Eero Lehtonen

Nephrin Forms a Complex with Adherens Junction Proteins and CASK in Podocytes and in Madin-Darby Canine Kidney Cells Expressing Nephrin

Mutations in the NPHS1 gene encoding nephrin lead to congenital nephrotic syndrome of the Finnish type. Nephrin is a key component of the glomerular slit diaphragms between epithelial foot processes, but its role in the pathogenesis of this disease is poorly understood. To further clarify the molecular mechanisms involved we investigated the interactions between nephrin and other components of the foot processes and filtration slits, especially adherens junction proteins, and searched for novel nephrin interacting proteins. Using co-immunoprecipitation and pull-down assays we show here that nephrin forms a multiprotein complex with cadherins and p120 catenin and with three scaffolding proteins, ZO-1, CD2AP, and CASK, in kidney glomeruli and when expressed in Madin-Darby canine kidney cells. CASK was identified as a novel binding partner of nephrin by mass spectrometry and was localized to podocytes in the glomerulus. CASK is a scaffolding protein that participates in maintenance of polarized epithelial cell architecture by linking membrane proteins and signaling molecules to the actin cytoskeleton. Our results support a model whereby the glomerular slit diaphragms are composed of cell adhesion molecules of the immunoglobulin and cadherin superfamilies that are connected to each other and to the actin cytoskeleton and signaling networks via the cytoplasmic scaffolding proteins CASK, CD2AP, and ZO-1.

Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7

The human OSBP related protein (ORP) family consists of 12 members, which can be divided into six subfamilies based on the genomic organization and amino acid homology. Here we performed basic characterization of subfamily III, which consists of three members: ORP3, ORP6, and ORP7. According to cDNA hybridization, the three genes are expressed in a tissue-specific manner. While ORP3 mRNA is most abundant in kidney, lymph nodes, and thymus, ORP6 shows highest expression in brain and skeletal muscle, and ORP7 in the gastrointestinal tract. Using monospecific peptide antibodies, we confirmed the presence of the three proteins in human and mouse tissues. ORP6 gene expression was induced upon differentiation of F9 embryonic carcinoma cells into parietal endoderm, while ORP3 and ORP7 mRNA levels were unchanged. In the F9 cells, endogenous ORP6 associated predominantly with the nuclear envelope. When expressed from the cDNA in cultured cells, the three proteins were distributed between the cytosol and endoplasmic reticulum (ER) membranes, with a minor portion found at the plasma membrane. Experiments with truncated constructs showed that the N-terminal portion of the proteins, containing a pleckstrin homology (PH) domain, has markedly strong plasma membrane targeting specificity, while the C-terminal half remains largely cytosolic. The expression data demonstrates that ORP3, -6, and -7 are not merely redundant gene products but show marked quantitative differences in tissue expression, suggesting tissue-specific aspects in their function. The dual targeting of the proteins indicates a putative role in communication between the ER and the plasma membrane.

SLC26A6 and SLC26A7 Anion Exchangers Have a Distinct Distribution in Human Kidney

Background: The anion transporters SLC26A6 (PAT1) and SLC26A7, transporting at least chloride, oxalate, sulfate and bicarbonate, show a distinct expression and function in different mammalian species. They are expressed in kidney, but their exact localization in human kidney has not been studied. We therefore examined SLC26A6 and A7 expression in human kidneys. Methods: The localization of SLC26A6 and A7 in different segments of human nephrons was studied by RT-PCR and immunohistochemistry by comparing to the tubular markers PNRA, CD10, Tamm-Horsfall antigen, high molecular weight cytokeratin, CK7, AQP2 and H+V-ATPase. Results: In human kidney, SLC26A6 is expressed in distal segments of proximal tubules, parts of the thin and thick ascending limbs of Henle’s loops, macula densa, distal convoluted tubules and a subpopulation of intercalated cells of collecting ducts. SLC26A7 is expressed in extraglomerular mesangial cells and a subpopulation of intercalated cells of collecting ducts. Conclusion: Our results show that in human kidney SLC26A6 and A7 have a distinct, partially overlapping expression in distal segments of nephrons. The distribution partly differs from that found previously in rodent kidneys.

CD2-associated protein in human urogenital system and in adult kidney tumours

We studied expression of CD2-associated protein (CD2AP) in human urogenital system and in adult kidney tumours. In the cortex of normal kidney, CD2AP was expressed in all types of tubules and in the glomeruli. Labelling was more intense in cytokeratin 7- and in Tamm–Horsfall-positive tubules than in proximal tubules. In the medulla, expression was observed in the collecting ducts. Urothelium and the epithelium of prostatic acini, seminal vesicles, seminiferous tubules, epididymal ducts, Fallopian tube, endometrium and endocervix as well as granulosa cells showed moderate to strong CD2AP positivity. In syncytiotrophoblast, the expression was weaker than in cytotrophoblast. Endometrial stroma was negative, but decidualised stroma was weakly positive. Clear cell renal cell carcinoma (RCC) (n=63) showed a weak expression. Type-I papillary RCCs (n=4) and papillary adenomas (n=3) were negative. The epithelium lining the cysts in multilocular cystic RCCs (n=3) and in cystic nephroma (n=1) was strongly positive. Chromophobe RCCs (n=2), oncocytomas (n=3) and urothelial carcinomas (n=2) were moderately positive. The results show that CD2AP displays a specific expression pattern in human urogenital organs and that distinct expression is shown in several types of kidney tumours but not in type-I papillary RCCs or in papillary adenomas.