C. Kashtan, M. Gregory, J. Savige, J. Ding, P. Thorner, D.R. Abrahamson, C. Antignac, K. Tryggvason, B. Hudson and J. H. Miner. Stem cell therapy for Alport syndrome: the hope beyond the hype. Nephrol Dial Transplant (2009) 24: 731–734 doi: 10.1093/ndt/gfn722
(11) G. Camussi, M.C. Deregibus, S. Bruno, V. Cantaluppi and L. Biancone. Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney International (2010) 78, 838–848; doi:10.1038/ki.