Podocyte in proteinuric renal diseases

Abstract

Introduction: Podocytes are highly specialized epithelial cells with complex morphology which cover the outer surface of the glomerular basement membrane and form the final barrier to protein loss during glomerular filtration. Podocyte injury is characterised by the presence of proteinuria, even without any detectable morphological changes, and there is compelling evidence that further damage to podocytes is central to the development of focal and complete glomerulosclerosis and development of chronic renal failure. It is an attractive idea that podocyturia could be a prognostic marker enabling high-risk patients to be targeted for specific therapy. Detection of urinary podocytes by immunofluorescence has indeed been reported to reflect glomerulonephritis activity in both animals and man, but most publications concerning patients are from a single group.

Aims: 1. To test the hypothesis that it would be prognostically useful to be able to identify podocytes in urine of patients with proteinuric renal disease, and that a reverse transcriptase polymerase chain reaction (RT-PCR) method would be an efficient and clinically applicable way to do this. 2. To create an animal model in which a graded podocyte injury could be induced, and examined alone or in combination with other pathology. This would be useful to test the hypothesis that treatment that protects the podocyte would ameliorate the disease.

Materials and Methods: 1. Total RNA was extracted from urine samples (20 ml each) of 70 proteinuric (urine protein >+++) renal patients at the renal outpatient department of Edinburgh Royal Infirmary. This was followed by RT-PCR for nephrin, podocalyxin (podocyte specific mRNAs) and p-actin (positive control) cDNAs. Immunofluorescence was conducted on urine samples of 100 patients with similar criteria using an anti-synaptopodin antibody (podocyte antibody).

2. 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 ß-actin results, and control

human kidney cDNA gave consistently positive results. Of 1000 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.

Discussion 1. The results of urine examination for podocytes contrast with those reported by Hara and colleagues (1995 and 1998). This could be partly because they examined selected high risk patients, but it may also be relevant that podocalyxin can be found on platelets and elsewhere. I looked at a clinically relevant population using clinically applicable tests. I did not find evidence that urinary podocyte excretion commonly occurs at detectable levels in these patients.

2. The tg21.1 and tg57.1 founders produced relatively adequate offspring at phase 1. So, the plan was to stop parents from breeding and preserve them, test some of their phase 1 offspring kidneys with anti-hEGF antibody to look for the expression of hDTR on podocytes and set the rest of their offspring to breed. The parents of tg47.1 and tg65.1 did not produce adequate offspring for anti-hEGF staining, so they are and their phase 1 offspring continue to active breed.

Conclusions 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.