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1 Department of Biology, Indiana University Purdue University at Indianapolis, 723 West Michigan Street, Indianapolis, IN 46202, USA 2 Department of Anatomy and Cell Biology, Indiana University School of Medicine, Indianapolis, IN 46202, USA3 Department of Pathology and Laboratory Medicine, Indiana University School of Medicine, Indianapolis, IN 46202, USA4 Division of Nephrology and Hypertension, Mayo Clinic, Rochester, MN 55905, USA Correspondence should be addressed to Bonnie L. Blazer-Yost, [email protected] Received 20 August 2010; Accepted 11 October 2010 Academic Editor: Lawrence Serfaty Copyright 2010 Bonnie L. Blazer-Yost et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Polycystic kidney disease (PKD) is a genetic disorder characterized by growth of fluid-filled cysts predominately in kidneytubules and liver bile ducts. Currently, the clinical management of PKD is limited to cyst aspiration, surgical resection or organtransplantation. Based on an observation that PPARγ agonists such as pioglitazone and rosiglitazone decrease mRNA levels of aCl− transport protein, CFTR (cystic fibrosis transmembrane conductance regulator), and the Cl− secretory response to vasopressinin cultured renal cells, it is hypothesized that PPARγ agonists will inhibit cyst growth. The current studies show that a 7- or 14-week pioglitazone feeding regimen inhibits renal and hepatic bile duct cyst growth in the PCK rat, a rodent model orthologous tohuman PKD. These studies provide proof of concept for the mechanism of action of the PPARγ agonists and suggest that this classof drugs may be eﬀective in controlling both renal and hepatic cyst growth and fibrosis in PKD.
Polycystic kidney disease (PKD) is a genetic disease with both autosomal dominant (ADPKD) and autosomal Agonists of the peroxisome proliferator activator receptor recessive (ARPKD) forms [7, 8]. ADPKD is the more gamma (PPARγ) have pleiotropic eﬀects on intermediary prevalent form, striking approximately 1 in 1000 in the metabolism. Two of these agents, rosiglitazone and piogli- adult population and presenting as slow growing, fluid-filled tazone, are approved for clinical use as insulin-sensitizing cysts which form predominately in the kidney. Progressive agents in the treatment of type II diabetes. One of the major cyst growth and fibrosis in the surrounding tissue generally side eﬀects of PPARγ treatment is fluid retention that can, compromise organ function after middle age and lead to in some instances, result in overt edema [1–3]. A renal end-stage renal disease after the fifth decade. ARPKD aﬀects collecting duct-specific knockout of PPARγ in rodent models children in the neonatal period and is characterized by abrogates the drug-induced fluid retention, suggesting that tubular dilation. Both forms of PKD have liver involvement the eﬀect arises from alterations in electrolyte and/or fluid with cysts that arise from cholangiocytes, an epithelial cell transport in the distal nephron [4, 5]. Studies in a cell culture type lining the hepatic bile ducts.
model of the principal cell type of the distal nephron have The genes that are mutated in PKD encode proteins demonstrated that PPARγ agonists inhibit cAMP-stimulated found in the primary cilia, cytoplasmic vesicles, endoplasmic anion transport and the mRNA expression of the cystic fibro- reticulum, and cell-cell and cell-extracellular matrix con- sis transmembrane conductance regulator (CFTR), a Cl− tacts. These proteins are either transient receptor potential channel found in the apical membrane of this cell type .
Ca2+ channels or proteins that regulate this class of Ca2+ channels [7, 8]. Disease-associated decreases in intracellular (Takeda Chemical Industries) was added to the food at Ca2+ concentrations modulate intracellular signaling path- concentrations estimated to provide 4 or 20 mg/kg/day. The ways including those regulating cAMP and Cl− channels.
control animals received unsupplemented diet. At the end of It is widely accepted that secretion of ions and fluid by each study, rats were anesthetized with ketamine 60 mg/kg the cyst-lining epithelial cells contribute to cyst expansion.
and xylazine 10 mg/kg, IP. Blood was obtained by cardiac Studies performed over a decade ago indicate that renal puncture for determination of plasma creatinine, BUN, cyst formation in PKD is driven by anion (Cl− or HCO− electrolytes, bilirubin, liver enzymes, and glucose. The right secretion [9–11], and more recent studies have shown a kidney, part of the liver, the pancreas, and the spleen were remarkably similar profile in freshly isolated bile duct epithe- placed into preweighed vials containing 10% formaldehyde lia . Inhibitor studies and electrophysiological analyses in phosphate buﬀer (pH 7.4). These tissues were embedded have shown that CFTR is the Cl− channel responsible for in paraﬃn for histological studies. The left kidney and part secretion in both kidney and biliary cysts [10, 12]. Based of the liver were immediately frozen in liquid nitrogen.
on the previous finding that PPARγ agonists inhibit CFTRexpression , the current studies were designed to test the 2.3. Histological Analyses eﬃcacy of a PPARγ agonist, pioglitazone, in inhibiting cystgrowth.
2.3.1. IUPUI. Medial transverse kidney sections were stained Of the several rodent models for PKD, we chose to use with hematoxylin, and eosin and cyst volumes were ana- the PCK rat model because the genetic mutation in this lyzed using point count stereology methods on deidentified animal is orthologous to human ARPKD, while the animals sections . Kidney and liver fibrosis was assessed with also express many of the phenotypic characteristics of human picrosirius red staining. Kidney fibrosis was graded on a ADPKD [13, 14]. The animals develop both kidney and liver scale of 1–4 (1-normal, 2-slight, 3-moderate, and 4-severe) fibrocystic diseases and live long enough to facilitate long- in both the cortex and the juxtamedullary areas of the kidney.
term treatment protocols. This model was used in preclinical Similarly, liver fibrosis was assessed on a scale of 1–4 (1- testing of both the renal V2 receptor antagonist and the less than 1% fibrosis, 2-less than 5% fibrosis, 3-less than somatostatin agonist as treatment options for PKD [15–17].
2.1. Animals. PCK rats were purchased from Charles River 2.3.2. Mayo. Four μm transverse tissue sections of the kid- Laboratories, Inc. (Wilmington, MA) or were bred in the ney, including cortex, medulla, and papilla, and of the liver colonies at Indiana University School of Medicine (IUSM) were stained with hematoxylin-eosin and picrosirius red.
or the Mayo Clinic. Institutional Animal Care and Use Com- Whole transverse tissue sections stained with hematoxylin- mittees at each institution approved all protocol procedures.
eosin were used to measure cyst volumes. Renal and hepatic The amount of pioglitazone added to the chow was based on fibrosis was scored using the picrosirius red staining. Image the estimated animal consumption of the base diets.
analysis procedures were performed without knowledge ofgroup assignments using Meta-Morph software (Universal 2.2. Study Design and Protocol Imaging, West Chester, PA). Stained sections were visualizedand digital images were acquired using a high-resolution 2.2.1. IUPUI. After weaning, at four weeks of age, rodents Nikon Digital camera and displayed on the monitor. Def- were randomly separated into treatment and control groups.
inition of interested structures and exclusion of fields too The treatment group was fed Purina no. 5002 Labdiet supple- damaged to be analyzed were achieved by interactively mented with pioglitazone calculated to provide 20 mg/kg BW applied techniques of enhancement. Colored thresholds were when provided ad libitum. Control rodents were fed unsup- applied at levels which separate cysts from noncystic tissue.
plemented Purina no. 5002 Labdiet. At week 18, rodents Kidney and liver fibrosis were graded as above.
were anesthetized with 100 mg/kg intraperitoneally injectedsodium pentobarbital. Blood was collected via intracardiac 2.4. Electron Microscopy Studies. Small pieces of the perfu- puncture and analyzed for plasma chemistries at the Clarion sion fixed kidney were processed for electron microscopic Pathology Laboratories of the Indiana University School of immunogold labeling in the IU School of Medicine Electron Medicine. After a laparotomy, the organs were flushed with saline, and the left kidney and right liver lobe were collected emcenter.php) using a standard postembedding labeling and frozen in liquid nitrogen. The remaining kidney and technique. Kidney pieces which had visible cysts were liver were infused with 4% paraformaldehyde, removed, and trimmed to 2 × 2 mm segments which were dehydrated stored in 4% paraformaldehyde for histology. Kidney and and embedded in Unicryl (Electron Microscopy Sciences, liver were embedded in paraﬃn and sectioned transversely Hatfield, PA). Thin sections (70–90 nm) were mounted on (4 μM sections).
Formvar/carbon coated nickel grids, rinsed with PBS, andgrids placed in blocking buﬀer (2% BSA, 0.1% Cold Water 2.2.2. Mayo. Treatment with pioglitazone was started at Fish Gelatin, and 0.1% Tween in PBS) for 30–45 minutes.
the time of weaning or three weeks of age. Pioglitazone The grids were then placed in the primary antibody (mouse monoclonal anti-CFTR antibody, 596, 1 : 5, kindly provided by John R. Riordan, University of North Carolina) overnightat 4◦C in a humid chamber. The grids were then rinsedwith PBS and floated on drops of the appropriate secondary antibody with attached 10 nm colloidal gold (AURION,Hatfield, PA) for 2 hours at room temperature and rinsedwith PBS. To more firmly adfix the antibody bindings, grids were placed in 2.5% Glutaraldehyde in 0.1 M Phosphatebuﬀer for 15 minutes. After rinsing in distilled water, thegrids were stained for contrast with uranyl acetate. The samples were viewed with a Tecnai Bio Twin transmissionelectron microscope (FEI, Hillsboro, OR). The primary andsecondary antibodies were diluted in an incubation buﬀercontaining 0.1% BSA-c (AURION), 0.05% Tween in PBS.
2.5. Statistical Analysis. Comparisons between treatment and gender groups were performed using the Student's t-testfor unpaired samples. Two-tailed P-values (P < .05) wereused to denote statistical significance.
Figure 1: Eﬀect of Pioglitazone on renal cysts in PCK rat model.
supplemented with pioglitazone to approximate a daily treatmentof 4 or 20 mg/kg BW as indicated on the figure.
The data presented below are the combination of studiesperformed independently in two institutions. The 7-weekfeeding study was conducted at the Mayo Clinic whilethe 14-week study was performed at Indiana University— eﬀect of pioglitazone was not consistently observed and Purdue University Indianapolis (IUPUI). The results of the reached statistically significance in only certain groups. In the independent studies were combined after completion of seven-week study, pioglitazone was eﬀective on renal disease each series of experiments. Slight diﬀerences in experimental in males on the low dose and on liver disease in females on protocols and measured parameters are a function of the the high dose, while in the fourteen week study the agonist independence of study design between the institutions.
was eﬀect on renal cortical but not medullary fibrosis in the PCK rats were fed either a control diet or a diet female rats (Tables 1 and 2).
supplemented with pioglitazone starting after weaning. Both Table 3 shows the results of serum analyses conducted on male and female animals were used in the 7-week study while the female animals that were fed for 14 weeks. No changes the 14-week study was conducted in female rats. The data in serum parameters were found except an increase in serum are analyzed in a gender-specific manner. The administration albumin. A similar profile was obtained at the 7th week time of pioglitazone at a dose of 20 mg/kg body weight was point (data not shown).
accompanied by a significant decrease in renal cyst burden in To elucidate a mechanism for the action of pioglitazone the male animals after only 7 weeks of pioglitazone treatment in cyst formation, the apical membrane expression of CFTR (Table 1; Figure 1). While the decrease in renal cyst burden in in epithelial cells surrounding cysts was examined using the female rats did not reach statistical significance after 7 weeks monoclonal antibody 596 . Figure 2 illustrates CFTR of feeding (P = .06), there was a significant decrease in the immunoreactivity in cholangiocytes lining the liver cysts.
pioglitazone-fed females after 14 weeks (Table 1; Figure 1).
The CFTR-positive apical membranous staining seen in the Pioglitazone was also eﬀective in decreasing the hepatic cyst control tissue was diminished in the pioglitazone-treated burden after 7 or 14 weeks in female rats, but had no animals. Insuﬃcient CFTR was present in the renal slices for detectable eﬀect on the milder liver cystic disease observed unambiguous detection of changes at the light level although in the male rats after 7 weeks.
the CFTR channel is clearly present based on functional Since PPARγ agonists exhibit dose-dependent, pleomor- studies [6, 9–11].
phic eﬀects, we also determined the eﬃcacy of a lower dose To provide a better quantitative approach, gold-labeled of pioglitazone (4 mg/kg BW) on the development of cystic immunoelectron microscopy was used to assess the density disease. Interestingly, this dose was as eﬀective as the higher of CFTR expression at the apical plasma membrane of the dose in reducing the renal cyst burden in both female and bile duct cysts (Figure 3). Sections from 7 drug-treated and 6 male animals (Table 2). The lower dose also decreased liver control rats such as the ones shown in figure 3 were subjected weight as a percentage of body weight in both male and to analysis by counting the number of gold particles bound female animals.
to the surface of the apical membrane and expressing this There was a trend for pioglitazone to reduce renal and quantity as the number of particles per linear μm of apical hepatic fibrosis. Possibly because fibrosis is mild at early surface. A total of 36.24 linear μm of apical surface was stages of the disease in this animal model, the antifibrotic examined in the treated animals and 35.08 μm was examined Figure 2: Immunostaining for CFTR in the liver of control and pioglitazone-treated PCK rats after 14 weeks of drug treatment. Control (a)or 20 mg/kg BW pioglitazone-treated (b) liver sections were immunostained for CFTR using mAb 596. Shown in both panels is the CFTRstaining (brown) in the apical membranes of cyst-lining biliary epithelial cells. 1000x magnification.
Table 1: Eﬀect of pioglitazone (20 mg/kg body weight) on fibrocystic disease in the PCK rat model.
7-week feeding protocol P-value and significance P-value and significance 239 ± 9.48 224 ± 5.04 347 ± 9.50 328 ± 9.45 Kidney weight (gr) 3.74 ± 0.22 3.27 ± 0.18 6.64 ± 0.37 5.42 ± 0.31 1.56 ± 0.04 1.46 ± 0.06 1.91 ± 0.08 1.65 ± 0.07 Renal cyst vol (ml) 0.39 ± 0.05 0.31 ± 0.09 0.72 ± 0.06 0.31 ± 0.04 Liver weight (gr) 13.4 ± 0.54 11.3 ± 0.43 16.80 ± 0.77 15.39 ± 0.81 5.59 ± 0.12 5.03 ± 0.11 4.83 ± 0.10 4.69 ± 0.18 1.69 ± 0.53 1.25 ± 0.42 2.00 ± 0.32 1.81 ± 0.46 2.44 ± 0.18 2.08 ± 0.38 2.30 ± 0.27 2.19 ± 0.26 14-week feeding protocol P-value and significance 315 ± 8.7 323 ± 9.7 Kidney weight (gr) 4.57 ± 0.13 3.91 ± 0.16 1.46 ± 0.06 1.21 ± 0.04 Renal cyst vol (ml) 0.66 ± 0.06 0.42 ± 0.09 Liver weight (gr) 20.2 ± 0.76 16.0 ± 0.05 6.47 ± 0.35 4.86 ± 0.11 Renal cortical fibrosis 2.69 ± 0.10 2.33 ± 0.11 Renal medullary fibrosis 2.17 ± 0.10 2.09 ± 0.10 2.46 ± 0.07 2.58 ± 0.04 PCK rats were fed on control or pioglitazone-supplemented diet (20 mg/kg BW) from weeks 3–10 (7 weeks) or 4–18 (14 weeks). The values given are averages± SEM. Abbreviations: KW% BW: total kidney weight as a percentage of total body weight; renal cyst vol: the estimated renal cystic volume; LW% BW:liver weight as a percentage of total body weight. Fibrosis was scaled on a 1–4 point scale using deidentified picrosirus red stained slides of transverse kidneysections as described in the methods section. P-values are for the comparison of control versus pioglitazone-supplemented diets by Students t-test. P lessthan .05 is considered significant. NS: not significant.
from the control animals. The pioglitazone-treated tissue animals on the control diet and an average of 1 per 6.04 μm showed a statistically significant decrease (P = .009) in the in the animals treated with pioglitazone.
number of apically localized immunolabeled CFTR antibody An overall decrease in CFTR labeling was also observed particles. The biliary cysts showed an average of 1 gold- in the immuno-gold labeled EM of renal cyst sections labeled particle per 0.23 linear μm of apical surface in the taken from animals treated with pioglitazone for 14 weeks.
Table 2: Eﬀect of pioglitazone (4 mg/kg body weight) on fibrocystic disease in the PCK rat model.
7-week feeding protocol P-value and significance P-value and significance 239 ± 9.48 236 ± 7.89 347 ± 9.50 342 ± 6.31 Kidney weight (gr) 3.74 ± 0.22 3.38 ± 0.19 6.64 ± 0.37 5.53 ± 0.20 1.56 ± 0.04 1.43 ± 0.06 1.91 ± 0.08 1.61 ± 0.05 0.39 ± 0.05 0.30 ± 0.03 0.71 ± 0.06 0.50 ± 0.03 Liver weight (gr) 13.4 ± 0.54 12.4 ± 0.59 16.80 ± 0.77 15.16 ± 0.49 5.59 ± 0.12 5.24 ± 0.11 4.83 ± 0.10 4.42 ± 0.12 1.69 ± 0.53 1.50 ± 0.27 2.00 ± 0.35 1.38 ± 0.44 2.44 ± 0.18 2.31 ± 0.37 2.30 ± 0.27 2.36 ± 0.24 PCK rats were fed on control or pioglitazone-supplemented diet (4 mg/kg BW) from weeks 3–10 (7 weeks). The values given are averages ± SEM.
Abbreviations: KW% BW: total kidney weight as a percentage of total body weight; renal cyst vol: the estimated renal cystic volume in ml; LW% BW: liverweight as a percentage of total body weight. Fibrosis was scaled on a 1–4 point scale using deidentified picrosirus red stained slides of transverse kidneysections as described in the methods section. P-values are for the comparison of control versus pioglitazone-supplemented diets by Students t-test. P lessthan .05 is considered significant. NS: not significant.
Table 3: Serum profiles from PCK female mice—20 mg/kg BW Pioglitazone weeks 4–18.
Pio diet n = 10 P-value and significance 140 ± 0.66 139 ± 0.76 4.92 ± 1.82 4.6 ± 0.53 99.7 ± 0.54 100.5 ± 0.72 10.3 ± 0.14 9.6 ± 0.36 13.6 ± 0.53 12.4 ± 0.48 0.33 ± 0.01 0.34 ± 0.03 162 ± 6.00 146.5 ± 4.59 Alkaline phos. U/L 315 ± 11.18 355 ± 26.67 Alanine aminotransferase U/L 62 ± 3.55 70.0 ± 6.71 136 ± 12.98 139.5 ± 20.5 0.41 ± 0.06 0.32 ± 0.04 Total protein gr/dL 5.5 ± 0.11 5.6 ± 0.11 1.13 ± 0.04 1.39 ± 0.03 Values are given as means ± SEM. Control: PCK rats on the control diet; PIO: PCK rats on a diet containing 20 mg/kg body weight pioglitazone.
n: number of animals P-values greater than .05 were considered nonsignificant (NS).
However, the lower abundance of CFTR expression in the serum concentrations at or below those used to treat insulin renal cysts from both the treated and untreated animals sensitivity . Based on these data, it was hypothesized that a precluded a statistical assessment for an alteration of CFTR PPARγ-mediated decrease in the number of CFTR channels immunoreactivity in the apical membrane.
in the epithelial cells that line PKD kidney and liver cystswould have the eﬀect of decreasing Cl− secretion into the cyst lumen, thereby decreasing the osmotic driving force for fluidaccumulation.
A variety of full and partial PPARγ agonists inhibit Previous studies have examined PPARγ agonist eﬀects vasopressin-stimulated Cl− secretion via CFTR in the on PKD in rodent models. A functional knockout of PKD1, MDCK-C7 (Madin Darby Canine Kidney, clone 7) cell the gene encoding the polycystin 1 protein, resulted in culture model of the principal cells lining the distal nephron.
an embryonic lethal phenotype in mice presenting with The dose response curves for agonist inhibition of transep- hydrops, cardiac conotruncal defects, and renal cystogenesis ithelial Cl− transport paralleled the EC50 for receptor trans- . The embryos show decreased cardiac levels of c-MYC, activation with a log unit leftward shift, suggesting that the and abnormal metabolism of E-cadherin and PECAM-1 in eﬀect on Cl− secretion is very sensitive and manifested within renal tubules, and hemorrhagic diathesis, the most serious Figure 3: Immunostaining for CFTR in the liver of control and pioglitazone-treated PCK rats after 14 weeks of drug treatment. Control (a)or 20 mg/kg BW pioglitazone-treated (b) liver sections were immunostained for CFTR with mAb 596 CFTR antibody followed by a gold-labeled secondary antibody. Shown in both panels is the CFTR staining (spherical dots) in the apical membrane sections of epithelial cellssurrounding the bile duct cysts. 49 000x magnification.
of which is hemorrhagic pericardial eﬀusions. Maternally the strength of the findings lies in the consistency of the final administered pioglitazone at high doses (80 mg/kg/day) improved survival of the embryos by several days, amelio- In the PCK model as little as 7 weeks of pioglitazone rated the cardiac defect and decreased the degree of renal reduced liver cyst burden in the female animals and renal cystogenesis by an unknown mechanism . In a similar cyst burden in the male animals. After 14 weeks on study based on the antineoplastic properties of pioglitazone, a pioglitazone-supplemented diet, there were significant the authors evaluated the drug's eﬀect in PC-Pkd1-KO mice, decreases in both kidney and liver in both sexes. Cross- a rapidly progressing model of ADPKD . Feeding the sectional images of the kidney from littermates on control nursing mothers, and then the pups, did not alter renal or pioglitazone-supplemented diets for 14 weeks indicate function, cell proliferation, apoptosis, or cyst formation.
that the decrease in cyst burden is predominately due to a However, the PPARγ agonist did increase survival and had decrease in cyst size. The "decrease" in cyst size is likely due an antihypertensive eﬀect. Dai and colleagues used the more to a slower growth of the cysts in the presence of pioglitazone.
slowly progressing, Han:SPRD rat model to show that rosigli- Similar to human ADPKD, the PCK rat exhibits some tazone (10 mg/kg BW/day) was able to delay the progression gender dimorphism in the severity of the renal and hepatic of kidney cysts and preserve renal function . The authors pathology . Up to 18 weeks, the pathological alterations suggested that the mechanism of action may be due to are relatively similar in both males and females. Starting PPARγ agonist downregulation of an abnormally activated at about 18 weeks of age, male PCK rats accelerate their β-catenin signaling pathway as well as the anti-inflammatory renal cystic enlargement resulting in a more severe renal and antifibrotic eﬀects of this class of drugs. Cyst formation dysfunction compared to the females [13, 23]. However, in this nonorthologous model may be diﬃcult to correlate female PCK rats develop a greater liver cyst volume density with human disease because the renal cysts are primarily starting at 18 weeks . The gender diﬀerences in eﬃcacy proximal in origin and the model does not demonstrate the during early pioglitazone treatment may be due to subtle, bile duct cyst formation that is common in human PKD.
earlier gender diﬀerences in disease progression.
Our studies, using an orthologous, slowly progressing The serum profiles after 14 weeks of feeding indicate that rat model, address the eﬀects of PPARγ agonist treatment kidney function in these animals is relatively well preserved early in the course of the disease but after the embryonic or despite the cystic burden. The blood urea nitrogen is within neonatal period. This preclinical model more closely mimics the normal range and is not significantly altered by treatment the situation in potential human treatment both in the with pioglitazone. While bilirubin levels are in the normal timing of when the agonists would be administered and in range, liver function as assessed by liver enzymes shows a the nature of the slow disease progression. The data represent compromised hepatic function and this is substantiated by two independent studies that were combined retrospectively.
the low levels of total protein and albumin in the serum.
Although there are diﬀerences in experimental protocols, Pioglitazone treatment did not correct the high levels of liver particularly with regard to the length of the feeding duration, enzymes but did increase the serum albumin concentration.
The eﬀect of pioglitazone on tissue fibrosis is more diﬃ- PKD. In bile duct cysts, the eﬀects were accompanied by cult to assess. In other diseases, the use of PPARγ agonists has a decrease in the apical expression of CFTR, indicating been correlated with decreases in inflammatory responses that the mechanism of action in vivo matches previous and tissue fibrosis, two related processes that contribute cell culture data that show a PPARγ-mediated decrease in to organ function decline in PKD. Recent studies have CFTR expression. In addition, in some studies, the degree shown that PPARγ agonists can reduce fibrosis in tissues as of fibrosis was diminished in pioglitazone-treated animals.
diverse as epidermis , lung [25, 26], kidney [27–30], and These studies provide proof-of-concept for the mechanism liver [31–34]. These agents are eﬀective in multiple fibrotic of action of the PPARγ agonists and suggest that these drugs diseases suggesting a commonality in the drug targets. Since may be eﬀective in controlling both renal and hepatic cyst fibrosis is mild in early stages of disease progression in this growth and fibrosis in PKD.
model of PKD, it is not surprising that our results regardingthe eﬀect of PPARγ agonists are somewhat inconsistent.
Longer term studies will be necessary to clarify potential eﬀects on fibrotic development.
The importance of CFTR activity in renal and bile duct The studies at IUPUI were supported by funds from an cyst formation is based on functional studies. Due to low IUPUI Research Support Fund Grant (B. L. Blazer-Yost), abundance of the protein, few studies have succeeded in and developmental funds from the Department of Pathology detecting the protein in the apical membrane of native renal and Laboratory Medicine (J.-H. Chen). The studies at cysts by immunohistochemical means. The demonstration the Mayo Clinic were supported by funds from Takeda that PPARγ agonists diminish CFTR levels was initially Pharmaceuticals North America, Inc. The authors thank Lee established in cultured cells . The current studies were able Ann Baldridge (Department of Pathology and Laboratory to extend the previous work to native tissue and demonstrate Medicine, Indiana University School of Medicine) for tech- that in biliary cysts, pioglitazone treatment results in a nical assistance with the histochemical and immunohisto- statistically significant lower amount of the CFTR protein chemical stains and to Caroline Miller for expert assistance in the apical membrane of the cholangiocytes lining the with the electron microscopy studies. The authors are cysts. These studies extend the cultured cell experiments and grateful to John R. Riordan (University of North Carolina) provide the proof-of-principle for a mechanism whereby the for kindly providing the CFTR monoclonal antibodies.
PPARγ agonists inhibit the progression of cyst growth.
The idea to use CFTR inhibitors to treat PKD is not new.
Several groups have proposed this approach [35, 36]. Therecent discovery that PPARγ agonists inhibit CFTR synthesis  S. Mudaliar, A. R. Chang, and R. R. Henry, "Thiazolidine- provides additional possibilities using drugs that have already diones, peripheral edema, and type 2 diabetes: incidence, undergone long-term testing in humans. Although the pathophysiology, and clinical implications," Endocrine Prac- PPARγ agonists can have side eﬀects, such as fluid retention tice, vol. 9, no. 5, pp. 406–416, 2003.
and weight gain via adipogenesis, these are relatively mild.
 J. Karalliedde and R. E. Buckingham, "Thiazolidinediones and However, strategies that minimize adverse events are very their fluid-related adverse eﬀects: facts, fiction and putative important when considering treatments for a chronic disease management strategies," Drug Safety, vol. 30, no. 9, pp. 741– such as PKD. Since renal cell culture studies indicate that ago- nist eﬀect on CFTR occurs at concentrations approximately  R. W. Nesto, D. Bell, R. O. Bonow et al., "Thiazolidinedione an order of magnitude lower than those necessary to stabilize use, fluid retention, and congestive heart failure: a consensus insulin action , lower drug doses may still be eﬀective with statement from the American Heart Association and Amer- minimal side eﬀects. Of note, the lower dose of pioglitazone ican Diabetes Association," Circulation, vol. 108, no. 23, pp.
was as eﬀective in our study as the higher dose.
PPARγ agonists alter the density of CFTR expression in  H. Zhang, A. Zhang, D. E. Kohan, R. D. Nelson, F. J. Gonzalez, the plasma membrane but do not interfere with the action of and T. Yang, "Collecting duct-specific deletion of peroxisome vasopressin on water transport or cause polyuria as observed proliferator-activated receptor γ blocks thiazolidinedione-induced fluid retention," Proceedings of the National Academy with vasopressin V2 receptor antagonists. Moreover, PPARγ of Sciences of the United States of America, vol. 102, no. 26, pp.
agonists appear to be eﬀective not only in the kidney but in 9406–9411, 2005.
other tissues where cyst formation and growth are dependent  Y. Guan, C. Hao, D. R. Cha et al., "Thiazolidinediones expand on CFTR-driven ion and fluid secretion. Therefore, their body fluid volume through PPARγ stimulation of ENaC- possible value, either alone or in combination with other mediated renal salt absorption," Nature Medicine, vol. 11, no.
promising therapies, in the treatment of PKD deserves 8, pp. 861–866, 2005.
 C. Nofziger, K. K. Brown, C. D. Smith et al., "PPARγ agonists inhibit vasopressin-mediated anion transport in the MDCK- C7 cell line," American Journal of Physiology, vol. 297, no. 1,pp. F55–F62, 2009.
The studies show that a 7- or 14-week pioglitazone feeding  V. E. Torres and P. C. Harris, "Autosomal dominant polycystic regimen inhibits renal and hepatic bile duct cyst growth kidney disease: the last 3 years," Kidney International, vol. 76, in the PCK rat, a rodent model orthologous to human no. 2, pp. 149–168, 2009.
 V. Patel, R. Chowdhury, and P. Igarashi, "Advances in the  G.-Y. Zhang, T. Cheng, M.-H. Zheng et al., "Activation of pathogenesis and treatment of polycystic kidney disease," peroxisome proliferator-activated receptor-γ inhibits trans- Current Opinion in Nephrology and Hypertension, vol. 18, no.
forming growth factor-β1 induction of connective tissue 2, pp. 99–106, 2009.
growth factor and extracellular matrix in hypertrophic scar  M. Ye and J. J. Grantham, "The secretion of fluid by renal cysts fibroblasts in vitro," Archives of Dermatological Research, vol.
from patients with autosomal dominant polycystic kidney 301, no. 7, pp. 515–522, 2009.
disease," The New England Journal of Medicine, vol. 329, no.
 T. Genovese, S. Cuzzocrea, R. Di Paola et al., "Eﬀect of rosigli- 5, pp. 310–313, 1993.
tazone and 15-deoxy-Δ12,14-prostaglandin J2 on bleomycin-  C. J. Davidow, R. L. Maser, L. A. Rome, J. P. Calvet, and J. J.
induced lung injury," European Respiratory Journal, vol. 25, no.
Grantham, "The cystic fibrosis transmembrane conductance 2, pp. 225–234, 2005.
regulator mediates transepithelial fluid secretion by human  J. E. Milam, V. G. Keshamouni, S. H. Phan et al., "PPAR-γ autosomal dominant polycystic kidney disease epithelium in agonists inhibit profibrotic phenotypes in human lung fibrob- vitro," Kidney International, vol. 50, no. 1, pp. 208–218, 1996.
lasts and bleomycin-induced pulmonary fibrosis," American  R. Mangoo-Karim, M. Ye, D. P. Wallace, J. J. Grantham, Journal of Physiology, vol. 294, no. 5, pp. L891–L901, 2008.
and L. P. Sullivan, "Anion secretion drives fluid secretion  J. E. Toblli, M. G. Ferrini, G. Cao, D. Vernet, M. Angerosa, and by monolayers of cultured human polycystic cells," American N. F. Gonzalez-Cadavid, "Antifibrotic eﬀects of pioglitazone Journal of Physiology, vol. 269, no. 3, pp. F381–F388, 1995.
on the kidney in a rat model of type 2 diabetes mellitus,"  M. N. Muchatuta, V. H. Gattone II, F. A. Wltzmann, and Nephrology Dialysis Transplantation, vol. 24, no. 8, pp. 2384– B. L. Blazer-Yost, "Structural and functional analyses of liver cysts from the BALB/c-cpk mouse model of polycystic kidney  M. S. Joy, D. S. Gipson, M. Dike et al., "Phase I trial of disease," Experimental Biology and Medicine, vol. 234, no. 1, rosiglitazone in FSGS: I. Report of the FONT Study Group," pp. 17–27, 2009.
Clinical Journal of the American Society of Nephrology, vol. 4,  D. J. Lager, Q. Qian, R. J. Bengal, M. Ishibashi, and V. E.
no. 1, pp. 39–47, 2009.
Torres, "The pck rat: a new model that resembles human  S. Ohtomo, Y. Izuhara, S. Takizawa et al., "Thiazolidinediones autosomal dominant polycystic kidney and liver disease," provide better renoprotection than insulin in an obese, Kidney International, vol. 59, no. 1, pp. 126–136, 2001.
hypertensive type II diabetic rat model," Kidney International,  P. C. Harris, "Molecular basis of polycystic kidney disease: vol. 72, no. 12, pp. 1512–1519, 2007.
PKD1, PKD2 and PKHD1," Current Opinion in Nephrology  T. Kawai, T. Masaki, S. Doi et al., "PPAR-γ agonist attenuates and Hypertension, vol. 11, no. 3, pp. 309–314, 2002.
renal interstitial fibrosis and inflammation through reduction  V. H. Gattone II, X. Wang, P. C. Harris, and V. E. Torres, "Inhi- of TGF-β," Laboratory Investigation, vol. 89, no. 1, pp. 47–58, bition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist," Nature Medicine, vol. 9,  F. Marra, R. DeFranco, G. Robino et al., "Thiazolidinedione no. 10, pp. 1323–1326, 2003.
treatment inhibits bile duct proliferation and fibrosis in a rat  V. E. Torres, X. Wang, Q. Qian, S. Somlo, P. C. Harris, and V.
model of chronic cholestasis," World Journal of Gastroenterol- H. Gattone II, "Eﬀective treatment of an orthologous model ogy, vol. 11, no. 32, pp. 4931–4938, 2005.
of autosomal dominant polycystic kidney disease," Nature  I. A. Leclercq, C. Sempoux, P. St¨arkel, and Y. Horsmans, Medicine, vol. 10, no. 4, pp. 363–364, 2004.
"Limited therapeutic eﬃcacy of pioglitazone on progression  T. V. Masyuk, A. I. Masyuk, V. E. Torres, P. C. Harris, and N. F.
of hepatic fibrosis in rats," Gut, vol. 55, no. 7, pp. 1020–1029, Larusso, "Octreotide inhibits hepatic cystogenesis in a rodent model of polycystic liver disease by reducing cholangiocyte  F. Marra, "Thiazolidinediones and hepatic fibrosis: don't wait adenosine 3,5-cyclic monophosphate," Gastroenterology, vol.
too long," Gut, vol. 55, no. 7, pp. 917–919, 2006.
132, no. 3, pp. 1104–1116, 2007.
 R. Belfort, S. A. Harrison, K. Brown et al., "A placebo-  J. R. Nyengaard, "Stereologic methods and their application in controlled trial of pioglitazone in subjects with nonalcoholic kidney research," Journal of the American Society of Nephrology, steatohepatitis," The New England Journal of Medicine, vol.
vol. 10, no. 5, pp. 1100–1123, 1999.
355, no. 22, pp. 2297–2307, 2006.
 L. Cui, L. Aleksandrov, X.-B. Chang et al., "Domain interde-  H. Li and D. N. Sheppard, "Therapeutic potential of cys- pendence in the biosynthetic assembly of CFTR," Journal of tic fibrosis transmembrane conductance regulator (CFTR) Molecular Biology, vol. 365, no. 4, pp. 981–994, 2007.
inhibitors in polycystic kidney disease," BioDrugs, vol. 23, no.
 S. Muto, A. Aiba, Y. Saito et al., "Pioglitazone improves the 4, pp. 203–216, 2009.
phenotype and molecular defects of a targeted Pkd1 mutant,"  B. Yang, N. D. Sonawane, D. Zhao, S. Somlo, and A. S.
Human Molecular Genetics, vol. 11, no. 15, pp. 1731–1742, Verkman, "Small-molecule CFTR inhibitors slow cyst growth in polycystic kidney disease," Journal of the American Society of  K. L. Raphael, K. A. Strait, P. K. Stricklett et al., "Eﬀect of Nephrology, vol. 19, no. 7, pp. 1300–1310, 2008.
pioglitazone on survival and renal function in a mouse modelof polycystic kidney disease," American Journal of Nephrology,vol. 30, no. 5, pp. 468–473, 2009.
 B. Dai, Y. Liu, C. Mei et al., "Rosiglitazone attenuates devel- opment of polycystic kidney disease and prolongs survival inHan:SPRD rats," Clinical Science, vol. 119, pp. 323–333, 2010.
 S. B. Mason, Y. Liang, R. M. Sinders et al., "Disease stage characterization of hepatorenal fibrocystic pathology in thePCK rat model of ARPKD," Anatomical Record, vol. 293, no.
Texas A&M University-Corpus Christi Environmental, Health & Safety Department Policy and Procedures for Using Controlled Substances in Non-Clinical Educational and Research Activities This internal policy regulates the use of controlled substances, controlled substance analogues, chemical precursors and certain chemical laboratory apparatus used in non-clinical educational training and research activities in the Texas A&M University-Corpus Christi Research, Physical and Environmental Science (PENS) and Life Sciences (LSCI) Departments. The Texas Department of Public Safety (DPS) and the Texas Higher Education Coordinating Board (THECB) signed a Memorandum of Understanding (MOU) that establishes responsibilities on institutions of higher education for implementing and maintaining a program for reporting information concerning controlled substances, controlled substance analogues, chemical precursors and chemical laboratory apparatus used in educational training and research activities. This document defines the requirements and procedures necessary for compliance with the MOU by the E,H&S. II.

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