<title>1. INTRODUCTION</title> <p>Thiazolidinediones (TZDs), such as rosiglitazone and pioglitazone,
are highly effective for the treatment of type 2 diabetes and are widely
prescribed. Unfortunately, fluid retention has emerged as the most common and
serious side effect of TZDs and has become the most frequent cause of
discontinuation of therapy. The incidence of TZD-induced fluid retention ranges
from 7% in monotherapy and to as high as 15% when combined with insulin [##REF##10926309##1##–##REF##11197573##3##]. The fluid
retention is often presented as peripheral edema, which can progress into pulmonary
edema and congestive heart failure. TZD use leads to a 6-7% increase in
blood volume in healthy volunteers [##UREF##1##4##, ##UREF##2##5##]. This blood
volume expansion can dilute the red blood cell concentration, producing a
reduced hematocrit. In fact, changes in hematocrit have been used as a
surrogate marker for TZD-induced plasma volume expansion. The fluid retention
is often resistant to loop diuretics but is reversed by withdrawing the drug. Many
aspects of TZD-induced fluid retention have been covered by excellent review
articles [##REF##18276926##6##–##UREF##3##12##]. This review will emphasize renal sodium retention and vascular
hyperpermeability as prominent mechanisms of TZD-induced fluid retention. We
will also introduce several possible treatment strategies.</p>
<title>5. CONCLUSIONS</title> <p>The fluid retention and rapid body weight
gain induced by TZD treatment are caused by increased fluid reabsorption in the
distal nephron as well as increased vascular permeability in adipose tissues (see ##FIG##2##Figure 3##).
The molecular mechanisms of the effects of TZDs in renal collecting duct and in
blood vessels remain unknown. Despite documentation of ENaC as a molecular target
of TZDs in the collecting duct, increasing evidence indicates ENaC-independent
mechanisms that may involve changes in paracellular transport. PKCß is shown to
mediate TZD-induced vascular permeability in adipose tissues. More studies are
required for determination of the signaling pathway responsible for PPAR<italic>γ</italic>-dependent
tissue-specific activation of PKCß. Currently,
there are no effective therapies for the side effects of TZDs except drug
withdrawal. A number of potential treatment strategies that target collecting
duct sodium transport (amiloride) and vascular permeability (PKC inhibitors)
have been developed from animal studies and should be evaluated by future clinical
trials.</p>
<p>Recommended by Jane Pinaire</p> <p>Thiazolidinediones (TZDs) are peroxisome proliferator-activated receptor subtype <italic>γ</italic> (PPAR<italic>γ</italic>) activators that are clinically used as an insulin sensitizer for glycemic control in patients with type 2 diabetes. Additionally, TZDs exhibit novel anti-inflammatory, antioxidant, and antiproliferative properties, indicating therapeutic potential for a wide variety of diseases associated with diabetes and other conditions. The clinical applications of TZDs are limited by the common major side effect of fluid retention. A better understanding of the molecular mechanism of TZD-induced fluid retention is essential for the development of novel therapies with improved safety profiles. An important breakthrough in the field is the finding that the renal collecting duct is a major site for increased fluid reabsorption in response to rosiglitazone or pioglitazone. New evidence also indicates that increased vascular permeability in adipose tissues may contribute to edema formation and body weight gain. Future research should therefore be directed at achieving a better understanding of the detailed mechanisms of TZD-induced increases in renal sodium transport and in vascular permeability.</p>
<title>2. RENAL MECHANISM</title> <p>The kidney is the key regulator of electrolyte balance and water conservation. Fluid
retention at the renal level is suggested by evidence that TZD-induced edema is
associated with reduced urinary sodium and water excretion. Song et al.
reported that chronic three-day administration of rosiglitazone to Sprague Dawley
rats significantly reduced urine volume (by 22%) and sodium excretion (by 44%) [##REF##14593089##13##]. These findings lead us to speculate that
renal mechanisms play a major role in TZD-induced fluid retention. TZDs may
cause renal fluid reabsorption directly by affecting tubular transport, renal
sodium retention, and vascular hyperpermeability or indirectly by affecting
renal hemodynamics or processes. Yang et al. examined the effect of a PPAR<italic>γ</italic>
agonist, GI262570 (farglitazar), on the glomerular filtration rate, effective
renal plasma flow, and renal filtration fraction in chronically
catheter-implanted conscious rats [##REF##12960690##14##]. In this
study, glomerular filtration rate was determined by using fluorescein
isothiocyanate (FITC)-inulin and renal blood
flow by using para-aminohippurate (PAH). A 10-day infusion of GI262570
decreased hematocrit, hemoglobin, and serum albumin (all <italic>P</italic> &lt; .05), indicating volume expansion, but did not alter
glomerular filtration rate, effective renal plasma flow, or renal filtration
fraction. This indicates that PPAR<italic>γ</italic> agonist-induced volume expansion is not
related to changes in renal hemodynamics [##REF##12960690##14##]. This
observation is reinforced by a human study in which the six-week administration
of pioglitazone to healthy volunteers led to sodium retention without a significant
effect on glomerular filtration rate or renal blood flow [##REF##15001599##15##]. This lack
of change in renal hemodynamics is, however, not universally reported. The three-day
administration of rosiglitazone in Sprague Dawley rates induced a 35% reduction
in creatinine clearance, an indirect measure of the glomerular filtration rate [##REF##14593089##13##]. It is
unclear whether or not this discrepancy is related to differences in glomerular
filtration rate measurement techniques or other experimental protocols.</p> <p>The lack of solid evidence to support
the alteration of renal hemodynamic parameters following treatment with PPAR<italic>γ</italic>
ligands suggests the possibility of a direct influence on tubular transport processes.
The regulation of NaCl reabsorption in the kidney can occur at the level of sodium
transport proteins lining the renal epithelia. These sodium transporters
include basolateral Na-K-ATPase, and the following apical transporters that
vary with individual nephron segments: the sodium hydrogenexchanger
subtype III (NHE3) and the sodium phosphate cotransporter subtype II (NaPi-2) in the proximal
convoluted tubule, the bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2
or BSC1) in the thick ascending limb, the thiazide-sensitive Na-Cl cotransporter (NCC or TSC)
in the distal convoluted tubule, and the amiloride-sensitive sodium channel
(ENaC) in the collecting duct. The major water channel proteins (aquaporins,
AQPs) in the kidney include AQP1-4, of which AQP1 and AQP2 function
on the apical membrane, and AQP3 and AQP4 on the basolateral
membrane [##REF##8743483##16##]. The study of Song et al. is the first to
provide a comprehensive examination of the effects of PPAR<italic>γ</italic> agonists on various
renal sodium and water transport proteins [##REF##14593089##13##]. In that study, a three-day rosiglitazone
treatment increasedthe whole kidney protein level of the <italic>α</italic>-1 subunit of Na-K-ATPase, NKCC2,
NHE3, AQP2, and AQP3 [##REF##14593089##13##]. These findings suggest that increases in
sodium transport may occur in the proximal convoluted tubule and the thick
ascending limb.</p> <p>The collecting duct reabsorbs approximately 2-3% of the filtered
sodium load primarily through ENaC, which is comprised of three subunits, <italic>α</italic>, <italic>β</italic>,
and <italic>γ</italic>. These proteins are vital to day-to-day adjustment of sodium reabsorption
and are regulated by the hormones aldosterone and insulin [##REF##10751213##17##–##REF##10510339##19##]. A key
mediator of aldosterone activation of ENaC is serum and glucocorticoid
regulated kinase 1 (SGK1) [##REF##12923071##20##, ##REF##12110505##21##]. Activated SGK1 prevents ENaC degradation by inactivating the ubiquitin
ligase Nedd4-2 [##REF##16192418##22##]. Nedd4-2
interacts with the PY motif of ENaC leading to endocytosis and degradation of
the channel [##REF##16192418##22##]. Prior to
the conditional knockout (KO) studies, three major lines of evidence indicated
that the activation of sodium transport processes in the distal nephron may
underlie TZD-induced fluid retention. First, within the kidney, PPAR<italic>γ</italic> is highly expressed in the renal medullary
collecting duct, with lower expression levels in glomeruli, proximal tubules,
and microvasculature. This was demonstrated by both RT-PCR and microdissection as well as by in situ hybridization techniques [##REF##9435691##23##–##REF##10600944##25##]. Second, in
a cultured human cortical collecting duct (CCD) cell line, PPAR<italic>γ</italic> agonists
increased levels of cell surface <italic>α</italic>-ENaC. This is paralleled by an increase in SGK1 mRNA, which is abolished by pretreatment with
a specific PPAR<italic>γ</italic> antagonist, leading to increased levels of cell surface <italic>α</italic>-ENaC.
Electrophoretic mobility shift assays further suggest that these effects are
caused by the binding of PPAR<italic>γ</italic> to a specific response element in the SGK1 promoter [##REF##12923071##20##]. Third, in vivo evidence shows that GI262570 stimulates
sodium and water reabsorption from the distal nephron in Sprague Dawley rats [##REF##15475592##26##]. This
evidence comes from increases in plasma sodium and chloride concentrations with
concomitant decreases in plasma potassium concentration. Reciprocal changes in
plasma NaCl and potassium levels are typically seen as a consequence of renal
mineralocorticoid activation promoting NaCl reabsorption and potassium
secretion in the distal nephron [##REF##15475592##26##]. Additionally,
mRNA levels for a group of genes involved in distal nephron sodium and water
absorption in the kidney medulla are changed with GI262570 treatment [##REF##15475592##26##].</p> <p>The involvement of the distal nephron in TZD-induced fluid retention has been assessed in two independent
studies using mice with a collecting duct-specific deletion of PPAR<italic>γ</italic> (CD PPAR<italic>γ</italic> KO) 
[##UREF##4##27##, ##REF##15956187##28##]. In both studies, the expression of Cre recombinase was driven by an
AQP2 promoter highly specific to the collecting duct. In these two studies, the
experimental approaches for assessment of fluid retention were quite different:
a combination of hematocrit, plasma aldosterone levels, and Evans blue (EB) dye-based
measurement of plasma volume in one study (see ##FIG##0##Figure 1##) [##REF##15956187##28##] and
determination of total water content in the other [##UREF##4##27##]. Remarkably,
both studies reported a similar phenotype in that the conditional PPAR<italic>γ</italic>
knockout mice proved to be resistant to the rosiglitazone- or
pioglitazone-induced body weight gain and plasma volume expansion found in mice
expressing PPAR<italic>γ</italic> in the collecting duct. As shown in ##FIG##0##Figure 1##, a nine-day
rosiglitazone treatment induced a gradual and significant increase in body
weight in floxed mice when compared to untreated floxed controls (2.74 ± 0.25 versus 1.05 ± 0.16 gram, on day 9, <italic>P</italic> &lt; .05). In contrast,
body weight gains between rosiglitazone-treated and untreated CD PPAR<italic>γ</italic> KO mice
were not significantly different (0.90 ± 0.25 versus 0.81 ± 0.19
gram, on day 9, <italic>P</italic> &gt; .05). Rosiglitazone treatment in the control mice
induced plasma volume expansion, which was reflected by a significantly
decreased hematocrit and plasma aldosterone levels as well as by a 32.2%
increase in plasma volume as assessed by the EB dye technique. In contrast,
rosiglitazone-treated CD PPAR<italic>γ</italic> KO mice exhibited nonsignificant trends toward
change in these parameters (see ##FIG##1##Figure 2##). These two studies also provided
evidence that exposure of primary collecting duct cells to PPAR<italic>γ</italic> ligands leads
to increased sodium transport as assessed by measurements of <sup>22</sup>Na<sup>+</sup> flux and transepithelial resistance.</p> <p>Guan et al. examined the effects of pioglitazone on the expression of <italic>α</italic>-, ß-, and <italic>γ</italic>-ENaC
subunits in cultured inner medullary collecting duct (IMCD) cells [##UREF##4##27##]. Notably, within one hour following treatment
of IMCDs with pioglitazone (1 <italic>μ</italic>M), <italic>γ</italic>-ENaC mRNA expression
increased roughly 10 folds
before gradually diminishing. This stimulatory effect appeared to be specific
for <italic>γ</italic>-ENaC mRNA, because <italic>α</italic>-ENaC and ß-ENaC mRNA levels did not show any change
in response to treatment with pioglitazone. Interestingly, PPAR response elements (PPREs) are identified in intron 1 but not in the
5′ flanking region of the <italic>γ</italic>-ENaC gene. Chromatin immunoprecipitation (ChIP) of
genomic DNAisolated from cultured
mouse IMCDs revealed a physical interaction between PPAR<italic>γ</italic> and <italic>γ</italic>-ENaC genomic DNA. Somewhat unexpectedly, the PPAR<italic>γ</italic> binding site
was shown to be located outside intron 1 of the <italic>γ</italic>-ENaC gene. Overall, these
data support <italic>γ</italic>-ENaC as a direct target gene of PPAR<italic>γ</italic> in the collecting duct
cells, although the exact mechanism remains to be elucidated.</p> <p>However, the role of ENaC as a direct target of PPAR<italic>γ</italic> has not always been demonstrable. Nofziger et al.
reported that, in collecting duct cell lines, PPAR<italic>γ</italic> agonists failed to enhance
basal or insulin-stimulated sodium transport as assessed by measurement of
short-circuit current (Isc) [##REF##16170524##29##]. This study
also did not find that PPAR<italic>γ</italic>-induced changes in the amount of SGK1 transcript or protein expression. Additionally,
there is no solid evidence for major changes in renal expression of any of the
ENaC subunits in response to PPAR<italic>γ</italic> ligands in vivo [##REF##14593089##13##, ##REF##15475592##26##, ##REF##17211457##30##]. More recently, Vallon et al. reported that collecting duct-specific gene
inactivation of <italic>α</italic>-ENaC in the mouse does not attenuate the rosiglitazone-induced
body weight gain [##UREF##5##31##]. In this
study, the Hoxb-7 promoter was used to inactivate <italic>α</italic>-ENaC in the collecting duct,
while leaving ENaC expression in the cortical connecting tubule (CNT) intact [##REF##12925696##32##]. As
expected, in the floxed control mice, rosiglitazone treatment (320 mg/kg diet) rapidly
increased body weight (ΔBW day 11: 4.5 ± 0.8% versus 1.1 ± 0.6%, <italic>P</italic> &lt; .05) and
lowered hematocrit (44 ± 1.0% versus 47 ± 1%, <italic>P</italic> &lt; .0005), while rosiglitazone treatment increased body weight (ΔBW: 7.3 ± 0.9% versus 0.9 ± 0.7%, <italic>P</italic> &lt; .0005) and lowered hematocrit (42 ± 2% versus 47 ±
1%, <italic>P</italic> &lt; .05) in <italic>α</italic>-ENaC collecting
duct knockout mice. These data may argue against collecting duct ENaC playing a
significant role in mediating the adverse effect of rosiglitazone. However,
involvement of ENaC activity in the CNT cannot be ruled out. To resolve this issue, AQP2-Cre mice could be used to
inactivate ENaC in the entire collecting duct system.</p> <p>The negative results discussed above
prompt consideration of alternative mechanisms for explaining PPAR<italic>γ</italic>-mediated
increases in distal tubular fluid reabsorption. There is a significant amiloride-insensitive
component in the rosiglitazone-induced increases in sodium transport [##REF##15956187##28##]. The
possibility exists that increased reabsorption may occur by way of a
paracellular route. For example, PPAR<italic>γ</italic> may regulate the tight junction leading
to altered permeability to sodium or other electrolytes. In an in vitromodel of differentiating normal human urothelial (NHU) cells, PPAR<italic>γ</italic> activation in conjunction with epidermal growth factor receptor (EGFR) blockade led to the de novo expression
of claudin 3 mRNA and protein and downregulation of claudin 2 transcription [##REF##16688762##33##]. These
results suggest a role for PPAR<italic>γ</italic> and EGFR signaling
pathways in regulating the tight junction formation in NHU cells. There is an
intriguing possibility that a similar mechanism may operate in renal epithelial
cells. Another possible mechanism is that PPAR<italic>γ</italic> may regulate transport of ions other than sodium. Further studies
are clearly needed to explore not only ENaC-dependent, but also
ENaC-independent mechanisms, for TZD-activated fluid reabsorption in the distal
nephron.</p> <title>3. VASCULAR MECHANISM</title> <p>PPAR<italic>γ</italic> is expressed in the vascular system [##REF##10696077##34##], including endothelial
cells [##REF##9610365##35##, ##REF##9920814##36##], vascular
smooth muscle cells (VSMC) [##REF##9655393##37##] as well as monocyte/macrophages
[##REF##9748221##38##, ##REF##9422508##39##]. Several
lines of evidence suggest that PPAR<italic>γ</italic> regulates various aspects of vascular
function, including capillary permeability. Increased capillary permeability
leads to extravasation of fluid and is thought to contribute to edema in
patients treated with TZDs. Donnelly et al. were the first to examine the
direct effect of rosiglitazone on endothelial barrier function using an in vitro system of pulmonary artery
endothelial cell monolayers. Transendothelial albumin flux was measured using EB
dye-labeled albumin. They found that exposure to high concentrations of rosiglitazone
for four hours increased transendothelial albumin flux dose-dependently, with a
noticeable effect at 10 <italic>μ</italic>M and a maximal effect at 100 <italic>μ</italic>M. This
hyperpermeability response to high concentrations of rosiglitazone was fully
reversible by washing rosiglitazone off the monolayer. After incubation for 24
to 48 hours, the effect of rosiglitazone began to subside. High concentrations
of rosiglitazone (0.1–1 mM) are also
needed to induce a vasodilator effect in isolated arteries [##REF##14744930##40##]. Future
studies, ideally employing gene knockout mice, may determine the extent of
PPAR<italic>γ</italic> mediation of the vascular response to high concentrations of TZDs. The
mechanism of TZD-induced capillary permeability is not well characterized but
may involve a number of factors, notably vascular endothelial growth factor (VEGF),
nitric oxide, and protein kinase C, each of which is discussed below.</p> <p>VEGF is a potent cytokine that augments vascular permeability in tumors, healing wounds, retinopathies, many
important inflammatory conditions, and certain physiological processes, such as
ovulation and corpus luteum formation [##REF##9893348##41##]. VEGF is estimated to be 50 times more potent than histamine in
enhancing vascular permeability [##REF##9893348##41##]. The gene transfer of naked plasmid DNA encoding the 165-amino acid isoform of VEGF in patents with peripheral artery
disease causes peripheral edema [##REF##10836914##42##]. Evidence
suggests an involvement of VEGF in TZD-induced edema. The study of Emorto et
al. was the first to report that plasma levels of VEGF are significantly 
increased in troglitazone-treated subjects (120.1 ± 135.0 pg/mL)
compared with those treated with diet alone (29.2 ± 36.1 pg/mL), sulfonylurea
(25.8 ± 22.2 pg/mL), or insulin (24.6 ± 19.0 pg/mL). The effect of
troglitazone on increased VEGF levels was further supported by plasma VEGF
levels in five patients before treatment (20.2 ± 7.0 pg/mL), after three months
of troglitazone treatment (83.6 ± 65.9 pg/mL), and three months after
discontinuation (28.0 ± 11.6 pg/mL). These authors further demonstrated that
troglitazone, as well as rosiglitazone, at the plasma concentrations observed
in patients, increased VEGF mRNA levels in 3T3-L1 adipocytes. The finding suggests that PPAR<italic>γ</italic> activation may
directly stimulate expression of VEGF that leads to tissue edema. However, it
is puzzling that several other studies show that PPAR<italic>γ</italic> negatively regulates
VEGF signaling. In transformed and primary endometrial cells rosiglitazone or 15-deoxy-delta
12,14-prostaglandin J<sub>2</sub> (15d-PGJ<sub>2</sub>) decreased VEGF protein
secretion [##UREF##6##43##]. In
transiently transfected Ishikawa cells, rosiglitazone repressed VEGF gene
promoter-luciferase activation with an IC [##REF##9655393##37##] approximately 50 nM. By using
truncated and mutated VEGF promoter constructs, this study further revealed
that the PPAR<italic>γ</italic>-regulated domain is a direct repeat (DR)-1 motif −443 bp
upstream of the transcriptional start site [##UREF##6##43##]. Similarly,
rosiglitazone attenuated VEGF-induced proliferation and migration of human
pulmonary valve endothelial cells (HPVECs) [##REF##16840174##44##].
Rosiglitazone also antagonized VEGF-induced nuclear factor translocation in activated T cells subtype c1 (NFATc1) [##REF##16840174##44##]. Furthermore,
rosiglitazone markedly decreased VEGF-induced tube formation and cell migration
in human umbilical vein endothelial cells [##REF##16297938##45##]. Taking these studies together, it seems likely that PPAR<italic>γ</italic> exerts a dual
effect on VEGF signaling, possibly depending on cell type.</p> <p>Nitric oxide (NO) is a ubiquitous,
naturally occurring molecule found in a variety of cell types and organ
systems. Endothelial cells are rich in NO, which has been shown to regulate
many aspects of vascular function, including vascular permeability. Polikandriotis
et al. report that 15d-PGJ<sub>2</sub> and ciglitazone increase cultured
endothelial cell NO release without increasing the expression of endothelial
nitric oxide synthase (eNOS) [##UREF##7##46##]. This study
provided further evidence that PPAR<italic>γ</italic> activation leads to eNOS ser1177
phosphorylation [##UREF##7##46##]. It seems
plausible that the stimulation of eNOS-derived NO may contribute to TZD-induced
edema. St-Pierre et al. examined the effect of rosiglitazone on muscle
vasopermeability and NO system in the fructose-fed rat model [##REF##15130775##47##]. In this
study, extravasation of EB dye in vivo
in specific muscle groups was used to assess vascular permeability. Fructose-fed
rats treated with rosiglitazone had a 30–50% increase in extravasation
of EB in the the Rectus femoris, soleus, gastrocnemius lateralis, vastus
lateralis, and tibialis cranialis skeletal muscles [##REF##15130775##47##]. In
homogenates of skeletal muscles (vastus lateralis) from fructose-fed rats,
rosiglitazone resulted in a significant increase in nitric oxide synthase (NOS)
activity and eNOS immunoreactive content compared to the control animals [##REF##15130775##47##].
Unexpectedly, the immunoreactive level of the most abundant muscle NOS
isoforms, neuronal NOS (nNOS), remained unchanged.</p> <p>Protein kinase C (PKC) plays a major role in determining vascular permeability through
phosphorylation of the cytoskeleton proteins that form the tight intercellular
junction [##REF##10854687##48##–##REF##1522136##51##]. In the study of Sotiropoulos et al., rosiglitazone treatment
selectively activated PKC in fat and retinal tissues in parallel with the
increased vascular permeability in these tissues [##REF##16672634##52##]. The activation of PKC is evaluated by
determining the enzyme activity together with tissue levels of diacylglycerol (DAG), a
strong PKC activator [##REF##16672634##52##]. These investigators tested the effect
of PKC<italic>β</italic> inhibition and gene knockout but did not determine specific PKC
isoforms. They found that posttreatment with ruboxistaurin (RBX), a PKC<italic>β</italic> inhibitor,
effectively attenuated the increases in capillary permeability, water content,
and weight of epididymal fat, as well as the increase in body weight associated
with rosiglitazone treatment; this finding was also confirmed by using PKC<italic>β</italic> KO mice
[##REF##16672634##52##].</p> <title>4. POTENTIAL THERAPIES</title> <title>4.1. Inhibition of sodium transport in the collecting duct</title> <p>The use of diuretics for management of TZD-induced fluid retention has been evaluated
by several case reports [##UREF##0##2##, ##REF##12396745##53##] and, 
recently, by a controlled trial [##REF##17093067##54##]. Most case reports show that the edema
is refractory to a loop diuretic (furosemide) and that the symptoms resolve
only after discontinuation of TZD. The recent controlled trial involved 381
patients with type 2 diabetes. It examined the effect of three diuretics that
act with different mechanisms on rosiglitazone-induced body weight gain and
plasma volume [##REF##17093067##54##]. The diuretics included furosemide,
which inhibits the Na-K-Cl cotransporter in the thick ascending limb of the loop
of Henle, hydrochlorothiazide (HCTZ), which acts to inhibit the Na-Cl
cotransporter in the distal convoluted tubule, and spironolactone (SPIRO), which
is an ENaC inhibitor in the collecting duct. The degree of fluid retention in
this study was evaluated by measuring changes in the hematocrit as an index of
changes in plasma volume, body weight, total body water, and extracellular
fluid changes determined by noninvasive bioelectrical impedance with an Akern
soft tissue analyzer. SPIRO and HCTZ both effectively reduced fluid retention
and body weight while furosemide had only a limited effect. The effectiveness
of SPIRO may be attributable to the ability of this diuretic to interfere with
the sodium retaining action of PPAR<italic>γ</italic> in the collecting duct. It is unclear
whether the same mechanism can explain the action of HCTZ. Thiazide diuretics act
primarily in the proximal part of the distal convoluted tubules where they
inhibit Na<sup>+</sup>/Cl<sup>−</sup> cotransport [##REF##3631283##55##, ##REF##7840252##56##], but they are also reported to inhibit
salt and water reabsorption in the medullary collecting duct [##REF##6876566##57##]. The reason for the lack of diuretic response
of TZD-treated diabetics to furosemide is not entirely clear, but one possible
explanation might be the lack of distal effect of this loop diuretic. Another possibility
is that TZD-induced fluid retention may be associated with impaired transport machinery
in the thick ascending limb. Possibly secondary to the volume expansion, the
plasma level of atrial natriuretic factor (ANF)
is elevated in TZD-treated diabetics [##REF##17093067##54##]. ANF inhibits NaCl reabsorption in the loop of Henle as well as in other sites of
nephron through the activation of guanylyl cyclase
receptors that release cyclic GMP [##REF##9551429##58##]. It also
remains possible that PPAR<italic>γ</italic> may negatively affect NaCl transport in the loop of
Henle.</p> <p>The experimental evidence favoring
ENaC as a potential target of PPAR<italic>γ</italic> in the distal nephron seems to provide a
rationale for the use of amiloride as a specific ENaC inhibitor for treatment
of TZD-induced fluid retention. Unfortunately, amiloride was not included in
this clinical trial [##REF##17093067##54##]. In the mouse, pretreatment with
amiloride effectively prevents body weight gain and fluid retention produced by
pioglitazone. However, in the rat model, posttreatment with amiloride
unexpectedly exacerbates the fluid retention induced by farglitazar. It is
unclear whether this discrepancy between the studies is due to species
differences, PPAR<italic>γ</italic> ligand activity, or the different timing of amiloride
treatment.</p> <title>4.2. Combination of a PPAR<italic>γ</italic> and a PPAR<italic>α</italic> agonist</title> <p>Boden et al. examined the effect of the
combined use of rosiglitazone and fenofibrate in patients with type 2 diabetes [##REF##17192489##59##]. Compared with rosiglitazone alone,
rosiglitazone/fenofibrate proved significantly more effective in lowering
fasting free fatty acid levels and tended to be more effective in achieving plasma
glucose control. Interestingly, rosiglitazone/fenofibrate completely prevented
the increase in body weight and body water content associated with
rosiglitazone. This study is the first to show that the combined use of a PPAR<italic>γ</italic>
and a PPAR<italic>α</italic> agonist can prevent rosiglitazone-induced fluid retention. The
investigators did not propose a mechanism to explain this phenomenon. The two
PPAR isoforms occur in different locations along the nephron. PPAR<italic>α</italic> mRNA is found predominately in the cortex and is
specifically localized in the proximal convoluted tubule (PCT). PPAR<italic>γ</italic> is
abundant in the renal inner medulla, specifically localized to the inner
medullary collecting duct [##REF##9435691##23##, ##REF##10600944##25##]. The
difference in nephron localization does not seem to favor the direct interaction
between the two PPAR isoforms. However, it remains possible that low PPAR<italic>α</italic>
activity in the collecting duct may antagonize the sodium-retaining action of
PPAR<italic>γ</italic>. Future studies are needed to investigate whether an interaction occurs
in the collecting duct or another location.</p> <p>Dual PPAR<italic>α</italic>/<italic>γ</italic> agonists
have been developed by several pharmaceutical companies, and some have
undergone or are currently undergoing clinical trials [##REF##16503761##60##–##REF##16239637##62##]. Unfortunately,
muraglitazar, the first dual PPAR<italic>α</italic>/<italic>γ</italic> agonist, has been associated with an
excessive incidence of major adverse cardiovascular events, including myocardial
infarction, stroke and transient ischemic attack, chronic heart failure and
death [##REF##16239637##62##]. This
finding raises significant safety concerns about the dual agonists as well as the combination of a PPAR<italic>γ</italic> and a PPAR<italic>α</italic> agonist. In the study of
Boden et al., rosiglitazone/fenofibrate appeared to be well tolerated [##REF##17192489##59##]. The safety issues may be related to
the ratio of PPAR<italic>γ</italic> to PPAR<italic>α</italic>. The ratios are fixed for the dual agonists, but can
be varied by changing the proportion of PPAR<italic>γ</italic> and PPAR<italic>α</italic> agonists. It should be
pointed out that Boden's study was limited to a small number of patients and a
short period of treatment [##REF##17192489##59##]. The safety issue regarding the
combined use of a PPAR<italic>γ</italic> and PPAR<italic>α</italic> agonist needs to be carefully evaluated in
larger-scale and longer-term clinical trials as well as animal studies.</p> <title>4.3. Inhibition of protein kinase C</title> <p>There is functional evidence suggesting
the involvement of vascular permeability in TZD-induced body weight gain and
fluid retention [##REF##16672634##52##]. Therefore, targeting vascular
permeability may provide a potential therapeutic strategy for this side effect
of the TZDs. In an animal study, the use of a PKC<italic>β</italic> inhibitor, RBX, to target
vascular permeability effectively attenuated the increases in TZD-induced body
weight gain [##REF##16672634##52##]. Is there any safety issue related to
RBX? In the animal models tested, including Zucker and lean fatty rats, and mice,
RBX reduced rosiglitazone-induced capillary permeability, but had no
significant effect on the baseline capillary permeability without rosiglitazone
treatment. In this short-term animal study, the compound appears to be well
tolerated. Another positive note is that RBX is being used in clinical trials
for diabetic microvascular complications. In these trials, as well as in animal
studies, RBX shows promise for treatment of diabetic retinopathy and nephropathy
without noticeable side effects [##REF##17693752##63##, ##UREF##8##64##].</p>
<title>ACKNOWLEDGMENTS</title> <p>Portions of this work were funded by NIH grants RO-1 HL079453,
RO-1 DK066592, R21 DK069490, and Veterans Affairs Merit Review (to T. Yang).</p>
<fig id="fig1" position="float"><label>Figure 1</label><caption><p>Body weight gains in untreated and rosiglitazone (RGZ)-treated PPAR<italic>γ</italic>
<sup>f/f</sup> mice (a) and CD PPAR<italic>γ</italic> knockout mice
(b) (adapted from [##REF##15956187##28##]).*, <italic>P</italic> &lt; .05
versus vehicle at the corresponding time point.</p></caption></fig> <fig id="fig2" position="float"><label>Figure 2</label><caption><p>Changes in plasma volume in PPAR<italic>γ</italic>
<sup>f/f</sup> and CD PPAR<italic>γ</italic> knockout mice following rosiglitazone
(RGZ) treatment (adapted from [##REF##15956187##28##]). (a) Hematocrit (Hct) in PPAR<italic>γ</italic>
<sup>f/f</sup> and
CD PPAR<italic>γ</italic> knockout mice before and after RGZ treatment. (b) Plasma aldosterone
levels in PPAR<italic>γ</italic>
<sup>f/f</sup> and CD PPAR<italic>γ</italic> knockout mice following RGZ
treatment. (c) Determination of plasma volume in PPAR<italic>γ</italic>
<sup>f/f</sup> and CD
PPAR<italic>γ</italic> KO mice by the Evans blue (EB) dye technique.</p></caption></fig> <fig id="fig3" position="float"><label>Figure 3</label><caption><p>The mechanism for thiazolidinedione- (TZD-) induced
edema. In the renal collecting duct, PPAR<italic>γ</italic> activation
increases sodium reabsorption through ENaC-dependent and independent
mechanisms. In the blood vessels of adipose tissues, PPAR<italic>γ</italic> ligands activate PKCß, VEGF, and NO, which together lead to
increased endothelial permeability. The increased renal sodium retention at the
level of the collecting duct in conjunction with increased vascular permeability
may determine edema development.</p></caption></fig>
<graphic xlink:href="PPAR2008-943614.001"/> <graphic xlink:href="PPAR2008-943614.002"/> <graphic xlink:href="PPAR2008-943614.003"/>
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PPAR Res. 2008 Sep 7; 2008:943614