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  • 1. BMC Nephrology BioMed CentralResearch article Open Access Podocyte specific knock out of selenoproteins does not enhance nephropathy in streptozotocin diabetic C57BL/6 mice Marsha N Blauwkamp1,2, Jingcheng Yu1, MaryLee A Schin3, Kathleen A Burke3, Marla J Berry4, Bradley A Carlson5, Frank C Brosius III3 and Ronald J Koenig*1,2Address: 1Division of Metabolism, Endocrinology and Diabetes, University of Michigan, Ann Arbor, MI 48109-5678, USA, 2Cell and Molecular Biology Graduate Program, University of Michigan, Ann Arbor, MI 48109-5619, USA, 3Division of Nephrology, University of Michigan, Ann Arbor, MI 48109-5676, USA, 4Department of Cell and Molecular Biology, University of Hawaii at Manoa, Honolulu, HI 96813, USA and 5Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, NationalInstitutes of Health, Bethesda, MD 20892, USA Email: Marsha N Blauwkamp - mousman@umich.edu; Jingcheng Yu - jingyu@umich.edu; MaryLee A Schin - mschin@umich.edu; Kathleen A Burke - kathleen_burke@brown.edu; Marla J Berry - mberry@hawaii.edu; Bradley A Carlson - carlsonb@mail.nih.gov; Frank C Brosius - fbrosius@umich.edu; Ronald J Koenig* - rkoenig@umich.edu * Corresponding author Published: 22 July 2008Received: 7 January 2008Accepted: 22 July 2008 BMC Nephrology 2008, 9:7doi:10.1186/1471-2369-9-7 This article is available from: http://www.biomedcentral.com/1471-2369/9/7 2008 Blauwkamp et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.AbstractBackground: Selenoproteins contain selenocysteine (Sec), commonly considered the 21stgenetically encoded amino acid. Many selenoproteins, such as the glutathione peroxidases andthioredoxin reductases, protect cells against oxidative stress by functioning as antioxidants and/orthrough their roles in the maintenance of intracellular redox balance. Since oxidative stress hasbeen implicated in the pathogenesis of diabetic nephropathy, we hypothesized that selenoproteinsprotect against this complication of diabetes.Methods: C57BL/6 mice that have a podocyte-specific inability to incorporate Sec into proteins(denoted in this paper as PodoTrsp-/-) and control mice were made diabetic by intraperitonealinjection of streptozotocin, or were injected with vehicle. Blood glucose, body weight,microalbuminuria, glomerular mesangial matrix expansion, and immunohistochemical markers ofoxidative stress were assessed.Results: After 3 and 6 months of diabetes, control and PodoTrsp-/- mice had similar levels of bloodglucose. There were no differences in urinary albumin/creatinine ratios. Periodic acid-Schiff stainingto examine mesangial matrix expansion also demonstrated no difference between control andPodoTrsp-/- mice after 6 months of diabetes, and there were no differences inimmunohistochemical stainings for nitrotyrosine or NAD(P)H dehydrogenase, quinone 1.Conclusion: Loss of podocyte selenoproteins in streptozotocin diabetic C57BL/6 mice does notlead to increased oxidative stress as assessed by nitrotyrosine and NAD(P)H dehydrogenase,quinone 1 immunostaining, nor does it lead to worsening nephropathy.Page 1 of 7(page number not for citation purposes)

2. BMC Nephrology 2008, 9:7 http://www.biomedcentral.com/1471-2369/9/7 Backgroundnull mice found that Gpx1 was not protective against Selenium, a trace element, is found in the amino acid renal injury [15]. selenocysteine (Sec) and is cotranslationally incorporated into the protein polypeptide chain via the codon UGA. C57BL/6 mice are relatively resistant to the development Although UGA generally signals translation termination, of diabetic nephropathy [16]. Given the evidence support- mRNAs that contain a conserved SECIS element in their 3'ing a role of oxidative stress in diabetic nephropathy and untranslated regions are able to decode UGA as Sec [1]. the role of selenoproteins in protecting against oxidative Sec is synthesized on a unique tRNA [2,3], termed stress, we postulated that podocyte selenoproteins protect tRNA[Ser]Sec because it is first aminoacylated by serine, against the development of diabetic nephropathy in which is then converted to selenocysteine. Since this is theC57BL/6 mice. To test this hypothesis, we created a podo- only tRNA that supports Sec incorporation into proteins,cyte specific knock out of all selenoproteins (PodoTrsp-/-) the absence of tRNA[Ser]Sec results in protein chain termi- in C57BL/6 mice, induced diabetes with STZ, and exam- nation instead of Sec incorporation. As discussed below,ined the mice for progression of diabetic nephropathy. selenoproteins are enzymes with Sec in the active site. Contrary to our hypothesis, we found the PodoTrsp-/- Therefore, even if a truncated protein lacking Sec is stable, mice did not develop increased nephropathy. it will not be biologically active. Thus, the absence of tRNA[Ser]Sec results in complete functional selenoprotein Methods deficiency. Whole mouse homozygous deletion of theTargeted Inactivation of the Selenocysteine tRNA[Ser]Sec tRNA[Ser]Sec gene Trsp is embryonic lethal [4]. However, thegene Trsp in Podocytes generation of mice carrying Trsp alleles flanked by loxPC57BL/6 transgenic mice expressing Cre recombinase sites has allowed the study of organ-specific deletion of driven by the 2.5 kb human podocin (NPHS2) promoter selenoprotein synthesis [5].were obtained from L.B. Holzman (University of Michi- gan, Ann Arbor, MI) [17]. The expression of Cre recombi- The human genome encodes 25 selenoproteins and thenase in these mice does not cause glomerular mouse genome 24 [6]. Many selenoproteins function asabnormalities [17-19]. Podocin-Cre mice were mated antioxidant enzymes or in redox signaling. Examples ofwith C57BL/6 mice in which both Trsp alleles are flanked selenoproteins with these activities include the glutath- by loxP sites, denoted TrspL/L [5]. The resulting podocin- ione peroxidases (Gpx) and thioredoxin reductases (Trxr)Cre;TrspL/+ mice were mated with TrspL/L mice to generate (reviewed in [7]). Other selenoproteins such as seleno- podocin-Cre;TrspL/L mice, denoted in this paper as phosphate synthetase and selenoprotein P indirectly sup-PodoTrsp-/-. Littermates of the genotype TrspL/L were used port those activities by functioning in Sec synthesis and as controls. Genotyping was performed by PCR using the selenium transport and storage [7]. following oligonucleotide primers for Trsp: 5'-CAA AAC CTC GCC TCC AAG TGA C-3' and 5'-TGT GAG ACG ACC Superoxide is a highly reactive and potentially toxic oxi-TTC TAT GCT CG-3'; and for Cre: 5'-GCG GTC TGG CAG dant produced during mitochondrial respiration and by TAA AAA CTA TC-3' and 5'-GTG AAA CAG CAT TGC TGT several cytoplasmic enzymes such as NAD(P)H oxidase.CAC TT-3'. The PCR program used for Trsp detection is as In diabetes, hyperglycemia induces overproduction offollows: step 1, 95C for 5 min; step 2, 95C for 15 sec; superoxide via the mitochondrial electron transport chain step 3, 64C for 30 sec; step 4, 68C for 2 min; step 5, 30 as well as by increased NAD(P)H oxidase activity [8] lead-repetitions of steps 2 to 4. The PCR program used for Cre ing to oxidative stress [9]. Oxidative stress is thought to detection is as follows: step 1, 95C for 5 min; step 2, play an important role in the progression of diabetic com-94C for 30 sec; step 3, 51C for 45 sec; step 4, 72C for plications, including nephropathy [10]. Podocyte1 min; step 5, 35 repetitions of steps 2 to 4. All animal care (glomerular epithelial cell) damage is central to the devel-and handling procedures were approved by the University opment of diabetic nephropathy [11]. An increase in of Michigan Committee on Use and Care of Animals. glomerular oxidative stress occurs early in diabetic neph- ropathy and enhanced mitochondrial and cytoplasmicInduction of diabetes oxidant stress leads directly to apoptosis in podocytes Ten week old male PodoTrsp-/- and TrspL/L (as controls) exposed to high extracellular glucose [12]. It has been mice were fasted for 4 hours and then injected intraperito- shown that injury to diabetic kidneys is reduced in trans-neally with 50 mg/kg STZ (Sigma) or vehicle for 5 consec- genic mice that over express superoxide dismutase utive days according to the low dose protocol of the (SOD1) [13]. In addition, streptozotocin (STZ) diabetic Animal Models of Diabetic Complications Consortium mice developed increased oxidative stress and kidneyhttp://www.amdcc.org. STZ was prepared in freshly made damage when subjected to a selenium deficient diet [14].100 mM sodium citrate buffer pH 4.5 at 7.5 mg/ml and However, a recent study of diabetic Gpx1 homozygous used within 15 minutes. For each experiment, the number of mice used is stated in the figure legend. Page 2 of 7 (page number not for citation purposes) 3. BMC Nephrology 2008, 9:7http://www.biomedcentral.com/1471-2369/9/7ImmunohistologyHistology and Morphometry Immunoperoxidase staining for Wilms' tumor homolog PLP-fixed tissue paraffin sections (3 m) were stained (Wt1) and Gpx1 was performed on 2% paraformalde- with periodic acid-Schiff's (PAS) reagent to identify kid- hyde-lysine-periodate (PLP) fixed kidney cryostat consec-ney structure and hematoxylin to visualize cell nuclei. utive sections (3 m). PLP-fixed tissue sections wereMicroscopic images of glomeruli (10 images per sample) incubated for 2 hrs at 90C in Retrieve-All 1 (Covance), were taken at 20 magnification and used for calculating followed by 3% hydrogen peroxide in methanol for 30the mesangial matrix cell volume per glomerular tuft. The mins and 1.5% normal rabbit serum in 3% BSA, 0.1%mean area of each glomerular tuft was measured by man- Tween 20 in 1 PBS for 20 mins accordin