Polycystic kidney disease (PKD) is a common disorder whose pathogenesis is incompletely understood. PKD is primarily characterized by the progressive development of renal epithelial cysts and in most instances results in end-stage renal disease. PKD can be inherited as an autosomal recessive (ARPKD) or autosomal dominant (ADPKD) trait with rapidly or slowly progressive clinical courses, respectively. In addition, PKD can be acquired, as seen in the majority of long-term dialysis patients (1). Two genes responsible for ADPKD have been identified. They encode proteins that are proposed to function in a macromolecular ion channel-receptor complex that transduces signals regulating the state of differentiation of renal epithelial cells (reviewed in [(2,3]). It is not known how mutations in either gene result in cyst formation or in loss of renal function.
Evidence has implicated reduced oxidant protection in the pathogenesis of PKD in several rodent models 4). Mice whose bcl-2 gene has been knocked out develop PKD (5), which may be related to the loss of the antioxidant properties of bcl-2 (6). Treatments that reduce renal antioxidant protection or alter renal redox metabolism can exacerbate PKD in the Han:SPRD-Cy rat model (7,8). Several antioxidant enzyme genes have also been reported as being underexpressed in cystic kidneys of the C57BL/6J-cpk mouse (9).
Antioxidant and detoxicant enzymes play important protective roles in the kidney (10). Primarily because of its transport functions, the kidney has a very active oxidative metabolism that results in the production of reactive oxygen species (ROS). If left unchecked, ROS can damage all major cellular components and lead to a state of oxidative stress. The primary antioxidant enzymes responsible for protection from ROS are superoxide dismutase, catalase, and glutathione peroxidase (GPx). Detoxicant enzymes such as glutathione S-transferase (GST) metabolize toxic electrophiles and are considered to be secondary antioxidant enzymes (11). Studies in which rats were fed an antioxidant-deficient, pro-oxidant diet have demonstrated the significance of an adequate antioxidant defense in both normal and injured kidneys (12,13). Oxidant injury is now recognized as playing a key role in the pathophysiologic pathways of a wide variety of progressive clinical and experimental renal diseases (14).
Reduced antioxidant enzyme expression in cystic kidneys might result in compromised protection against oxidative injury that could lead or contribute to renal dysfunction and progression to renal failure. To test this hypothesis, we assessed markers of oxidative stress and oxidant injury, and we examined the mRNA and protein expression of extracellular GPx (EGPx) during the development of PKD in the cpk mouse and the Cy rat. These models, with different modes of transmission and rates of disease progression, have been studied extensively as representative of human ARPKD and ADPKD (15,16). The study presented here demonstrates that cystic kidneys of cpk mice and Cy rats have reduced enzymatic protection against oxidative attack and that they experience oxidative damage. These results suggest that reduced renal antioxidant protection and oxidant stress are pathogenetic mechanisms common to all forms of PKD.
Materials and Methods
Colonies of C57BL/6J-cpk mice and Han:SPRD-Cy rats are maintained at the University of Kansas Medical Center. Animal treatment was in accord with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Tissue Collection and Processing
Plasma from heparinized blood was used for EGPx enzyme assays and Western blot analyses. After perfusion with phosphate-buffered saline, kidneys or liver were removed and homogenized in either guanidinium thiocyanate buffer (RNA isolation) (17), in 5 times the organ weight of 20 mM Tris, pH 7.5, 5 mM ethylenediaminetetraacetate (EDTA), and 0.1 mM phenylmethylsulfonyl fluoride (renal EGPx Western blot test), or in 50 mM KPO4, pH 7.0, and 1 mM EDTA (liver GPx enzyme assay). Quantitation of protein was by the Bio-Rad DC assay kit (Bio-Rad, Hercules, CA) after solubilization of extracts by addition of sodium dodecyl sulfate to 2%.
Northern Blot Analyses
Total RNA was isolated by the method of Chomczynski and Sacchi (17). RNA samples (5 to 20 μg) were electrophoresed in formaldehyde gels and blotted for hybridization with antisense RNA probes as previously described (18). The mouse EGPx cDNA clone was isolated as described previously (18). GST-Ya, catalase, and MnSOD cDNA were produced by reverse transcriptase–PCR that used mouse kidney RNA and were confirmed by sequencing. The mouse heme oxygenase-1 (HO-1) cDNA clone was obtained from S. Sakiyama. As a quantitation control, all blots were rehybridized with an oligonucleotide probe for 18S rRNA as described previously (18).
Western Blot Analyses
EGPx immunoanalyses were performed by means of an anti-rat EGPx antibody as described previously (18). Equal amounts of total kidney protein or equal volumes of blood plasma from individual animals of a specific age were electrophoresed in duplicate sodium dodecyl sulfate–polyacrylamide gels. One gel was stained with Coomassie blue to assess protein loading; the other gel was electroblotted, probed with the anti-EGPx antibody, and developed with the chemiluminescent substrate CDP-Star (Tropix, Bedford, MA). Each age group, representing a single litter of rats or mice, was analyzed separately.
Paraffin-embedded sections of paraformaldehyde-fixed kidneys were used. After the paraffin was removed, the sections were incubated with H2O2 to remove endogenous peroxidase activity, then blocked with 2% dry milk/phosphate-buffered saline or with Terminator Block (BioCare, Walnut Creek, CA; anti-Mn SOD only). Sections were incubated with diluted rabbit anti–HO-1 (1:1000; Stressgen, Victoria BC, Canada), anti–GST-α (1:1000; Alpha Diagnostics, San Antonio, TX), or anti-MnSOD (1:2000; Stressgen) serum in 3% normal goat serum overnight at 4°C. Sections were incubated with biotinylated secondary antibody (Histostain-SP kit; Zymed, San Francisco, CA) and streptavidin–horseradish peroxidase conjugate, then developed with aminoethyl carbazole followed by hematoxylin counterstaining. For controls, the primary antibody was replaced with an appropriate dilution of normal rabbit serum (all antibodies) or was competed by preincubation with recombinant HO-1 protein (anti–HO-1 only). Controls were consistently negative.
GPx Enzymatic Assay
EGPx activity in plasma samples (10 to 15 μl) was measured at 37°C by following the oxidation of nicotinamide adenine dinucleotide phosphate at 340 nm in an SLM Aminco 3000 Array spectrophotometer (Milton Roy) with either H2O2 or tert-butylperoxide as substrate as described by Avissar et al. (19). Cellular GPx activity was measured in 100,000 × g liver supernatants by use of H2O2 as described by Lawrence and Burk (20). Assay reagents were from Sigma Chemical Co. (St. Louis, MO).
Lipid Peroxidation Assay
Combined levels of malondialdehyde (MDA) and 4-hydroxy-2(E)-nonenal (HNE) were measured in plasma and kidney extracts from mice by means of a lipid peroxidation assay kit (Calbiochem, La Jolla, CA) according to the manufacturer’s instructions. Blood was collected; EDTA was used as the anticoagulant. Plasma was prepared and used in the assay within 1 h of collection. Whole kidneys from animals perfused with phosphate-buffered saline were homogenized in 20 mM Tris-HCl, pH 7.4, and supernatants from a 3000 × g spin were used in the assay.
For quantitation, autoradiographic films and stained protein gels were scanned and analyzed by NIH Image software (available at http://rsb.info.nih.gov/nih-image/). Statistical analyses included unpaired t test with F test for equality of variances (enzymatic activity) and two-way (age × phenotype) ANOVA with Levene’s test of error variances (protein measurements). Spearman’s rank correlation was used to study the relationship between renal and plasma EGPx. P < 0.05 was considered significant.
Previous observations suggested that cystic kidneys have an altered redox metabolism and may be in a state of chronic oxidative stress (8,21). To assess oxidative stress, the expression of HO-1, a widely accepted marker of oxidative stress (22), was examined in normal and cystic kidneys of both the C57BL/6J-cpk mouse and the Han:SPRD-Cy rat. Cystic disease in these animal models is most likely the result of mutations in different genes (23,24), and disease severity is determined by gene dosage. Homozygous cpk/cpk mice develop cystic kidneys rapidly after birth and die of azotemia by 3 to 4 wk of age (15), but heterozygous cpk/+ mice experience no renal abnormalities (25). Homozygous cystic (Cy/Cy) rats also develop a rapidly progressive form of PKD, resulting in death at approximately 3 wk of age; heterozygous cystic (Cy/+) rats develop a more slowly progressive PKD that is sexually dimorphic (16). Cystic disease in male Cy/+ rats is more severe, characterized by larger kidneys, earlier development of azotemia (approximately 8 wk of age), and death at approximately 1 yr of age, in contrast to Cy/+ females, which do not die as a result of renal failure until 2 yr of age (26).
Northern blot analysis showed that HO-1 mRNA levels are increased in cystic cpk kidney at 14 and 21 d of age, and possibly as early as 9 d (Figure 1, A and B). HO-1 mRNA levels are also dramatically increased in Cy/Cy rat kidney at 2 and 3 wk of age and in male Cy/+ kidney at 8 wk of age (Figure 1, C and D). In contrast, HO-1 mRNA levels are only slightly increased in female Cy/+ kidney at 8 wk of age. Thus, HO-1 induction indicates that oxidant stress occurs in cystic kidneys and serves as a measure of disease severity in these two models.
A state of oxidative stress can develop when there is an excessive generation of ROS or an inadequate antioxidant defense. Previous work suggested that the expression of numerous protective enzyme genes was reduced in cystic kidneys of cpk mice (9). Therefore, mRNA levels of the secreted GPx (EGPx), the mitochondrial Mn superoxide dismutase (MnSOD), the peroxisomal catalase, and the cytosolic α-class GST (GST-Ya) were examined in normal and cystic kidney of cpk mice (Figure 2). Expression of each of the mRNAs is significantly reduced in cystic kidney by 14 d of age and is further reduced by 21 d (Figure 2E). To determine whether the reduced expression of these genes is kidney specific, GST-Ya, MnSOD, and catalase mRNA were examined in liver from 3-wk-old normal and cystic mice. No differences in hepatic levels of these mRNA were detected (data not shown).
To determine whether reduced expression of these protective enzymes is a feature common to both rapidly and slowly progressive PKD, their expression was examined in Cy rat kidney (Figure 3). Cystic kidneys from male and female Cy/Cy rats have dramatically reduced levels of EGPx, MnSOD, catalase, and GST-Ya mRNA at 3 wk of age. EGPx and GST-Ya mRNA are also decreased at 2 wk (data not shown; MnSOD and catalase were not examined). In the kidneys of Cy/+ rats, reduced expression of all 4 mRNA is detectable as early as 3 wk in males. At 8 wk, all 4 antioxidant mRNA are reduced in kidney from male and female Cy/+ rats; however, the reduction is more evident in males. Thus, in the early stages of cystic disease in the rat, these protective enzymes are underexpressed, and their degree of underexpression appears to correlate with the severity of PKD. Furthermore, these results demonstrate that reduced antioxidant enzyme expression is not simply a reflection of the tubule segment affected because cysts develop in different segments in these models—that is, the collecting ducts of the cpk/cpk mouse (15), the proximal tubules of the Cy/+ rat, and all tubule segments of the Cy/Cy rat (26).
To gain insight into where the changes in HO-1 and antioxidant enzyme gene expression are occurring, cystic kidneys from 2-wk-old cpk mice and from 8-wk-old Cy/+ rats were immunohistochemically stained for HO-1, GST, or MnSOD proteins (Figure 4). In cpk kidney, HO-1 is expressed in some smaller cortical cysts (most likely proximal tubular cysts) and in many nondilated cortical tubules, but is largely absent from the large collecting duct cysts. A similar staining pattern is observed for GST (Figure 4B) and MnSOD (data not shown) in the cpk kidney. Within Cy/+ rat kidneys, HO-1 protein is found in the majority of nondilated cortical tubules and in cysts, but it is reduced or absent in cystic epithelial cells overlying a thickened basement membrane (Figure 4C). GST (data not shown) and MnSOD (Figure 4D) staining is similar to that for HO-1. This immunoanalysis demonstrates that in cystic kidney of both the cpk mouse and the Cy rat, changes in HO-1 and antioxidant protein expression primarily involve the cystic epithelium with a loss or reduction of staining within many cysts. In addition, a dramatic upregulation of HO-1 is observed in normal-appearing tubules.
To determine whether decreased antioxidant mRNA levels are accompanied by reductions in protein, we selected EGPx, a secreted enzyme that catalyzes the reduction of hydrogen peroxide, alkyl hydroperoxides, and phospholipid hydroperoxides (reviewed in ). EGPx activity is present in many extracellular fluids, including plasma. Studies in anephric humans and rats have demonstrated that at least 70% of the EGPx plasma enzymatic activity is produced by the kidney (28). As such, plasma levels of EGPx should provide an additional indication of the renal production of this enzyme. The levels of EGPx protein were examined by Western blot analysis in kidney and plasma of normal and cystic mice and rats at various ages. In mice, an overall significant effect of phenotype is observed for renal (P < 0.0001) and for plasma (P = 0.0029) EGPx protein levels (Figure 5). Plasma EGPx levels reflect renal levels, as shown by a significant positive correlation (Figure 5C; rs = 0.728, P = 0.0036), consistent with a reduced renal production in the cystic mice. Renal and plasma EGPx protein levels were reduced in cystic rats at 6, 8, and 10 wk of age (Figure 6), and there was an overall significant effect of phenotype for both (P < 0.01).
To determine whether the reduced levels of EGPx protein result in decreased enzymatic activity, EGPx enzyme assays were performed with plasma from 20-d-old mice (Table 1). Plasma EGPx activity is significantly reduced in cystic mice. GPx family members are selenium-dependent enzymes, and selenium bioavailability has been shown to affect GPx mRNA, protein, and activity levels (reviewed in [(27]). To rule out defective selenium metabolism as a mechanism for reduced EGPx production, we examined hepatic enzymatic activity and renal and hepatic mRNA levels of the ubiquitous, cellular form of GPx (cGPx), a sensitive biomarker of selenium status. Although cGPx mRNA was reduced in cystic kidneys, cGPx mRNA and enzymatic activity levels were not affected in livers of 19- to 21-d-old cystic mice (data not shown).
Reduced antioxidant enzyme mRNA, protein and activity levels, and increased HO-1 mRNA levels suggest that there is increased ROS and resulting oxidative damage in cystic kidneys. Membrane lipids are a frequent target of ROS and can be oxidized to form lipid hydroperoxides. If not reduced by enzymes (such as GPx), lipid hydroperoxides can undergo further reactions to produce various aldehydes, including MDA and HNE (29). The combined MDA + HNE levels were measured in cpk mice (Figure 7) and were found to be greatly elevated in cystic kidney tissue from mice at 17 and 19 d of age and in plasma from cystic mice at 19 d of age. These results demonstrate that oxidative damage is occurring in cystic mice.
The study presented here establishes that there is reduced antioxidant enzyme protection and increased oxidative stress in two different models of cystic disease and thereby solidifies the view that oxidant or xenobiotic stress and tissue injury are pathogenetic factors in the progression of PKD (4,30). Early observations of the development of cystic disease in long-term dialysis patients (1) and in deconditioned germfree rats treated with nordihydroguaiaretic acid (31) led to the idea that environmental insults can result in renal cyst formation. Later observations in numerous animal models suggested that oxidant mechanisms may be at work in cystic disease (4). Cystic kidneys from Cy/+ rats and bcl-2 knockout mice have elevated markers of oxidative stress (7,21,32). Treatments that increase renal production of oxidants (33,34) exacerbate PKD in the Cy rat (8,35).
Other studies have made connections between reduced antioxidant protection and the development (36) or progression (7) of PKD in the rat. Antioxidant promoting treatments (37) have been shown to attenuate cystic disease in mouse (38) and rat (39,40) models. Finally, connections between human PKD and reduced protection or oxidant stress exist. ADPKD patients are reported to have reduced plasma EGPx protein and enzyme activity (41) and increased plasma lipid peroxidation products (42). Cultured ADPKD renal epithelial cells are more sensitive to oxidant treatment than normal renal epithelial cells (43). In light of the data presented in this report, it is likely that this increased oxidant sensitivity is the result of decreased antioxidant enzyme expression in the ADPKD cells.
The study presented here provides direct evidence for oxidative stress, as shown by induction of the HO-1 gene in the cystic kidneys of two different animal models of ARPKD and ADPKD and by elevated lipid peroxidation levels in cpk mice. Levels of HO-1 induction correlate with gene dosage and severity of cystic disease in that HO-1 induction is higher in homozygous cystic rats and mice with rapidly progressive disease and lower in heterozygous cystic rats with more slowly developing disease. Female Cy/+ rats that develop relatively mild PKD have the lowest HO-1 induction levels. The fact that HO-1 induction is evident at early stages of PKD suggests that oxidative stress plays a role relatively early in disease progression. As such, the results presented here, together with the aforementioned studies, strongly suggest that oxidative stress may be a general pathogenetic feature in all cystic diseases.
How might oxidative stress be involved in the pathogenesis of PKD? Renal oxidative stress due to decreased oxidant protection has been shown to result in increased cell proliferation, increased extracellular matrix synthesis, increased inflammatory cell infiltration, and increased apoptosis (44), pathogenic features all commonly attributed to cystic kidneys (reviewed in [1,3]). As such, one could envision that the level of oxidative stress and resulting macromolecular damage could determine the rate of progression of PKD and resultant loss of renal function. In extrapolating our observations to human ADPKD, where cystogenesis is proposed to result from a second somatic mutation in one of the PKD genes (reviewed in [2,45]), we speculate that renal oxidative stress could result in DNA damage, which might increase the rate of second hits and thereby the severity of PKD.
We have investigated the possibility that reduced renal antioxidant enzyme protection may contribute to oxidative stress in cystic kidneys. Renal levels of EGPx, MnSOD, catalase, and GST-Ya mRNA are reduced in cystic mice and rats. The levels of antioxidant enzyme mRNA reduction appeared to reflect the severity of cystic disease and to coincide with the induction of HO-1 mRNA. EGPx is also reduced at the protein level in the plasma and kidneys of both cystic mice and rats, and at the enzymatic activity level in plasma from cystic mice. Primarily thought of as a plasma enzyme, whose major site of production and secretion are renal proximal tubules, recent studies have demonstrated a protective function for EGPx within the kidney. Treatments that reduced renal EGPx protein production were shown to result in oxidative damage in the kidney (46). Renal EGPx overproduction in transgenic mice protected their kidneys from damage in ischemia/reperfusion experiments as evidenced by decreased blood urea nitrogen levels, decreased tubular necrosis and apoptosis, and decreased lipid peroxidation (47). In the study presented here, elevated levels of MDA + HNE are observed in the kidney and plasma of cystic mice. Because lipid hydroperoxides are substrates for EGPx, and are precursors of MDA and HNE, it is quite possible that there is a link between reduced EGPx and the increased lipid peroxidation observed in cystic mice.
The results in this report imply that lack of appropriate expression of protective enzyme genes and oxidative stress are general pathologic mechanisms of cystic disease progression. Because reduced antioxidant enzyme expression and increased HO-1 expression were observed in two different animal species with cystic disease caused by mutations in different genes and affecting different tubule segments, it appears that these changes are general consequences of renal cyst formation, possibly as the result of widespread changes in renal tubular metabolism and function activated by the cystogenic process. This idea is supported by the observed loss of antioxidant enzyme expression in cystic tubules and the observed induction of HO-1 protein in cystic and nondilated tubules of polycystic kidneys.
What could be responsible for the reduction of antioxidant protection in cystic kidneys? One important feature common to all forms of PKD is that cystic epithelial cells are abnormally or incompletely differentiated (30). A number of antioxidant enzymes are developmentally expressed in the rodent kidney, first appearing in fetal kidney tubules that are somewhat differentiated and then increasing in expression during postnatal development (18,48,49). The differential staining for antioxidant enzymes within cysts, which was particularly evident in the previously described, immature cystic epithelial cells overlying thickened basement membrane in the rat kidneys (50), suggests that cellular immaturity may result in loss of antioxidant enzyme expression. It is possible that the primary gene defect in inherited PKD results in abnormally differentiated renal epithelial cells that are incapable of producing or maintaining the normal complement of protective enzymes. Such a situation could then lead to renal oxidative stress as the cystic kidney attempts to meet its metabolic demands, resulting in a vicious cycle of oxidative damage, renal injury, and injury-induced dedifferentiation, eventually leading to apoptosis, loss of renal function, and kidney failure.
This work was supported by the Polycystic Kidney Disease Foundation (grant 99008 to RLM) and the National Institutes of Health (grant DK57301 to RLM and JPC). We thank Benjamin D. Cowley Jr. for providing Cy rat Northern blot analyses for initial analysis, S. Sakiyama for the mouse HO-1 cDNA clone, S. Yoshimura for the chicken anti-rat EGPx antibody, Rosetta Barkley for expert histology work, Jared J. Grantham for critical discussions, and John M. Belmont for statistical analysis and editorial assistance.
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