Beese, M.

Borley, K.

Elger, M.

Haller, H.

Hentschel, H.

Kirsch, T.

Litteral, J.

Wortmann, J.

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Evaluating mechanisms of nephrogenesis on tissue culturesfrom adult Little Skate, Leucoraja erinacea kidneyJennifer Litteral1, Torsten Kirsch2, Jessica Wortmann2, Michaela Beese2,Kimberly Borley3, Hartmut Hentschel4, Hermann Haller2, and Marlies Elger2.1Mount Desert Island Biological Laboratory, Salisbury Cove, Maine2Department of Nephrology, Hannover Medical School, Germany3Ohio University, Athens, Ohio4Max-Planck-Institute for Molecular Physiology, Dortmund, GermanyThe Little Skate, Leucoraja erinacea possesses a nephrogenic zone in the adult kidney, whichresembles the embryonic metanephric kidney and contains stem cell-like mesenchymal cells1.Although understanding the underlying mechanisms of renal regeneration in Leucoraja erinacea ishighly important, our aim was to establish a serviceable animal model for nephrogenesis.  We want toutilize kidney cultures from the Little Skate and ultimately evaluate outside stimulation on thisnephrogenic zone. We first evaluated differences in culturing media and established a method toevaluate these effects. We have previously shown that nephrogenic tissue survives well in culture andthat proliferation of the developing stages and young tubules was vigorous as seen by the incorporationof 5-bromo-2-deoxy uridine2. We have also shown similarity between the adult skate nephrogenic zoneand the developing mammalian kidney by cloning a fragment of a gene known to be important in earlymammalian nephrogenesis from skate, the extracellular signaling factor Wnt43. To evaluate the effectsof different media on the nephrogenic zone of the little skate we compared the relative expressionlevels of Wnt4 with cultured and non-cultured kidney tissue using quantitative real-time PCR.Small tissue pieces were micro-dissected from the kidney of Little Skate, Leucoraja erinacea. Thefirst piece of tissue was flash frozen in liquid nitrogen to ensure we had a base line for each tissuepiece that was an experiment. These experimental pieces were then washed in culture medium (VCM)(Valentich, J. Tissue Cult. Method 13:149-162, 1991) containing: 1:1 DME (Delbecco’s ModifiedEagle’s Medium), 100mM NaCl, 3.9mM CaCl2, 2.5mM MgCl2, 350mM Urea, 72mM TMAO, 8mMNaHCO3, pH of 7.8 and an osmolarity of 1000 mOsm/kg. The washing medium was supplementedwith 1X antibiotic antimycotic solution containing penicillin, streptomycin, and amphotericin B(Sigma).  After washing, the tissue pieces were transferred into sterile six well tissue culture plateswith 5 ml of medium. The first medium (V) contained VCM only. The second medium (VI), 10XInsulin-Transferrin-Sodium Selenite liquid media supplement (ITS, Sigma) was added to VCM.Medium three (VS), filtered shark serum was added to VCM. The forth medium (VIS), both ITS andshark serum was added to VCM. The plates were placed at 17˚C in a Thermo Forma CO2/O2 incubatorwith a water jacket and a re-circulating refrigerated water bath and the tissues were gassed with 1%CO2 and ambient O2 levels.  The medium was changed every 48 hours.  After one week the cultureswere flash frozen in liquid N2. RNA was extracted using Trizol, Dnase Digested (Ambion) andtranscribe into cDNA (Promega). We utilized the ABI Prism 7700 Taqman Real-Time PCR to analyzethe relative mRNA expression.  Specific primers from Wnt4 were previously designed and tested.  Theamount of cDNA in the PCR reaction was quantified using SYBR Green chemistry (Invitrogen). Fornormalization of the results, the expression of B-actin was used as a reference point. Standard thermalcycle conditions for the ABI Prism 7700 Taqman were followed. The fluorescence intensity wasgenerated in real-time data during PCR amplification. The data was then imported into an Excelspreadsheet and analyzed.00.10.20.30.40.50.60.70.80.911Wnt4 Cultures With Media Only2v2vi2vs2vis2baseFig 1. Real-time PCR data of Wnt4 expression from skate developing kidney cultures in different culture media. 2V: VCMmedia only, 2VI: VCM plus ITS, 2VS: VCM with shark serum, 2VIS: VCM media with ITS and shark serum 2Base: Baseline was frozen tissue not cultured.Our preliminary data showed that there is very little difference between these four media.  The dataalso showed the highest level of expression of Wnt4 in the baseline tissues. Since the expression ofWnt4 decreases after it is placed in media that has been shown to sustain kidney tissue other genesshould also be investigated. In conclusion, we are able to asses effects on tissue cultures using real-time PCR and specific gene expression levels.Support from NSF DBI-0139190.1. Elger M, Hentschel H, Litteral J, Wellner M, Kirsch T, Luft F, Haller H. Neprhogenesis is induced by partial    nephrectomy in the elasmobranch Leucoraja erinacea. J Am Soc Nephrol, 14:1506-1518, 2003.2. Elger M, Litteral J, Renfro JL, Waldron P, Sheldon A, Haller H, Hentschel H. In vitro culture of  renal tissue of the little    skate, Leucoraja erinacea. MDIBL Bulletin 42: 105-106, 2003.3. Litteral J, Kirsch T, Elger M, Hentschel H, Weber G, Raymond R, Sheldon A, Waldron P, Haller H. Expression and    distribution of renal growth factors in Squalus acanthias and Leucoraja erinacea.  MDIBL Bulletin 42:76-79, 2003.

Evaluating mechanisms of nephrogenesis on tissue cultures from adult Little Skate, Leucoraja erinacea kidney

Evaluating mechanisms of nephrogenesis on tissue cultures from adult Little Skate, Leucoraja erinacea kidney

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