Novel expression of claudin-5 in glomerular podocytes.

Tight junctions are the main intercellular junctions of podocytes of the renal glomerulus under nephrotic conditions. Their requisite components, claudins, still remain to be identified. We have measured the mRNA levels of claudin subtypes by quantitative real-time PCR using isolated rat glomeruli. Claudin-5 was found to be expressed most abundantly in glomeruli. Mass spectrometric analysis of membrane preparation from isolated glomeruli also confirmed only claudin-5 expression without any detection of other claudin subtypes. In situ hybridization and immunolocalization studies revealed that claudin-5 was localized mainly in glomeruli where podocytes were the only cells expressing claudin-5. Claudin-5 protein was observed on the entire surface of podocytes including apical and basal domains of the plasma membrane in the normal condition and was inclined to be concentrated on tight junctions in puromycin aminonucleoside nephrosis. Total protein levels of claudin-5 in isolated glomeruli were not significantly upregulated in the nephrosis. These findings suggest that claudin-5 is a main claudin expressed in podocytes and that the formation of tight junctions in the nephrosis may be due to local recruitment of claudin-5 rather than due to total upregulation of the claudin protein levels.

Mesangial cells connected by the N-cadherin-catenin system in the rat kidney.

BACKGROUND/AIMS: Mesangial cells bear the tensional forces generated in the glomerular capillary wall. Not only mesangial cells per se, but also their intercellular junctions should be physically stable against the tensional forces; this prompted the search for actin filament-reinforced adherens junctions of mesangial cells. We previously reported alpha- and beta-catenins localized at the cell-cell contact sites of mesangial cells in the rat. Classical cadherin expressed by mesangial cells, however, remains to be elucidated. METHODS: Expression of classical cadherins, especially N-cadherin, was examined in rat glomeruli by ribonuclease protection assay, Western blot analysis and immunofluorescence and immunoelectron microscopy. RESULTS: Ribonuclease protection assay detected significant expression of N-cadherin in rat glomeruli. Western blot analysis showed that rabbit and murine antibodies against N-cadherin reacted with a specific band in isolated glomeruli. Immunofluorescence microscopy revealed that both antibodies reacted only with the mesangium in glomeruli. Immunoelectron microscopy demonstrated that the immunogold particles for N-cadherin were found predominantly at cell-cell contact sites of mesangial cells where actin filaments concentrated. CONCLUSION: N-cadherin interconnects mesangial cells, suggesting that the cadherin-catenin-actin filament system in the mesangium may play a role in the counteraction of the hydraulic pressure gradient across the capillary wall.

Identification and localization of novel genes preferentially expressed in human kidney glomerulus.

AIM: To find novel genes abundantly and preferentially expressed in human glomerulus, we constructed a glomerular cDNA library and verified the reliability of our database by comparison with the Stanford Microarray Database (SMD), followed by reverse transcription polymerase chain reaction (RT-PCR) and in situ hybridization (ISH). METHODS: RNA was extracted from normal human glomeruli, and the cDNA library was constructed by plasmid cloning. Out of 5 x 10(3) clones from the library, 91 UniGene clusters of more than three clones were identified as 'glomerular-abundant genes'. All these genes were referred to the SMD, and 18 genes were defined as 'glomerular preferential genes'. Four unknown genes -IFI27, CRHBP, FLJ10154 and SEMA5B- were selected for RT-PCR to compare expression in the glomerulus with that in the cortex and medulla, and for ISH to examine glomerular localization. Also, three unknown genes that were glomerular abundant but not listed in the SMD -DDX5, HSPC138, and MGC10940- were selected for RT-PCR and ISH. Finally, a kidney biopsy specimen of crescentic glomerulonephritis was used for ISH to examine glomerular expression for CRHBP mRNA. RESULTS: Among the selected seven glomerular-abundant genes, six were confirmed as 'glomerular preferential genes' by RT-PCR. By ISH, all these genes were demonstrated in podocytes. The expression of CRHBP mRNA in a single living podocyte was not changed between normal and crescentic glomerulus. CONCLUSION: Glomerular preferential expression and podocyte localization of these novel genes have been demonstrated for the first time. Because some of these genes were not listed in SMD, our database can be a useful tool to find novel human glomerular genes.

Claudin-6 localized in tight junctions of rat podocytes.

Tight junctions rarely exist in podocytes of the normal renal glomerulus, whereas they are the main intercellular junctions of podocytes in nephrosis and in the early stage of development. Claudins have been identified as tight junction-specific integral membrane proteins. Those of podocytes, however, remain to be elucidated. In the present study, we investigated the expression and localization of claudin-6 in the rat kidney, especially in podocytes. Western blot analysis and RT-PCR revealed that the neonatal kidney expressed much higher levels of claudin-6 than the adult kidney. Immunofluorescence microscopy showed intense claudin-6 staining in most of the tubules and glomeruli in neonates. The staining in tubules declined distinctly in adults, whereas staining in glomeruli was well preserved during development. Claudin-6 in glomeruli was distributed along the glomerular capillary wall and colocalized with zonula occludens-1. The staining became conspicuous after kidney perfusion with protamine sulfate (PS) to increase tight junctions in podocytes. Immunoelectron microscopy showed that immunogold particles for claudin-6 were accumulated at close cell-cell contact sites of podocytes in PS-perfused kidneys, whereas a very limited number of immunogold particles were detected, mainly on the basal cell membrane and occasionally at the slit diaphragm and close cell-cell contact sites in normal control kidneys. In puromycin aminonucleoside nephrosis, immunogold particles were also found mainly at cell-contact sites of podocytes. These findings indicate that claudin-6 is a transmembrane protein of tight junctions in podocytes during development and under pathological conditions.