Altered podocyte structure in GLEPP1 (Ptpro)-deficient mice associated with hypertension and low glomerular filtration rate.

Glomerular epithelial protein 1 (GLEPP1) is a receptor tyrosine phosphatase present on the apical cell surface of the glomerular podocyte. The GLEPP1 gene (PTPRO:) was disrupted at an exon coding for the NH(2)-terminal region by gene targeting in embryonic stem cells. Heterozygote mating produced the expected genotypic ratio of 1:2:1, indicating that the Ptpro(-/-) genotype does not lead to embryonic or neonatal lethality. Kidney and glomerular structure was normal at the gross and light microscopic levels. Scanning and transmission electron microscopy showed that Ptpro(-/-) mice had an amoeboid rather than the typical octopoid structure seen in the wild-type mouse podocyte and that there were blunting and widening of the minor (foot) processes in association with altered distribution of the podocyte intermediate cytoskeletal protein vimentin. Reduced filtration surface area in association with these structural changes was confirmed by finding reduced glomerular nephrin content and reduced glomerular filtration rate in Ptpro(-/-) mice. There was no detectable increase in the urine albumin excretion of Ptpro(-/-) mice. After removal of one or more kidneys, Ptpro(-/-) mice had higher blood pressure than did their wild-type littermates. These data support the conclusion that the GLEPP1 (Ptpro) receptor plays a role in regulating the glomerular pressure/filtration rate relationship through an effect on podocyte structure and function.

Effect of surgical subspecialization on breast cancer outcome.

BACKGROUND: There is increasing pressure for specialization of medical services. The effect of surgical specialization on the outcome of breast cancer in Bedford has been assessed. METHODS: The Bedford Breast Cancer Registry, which contains prospective diagnostic, treatment and follow-up data on all breast cancers treated in North Bedfordshire, was analysed to compare breast cancer outcome between 1990-1992 and 1993-1996, that is before and after the advent of surgical subspecialization. All 784 patients were analysed, including patients with metastases (4 per cent) and those treated by tamoxifen alone (8 per cent). Outcome was compared in terms of disease-free survival (DFS), locoregional and all (locoregional and metastases) recurrence rates assessed by Cox proportional hazard and Kaplan-Meier analyses. RESULTS: Overall DFS was 75 per cent and the locoregional recurrence rate was 8 per cent at 3 years. The tumour stage and grade at presentation and the proportion of screen-detected cancers were similar for both intervals. The outcome for patients before specialization (1990-1992; n = 329) was worse: hazard ratio (HR) for DFS 1.5 (95 per cent confidence interval 1.2-2.0) and HR for locoregional recurrence 2.0 (1.2-3.5). After subspecialization (1993-1996, n = 455) DFS improved from 70 to 79 per cent (P = 0.009) and the all recurrence rate fell from 22 to 12 per cent (P = 0.0004) at 3 years. The improvement in outcome was mainly in younger patients (aged less than 70 years), in whom DFS improved from 72 to 81 per cent (P = 0.02) and the all recurrence rate fell from 24 to 12 per cent (P = 0.001) at 3 years. The improvement was associated with increased axillary surgery (47 to 74 per cent; P < 0.0001), and more frequent use of tamoxifen (74 to 84 per cent; P = 0.004) and chemotherapy (10 to 27 per cent; P < 0.0001) in this age group. CONCLUSION: There was a significant improvement in outcome for patients with breast cancer after surgical subspecialization in Bedford. This may relate to the more frequent use of appropriate systemic therapy.

Age-related variation in the treatment and outcomes of patients with breast carcinoma.

BACKGROUND: There is controversy regarding the effect of age on breast carcinoma, and previous analyses have often excluded the most elderly patients as well as those with advanced cancers. This study assessed treatment variations and outcome in relation to age for a complete series of patients who presented with breast carcinoma within a defined geographic district. METHODS: Data were collected prospectively for all 784 patients who presented with breast carcinoma in North Bedfordshire, United Kingdom, from 1990 to 1996. Stage of disease, treatment, and outcome were compared for different age groups. RESULTS: Older patients had more advanced cancers: 14% of lymph node negative patients age > or = 60 years had T3 or T4 tumors compared with 4% of younger lymph node negative patients (P < 0. 0001). Treatment varied with age: 94% of lymph node negative patients age > or = 60 years received tamoxifen compared with 73% of younger lymph node negative patients (P < 0.0001). For lymph node positive patients, outcome was unaffected by age; however, for lymph node negative patients, outcome was best for patients ages 60-69 years: disease free survival was 91% compared with 78% for other ages at 3 years (P = 0.008), and the locoregional recurrence rate was 2% compared with 7% at 3 years (P = 0.04). The improvement in the locoregional recurrence rate applied to all lymph node negative patients age > or = 60 years: the recurrence rate was 2% compared with 9% for younger patients at 3 years (P = 0.04). CONCLUSIONS: These data support the theory that breast carcinomas are more aggressive in younger patients. The high prescription rate of tamoxifen for older lymph node negative women may have contributed to the low locoregional recurrence rate for this group.

Molecular cloning and characterization of human podocalyxin-like protein. Orthologous relationship to rabbit PCLP1 and rat podocalyxin.

Human renal cortex and heart cDNA libraries were screened for a human homolog of rabbit PCLP1 using the rabbit PCLP1 cDNA as a probe. Clones spanning 5869 base pairs with an open reading frame coding for a 528-amino acid peptide were obtained. The putative peptide contains a potential signal peptide and a single membrane-spanning region. The extracellular domain contains multiple potential sites for N- and O-linked glycosylation and 4 cysteines for potential disulfide bonding similar to rabbit PCLP1. On Northern blot a major transcript is seen at 5.9 kilobases. Antibodies to this protein show a doublet at 160/165 kDa on Western blots of human glomerular extract and a pattern of intense glomerular staining and vascular endothelial staining on immunofluorescence of human kidney sections. Comparison of the rabbit and human peptide sequences shows a high degree of identity in the transmembrane and intracellular domains (96%) with a lower degree of identity in the extracellular domain (36%). An antibody to the intracellular domain reacted across species (human, rabbit, and rat) and recognized both rabbit PCLP1 and rat podocalyxin. An interspecies Southern blot probed with a cDNA coding for the intracellular domain showed strong hybridization to all vertebrates tested in a pattern suggesting a single copy gene. We conclude that this cDNA and putative peptide represent the human homolog of rabbit PCLP1 and rat podocalyxin.

Molecular cloning, expression, and characterization of podocalyxin-like protein 1 from rabbit as a transmembrane protein of glomerular podocytes and vascular endothelium.

Podocytes are responsible in part for maintaining the size and charge filtration characteristics of the glomerular filter. The major sialoprotein of the podocyte foot process glycocalyx is a 140-kDa sialoprotein named podocalyxin. Monoclonal antibodies raised against isolated rabbit glomeruli that recognized a podocalyxin-like protein base upon size, Alcian blue staining, wheat germ agglutinin binding, and distribution in renal cortex were used to expression clone cDNAs from a rabbit glomerular library. On Northern blot the cDNAs hybridized to a 5.5-kilobase pair transcript predominantly present in glomerulus. The overlapping cDNAs spanned 5,313 base pairs that contained an open reading frame of 1,653 base pairs and were not homologous with a previously described sequence. The deduced 551-amino acid protein contained a putative 21-residue N-terminal signal peptide and a 26-amino acid transmembrane region. The mature protein has a calculated molecular mass of 55 kDa, an extracellular domain that contains putative sites for N- and O-linked glycosylation, and a potential glycosaminoglycan attachment sites. The intracellular domain contains potential sites for phosphorylation. Processing of the full-length coding region in COS-7 cells resulted in a 140-kDa band, suggesting that the 55-kDa core protein undergoes extensive post-translational modification. The relationship between the cloned molecule and the monoclonal antibodies used for cloning was confirmed by making a fusion protein that inhibited binding of the monoclonal antibodies to renal cortical tissue sections and then raising polyclonal antibodies against the PCLP1 fusion protein that also recognized glomerular podocytes and endothelial cells in tissue sections in a similar distribution to the monoclonal antibodies. We conclude that we have cloned and sequenced a novel transmembrane core glycoprotein from rabbit glomerulus, which has many of the characteristics of podocalyxin. We have named this protein podocalyxin-like protein 1.

Molecular cloning of cDNAs encoding human GLEPP1, a membrane protein tyrosine phosphatase: characterization of the GLEPP1 protein distribution in human kidney and assignment of the GLEPP1 gene to human chromosome 12p12-p13.

Human glomerular epithelial protein 1 (GLEPP1), a receptor-like membrane protein tyrosine phosphatase (PTPase), was cloned and sequenced from a human renal cortical cDNA library. The human nucleotide and derived amino acid sequences were, respectively, 90 and 97% identical to those of rabbit. Human GLEPP1 is predicted to contain 1188 amino acids. The predicted mature protein is 1159 amino acids long and contains a large extracellular domain, a single transmembrane domain, and a single intracellular PTPase domain. Monoclonal and polyclonal antibodies raised against a human GLEPP1 fusion protein recognized a protein with distribution restricted to the glomerulus in human kidney and with an apparent molecular weight of approximately 200 kDa. The GLEPP1 gene was assigned to human chromosome 12p12-p13 by fluorescence in situ hybridization.

GLEPP1, a renal glomerular epithelial cell (podocyte) membrane protein-tyrosine phosphatase. Identification, molecular cloning, and characterization in rabbit.

Podocytes are specialized epithelial cells with delicate interdigitating foot processes which cover the exterior basement membrane surface of the glomerular capillary. They are in part responsible for the extraordinary charge and size filtration characteristics of the glomerulus. To better understand disease processes affecting the glomerular filter, we searched for proteins with relative specificity to the podocyte using a monoclonal antibody strategy. The first such protein characterized (designated glomerular epithelial protein 1 (GLEPP1)) is a membrane protein-tyrosine phosphatase (PTPase) with a large extracellular domain containing eight fibronectin type III-like repeats, a hydrophobic transmembrane segment, and a single PTPase domain. The GLEPP1 PTPase domain shows homology with two other single domain transmembrane PTPases (PTP beta and Drosophila central nervous system PTP10D). This homology includes 2 cysteines in the PTPase domain not present in intracellular or tandem domain membrane PTPases. GLEPP1 PTPase protein is distributed to the podocyte foot processes themselves. RNase protection assay shows that GLEPP1 mRNA is also present in brain. By analogy with the CD45 PTPase of T cells, we expect that this receptor might play a role in maintaining foot process structure and/or function by regulating tyrosine phosphorylation of podocyte proteins.