Nephrol Dial Transplant (2015) 30: iv60–iv67
doi: 10.1093/ndt/gfv264
Full Review
New developments concerning the proximal tubule in diabetic
nephropathy: in vitro models and mechanisms
Jennifer Slyne, Craig Slattery, Tara McMorrow and Michael P. Ryan
Renal Disease Research Group, School of Biomolecular and Biomedical Sciences, UCD Conway Institute, University College Dublin,
Belfield, Dublin 4, Ireland
Correspondence and offprint requests to: Michael P. Ryan; E-mail: [email protected]
Keywords: albumin handling, diabetic nephropathy, in vitro
models, proximal tubule, RPTEC/TERT1 cells
A B S T R AC T
The incidence of Type 2 diabetes is increasing rapidly worldwide, and understanding the mechanisms of its complications
including diabetic nephropathy (DN) is important in the discovery of early biomarkers, understanding the causative mechanisms of its complications and identifying therapeutic
targets. DN is characterized by glomerulosclerosis, tubulointerstitial fibrosis and tubular atrophy. The tubular component of
the disease is important in progression of disease. In vitro models are a valuable alternative to animal studies and an effective
way to explore mechanisms of human disease. Several proximal
tubular cell lines have been used in studying mechanisms of
DN. Key extracellular conditions that contribute to damage to
the proximal tubule in DN include hyperglycaemia, proteinuria,
and hypoxia and inflammation. According to current knowledge, these exert their effects through changes in transforming
growth factor beta signalling, the renin–angiotensin system, dysregulation of pathways such as the polyol pathway, hexosamine
pathway and protein kinase C pathway and through formation of
advanced glycation end products. Studies in cell culture models
have been instrumental in the delineation of these processes.
However, all of the existing cell culture models have limitations
including dedifferentiation. To bring research forward along
with technological advances, such as major advances in ‘omics’
methodologies, a more suitable model is necessary. The RPTEC/
TERT1 cell line is a promising alternative to previous proximal
tubular epithelial cell lines due to features that resemble the cell
type in vivo, such as its epithelial characteristics, maintenance of
functional capabilities, glucose handling, expression of the primary cilium and transport activity including albumin. This cell
line will facilitate identification of mechanisms of DN with
potential to identify new therapeutic targets.
© The Author 2015. Published by Oxford University Press
on behalf of ERA-EDTA. All rights reserved.
C L I N I C A L I M P O R TA N C E O F T H E P R O X I M A L
T U B U L E I N D I A B E T I C N E P H R O P AT H Y
It has been estimated that 382 million people worldwide have
diabetes mellitus, a figure that is expected to rise to 592 million
by 2035 [1]. Diabetic nephropathy (DN) is the leading cause of
end-stage renal disease, with up to 40% of Type 1 or Type 2 diabetic patients developing nephropathy. Clinically, DN is characterized by the development of albuminuria along with decline
in glomerular filtration rate (GFR). Features of DN include glomerulosclerosis, tubulointerstitial fibrosis (TIF) and atrophy.
Glomerular lesions that occur in glomerulosclerosis involve
basement membrane thickening, mesangial expansion and matrix formation and formation of Kimmelstiel–Wilson lesions.
A publication by Tervaert et al. [2] has provided a classification
of DN (Class I–IV) based on the degree of severity of these
changes. While much research in DN has focused on the glomerulus, there is increasing evidence for the importance of the
tubules in progression of the disease. In DN, TIF is the final
common pathway and correlates with the progression of disease
better than glomerular damage [3, 4]. Given that the tubulointerstitial compartment represents up to 90% of the parenchymal
mass of the kidney, it is not surprising that it has importance in
the development of the disease. TIF features interstitial matrix
deposition, inflammation, fibroblast activation, microvascular
rarefaction and tubular cell loss [5]. Emerging evidence has provided further support for the tubular hypothesis; a study by
Grgic et al. [6] showed that targeted proximal tubule drives
an inflammatory response that triggers not only TIF and
iv60
tubular atrophy but also potentially glomerulosclerosis. The
clinical importance of the tubules in DN progression is also evident in the discovery of kidney injury biomarkers of proximal
tubular origin, such as kidney injury molecule 1 (KIM-1), neutrophil gelatinase-associated lipocalin and N-acetyl-β-D-glucosaminidase (NAG) [7, 8]. Regression of microalbuminuria in
Type 1 diabetes mellitus was found to be associated with
lower levels of KIM-1 and NAG [9].
Our knowledge of DN comes from a range of investigations
including clinical and epidemiological studies, animal models
and cell culture studies. While animal models have provided
useful insights, there is a problem in that the present in vivo animal models only reproduce some of the histological features of
human DN [10]. This may be due, in part, to species variation.
IN VITRO MODELS
Table 1. Number of studies related to DN in different proximal tubular
epithelial primary and immortalized cell lines
Cell line
Species
Method of cell line
immortalization
Number of
publications
Primary PTECs
Not immortalized
42
HK-2 (human)
HKC8 (human)
LLC-PK1
Human and
other species
Human
Human
Pig
68
1
34
OK
Opossum
NRK-52E
Rat
IRPTC
MCT cells
Rat
Mouse
HPV-16 transformed
SV40 transformed
Spontaneously
immortalized
Spontaneously
immortalized
Spontaneously
immortalized
SV40 transformed
SV40 transformed
8
29
3
3
TGF-β and TIF
TGF-β has long been known as a profibrotic mediator.
TGF-β stimulates matrix protein production while decreasing
activity of enzymes that degrade ECM, leading to deposition
of ECM proteins such as collagens I and IV and fibronectin,
thus promoting fibrosis in PTECs. Activation of the TGF-β receptor leads to phosphorylation of Smad transcription factors,
which results in gene regulation associated with several processes including growth arrest, cell differentiation and epithelial–mesenchymal transition (EMT) [14]. The phases of TIF
have been described very clearly by Eddy [15], comprising
(i) activation of tubular, perivascular and mononuclear cells and
release of proinflammatory molecules; (ii) production of fibrosispromoting factors [e.g. TGF-β, connective tissue growth factor
(CTGF), Ang II and platelet-derived growth factor (PDGF)];
(iii) ECM production is increased and matrix degradation is decreased and (iv) decline in the number of intact nephrons.
Epithelial–mesenchymal transition
During development of TIF, matrix producing fibroblasts
are a source of ECM proteins in fibrosis, and there is evidence
that a proportion of them originate from EMT [16]. While the
precise contribution of EMT to renal fibrosis is somewhat controversial, some of the conflicting evidence on the role of EMT,
as recently reviewed, may be related to the particular strain of
animal and type of disease model used in studies [17]. Cell culture studies have been useful in exploring mechanisms of EMT.
EMT is characterized by loss of epithelial cell adhesion molecules [including E-cadherin and zona occludens protein 1
(ZO-1)], de novo alpha-smooth muscle actin (α-SMA) expression along with actin reorganization, tubular basement membrane disruption and finally enhanced cell migration [18].
Early studies from our laboratory demonstrated that an
immune-mediated model involving activated leukocytes could
result in EMT in HK-2 cells [19]. Follow-up studies using a
proteomic approach identified additional protein changes
in the tubular cells under these conditions [20]. HG has been
demonstrated in vitro to induce EMT in HK-2 cells via p38
mitogen-activated protein kinase (MAPK) signalling and activator protein 1 activation, as well as reactive oxygen species
(ROS), phosphoinositide 3-kinase/Akt, glycogen synthase
kinase 3 beta, Snail and β-catenin [21, 22]. Similarly, in primary
Diabetic nephropathy: in vitro models and mechanisms
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FULL REVIEW
Although cell culture systems are reductive and lack the complexity of the intact kidney system, they facilitate the exploration
of possible intracellular pathways and signalling processes when
cells are challenged with conditions mimicking the clinical situation. Furthermore, identification of the mechanisms and pathways provides an understanding which may lead to novel
therapeutic strategies. Cell culture studies also aid in identification of potential biomarkers which are linked to the mechanisms involved.
We have reviewed the literature on studies that specifically
used in vitro renal proximal tubular cell models to explore
DN. A PubMed search yielded over 100 studies with many
proximal tubular epithelial cell lines (PTECs), both primary
and immortalized, from different species. The numbers of publications, species and details of cell line establishment are listed
in Table 1. It should be stressed that the search was for studies
that specifically addressed DN. There are multiple studies
on other aspects of renal proximal tubular cells. Exposure
of PTECs to pathophysiological mediators mimicking DN
including high glucose (HG), elevated transforming growth
factor beta (TGF-β), glycated proteins and angiotensin II
(Ang II), proteinuria and hypoxia have been commonly used
in in vitro modelling of DN.
Hyperglycaemia
Hyperglycaemia is a major contributor to damage to PTECs
in DN. High glucose content in the PTECs results from both
increased glucose in plasma and also from excess glucose filtered by the glomeruli, resulting in an excessive glucose load
in the tubule. Early in vitro modelling of DN in PTECs focused
on culturing of PTECs in HG conditions. Murine PTECs cultured in HG resulted in cellular hypertrophy and increased collagen transcription, and these effects are caused by stimulation of
TGF-β expression [11, 12]. In human PTECs, exposure to HG
led to accumulation of extracellular matrix (ECM) proteins collagen and fibronectin; this was associated with decreased degradation of ECM proteins, through upregulation of tissue inhibitors
of metalloproteinase 1 and 2 (TIMP1 and TIMP2) [13].
FULL REVIEW
cultured PTECs, HG induced EMT, and this effect was reversed
by the peroxisome proliferator-activated receptor gamma
(PPARγ) agonist troglitazone [22].
Angiotensin II
Another mediator of hyperglycaemia-mediated damage in
DN is Ang II. In early studies in mouse PTECs, HG-mediated
cellular hypertrophy was shown to be enhanced when also
exposed to Ang II; this hypertrophy occurs via TGF-β activation [23, 24]. Ang II caused an increase in Type IV collagen
in murine proximal tubular (MCT) cells, mediated by TGF-β
[25]. Ang II stimulates Type I angiotensin (ATR1) receptors, activating nuclear factor kappa-light-chain-enhancer of activated
B cells (NF-κB) signalling; AT1 receptor blockers (ARBs) are a
treatment in use for DN, which work partly by inhibition of
NF-κB. In fact, studies in primary RPTECs show that glucose
treatment significantly increased Ang II concentrations in cell
lysates and that ARB treatment significantly reduced this effect
[26, 27]. An increase in oxidative stress is evident in DN. A study
done in MCT cells demonstrated that exposure to Ang II promotes vascular endothelial growth factor (VEGF) translation
and that this was mediated by ROS [28]. Ang II is also a key mediator in EMT in DN by signalling through ATR1 receptors,
which are also mediators in HG-induced EMT [29, 30]. In immortalised rat proximal tubular cells (IRPTC) cells, it was
shown that Ang II works through Janus kinase/signal transducers and activators of transcription signalling in tubular cell
proliferation and regeneration following injury [31]. It has
been recently suggested that miR-29b plays a role in Ang
II-induced EMT in NRK-52E cells; studies that inhibited the
microRNA (miRNA) saw an increase in TGF-β signalling and
increased expression of α-SMA and collagen I [32].
Proteinuria
Proteinuria is usually considered one of the earliest clinical
indicators of renal damage although some studies have shown
that renal impairment in diabetes may occur without prior
proteinuria in up to 51% of patients, lending to the concept
that albuminuria and reduced GFR have different underlying
pathologies [33]. In fact, recent studies have found tubular proteinuria to be a predictor of progression of DN [34]. It was demonstrated in a diabetic rat model that the proximal tubule
plays an important role in the development of proteinuria [35].
Proteinuria has long been viewed to result from glomerular damage and leakage of excess albumin into the tubules. An important
feature of PTECs in vivo is the functionality of the megalin–
cubilin protein reabsorption pathway. While the glomerular
filtration barrier normally prevents a high proportion of serum
proteins from filtering into Bowman’s space, small but significant
quantities of serum proteins ( principally serum albumin) do
reach the tubular lumen and must be reabsorbed by tubular epithelial cells to prevent loss in the urine [36]. There is significant
evidence that disruption of tubular albumin reabsorption is an
important event during DN development causing PTEC dysfunction and proteinuria [37]. This pathway has been elucidated
and involves the megalin–cubilin receptor complex, clathrinmediated endocytosis and lysosomal degradation [38, 39]. Further
molecular characterization of this pathway, and investigation of
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functionality under normal and pathophysiological conditions,
has been problematic due to lack of reflective in vitro model systems. The vast majority of in vitro work to date has been carried
out in non-human PTEC lines such as the OK cell line and
LLC-PK1 cells [40–42]. More recently, a number of studies
have been performed in HK-2 cells; however, Slattery et al. [43]
demonstrated that full establishment of the megalin–cubilin albumin reabsorption pathway required highly specific culture conditions, and that under such conditions, other desirable aspects of
the HK-2 model were detrimentally affected leading to a compromised epithelial phenotype and limiting the relevance to in vivo
situations. Filtered proteins that may cause toxic effects on the tubule include albumin, immunoglobulin G, lipoproteins, transferrin and complement proteins. There is evidence in cultured
PTECs that exposure to high levels of albumin causes activation
of PTECs, resulting in synthesis of chemokines and inflammatory
molecules including interleukin 1β (IL-1β), interleukin 6 (IL-6),
interleukin 8 (IL-8), chemokine (C–C motif) ligand 2 and chemokine (C–C motif) ligand 5 [22, 44, 45]. This effect can be further fuelled by components of the complement system, such as
complement component 3 (C3), complement component 3a
(C3a), complement component 5a (C5a) and complement regulatory protein (Crry), which have chemotactic properties, causing
an additional inflammatory response [46]. Studies have recently
shown that albumin overload on cultured PTECs results in EMT
as demonstrated by loss of E-cadherin along with overexpression
of α-SMA, fibronectin and collagen IV [47].
Advanced glycation end products
Among the many changes that occur in DN as a result of
hyperglycaemia is formation of advanced glycation end products
(AGEs), which are formed through non-enzymatic glycation of
proteins, lipids and nucleic acids. AGEs including glycated albumin (GA) and glycosylated haemogloblin (HbA1c) have been
proved as reliable diagnostic markers in diabetic patients [48].
The proximal tubule is an important site of action of AGEs as
it is the primary site for reabsorption of filtered AGEs. One of
the ways that AGEs contribute to fibrosis in DN is through
modulation of ECM proteins. PTECs stimulated with AGEmodified bovine serum albumin displayed increased metalloproteinase 2 (MMP2) and ROS. This effect was reversed by
treatment with ramiprilat, an active metabolite of the ACE inhibitor ramipril [49]. AGEs cause activation and increased expression
of a number of disease mediators that have been implicated in
DN including NF-κB and protein kinase C (PKC). For example,
exposure of cultured PTECs to AGE-albumin resulted in activation of NF-κB, which was associated with release of IL-6 into the
supernatant [50]. AGEs also contribute to release of proinflammatory cytokines, oxidative stress and expression of growth factors and adhesion molecules that have been implicated in DN as
well as activation of signalling proteins such as Src, Akt and
ERK1/2 [51]. A study in cultured PTECs demonstrated increased
generation of intracellular ROS and upregulation of TGF-β after
exposure to AGE-albumin [52]. Tang et al. [53] demonstrated
that proximal tubular cells exposed to GA had increased expression of IL-8 and intercellular adhesion molecule 1 while affecting
NF-κB and MAPK signalling. Other proinflammatory molecules
that are induced by AGEs include VEGF, CTGF, insulin-like
J. Slyne et al.
growth factor 1, PDGF, tumour necrosis factor alpha (TNF-α)
and IL-1β [54].
Hypoxia
Hypoxia is another described mechanism of damage, representing an early event in the progression of DN. Hypoxia may
occur in the proximal tubule in response to (i) enhanced tubular oxygen consumption and (ii) intrarenal vasoconstriction
due to activation of the renin–angiotensin system or through
structural impairment of blood flow secondary to interstitial
fibrosis. Mechanisms through which hypoxia causes tubular
damage include oxidative stress, inflammation and matrix production. Early studies by Orphanides et al. [55] demonstrated
that exposure of PTECs to hypoxia stimulates ECM accumulation (increased collagen production, decreased MMP2 activity
and increased TIMP1 expression). Hypoxia induces plasminogen activator inhibitor-1 (PAI-1) via nuclear accumulation of
hypoxia inducible factor 1, alpha subunit (HIF-1α) and NF-κB
activation and this effect is synergistically enhanced by TNF-α,
suggesting the synergistic relationship between inflammation
and hypoxia in tubular damage in DN [56]. HIF-1α, which mediates the renal response to hypoxia, is regulated by propyl hydroxylase domain-containing protein 1 [57]. More recently, studies
have shown that hypoxia induces apoptosis and enhances production of TNF-α and IL-8 and that this effect is dependent
on toll-like receptor 4 [58].
HORIZON SCANNING AND FUTURE
PERSPECTIVES
MicroRNA
In recent years, miRNA research is emerging as a major area
of investigation in DN, as well as several other conditions. miRNAs are small non-coding RNAs that regulate gene expression
through inhibiting mRNA translation. In the last years, several
miRNAs have been linked with processes involved in the pathogenesis of DN. For example, microRNA-29 (miR-29) expression is reduced by TGF-β1 treatment in NRK-52E cells.
Ectopic expression of miR-29 represses expression of collagens
I, III and IV and increased E-cadherin expression [65].
miR-200a was found to be suppressed by TGF-β1 and TGFβ2 in NRK-52E cells. Expression of miR-200a downregulated
smad-3 and expression of several matrix proteins including collagens I and IV, and fibronectin reduced expression of α-SMA,
while increasing expression of E-cadherin, zinc finger E-boxbinding homeobox 1 and 2 (ZEB1 and ZEB2) [66]. Therefore,
miR-29 and miR-200a are downstream mediators of TGF-β1
signalling in the process of fibrosis. miR-29 and miR-200a are
among many miRNAs that have been implicated in DN, and
this emerging field of research is promising to identify many
more miRNAs related to DN in future years, with cell culture
models providing a suitable platform to elucidate mechanisms
of their pathways.
Issues with traditional proximal tubular cell
culture models
Although the studies reviewed above on in vitro models of
proximal tubular injury have contributed significantly to the
understanding of mechanisms of tubular damage in DN, a
few points of caution must be noted. First, some of these cell
lines originate from other species, including mouse, rat and
opossum, and it is uncertain how data obtained in another species translate to human disease. Ideally, a cell culture model is of
human origin to limit these issues. Second, primary cells,
though they pose the advantage that they are unaltered biochemically and their cellular structure is similar to their state
in vivo, have been shown to dedifferentiate within a short period
of time [67]. Immortalized cells lines, derived by genetic modification of primary cells, are beneficial in that they can be cultured for some time. However, many of these cell lines will have
lost the characteristics of their parental cell strains, in part due
to the methods used to immortalize the cell line. Differences
that may occur as a result of immortalization include changes
in expression of intracellular proteins, changes in cellular
morphology (such as lack of tight junctions and microvilli, cellular receptors, transporters or ligands), loss of cell polarity or
loss of contact inhibition [67]. Human cell lines that relied on
viral oncogenes include HK-2 cells immortalized using the viral
oncogene human papillomavirus 16 (HPV-16) E6/E7, while
HKC cells were immortalized using a hybrid adeno-12-Simian
virus 40 (SV40) virus [68, 69].
Diabetic nephropathy: in vitro models and mechanisms
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FULL REVIEW
Other pathways
Several other pathways have been implicated in the pathogenesis of DN including the polyol pathway, hexosamine pathway
and PKC pathway, which may work independently or together
in the progression of disease. During hyperglycaemia, increased
flux through the polyol pathway occurs. The polyol pathway reduces glucose to polyalcohol sorbitol by aldose reductase (AR).
Sorbitol is oxidized to fructose by sorbitol dehydrogenase. AR
uses nicotinamide adenine dinucleotide phosphate (NADPH)
as a cofactor, so during hyperglycaemia when more glucose is
channelled through the pathway, NADPH is depleted, resulting
in oxidative stress and possibly causing ECM protein changes.
For example, one study in LLC-PK1 cells demonstrated that fibronectin generation in response to HG is mediated by the polyol
pathway [59]. The hexosamine pathway, a minor branch of glycolysis, converts fructose-6-phosphate into glucosamine-6phosphate. In IRPTC cells, HG caused cellular hypertrophy
and increased angiotensinogen gene expression and this was
partly via activation of the hexosamine pathway [60]. Other studies have shown that flux of glucose through the hexosamine pathway mediates ECM production via stimulation of TGF-β [61].
PKC signalling is central to many mechanisms of pathogenesis
in DN. PKC is activated by DAG that is formed from excess
glyceraldehyde-3-phosphate in hyperglycaemia, and also by increased glucose flux through the polyol pathway [62]. PKC activation has been implicated in increased ECM expression, at some
time points through TGF-β overexpression. PKC-induced overexpression of PAI-1 and activation of NF-κB signalling also contribute to the inflammatory response and fibrosis [63]. PKC
activation interacts with several other pathways in exerting its effects including (ERK)1/2, p38 MAPK and NADPH oxidase [64].
A summary of the mechanisms investigated in the cell culture
models is provided in Figures 1 and 2.
F I G U R E 1 : A diagrammatic overview of the involvement of the renal proximal tubule in DN indicating the role of altered glomerular filtrate and
FULL REVIEW
disrupted tubular epithelial cell function.
F I G U R E 2 : Illustration of possible pathways and interactions in mediating tubular dysfunction in DN.
Novel human renal proximal tubular
cell—RPTEC/TERT1
An alternative method to oncogene insertion is the introduction of the catalytic subunit of human telomerase (hTERT),
which was used in the development of the RPTEC/TERT1 cell
iv64
line from primary human cells [70]. This cell line is a highly differentiated cell line, which maintains many characteristics of
primary cells and the in vivo human proximal tubule. Unpublished work from our laboratory had shown normal chromosomal number and nuclear stability.
J. Slyne et al.
Table 2. Advantages of RPTEC/TERT1 cells in the investigation of DN
Advantage/characteristic
Reference
1. Human renal proximal tubular origin
2. No oncogene transformation, immortalized with
expression of catalytic subunit of hTERT
3. Maintained in defined medium with no serum and low
glucose which enhances suitability for mimicking diabetic
conditions in vitro
4. Maintains proximal tubular characteristics including
parathyroid hormone receptors and transporters such as
OCT2, OCT3, OCTN2, MATE1, MATE2, OAT1,
OAT3 and OAT4
5. Maintains proximal tubular transport capabilities which
can be measured including TEER
6. Expresses primary cilia of relevance to proximal tubular
function which can also be used to investigate dysfunction
in vitro
7. Expresses an intact megalin–cubilin transport system
providing a model to explore proximal tubular transport
of albumin in vitro
8. Maintains a high level on betaine in vitro consistent with
high abundance in renal tissue in vivo
9. Metabolic and genomic stability when fully differentiated
in culture enhancing suitability for metabolomic,
proteomic and transcriptomic investigations in vitro
[70]
[70]
[70, 71, 74,]
[74]
[70]
[75]
[70]
[71]
[71, 75–77]
OAT3 and OAT4 and multidrug and toxin extrusion proteins
1 and 2 (MATE1 and MATE2) [74].
Another useful feature of RPTEC/TERT1 cells, relevant to the
in vivo kidney, is expression of primary cilia [70]. The primary
cilium plays roles in chemo- and mechanosensation, signal transduction, phototransduction and developmental patterning. In
the kidney, the primary cilium extends into the lumen of the
renal tubule and is proposed to play a major sensory role. Recent
studies from our laboratory have suggested that the primary cilium can be used as a measure of normal function and effector of
chemical toxicity. A study by Radford et al. [75] demonstrated
that the carcinogens ochratoxin A and potassium bromate induce loss of the primary cilium in RPTEC/TERT1 cells. The
cilia in RPTEC/TERT1 cells may also be useful for biomarker investigation as some biomarkers such as KIM-1 originate from the
cilia. Other work in our laboratory, unpublished to date, indicate
that the RPTEC/TERT1 cells respond to conditions mimicking
diabetes such as HG and AGE-albumin exposure with altered
cellular signalling and functional responses.
SUMMARY
Cell culture models are increasingly used in the search for biomarkers. The RPTEC/TERT1 cell line is useful to study biomarkers for several reasons. These cells have a highly differentiated
phenotype with similarities to proximal tubular cells in vivo.
This cell type therefore has the potential to be used alongside
clinical studies in DN research. The cell line has been shown
to display a stable phenotype when differentiated and shows
only modest changes in the transcriptome over time [72].
The use of this cell model is suitable for molecular screening
using ‘omics’ methodology. The cell line has been used successfully in integrated use of transcriptomics, metabolomics and
Diabetic nephropathy: in vitro models and mechanisms
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FULL REVIEW
RPTEC/TERT1 cells maintain the same epithelial characteristics as proximal tubular cells in vivo. The cells maintain an intact barrier and cell polarization as evidenced by the formation
of characteristic domes. These features are required for vectorial
transport of water and solutes, in keeping with the functions of
proximal tubular cells. The cell line also expresses biochemical
markers of RPTECs including aminopeptidase N and gammaglutamyl transpeptidase, enzymes located in the brush border.
Tight junction formation is another key feature of RPTEC/
TERT1 cells; E-cadherin, ZO-1 and occludin are localized around
each individual cell. Transepithelial electrical resistance (TEER)
is used to measure barrier function; RPTEC/TERT1 cells establish a stable TEER after 10–15 days, confirming their ability to
form an intact functional barrier [70, 71].
There are certain metabolic features of RPTEC/TERT1 cells
that make it particularly beneficial for studying DN. When
RPTEC/TERT1 cells have differentiated in culture, a metabolic
switch occurs. Lactate production decreases while glucose
consumption decreases, with both remaining stable from Day
4/5 of culture. This alteration in glycolysis was confirmed by
Aschauer et al. [72], as demonstrated by altered expression in
genes involved in the process. This means that the amount of
glucose needed to maintain the cells in culture is low in marked
contrast to other cells lines. This is favourable for a DN model
where cells are exposed to HG as the effect is more likely to reflect the in vivo situation. In a detailed analysis of the metabonome of RPTEC/TERT1 cells, we confirmed the low glucose
requirement for the cells. We also demonstrated that these
cells, in contrast to other existing renal cell lines, had high levels
of betaine with myo-inositol and glycerophosphocholine that
are typical of renal tissue [71]. Another key advantage of
RPTEC/TERT1 cells is that they can be maintained in a defined
medium without the need for addition of serum and therefore
avoiding effects of unknown factors.
Proximal tubular transport activity is also retained in
RPTEC/TERT1 cells. These cells express the SLC34A3 sodiumdependent phosphate transporter that was shown to be functioning properly, along with the megalin–cubilin transporter
system, important in processing of albumin. The megalin–
cubilin transport system has been demonstrated to work well
using fluorescence-labelled aprotinin, a ligand for megalin
[70]. Another major ligand of megalin and cubilin receptors
is albumin, an important marker of renal injury and mediator
of proximal tubular damage as discussed above. Reabsorption
of albumin from the glomerular fitrate is known to occur via
receptor-mediated endocytosis in the proximal tubule. This
process begins when albumin binds in apical clathrin-coated
pits, when endocytosis occurs and lysosomal degradation ensues. Studies have shown that megalin and cubilin are important endocytotic receptors in this process [73]. The finding that
megalin–cubilin transport is intact in the RPTEC/TERT1 cell
line suggests that it would be an excellent model for albumin
handling studies relating to kidney disease. Studies in our laboratory indicate that RPTEC/TERT1 cells have an efficient albumin uptake system and GA can interact with the system.
Other transport systems that are expressed in the cell line include the organic cation transporters (OCTs) OCT2, OCT3
and OCTN2; the organic anion transporters (OATs) OAT1,
proteomics to investigate mechanisms of drug toxicity in recent
years [76, 77]. Recently, transcriptomic profiling was performed
in a model of renal injury in RPTEC/TERT1 cells to identify
markers of renal damage. Interleukin 19 (IL-19) was identified
as the most dysregulated gene and this correlated with renal
function in chronic kidney disease urine samples [78]. This
cell line may be useful, using these methodologies, in the prediction of biomarkers of several other renal diseases, including
DN. The characteristics of RPTEC/TERT1 cells which make
them suitable for investigation of DN are summarized in
Table 2.
AC K N O W L E D G E M E N T
This study was funded by the EU Seventh Framework grant
‘Systems biology towards novel chronic kidney disease diagnosis
and treatment—SysKid’.
C O N F L I C T O F I N T E R E S T S TAT E M E N T
FULL REVIEW
None declared.
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