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Title: The podocyte in proteinuric renal diseases
Author: Ihmoda, Ihmoda Ahmad
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 2006
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Materials and Methods: 1. Total RNA was extracted from urine samples (20 ml each) of 70 protinuric renal patients at the renal outpatient department of Edinburgh Royal Infirmary. This was followed by RT-PCR for nephrin, podocalyxin (podocyte specific mRNAs) and β-actin (positive control) cDNAs. Induction of specific podocyte injury with diphtheria toxin (DT) in a transgenic mice expressing human diphtheria toxin receptor (hDTR) on podocytes. Mice DTR is normally resistant to the effects of DT. These transgenic mice were generated by male pronuclear microinjection of murine fertilized ova with the plasmid (pIN); a construct contains murine nephrin promoter (podocyte promoter) and hDTR gene which is human Heparin Binding-Epidermal Growth Factor cDNA (hHB-EGF cDNA). Results: 1. All of the 70 urine samples were negative for podocyte protein mRNAs by RT-PCR, although many samples gave positive β-actin results, and control human kidney cDNA gave consistently positive results. Of 100 urine samples examined by immunofluorescence, only one (1%) gave a positive result. The technique was tested with human cultured podocytes and found to detect 10-20% of the actual number of podocytes in urine, and a similar proportion of control cells. 2. Two trials of male pronuclear microinjection of fertilized murine ova with podocyte construct were undertaken. The first microinjection trial was unsuccessful but four hDTR transgenic founders (tg21.1, tg47.1, tg57.1, tg65.1) were established with the second round of microinjections. They gave identical results in two genotyping PCRs. These founders have shown the capability of passing the transgene to their phase 1 offspring. Conclusion: 1. Looking for urinary podocytes is not a clinically useful technique in patients with proteinuria. 2. Podocyte construct (pIN) has proved its validity by generating four transgenic founders by male pronuclear microinjection and furthermore, all of them have passed the transgene to their offspring.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (M.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available