Discussion
Previous renal MPS models have
targeted specific structures of the nephron, but regulation of
xenobiotics and substrates in the blood is a concerted action of both
filtration in the glomeruli and active reabsorption by the proximal
tubule. The challenge in establishing an effective nephron MPS
originates from fulfilling all required characteristics of kidney MPS:
incorporation of renal cellular models (tubular structures or in
vitro barrier models) (Jang et al.,
2013; C. Sakolish et al., 2020;
C. Sakolish et al., 2018;
C. M. Sakolish et al., 2016;
Tourovskaia et al., 2014;
Weber et al., 2016), functional
(filtration, absorption, and secretion) processes, and drug toxicity/
recovery testing. In this study, the glomerulus- and PCT- MPS addresses
most of the features of a renal MPS, while also maintaining in
vivo shear stress across the apical PCT (0.4-1.5
dyne-s/cm2) and recirculating 64.0% of flow into the
bloodstream and 36.0% into the filtrate output. The tri-culture system
of CIHP-1, HUVECs and HK-2 cells provides a novel, realistic approach by
recapitulating the critical functions in both the glomerulus and PCT for
long-term, overcoming the limitations of existing glomerulus-on-a-chip
and PCT-on-a-chip designs, and assessing for drug toxicity.
Geometrically, many vital attributes contribute to the innovation of the
glomerulus and PCT- MPS that have never been modeled previously in
vitro . The MPS presents a dual in vitro barrier model in the
glomerular filtration chamber and the PCT reabsorption device. Within
the glomerular unit, the 30nm pore size PES membrane was selected based
on the physiological size-exclusive filtration found on the human
glomerular capillary walls (C. M. Sakolish
& Mahler, 2017; C. M. Sakolish et al.,
2019). The PCT barrier model highlights a multi-layered fluidic
platform separated by a porous membrane, creating a dual channel system
(apical and basolateral). Proximal tubule cells on the membrane
selectively resorb solutes from the apical filtrate formation line to
the basolateral bloodstream, reenacting the renal active and passive
transport processes. This recirculation of flow was established through
the T-Junction component, crucial in bloodstream circuit reservoir to
simulate the removal of compounds that have been reabsorbed by the renal
tubule. Additionally, the PCT device was constructed in a polycarbonate
material instead of the traditional polydimethylsiloxane (PDMS), meaning
that the devices were highly robust, autoclavable, and reused up to 10
times. Failure points, such as leakage, occurred in the presence of
long-term exposure to humidity, high temperatures, and shear stress.
Yet, the MPS design fosters low-cost manufacturing and minimizes wasted
resources in single-use devices.
Throughout the MPS experiments, comparisons of the single and
tri-culture systems determined the significance of modeling concerted
structures of the nephron. The single culture is illustrative of the
conventional PCT devices (Jang et al.,
2013; Ng et al., 2012;
Raghavan et al., 2014;
C. M. Sakolish et al., 2019;
Weber et al., 2016), but the unique
attributes of the glomerulus and MPS system include three cell types,
selected based on its key functions in filtration, reabsorption, and
secretion. Filtration is operated through the glomerular filtration
barrier mainly controlled by the glomerular and endothelial cells
(Li et al., 2016). Co-culturing these
cells have previously proved challenging through the technical
difficulties of glomerular endothelial cells and podocytes. Since both
cell types are highly differentiated, specialized, and interdependent
cell types (Xinaris et al., 2016;
Xinaris et al., 2012), selection of the
cells in the model is vital in evaluating the MPS. CIHP-1 cells,
representative of the podocytes, were selected based on its
immortalized, homogenous characteristics. To ensure that the MPS
establishes efficacy, homogeneity minimizes outliers and
inconsistencies. Similarly, HUVECs were selected based on prevalent use
in endothelial cell studies (Caulfield &
Farquhar, 1974; Venturoli & Rippe,
2005) and derivation from humans. Reabsorption is mostly contributed by
the proximal tubule cells as denoted by the HK-2 cells, which were
selected based on extensive use previous work
(Jang et al., 2013;
C. M. Sakolish et al., 2016;
C. M. Sakolish & Mahler, 2017;
C. M. Sakolish et al., 2019). From the
comparative results, the single culture and tri-culture were indicative
of the significance of establishing a multi-cellular model in future
kidney research. The differences in velocity flow rate and protein
concentrations validate how the glomerulus and PCT MPS greatly impact
the retention of functional cell-cell and cell-tissue interactions.
To
achieve animal-byproduct free culture conditions, multiple media types
were tested. Based on the results of the viability and proliferation
testing (Figure S1 ), all three cell types were adapted to an
optimal common cell culture medium (ESFM) prior to dynamic
experimentation. For the glomerular functional assay, human serum
albumin was used (FITC-HSA, 67.0 kDa). Cells within the glomerulus and
PCT-MPS demonstrated an in vivo - like functional activity through
the retention of HSA (~67 kDa) from circulating in the
bloodstream pathway, while resorbing concentrations of glucose in
filtrate output. The protein concentrations were depicted in both the
plate reader fluorescence readings, but also in the confocal images,
where there was an uptake of the green fluorescent protein in the
podocytes (Figure S2 ). Previous works
(Jang et al., 2013;
Ng et al., 2012) found that dynamic
conditions results in cytoskeletal reorganization and junctional
reformation in PCT-derived cells. These outcomes were not clearly
observed in the glomerulus and PCT MPS due to the low shear stress to
exhibit healthy renal conditions and longer culture time. While human
serum albumin results in this study do not distinguish between
glomerular filtration and tubular reabsorption, a previous glomerulus
and PCT MPS iteration (C. M. Sakolish &
Mahler, 2017) featured transport with other proteins sizes. The MPS
with only endothelial (HUVECs) and proximal tubule (HK-2) cells was
subjected with 0.1 mg/mL of FITC-bovine serum albumin (FITC-BSA, 66.4
kDa) and FITC-ovalbumin (45.0 kDa). The HUVECs demonstrated more BSA
localization in the junctions of the cell monolayer, whereas the HK-2
had an increase in BSA in vesicles, suggesting a presence of passive and
active transport, respectively. Future works can incorporate different
sized fluorescent compounds (i.e. dextran) to characterize the
specificity of trans-glomerular filtration and tubular reabsorption
(Caulfield & Farquhar, 1974;
Ng et al., 2012;
Venturoli & Rippe, 2005;
Xinaris et al., 2016;
Xinaris et al., 2012). For imaging in
post-dynamic experiments, immunocytochemistry would typically be used to
display functional biomarkers, such as VE-cadherin, clathrin, or nephrin
(Musah et al., 2017;
Petrosyan et al., 2019;
Weber et al., 2016). In this study,
alternate stains were used to avoid the use of animal products (Calcein
AM, Hoescht 3342, and phallodin 568). Largely, the glomerulus- and PCT-
MPS offers an effective tool for assessing preclinical drug adsorption,
distribution, metabolism, elimination, and toxicity (ADMET) testing,
modeling disease for drug treatment regime trials, and understanding
cellular crosstalk.
Additional work is required to ensure confluent endothelial cell
adhesion by day 7 on the PES membrane.
Previous
works have identified Collagen IV and laminin as a more physiological
relevant extracellular matrix in renal studies
(D. R. Abrahamson, 1991;
D. R. Abrahamson, Hudson, Stroganova,
Borza, & John, 2009; John & Abrahamson,
2001; Li et al., 2016;
Mauer, 1994;
Pavenstadt, Kriz, & Kretzler, 2003;
C. Sakolish et al., 2018;
Weber et al., 2016).
Initially, glomerular endothelial
and epithelial cells generate a glomerular basement membrane (GBM), a
cytoskeletal template for renal cell adhesion. Following, fully
differentiated podocytes further assemble matrix molecules into a highly
intricate meshwork of collagen IV, laminin, heparin sulfate, entactin,
agrin, and perlecan (D. Abrahamson, 1987;
D. R. Abrahamson, 1985;
Adler, 1992;
John & Abrahamson, 2001;
Miner, 1998;
Sariola, 1984). Of those extracellular
proteins, type IV collagen and laminin have ensured endothelial
monolayers and better preservation of the podocyte phenotype based on
the F-actin distribution, nephrin, and synaptopodin
(Li et al., 2016). Recreating a kidney
diseased model, such as chronic kidney disease and drug-induced kidney
injuries (DIKI), on a molecular scale is essential in innovating safer
therapeutic agents. Establishing predictive models of DIKI avoid
unnecessary risk for patients and reduce financial burden of compound
attrition in pharmaceutical development. Further investigation of renal
drug candidates, such as cisplatin, gentamicin, perfluorooctanoic acid
(PFOA), and angiotensin-converting enzyme inhibitors (ACEI) would permit
a wider scope into the pharmacokinetics within the glomerular and
proximal tubule MPS.
In Sakolish et. al, 2019, a
similar glomerulus and PCT MPS with only HK-2 cells was implemented to
test altered glomerular filtration rate (GFR), hyperglycemia,
nephrolithiasis, and drug-induced nephrotoxicity. Four conditions were
subjected in the MPS with their respective disease model: (1)
physiological flow of 0.8 dyne-s/cm2 and high shear
flow of 5 dyne-s/cm2, (2) KSFM and glucose-treated
SFM, (3) 0.3mg/ml of calcium oxalate monohydrate crystals (COM), and (4)
1.4mM of cisplatin and 0.27 mM of cyclosporine exposures for 72 hrs at
24 hr intervals. Based on the significant changes in cellular responses
of these single culture MPS compared to the static conditions
(Figure S3 ), this operates as a precursor for the current
glomerulus and PCT tri-culture MPS’s potential application in assessing
DIKI. More functional assays would build confidence in the
physiologically credible characteristics. MPS under fluid-driven flow
would have increased mechanical signaling in the primary cilia,
microvilli, or glycocalyx, increased trafficking and expression of
apical and basolateral transporters, and increased epithelial barrier
function (Koepsell, 2013;
Nieskens et al., 2016;
Nieskens & Wilmer, 2016;
Phillips et al., 2020).