FilGAP controls cell-extracellular matrix adhesion and process formation of kidney podocytes.

The function of kidney podocytes is closely associated with actin cytoskeleton regulated by Rho small GTPases. Loss of actin-driven cell adhesions and processes is connected to podocyte dysfunction, proteinuria, and kidney diseases. FilGAP, a GTPase-activating protein for Rho small GTPase Rac1, is abundantly expressed in kidney podocytes, and its gene is linked to diseases in a family with focal segmental glomerulosclerosis. In this study, we have studied the role of FilGAP in podocytes in vitro. Depletion of FilGAP in cultured podocytes induced loss of actin stress fibers and increased Rac1 activity. Conversely, forced expression of FilGAP increased stress fiber formation whereas Rac1 activation significantly reduced its formation. FilGAP localizes at the focal adhesion (FA), an integrin-based protein complex closely associated with stress fibers, that mediates cell-extracellular matrix (ECM) adhesion, and FilGAP depletion decreased FA formation and impaired attachment to the ECM. Moreover, in unique podocyte cell cultures capable of inducing the formation of highly organized processes including major processes and foot process-like projections, FilGAP depletion or Rac1 activation decreased the formation of these processes. The reduction of FAs and process formations in FilGAP-depleted podocyte cells was rescued by inhibition of Rac1 or P21-activated kinase 1 (PAK1), a downstream effector of Rac1, and PAK1 activation inhibited their formations. Thus, FilGAP contributes to both cell-ECM adhesion and process formation of podocytes by suppressing Rac1/PAK1 signaling.

Dynamic changes of podocytes caused by fibroblast growth factor 2 in culture.

Fibroblast growth factor 2 (FGF2) augments podocyte injury, which induces glomerulosclerosis, although the mechanisms remain obscure. In this study, we investigated the effects of FGF2 on cultured podocytes with interdigitating cell processes in rats. After 48 h incubation with FGF2 dynamic changes in the shape of primary processes and cell bodies of podocytes resulted in the loss of interdigitation, which was clearly shown by time-lapse photography. FGF2 reduced the gene expressions of constituents of the slit diaphragm, inflections of intercellular junctions positive for nephrin, and the width of the intercellular space. Immunostaining for the proliferation marker Ki-67 was rarely seen and weakly stained in the control without FGF2, whereas intensely stained cells were frequently found in the presence of FGF2. Binucleation and cell division were also observed, although no significant increase in cell number was shown. An in vitro scratch assay revealed that FGF2 enhanced migration of podocytes. These findings show that FGF2 makes podocytes to transition from the quiescent state into the cell cycle and change their morphology due to enhanced motility, and that the culture system in this study is useful for analyzing the pathological changes of podocytes in vivo.

Distinct differences between cultured podocytes and parietal epithelial cells of the Bowman's capsule.

Phenotypic changes in culture hamper the identification and characterization of cultured podocytes and parietal epithelial cells of the Bowman's capsule (PECs). We have recently established culture conditions that restore podocytes to their differentiated phenotypes. We compared podocytes and PECs cultured under the same conditions to determine the unique characteristics of the two cell types. Performing this comparison under the same conditions accentuated these differences. Podocytes behaved like non-epithelial cells by extending cell processes even at confluence. By contrast, PECs behaved like typical epithelial cells by maintaining a polygonal appearance. Other differences were identified using immunostaining and RT-PCR; podocytes expressed high levels of podocyte-specific markers while PECs expressed high levels of PEC-specific markers. However, while podocytes expressed low levels of PEC markers, PECs expressed low levels of podocyte markers. Therefore, the identification of podocytes and PECs in culture requires the evaluation of respective cell markers and the expression of markers for other cell types.

Ontogeny of renin gene expression in the chicken, Gallus gallus.

Renin or a renin-like enzyme evolved in ancestral vertebrates and is conserved along the vertebrate phylogeny. The ontogenic development of renin, however, is not well understood in nonmammalian vertebrates. We aimed to determine the expression patterns and relative abundance of renin mRNA in pre- and postnatal chickens (Gallus gallus, White Leghorn breed). Embryonic day 13 (E13) embryos show renal tubules, undifferentiated mesenchymal structures, and a small number of developing glomeruli. Maturing glomeruli are seen in post-hatch day 4 (D4) and day 30 (D30) kidneys, indicating that nephrogenic activity still exists in kidneys of 4-week-old chickens. In E13 embryos, renin mRNA measured by quantitative polymerase chain reaction in the adrenal glands is equivalent to the expression in the kidneys, whereas in post-hatch D4 and D30 maturing chicks, renal renin expressions increased 2-fold and 11-fold, respectively. In contrast, relative renin expression in the adrenals became lower than in the kidneys. Furthermore, renin expression is clearly visible by in situ hybridization in the juxtaglomerular (JG) area in D4 and D30 chicks, but not in E13 embryos. The results suggest that in chickens, renin evolved in both renal and extrarenal organs at an early stage of ontogeny and, with maturation, became localized to the JG area. Clear JG structures are not morphologically detectable in E13 embryos, but are visible in 30-day-old chicks, supporting this concept.

Induction of interdigitating cell processes in podocyte culture.

Highly organized cell processes characterize glomerular podocytes in vivo. However, podocytes in culture have a simple morphology lacking cell processes, especially upon reaching confluence. Here, we aimed to establish culture conditions under which cultured podocytes extend cell processes at confluence. Among various culture conditions that could possibly cause phenotypic changes in podocytes, we examined the effects of heparin, all-trans retinoic acid, fetal bovine serum, and extracellular matrices on the morphology of podocytes in rat primary culture. Consequently, long arborized cell processes were observed to radiate extensively from the cell body only when cells were cultured in the presence of heparin and all-trans retinoic acid on laminin-coated dishes with decreasing concentrations of fetal bovine serum. Primary processes branching repeatedly into terminal processes and cell process insertion under adjacent cell bodies were evident by electron microscopy-based analysis. Immunostaining for podocin showed conspicuous elongations of intercellular junctions. Under these conditions, the expression levels of podocyte-specific proteins and genes were markedly upregulated. Thus, we succeeded in establishing culture conditions in which the cultured podocytes exhibit phenotypes similar to those under in vivo conditions.

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding.

Formalin-fixed paraffin-embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin-induced alternations on a proteome-wide level. We compared LC-MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2-6% of all peptide-spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.

Deep proteome mapping of mouse kidney based on OFFGel prefractionation reveals remarkable protein post- translational modifications.

Performing a comprehensive nonbiased proteome analysis is an extraordinary challenge due to sample complexity and wide dynamic range, especially in eukaryotic tissues. Thus, prefractionation steps conducted prior to mass spectrometric analysis are critically important to reduce complex biological matrices and allow in-depth analysis. Here we demonstrated the use of OFFGel prefractionation to identify more low abundant and hydrophobic proteins than in a nonfractionated sample. Moreover, OFFGel prefractionation of a kidney protein sample was able to unveil protein functional relevance by detecting PTMs, especially when prefractionation was augmented with a targeted enrichment strategy such as TiO2 phospho-enrichment. The OFFGel-TiO2 combination used in this study was comparable to other global phosphoproteomics approaches (SCX-TiO2, ERLIC-TiO2, or HILIC-TiO2). The detailed mouse kidney proteome with the phosphopeptide enrichment presented here serves as a useful platform for a better understanding of how the renal protein modification machinery works and, ultimately, will contribute to our understanding of pathological processes as well as normal physiological renal functions.

Basics and recent advances of two dimensional- polyacrylamide gel electrophoresis.

Gel- based proteomics is one of the most versatile methods for fractionating protein complexes. Among these methods, two dimensional- polyacrylamide gel electrophoresis (2-DE) represents a mainstay orthogonal approach, which is popularly used to simultaneously fractionate, identify, and quantify proteins when coupled with mass spectrometric identification or other immunological tests. Although 2-DE was first introduced more than three decades ago, several challenges and limitations to its utility still exist. This review discusses the principles of 2-DE as well as both recent methodological advances and new applications.

Decreased urinary calbindin 1 levels in proteinuric rats and humans with distal nephron segment injuries.

BACKGROUND: Several proteins have been proposed as new urinary biomarkers of kidney injuries, but they are not always capable of identifying the kidney nephron segment that has been injured. Since calbindin 1 protein is exclusively localized in the kidney distal nephron segment, it is presumed that its expression is altered during distal nephron segment injuries, resulting in changes in its urinary excretion. METHODS: Calbindin 1 expression in normal rat kidneys was compared with that in the kidneys of rats that had suffered distal nephron segment injuries (unilateral ureteral obstruction [UUO] or anti-glomerular basement membrane glomerulonephritis [anti-GBM GN]) using immunohistochemical examinations and real-time polymerase chain reaction. The urinary calbindin 1 protein concentration of normal rats was also compared with that of anti-GBM GN rats and of cisplatin nephropathy rats using Western blotting. We also compared the kidney and urinary calbindin 1 protein concentrations of normal human subjects with those of proteinuric patients [immunoglobulin (Ig)A nephropathy; IgAN] with distal nephron segment injuries. RESULTS: Calbindin 1 mRNA expression in the renal cortices and calbindin 1 protein expression in the kidney distal nephron segments were decreased in the UUO and anti-GBM GN rat kidneys. The urinary calbindin 1 protein levels of the anti-GBM GN rats were also markedly decreased, whereas those of the cisplatin nephropathy rats were slightly decreased. The human IgAN patients displayed decreased renal calbindin 1 protein expression in their dilated distal tubules, and some patients displayed decreased urinary calbindin 1 levels. CONCLUSION: Since it has been demonstrated that decreased urinary calbindin 1 levels are indicative of decreased calbindin 1 kidney expression due to distal nephron segment injuries, calbindin 1 might be a useful urinary biomarker for identifying distal nephron segment injuries.

Heparin increasing podocyte-specific gene expressions.

AIM: Heparin, a highly sulfated glycosaminoglycan, has been shown to have a renoprotective effect on renal diseases, but its mechanisms remain to be elucidated. In this study, we examined the effect of heparin on podocytes by using primary cultured podocytes positive for podocyte-specific markers including podocin and podocalyxin. METHODS: Podocytes were cultured from highly purified glomeruli isolated by the method with renal perfusion with magnetic beads and digestion of collagenase. Podocyte-specific gene expressions and proteins were examined by real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence microscopy. RESULTS: Real-time PCR showed that addition of heparin to the culture media significantly upregulated most of the podocyte-specific genes in a dose-dependent and time-dependent manner. Western blotting showed a marked increase in protein levels of nephrin and podocin. Podocin localization at cell-cell contact sites became conspicuous in the presence of heparin. The effect of heparin was observed even in culture media deprived of bovine foetal serum. Heparan sulfate, less sulfated than heparin, and hyaluronan did not show such effects, but sulfated dextran did markedly. CONCLUSION: Heparin acts on cultured podocytes to increase podocyte-specific gene expressions. A high degree of sulfation is crucial for the effect of heparin.

Profiling and annotation of human kidney glomerulus proteome.

BACKGROUND: The comprehensive analysis of human kidney glomerulus we previously performed using highly purified glomeruli, provided a dataset of 6,686 unique proteins representing 2,966 distinct genes. This dataset, however, contained considerable redundancy resulting from identification criteria under which all the proteins matched with the same set of peptides and its subset were reported as identified proteins. In this study we reanalyzed the raw data using the Mascot search engine and highly stringent criteria in order to select proteins with the highest scores matching peptides with scores exceeding the "Identity Threshold" and one or more unique peptides. This enabled us to exclude proteins with lower scores which only matched the same set of peptides or its subset. This approach provided a high-confidence, non-redundant dataset of identified proteins for extensive profiling, annotation, and comparison with other proteome datasets that can provide biologically relevant knowledge of glomerulus proteome. RESULTS: Protein identification using the Mascot search engine under highly stringent, computational strategy generated a non-redundant dataset of 1,817 proteins representing 1,478 genes. These proteins were represented by 2-D protein array specifying observed molecular weight and isoelectric point range of identified proteins to demonstrate differences in the observed and calculated physicochemical properties. Characteristics of glomerulus proteome could be illustrated by GO analysis and protein classification. The depth of proteomic analysis was well documented via comparison of the dynamic range of identified proteins with other proteomic analyses of human glomerulus, as well as a high coverage of biologically important pathways. Comparison of glomerulus proteome with human plasma and urine proteomes, provided by comprehensive analysis, suggested the extent and characteristics of proteins contaminated from plasma and excreted into urine, respectively. Among the latter proteins, several were demonstrated to be highly or specifically localized in the glomerulus by cross-reference analysis with the Human Protein Atlas database, and could be biomarker candidates for glomerular injury. Furthermore, comparison of ortholog proteins identified in human and mouse glomeruli suggest some biologically significant differences in glomerulus proteomes between the two species. CONCLUSIONS: A high-confidence, non-redundant dataset of proteins created by comprehensive proteomic analysis could provide a more extensive understanding of human glomerulus proteome and could be useful as a resource for the discovery of biomarkers and disease-relevant proteins.

Profiling of kidney vascular endothelial cell plasma membrane proteins by liquid chromatography-tandem mass spectrometry.

BACKGROUND: Vascular endothelial cells (VECs) play crucial roles in physiological and pathologic conditions in tissues and organs. Most of these roles are related to VEC plasma membrane proteins. In the kidney, VECs are closely associated with structures and functions; however, plasma membrane proteins in kidney VECs remain to be fully elucidated. METHODS: Rat kidneys were perfused with cationic colloidal silica nanoparticles (CCSN) to label the VEC plasma membrane. The CCSN-labeled plasma membrane fraction was collected by gradient ultracentrifugation. The VEC plasma membrane or whole-kidney lysate proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and digested with trypsin in gels for liquid chromatography-tandem mass spectrometry. Enrichment analysis was then performed. RESULTS: The VEC plasma membrane proteins were purified by the CCSN method with high yield (approximately 20 µg from 1 g of rat kidney). By Mascot search, 582 proteins were identified in the VEC plasma membrane fraction, and 1,205 proteins were identified in the kidney lysate. In addition to 16 VEC marker proteins such as integrin beta-1 and intercellular adhesion molecule-2 (ICAM-2), 8 novel proteins such as Deltex 3-like protein and phosphatidylinositol binding clathrin assembly protein (PICALM) were identified. As expected, many key functions of plasma membranes in general and of endothelial cells in particular (i.e., leukocyte adhesion) were significantly overrepresented in the proteome of CCSN-labeled kidney VEC fraction. CONCLUSIONS: The CCSN method is a reliable technique for isolation of VEC plasma membrane from the kidney, and proteomic analysis followed by bioinformatics revealed the characteristics of in vivo VECs in the kidney.

Restoration of Podocyte Phenotype in Culture.

The most distinctive characteristic of podocytes in the kidney is the presence of interdigitating cell processes with nephrin and podocin that are concentrated at sites of cell-cell contact. Unfortunately, these defining features are easily lost in culture. We previously reported culture conditions that can restore the differentiated phenotypes in primary cultures of rat podocyte. Since then, some of the materials used have been discontinued or improved. In this chapter, we therefore provide our most recent protocol for the restoration of the podocyte phenotype in culture.

Megalin is involved in angiotensinogen-induced, angiotensin II-mediated ERK1/2 signaling to activate Na + -H + exchanger 3 in proximal tubules.

BACKGROUND: Kidney angiotensin (Ang) II is produced mainly from liver-derived, glomerular-filtered angiotensinogen (AGT). Podocyte injury has been reported to increase the kidney Ang II content and induce Na + retention depending on the function of megalin, a proximal tubular endocytosis receptor. However, how megalin regulates the renal content and action of Ang II remains elusive. METHODS: We used a mass spectrometry-based, parallel reaction-monitoring assay to quantitate Ang II in plasma, urine, and kidney homogenate of kidney-specific conditional megalin knockout (MegKO) and control (Ctl) mice. We also evaluated the pathophysiological changes in both mouse genotypes under the basal condition and under the condition of increased glomerular filtration of AGT induced by administration of recombinant mouse AGT (rec-mAGT). RESULTS: Under the basal condition, plasma and kidney Ang II levels were comparable in the two mouse groups. Ang II was detected abundantly in fresh spot urine in conditional MegKO mice. Megalin was also found to mediate the uptake of intravenously administered fluorescent Ang II by PTECs. Administration of rec-mAGT increased kidney Ang II, exerted renal extracellular signal-regulated kinase 1/2 (ERK1/2) signaling, activated proximal tubular Na + -H + exchanger 3 (NHE3), and decreased urinary Na + excretion in Ctl mice, whereas these changes were suppressed but urinary Ang II was increased in conditional MegKO mice. CONCLUSION: Increased glomerular filtration of AGT is likely to augment Ang II production in the proximal tubular lumen. Thus, megalin-dependent Ang II uptake should be involved in the ERK1/2 signaling that activates proximal tubular NHE3 in vivo , thereby causing Na + retention.

Phosphate overload induces podocyte injury via type III Na-dependent phosphate transporter.

Uptake of P(i) at the cellular membrane is essential for the maintenance of cell viability. However, phosphate overload is also stressful for cells and can result in cellular damage. In the present study, we investigated the effects of the transgenic overexpression of type III P(i) transporter Pit-1 to explore the role of extracellular P(i) in glomerular sclerosis during chronic renal disease. Pit-1 transgenic (TG) rats showed progressive proteinuria associated with hypoalbuminemia and dyslipidemia. Ultrastructural analysis of TG rat kidney by transmission electron microscopy showed a diffuse effacement of the foot processes of podocytes and a thickening of the glomerular basement membrane, which were progressively exhibited since 8 wk after birth. TG rats died at 32 wk of age due to cachexia. At this time, more thickening of the glomerular basement membrane and segmental sclerosis were observed in glomeruli of the TG rats. Immunohistochemical examination using anti-connexin 43 and anti-desmin antibodies suggested the progressive injury of podocytes in TG rats. TG rats showed higher P(i) uptake in podocytes than wild-type rats, especially under low P(i) concentration. When 8-wk-old wild-type and TG rats were fed a 0.6% normal phosphate (NP) or 1.2% phosphate (HP) diet for 12 wk, HP diet-treated TG rats showed more progressive proteinuria and higher serum creatinine levels than NP diet-treated TG rats. In conclusion, our findings suggest that overexpression of Pit-1 in rats induces phosphate-dependent podocyte injury and damage to the glomerular barrier, which result in the progression of glomerular sclerosis in the kidney.

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.

Comparison of human glomerulus proteomic profiles obtained from low quantities of samples by different mass spectrometry with the comprehensive database.

BACKGROUND: We have previously constructed an in-depth human glomerulus proteome database from a large amount of sample for understanding renal disease pathogenesis and aiding the biomarker exploration. However, it is usually a challenge for clinical research to get enough tissues for large-scale proteomic characterization. Therefore, in this study, we focused on high-confidence proteomics analysis on small amounts of human glomeruli comparable to those obtained from biopsies using different mass spectrometers and compared these results to the comprehensive database. RESULTS: One microgram of human glomerular protein digest was analyzed each on five LC- combined mass spectrometers (LIT-TOF, LTQ-Orbitrap, Q-TOF, LIT and MALDI-TOF/TOF) yielding 139, 185, 94, 255 and 108 proteins respectively identified with strict criteria to ensure high confidence (> 99%) and low false discovery rate (FDR) (< 1%). An integrated profile of 332 distinct glomerular proteins was subsequently generated without discerned bias due to protein physicochemical properties (pI and MW), of which around 60% were detected commonly by more than two LC-MS/MS platforms. Comparative analysis with the comprehensive database demonstrated 14 proteins uniquely identified in this study and more than 70% of identified proteins in small datasets were concentrated to the top abundant 500 in the comprehensive database which consists of 2775 non-redundant proteins. CONCLUSION: This study showed representative human glomerulus proteomic profiles obtained from biopsies through analysis of comparable amounts of samples by different mass spectrometry. Our results implicated that high abundant proteins are more likely to be reproducibly identified in multiple mass spectrometers runs and different mass spectrometers. Furthermore, many podocyte essential proteins such as nephrin, podocin, podocalyxin and synaptopodin were also identified from the small samples in this study. Bioinformatic enrichment analysis results extended our understanding of the major glomerular proteins about their subcellular distributions and functions. The present study indicated that the proteins localized in certain cellular compartments, such as actin cytoskeleton, mitochondrial matrix, cell surface, basolateral plasma membrane, contractile fiber, proteinaceous extracellular matrix and adherens junction, represent high abundant glomerular proteins and these subcellular structures are also highly significantly over-represented in the glomerulus compared to the whole human background.

Differential proteomic shotgun analysis elucidates involvement of water channel aquaporin 8 in presence of α-amylase in the colon.

Aquaporin (AQP) family plays a pivotal role in fluid secretion and absorption, especially in the digestive system and secretory glands. Within this family, AQP8 was reported to be widely expressed in the epithelia of the digestive tract, liver, and pancreas. In two parallel experimental platforms with different analytical and comparative approaches, in-gel tryptic digestion with macro-embedded spreadsheet analysis and in-solution tryptic digestion with LC-MS alignment based approach, we compared wild-type and AQP8 knockout mice colon proteomes. Shared result between both experiments revealed down-regulation of α-amylase 2 in AQP8-deleted mice model. Verification on both transcriptional and translational levels confirmed the involvement of AQP8 in α-amylase 2 regulation. Given the profound role of AQP8 as a water and solutes transporter, it might be important in modulating α-amylase 2 synthesis by colonic epithelial cells as well. Here, we also proved the capability of our coupled approaches for selecting the most reliable and significant candidates, an applicable process for initial screening of biological biomarkers in complex specimens and tissue extracts.

Glomerular proteins related to slit diaphragm and matrix adhesion in the foot processes are highly tyrosine phosphorylated in the normal rat kidney.

BACKGROUND: Tyrosine phosphorylation of proteins has been a focus of extensive studies since it plays crucial roles in regulation of diverse biological reactions. To understand the involvement of tyrosine phosphorylation in kidney functions, a comprehensive proteomic study for tyrosine-phosphorylated proteins was performed in the normal rat kidney. METHODS: Two-dimensional gel electrophoresis and immunoprecipitation using anti-phosphotyrosine antibodies were employed to detect tyrosine-phosphorylated proteins. The proteins were analysed by mass spectrometry and validated by immunological analyses using specific antibodies. RESULTS: Most of tyrosine-phosphorylated proteins were confined to the glomerulus and predominantly localized along the glomerular capillary wall, especially in the foot processes of podocytes. Our systematic proteomic analysis identified nephrin, SHPS-1 (tyrosine-protein phosphatase non-receptor-type substrate 1), FAK1 and paxillin as major tyrosine-phosphorylated proteins and Neph1, talin and vinculin as minor tyrosine-phosphorylated proteins. In the present study, SHPS-1 was identified as a novel tyrosine-phosphorylated protein in the glomerulus and was also predominantly localized at the foot processes. Mass spectrometric analysis identified in vivo phosphorylation sites of SHPS-1 on Y460, Y477 and Y501. CONCLUSION: This study identified tyrosine-phosphorylated proteins in normal rat kidney, which were prominently rich in the glomerulus and localized at the podocyte foot processes. These proteins were categorized as cell-to-cell or cell-to-matrix adhesion complex-related molecules, suggesting their pivotal roles in the glomerular ultrafiltration.

Expression and localization of insulin-like growth factor binding proteins in normal and proteinuric kidney glomeruli.

AIM: Insulin-like growth factor I (IGF-I) acts on target cells in an endocrine and/or local manner through the IGF-I receptor (IGF-IR), and its actions are modulated by multiple IGF binding proteins (IGFBP). To elucidate the roles of local IGFBP in kidney glomeruli, the expression and localization of their genes were examined and compared with normal and proteinuric kidney glomeruli. METHODS: A cDNA microarray database (MAd-761) was constructed using human kidney glomeruli and cortices. The gene expression levels of IGF-I, IGF-1R and IGFBP (1-10) were examined in glomeruli and cortices by polymerase chain reaction (PCR) and in situ hybridization (ISH), and the expression levels of IGFBP that were abundantly found in the glomerulus were compared between normal and proteinuric kidneys in rats and humans. RESULTS: IGFBP-2, -7 and -8 were demonstrated to be abundantly and preferentially expressed in the glomerulus. In PCR, the expression levels of the IGFBP-2, -7, -8 and -10 genes in glomeruli were shown to have more than doubled compared with their levels in the cortices. In ISH, the IGFBP-2, -7, -8 and -10 genes were found to be localized in glomerular cells including podocytes, and their increased expression was observed in inflammatory glomeruli. IGF-I gene expression was localized in glomerular podocytes, whereas the IGF-IR gene was expressed in glomerular podocytes and cortical tubular cells. In nephrotic rats, the expression of the IGFBP-10 gene was increased in glomerular podocytes; however, the expression levels of IGFBP-2, -7 and -8 did not change. CONCLUSION: IGFBP-2, -7, -8 and -10 are produced by normal and injured glomerular podocytes and may regulate local IGF-I actions in podocytes and/or cortical tubular cells in the kidney.