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International Journal of Nephrology
Volume 2011, Article ID 957164, 9 pages
1Department of Applied Molecular Medicine, Niigata University Graduate School of Medical and Dental Sciences,
1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan
2Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical and Dental Sciences,
1-757 Asahimachi-dori, Chuo-ku, Niigata 951-8510, Japan
Correspondence should be addressed to Akihiko Saito, firstname.lastname@example.org
Received 6 September 2010; Accepted 5 November 2010
Academic Editor: Mitchell H. Rosner
Copyright © 2011 Akihiko Saito et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
microalbuminuric stages with normal glomerular filtration rates. Proximal tubule cells (PTCs) mediate metabolism and urinary
substances from circulation or synthesize them for release into the circulation. PTCs are also involved in the uptake of sodium and
phosphate, which are critical for hemodynamic regulation and maintaining the mineral balance, respectively. Dysregulation of
PTC functions in CKD is likely to be associated with the development of CVD and is linked to the progression to end-stage renal
disease. In particular, PTC dysfunction occurs early in diabetic nephropathy, a leading cause of CKD. It is therefore important to
elucidate the mechanisms of PTC dysfunction to develop therapeutic strategies for treating cardiorenal syndrome in diabetes.
Chronic kidney disease (CKD) is a worldwide public health
problem, and the incidence of end-stage renal disease
(ESRD) with poor outcomes and associated high costs is
increasing. Patients with CKD are also at high risk of
developing cardiovascular disease (CVD). It is therefore
important to elucidate the pathogenesis of CKD and the
mechanisms underlying its role in the development of CVD.
Albuminuria/proteinuria is a distinctive clinical sign
in patients with CKD. Although a decrease in glomerular
filtration rate (GFR) correlates with an increase in incidence
of CVD, patients showing normal GFR with even mild
albuminuria/proteinuria are also at risk of developing CVD
[1, 2]. The link between albuminuria/proteinuria and CVD
has generally been attributed to vascular endothelial injury
endothelial injury may not only be a cause of CKD,
but also a consequence of the disease. In addition, the
vascular pathology of CKD is characterized by medial layer
calcification that may be mediated by calcium-phosphate
dysregulation . Therefore, to clarify the mechanisms of
CVD in patients with CKD, it is important to investigate the
renal factors that cause albuminuria/proteinuria and those
that are involved in the induction of vascular endothelial
injury and calcification.
The aim of this paper is to hypothesize and verify on
the basis of the available evidence that proximal tubule cell
(PTC) dysfunction explains well the link between the devel-
opment of albuminuria/proteinuria and cardiovascular risk,
especially in diabetic nephropathy which is a leading cause of
CKD and is highly associated with the development of CVD.
2.OverallFunctions of PTCs
The various functions of PTCs include (1) reabsorption
such as proteins, peptides, glucose, amino acids, uric acid,
sodium, potassium, phosphate, and water via apical mem-
brane receptors, transporters, and channels; (2) uptake of
membrane transporters followed by metabolism or secretion
2International Journal of Nephrology
Figure 1: Normal functions of proximal tubule cells (PTCs) and structural changes around the cells in the early stages of diabetic
nephropathy. Normal functions of PTCs include (1) reabsorption and intracellular processing of glomerular-filtered substances via apical
membrane receptors, transporters, and channels; (2) uptake of substances via basolateral membrane transporters followed by metabolism or
in diabetic nephropathy even at the early stages in which PTCs are hypertrophied with increased metabolic demands and are phenotypically
altered. In addition, tubular basement membranes (TBMs) are thickened, and interstitial spaces are expanded with fibrosis, alienating PTCs
from interacting with peritubular capillaries.
to the urinary space; (3) synthesis of substances that are
of these diverse functions is likely to affect systemic hemo-
dynamic and metabolic homeostasis and may mediate the
development of CVD as discussed below.
3.Dysfunctionof PTCs in
In the early stages of diabetic nephropathy, PTCs are
hypertrophied because of increased metabolic demands and
phenotypically changed to express cytokines or chemokines
. Tubular basement membranes are thickened and inter-
stitial spaces are expanded with fibrosis, isolating the PTCs
structural changes and increased metabolic demands on
PTCs are likely to cause ischemia in the cells. At more
advanced stages, interstitial fibrosis is increased, peritubular
capillaries become dispersed, and PTCs undergo atrophy,
which further diminishes interaction between the cells and
surrounding capillaries. Similar phenotypic changes of PTCs
are also observed in patients with obesity or metabolic
syndrome. In other glomerular diseases, tubulointerstitial
damage also follows as a final common pathway for progres-
sion to ESRD .
4.Megalinand Cubilin:Two Endocytic
Receptors inApicalPTC Membranes
Glomerular-filtered substances are reabsorbed by megalin
and cubilin, two endocytic receptors expressed in apical
PTC membranes (Figure 2). Megalin is a large (∼600kDa)
glycoprotein member of the low-density lipoprotein receptor
family [6, 7] that is primarily expressed in clathrin-coated
pits . Megalin-ligand complexes are internalized by
invagination of clathrin-coated pits mediated by multiple
adaptor proteins and motor molecules, forming endosomal
vesicles. Acidification of the intravesicular lumen dissociates
the ligands from megalin, and they are transported to
lysosomes for degradation or storage, or secreted into the
cytosol for further processing or transport. Megalin is
recycled to the apical membranes through a recycling com-
partment. Megalin thus plays a critical role in reabsorption
and metabolism of glomerular-filtered substances including
albumin and low molecular weight proteins. Megalin
knockout mice display low molecular weight proteinuria and
and facio-oculo-acoustico-renal syndromes, caused by
tion of albumin and low molecular weight proteins .
Cubilin is a 460kDa peripheral glycoprotein that lacks
transmembrane and intracellular segments but is anchored
to apical membranes in PTCs. It was originally identified
as the receptor for intrinsic factor-vitamin B12 complex
[11, 12]. Cubilin gene defects are the cause of hereditary
megaloblastic anaemia 1 or Imerslund-Gr¨ asbeck syndrome,
known as selective vitamin B12malabsorption with protein-
protein ligands present in glomerular filtrates, including
albumin, transferrin, and vitamin D-binding protein (DBP)
. Cubilin requires interaction with megalin to regulate
its endocytic functions [14, 15]; however, it is bound more
firmly by a protein called amnionless, forming a complex
named CUBAM [16, 17] (Figure 2). Amnionless, a 38–
50kDa membrane protein with a single-transmembrane
domain, was initially identified as a component required for
normal development of the trunk mesoderm derived from
the middle streak . In addition, defects of the amnionless
gene cause hereditary megaloblastic anaemia .
Decreased megalin expression in PTCs has been found in
the early diabetic stages of experimental animals [20, 21].
International Journal of Nephrology3
Figure 2: Endocytic receptors and transporters involved in the uptake of substances at the apical membranes of proximal tubule cells
(PTCs). At apical membranes of PTCs, megalin and the cubilin-amnionless complex are involved in endcytosis of protein ligands. Megalin
facilitates uptake of various ligands including vitamin D/vitamin D-binding protein (DBP), vitamin B12/transcobalamin (TC), folate/folate-
binding protein (FBP) complexes, and selenoprotein P. Similarly, cubilin facilitates uptake of the vitamin D/DBP complex. Type IIa
Na/Pi cotransporter (NaPi-IIa) and Na+/H+exchanger isoform 3 (NHE3) are primarily involved in the uptake of phosphate and sodium,
respectively. Homocysteine (Hcy) and asymmetric dimethylarginine (ADMA) may be taken up by cationic amino acid transporters (CATs)
and metabolized in PTCs. Dysregulation of the uptake or metabolism of these substances in PTCs in patients with CKD, especially with
diabetic nephropathy, is likely to be involved in the mechanism that promotes the development of CVD.
It has also been suggested that the functions of megalin
are impaired in patients during the early stages of diabetic
nephropathy, since low molecular weight proteinuria is
frequently observed in patients at these stages [22, 23].
Therefore, the altered regulation of megalin expression and
its functions must be responsible for the early development
of proteinuria/albuminuria in diabetic patients. The func-
tions of cubilin, a direct receptor for albumin, may also
be impaired in the early stages of diabetic nephropathy as
urinary excretion of transferrin, another endocytic ligand of
. The functions of both megalin and cubilin are likely
to be further affected as tubulointerstitial injury in CKD
Cellular expression of megalin was found to be downreg-
in cultured PTCs is upregulated following treatment with
insulin or high-concentration glucose. Conversely, it is
downregulated by angiotensin II . Furthermore, we
demonstrated that there is competitive crosstalk between
angiotensin II type 1 receptor- and insulin-mediated sig-
naling pathways in the regulation of megalin expression
in the cells . Anigotensin II may be a major factor in
suppressing megalin expression in the early stages of diabetic
nephropathy since intrarenal RAS is activated in the disease
Decreased expression or functioning of megalin and/or
cubilin results in reduced reabsorption of their glomerular-
filtered ligands. Impaired reabsorption of some ligands of
these receptors may be associated with the development of
CVD, as described next.
Ligands That May Promote Development of
CVD When Depleted
6.1. Vitamin D. Megalin and cubilin take up the 25(OH)D3/
DBP complex from glomerular filtrates [27, 28] (Figure 2).
In PTCs, 25(OH)D3 is dissociated from DBP and con-
verted by 1α-hydroxylase to 1,25(OH)2D3, a biologically
active form, which is released to the peritubular capillaries.
Therefore, dysfunction of these endocytic receptors is an
important cause of deficiency of both 25(OH)D3 and
1,25(OH)2D3 in CKD in addition to other factors such
as decreased 1α-hydroxylase activity. Vitamin D deficiency
develops very early in the course of CKD, especially in
diabetic nephropathy, and is associated with the develop-
ment of CVD or mortality in patients at predialysis stages
[29, 30]. Treatment with the activated vitamin D analogue
calcitriol was significantly associated with improved survival
of patients with CKD [31, 32]. In addition, vitamin D
deficiency may also be associated with an increased risk of
CVD in the general population , although the effects of
vitamin D supplementation on the CVD-related mortality in
the population remain controversial.
Many studies have investigated vitamin D deficiency-
associated mechanisms of vascular calcification and cardiac
dysfunction. Vitamin D acts on vascular smooth muscle cells
4 International Journal of Nephrology
Figure 3: Intracellular synthesis and metabolism of homocysteine (Hcy) and asymmetric dimethylarginine (ADMA) and their biochemical
link. Vitamin B12serves as a cofactor for the formation of methionine (Met) from homocysteine (Hcy) by methionine synthase using 5-
and serves as the methyl donor to form S-adenosylhomocysteine (AdoHcy). Hcy is either remethylated to Met or transsulfurated to cysteine.
Asymmetric dimethylarginine (ADMA) is an endogenous competitive inhibitor of endothelial nitric oxide synthase (eNOS). ADMA is
formed by methylation of arginine residues in proteins with protein methyltransferase (PRMT) and released after proteolysis. Metabolism of
ADMA is mediated by dimethylarginine dimethylaminohydrolases (DDAHs), which are downregulated by reactive oxygen species and Hcy.
to inhibit activators of vascular calcification, such as core
binding factor-1 (Cbfa1), bone morphogenic protein-2, type
I collagen, interleukin-1b, interleukin-6, and transforming
growth factor-ß, and to stimulate inhibitors of vascular
calcification, such as matrix Gla protein and osteopontin
. Furthermore, decreased vitamin D-receptor activity
increases circulating renin levels and blood pressure which
results in left ventricular and myocyte hypertrophy .
6.2. Vitamin B12 and Folate. Vitamin B12 is a cofactor
involved in the formation of methionine (Met) from homo-
cysteine (Hcy) by cytoplasmic methionine synthase using
5-methyl-tetrahydrofolate (5-MTHF), the dominant folate
form in serum, as a one-carbon donor  (Figure 3).
Therefore, vitamin B12and/or folate deficiency results in the
accumulation of Hcy that is associated with the development
of CVD. Following absorption from the intestine with
intrinsic factor, vitamin B12 is bound in the serum with
transcobalamin, a 45kDa serum protein, for transport to
target tissues. The transcobalamin-vitamin B12 complex is
(Figure 2), which explains why vitamin B12deficiency can be
induced by decreased megalin function.
Folate binds to a carrier protein termed folate-binding
protein and also to other proteins including albumin.
Alternatively, it exists in free form in serum. After being
filtered by glomeruli, protein-bound folate is reabsorbed by
PTCs through megalin-mediated endocytosis while the free
form is likely taken up by folate receptors  (Figure 2).
Dysfunction of PTCs therefore results in decreased renal
retrieval of folate, which subsequently leads to its deficiency.
6.3. Selenoprotein P. Megalin is also involved in the reab-
sorption of selenoprotein P, a selenium-carrier protein from
glomerular filtrates [38, 39] (Figure 2). Selenium is released
from selenoprotein P and used in PTCs to synthesize
enzyme . GPx3 is involved in maintaining the vascular
bioavailability of nitric oxide, a major vasorelaxant, as well as
inhibiting platelet function . Therefore, reduced uptake
may result in decreased GPx3 synthesis which may be asso-
ciated with the development of vascular diseases. Notably, a
recent proteome analysis revealed that serum GPx3 levels are
significantly decreased in patients at the microalbuminuric
stage of type 2 diabetes and even further at the progressive
stages . In fact, familial GPx3 deficiency has been
Also, there have been reports that demonstrate decreased
GPx 3 activity among patients with coronary artery disease,
supporting a broader effect of this defect in the vascular
7.IncreasedPhosphate Reabsorption inPTCs
Hyperphosphatemia is significantly associated with the
development of CVD and high mortality in patients with
CKD, independent of estimated creatinine clearance .
International Journal of Nephrology5
Inorganic phosphate appears to act directly on cultured
vascular smooth muscle cells to express the osteogenic
markers Cbfa1 and osteocalcin, with subsequent mineral-
ization of the extracellular matrix . Serum phosphate
concentration is regulated by intestinal absorption from
dietary phosphate intake, but more importantly, by
glomerular filtration and reabsorption of phosphate via type
II Na/Pi cotransporters (NaPi-IIa and NaPi-IIc) in the apical
membranes of PTCs. In particular, NaPi-IIa plays a central
role in phosphate reabsorption in the kidney (Figure 2). The
presence of hyperphosphatemic patients with CKD whose
GFR is normal is well explained by a hypothesis that Na/Pi
cotransporters in PTCs may be inappropriately upregulated.
The functions of NaPi-IIa are regulated by various hormones
and hormone-like substances, such as parathyroid hormone,
fibroblast growth factor 23, and Klotho that all downregulate
NaPi-IIa and induce phosphaturia. Regulation of NaPi-
IIa is almost exclusively mediated via receptor-mediated
endocytosis and lysosomal degradation of NaPi-IIa .
Because megalin mediates the endocytic pathway for
degradation of NaPi-IIa , decreased megalin function
may result in hypophosphaturia or hyperphosphatemia even
in cases with normal GFR.
Proximal tubular uptake of sodium is increased in patients
with diabetic nephropathy [52–54] and metabolic syndrome
[55, 56] and is associated with the development of hyperten-
sion, another potent factor for CVD . Na+/H+exchanger
isoform 3 (NHE3) is the main NHE isoform in PTCs and
mediates isotonic reabsorption of approximately two-thirds
of filtered NaCl and water, reabsorption of bicarbonate,
and secretion of ammonium ions  (Figure 2). Enhanced
NHE3 activity is assumed to play a leading role in increased
sodium reabsorption in diabetes while intrarenal RAS acti-
vation is also thought to be involved in the process .
Increased action of sodium glucose cotransporter SGLT2 is
yet another factor promoting increased sodium uptake in
PTCs in diabetes .
SubstancesTaken up viaApical
9.1. Homocysteine (Hcy). Hcy is a sulfhydryl amino
acid formed by demethylation of Met (Figure 3). S-
adenosylmethionine (AdoMet) is the intermediate in
this reaction and serves as the methyl donor to form S-
to Met or transsulfurated to cysteine. Approximately 75%
of total plasma Hcy is bound to protein, primarily albumin,
via a disulfide bond (bound Hcy), while the remaining 25%
exists in a free-form unbound state (free Hcy) in humans.
When patients with extreme hyperhomocysteinemia
due to genetic enzyme defects were found to suffer from
premature atherosclerosis and venous thrombosis, Hcy was
hypothesized to be a direct vasculotoxic agent . Subse-
quently, it was shown that plasma Hcy is strongly associated
with renal function, and that 85%–100% of ESRD patients
have elevated Hcy levels . Hyperhomocysteinemia is
recognized as a risk marker for CVD in patients with ESRD
unless their conditions are complicated with malnutrition or
inflammation that induces hypoalbuminemia and apparent
low plasma Hct levels [62, 63].
The kidney probably plays an important role in Hcy
clearance and metabolism. It is highly likely that free
Hcy is filtered by glomeruli and taken up via cationic
amino acid transporters in the apical membranes of PTCs
[64, 65] (Figure 2). However, renal uptake of Hcy derived
from bound Hcy may be mediated by basolateral tubular
transporters. It is therefore assumed that impaired uptake
and/or metabolism of Hcy in PTCs are associated with
hyperhomocysteinemia in patients with CKD or ESRD and
the development of CVD.
Hyperhomocysteinemia is also associated with an
increase in AdoHcy, which is considered another predictor
of cardiovascular events. AdoHcy is a powerful competitive
inhibitor of protein as well as DNA methyltransferases.
Increased intracellular AdoHcy can be expected to result in
hypomethylation of proteins and genes, which will in turn
induce protein dysfunction and epigenetic dysregulation,
respectively [66, 67].
9.2. Asymmetric Dimethylarginine (ADMA). ADMA, a natu-
rally occurring L-arginine analogue, is an endogenous com-
petitive inhibitor of nitric oxide synthase and an important
inducer of endothelial dysfunction. ADMA is formed by the
methylation of arginine residues in peptides with protein
In this reaction, AdoMet is the methyl donor, and AdoHcy is
the demethylated product. Formations of ADMA and Hcy
are therefore biochemically linked (Figure 3).
An increased plasma concentration of ADMA is
associated with the development of CVD . In patients
with nondiabetic CKD, blood concentrations of ADMA are
markedly increased at an early stage, even when GFR is still
within the normal range . Increased plasma ADMA
levels are also closely associated with the development and
progression of nephropathy in patients with type 2 diabetes
[71, 72], which is eliminated from circulation by both renal
excretion and metabolic degradation. Renal uptake of
ADMA is very likely mediated by cationic amino acid
transporters that are predominantly expressed in the apical
membranes of PTCs . ADMA metabolism is mediated
by dimethylarginine dimethylaminohydrolases (DDAHs),
which are posttranscriptionally downregulated by reactive
oxygen species and Hcy [74, 75]. Two isoforms of DDAH
ney, DDAH I is abundantly expressed in PTCs, while DDAH
II is located in glomeruli, afferent arterioles, macula densa,
and distal nephrons . Recent studies have indicated
that DDAH I is mainly involved in the regulation of plasma
ADMA levels . In addition, ADMA is formed by the
activity of PRMT that is highly expressed in PTCs. In subto-
tally nephrectomized rats showing increased plasma ADMA
6 International Journal of Nephrology
levels, DDAH protein levels were decreased while expression
of PRMT was increased in the kidney . Such effects
are likely to mediate the mechanism of increasing plasma
ADMA levels. Streptozotocin-induced rat diabetic kidneys
also showed decreased DDAH I expression, which was
reversed by telmisartan, an angiotensin II-receptor blocker
Glycation, Oxidation, and Nitration Products. Megalin medi-
ates proximal tubular uptake of AGEs, a potent factor of
vascular injury . It remains unclear how effectively AGEs
are metabolized in PTCs, but this metabolic process may be
affected in damaged PTCs. AGE precursors which include
glycation, oxidation, and nitration free adducts are also
excreted or metabolized in the kidney . Methylglyoxal,
one such dicarbonyl adduct, is a potent glycating agent
associated with oxidative stress and vascular injury 
and is increased in the serum of patients with CKD or
uremia, probably because of reduced renal metabolism .
Methylglyoxal is metabolized by glyoxalase I that is usually
expressed in PTCs but is downregulated in the rat model
of renal injury . This suggests that decreased enzymatic
activities in PTCs may be a cause of increased serum
methylglyoxal in CKD.
10.ImpairedUptake, Metabolism,or Urinary
Excretionof Vasculotoxic Substancesvia
Basolateral PTC TransportersinPTCs
10.1. Indoxyl Sulfate and Other Protein-Bound Uremic Toxins.
Indoxyl sulfate is a protein-bound uremic toxin that results
from the metabolism of dietary tryptophan. Increase of
both the development of CVD and mortality . Indoxyl
sulfate is excreted in urine via the organic anion transporters
OAT1 and OAT3 that are predominantly expressed in the
basolateral membranes of PTCs . These transporters are
important as they are also involved in urinary excretion
of other protein-bound uremic toxins such as 3-carboxy-4-
methyl-5-propyl-2-furanpropionate, indoleacetate, and hip-
purate, which may also be associated with the development
of CVD in patients with CKD .
10.2. Guanidino Succinate, Transaconitate, and ADMA.
SLCO4C1 is a human kidney-specific organic anion trans-
porting polypeptide that was first identified as a digoxin
transporter . In renal failure, basolateral SLCO4C1
expression in PTCs is decreased; however, the expression
level of multidrug resistance protein 1 that mediates the
tubular secretion of digoxin in the apical membranes of
PTCs is not changed . A kidney-specific transgenic rat
line overexpressing human SLCO4C1 in PTCs was shown to
significantly eliminate the uremic toxins guanidino succinate
and trans-aconitate as well as ADMA from circulation, even
when renal failure was induced by 5/6 nephrectomy .
In this study, pravastatin was also found to upregulate
the expression of SLCO4C1 and facilitate the removal of
As mentioned earlier, vasculoprotective substances such
as 1,25(OH)2D3 and GPx3 are synthesized by PTCs and
secreted into circulation. In addition, renalase, a circulating
by the PTCs and regulates various cardiac functions and
blood pressure . Plasma concentrations of these factors
are reduced in patients with CKD most likely because of
decreased synthesis in the PTCs.
12.Therapeutic Strategies for
Targeting PTC Dysfunction
Given the diverse and complex functions of PTCs, it is
important to establish comprehensive therapeutic strategies
to preserve PTC viability and maintain their broad range
of functions in diabetic nephropathy and other disorders
related to CKD. Therefore, it may not be sufficient to
compensate only for specific functions of the cells; in fact,
such an approach may explain why the outcomes of recent
supplemental trials that used vitamin B12and folate to target
Hcy levels were controversial [89, 90]. In addition, vitamin
B12and folate deficiencies due to decreased PTC uptake may
be masked by reduced GFR in advanced stages of CKD.
Therefore, supplementation with these vitamins could lead
to overdose and adverse side effects. Inhibitors of the renin-
angiotensin II system and statins may effectively alleviate
PTC dysfunction; however, the mechanisms of these agents
acting on PTCs remain to be elucidated as the phenotypes
or pharmacological responsiveness of PTCs may change
according to pathogenic stages. Therefore, it is also necessary
to develop effective biomarkers to evaluate and monitor the
stages of PTC dysfunction.
Dysregulation of PTC functions is likely to mediate the
multifactorial mechanisms of the development of CVD as
well as progression to ESRD and therefore plays a role in
cardiorenal syndrome. In particular, PTC dysfunction occurs
at the early stages of diabetic nephropathy, a leading cause of
CKD. It is important to elucidate the mechanisms of PTC
dysfunction and establish therapeutic strategies that protect
against PTC dysregulation.
This work was supported by a Grant-in-Aid for Scientific
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