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Biomedical subjects

M Kretzler

Publications and source records attributed to M Kretzler.

At least 19 recordsLinked to original sources
PubMed2002

[Gene expression profiling in prostatic cancer].

Basic aspects of the biology and molecular alterations in prostate carcinoma remain poorly understood. New diagnostic and prognostic markers for prostate carcinoma may add additional information to current histopathological diagnosis. In order to achieve these goals, a comprehensive gene expression analysis was performed on non-metastasizing, untreated prostate cancer tissues. RNA expression profiles of approximately 12,600 sequences from 26 human prostate tissues (17 adenocarcinomas and 9 normal adjacent to cancer tissues) were investigated using high-density oligonucleotide microarray technology (Affymetrix). We identified 63 genes which were significantly increased (at least 2.5-fold) and 153 genes which were decreased (at least 2.5-fold). Upregulated genes included several which had not yet been described, such as the genes encoding the specific granule protein (SGP28), several members of the histone family, and the alpha-methylacyl-CoA racemase, but also previously reported ones such as hepsin, LIM domain kinase 2, and carcinoma-associated antigen GA733-2. Laser capture-microdissection of epithelial and stromal compartments from cancer and histologically normal specimens followed by an amplification protocol for low amounts of RNA (< 0.1 microgram) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in bulk tumor material as upregulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. DNA microarray data for up- and downregulated genes were confirmed by quantitative RT-PCR. We demonstrated that development of prostate cancer is associated with downregulation as well as upregulation of genes that show complex differential regulation in epithelia and stroma. Some of the alterations in gene expression identified in this study may prove useful in development of novel diagnostic and therapeutic strategies. Gene expression profiling of microdissected tumor cells in prostate biopsies may supplement histopathologic diagnosis.

Adenocarcinoma↗
PubMed2000

Molecular cloning, expression, and distribution of glomerular epithelial protein 1 in developing mouse kidney.

BACKGROUND: Glomerular epithelial protein 1 (GLEPP1) is a receptor-like membrane protein tyrosine phosphatase (RPTP) with a large ectodomain consisting of multiple fibronectin type III repeats, a single transmembrane segment, and a single cytoplasmic phosphatase active site sequence. In adult human and rabbit kidneys, GLEPP1 is found exclusively on apical membranes of podocytes and especially on surfaces of foot processes. Although neither ligand nor function for this protein is known, other RPTPs with similar topologies have been implicated in mediating adherence behavior of cells. METHODS: To evaluate potential roles of GLEPP1 further, we cloned the full-length mouse GLEPP1 cDNA and examined its expression patterns in developing kidney by Northern blot analysis, in situ hybridization, and immunofluorescence microscopy. RESULTS: Nucleotide sequencing showed that mouse GLEPP1 was approximately 80% identical to rabbit and human GLEPP1 and approximately 91% identical at the amino acid level. The membrane-spanning and phosphatase domains of mouse GLEPP1 shared> 99% homology with PTPphi, a murine macrophage cytoplasmic phosphatase. Northern analysis identified a single GLEPP1 transcript of approximately 5.5 kb in fetal kidney that became approximately threefold more abundant in adults. In situ hybridization of newborn mouse kidney revealed GLEPP1 mRNA in visceral epithelial cells (developing podocytes) of comma- and S-shaped nephric figures, and expression increased in capillary loop and maturing stage glomeruli. Beginning on embryonic day 14, GLEPP1 protein was first observed on cuboidal podocytes of capillary loop stage glomeruli, but nascent podocytes of earlier comma- and S-shaped nephric figures were negative. At later stages of glomerular maturation, where foot process elongation and interdigitation occurs, GLEPP1 immunolabeling intensified on podocytes and then persisted at high levels in fully developed glomeruli. CONCLUSION: Our findings are consistent with a role for GLEPP1 in mediating and maintaining podocyte differentiation specifically.

Amino Acid Sequence↗
PubMed2000

Expression of chemokines and their receptors in nephrotoxic serum nephritis.

BACKGROUND: Chemokines play a major role in leukocyte infiltration in inflammatory kidney diseases. The specificity of the chemokine action is determined by the restricted expression of the corresponding receptors on leukocytes. We therefore simultaneously studied the expression of CC-chemokine and CC-chemokine receptor 1-5 (CCR 1-5) mRNA in an accelerated model of nephrotoxic nephritis in CD-1 mice. METHODS: Kidneys were harvested at day 0, 2 and 7. Induction of nephritis was confirmed by assessment of albuminuria by ELISA and by histological evaluation. RNA was prepared from cortex and isolated glomeruli. RNase protection assays were performed to study the expression of chemokines, RNase protection assays as well as quantitative RT-PCR assays to study the expression of chemokine receptors. RESULTS: In the cortex of nephritic kidneys mRNA for MCP-1 was increased 5-fold on day 2 and increased 4-fold on day 7 as compared to controls. mRNA for RANTES was increased 5-fold on day 7 and mRNA for IP-10 6-fold on day 7. The increase of mRNA for the chemokine receptors CCR1 and 5 was between 2-fold and 3-fold determined by RNase protection assay and for CCR1, 2 and 5 between 2- and 4-fold as determined by RT-PCR. In isolated glomeruli we found by RT-PCR an increase of CCR1, CCR2 and CCR5 of between 3 and 12-fold. CONCLUSION: These results show that chemokines and their specific chemokine receptors are increased in parallel in this model of glomerulonephritis, consistent with the potential role of the chemokine system in leukocyte recruitment to the immune injured kidney.

Animals↗
PubMed2000

The apoptosis mediator mDAP-3 is a novel member of a conserved family of mitochondrial proteins.

Programmed cell death is essential for organ development and regeneration. To identify molecules relevant for this process, full length cDNA cloning of a short, developmentally regulated murine cDNA fragment, MERM-3, was performed and showed a 1.7 kb mRNA encoding a 45 kDa protein with an ATP/GTP binding motive (P-loop). Sequence analysis revealed an 82% amino acid identity to the human death associated protein 3 (hDAP-3), a positive mediator of apoptosis. The full length sequence being the murine orthologue of hDAP-3 is therefore referred to as mDAP-3. In situ hybridization and northern blot analysis showed an abundant mRNA expression with a pronounced expression in highly proliferative epithelial compartments. For mDAP-3, cytochrome c release and induction of cell death could be demonstrated by overexpression of a mDAP-3/EGFP fusion protein. DAP-3 mediated apoptosis was shown to depend on a functional P-loop. Intracellular localization studies using the mDAP-3/EGFP fusion protein, cell fractionation and protease protection experiments localized mDAP-3 to the mitochondrial matrix. DAP-3, in contrast to cytochrome c, retained its mitochondrial localization during apoptosis induction. A mutant of a putative yeast orthologue of mDAP-3, YGL129c, here referred to as yDAP-3, has been shown to exhibit disrupted mitochondrial function. yDAP-3 deficient mutants could be shown to progressively loose mitochondrial DNA. Loss of mitochondrial DNA in yDAP-3 was partially prevented by transfection of the yDAP-3 deficient mutant with mDAP-3, indicating functional complementation by murine DAP-3 in the yeast system. These data identify mDAP-3 as one of the first proapoptotic factors in the mitochondrial matrix and provide evidence for a critical, evolutionary conserved role of members of the DAP-3 protein family for mitochondrial biogenesis.

Adaptor Proteins, Signal Transducing↗
PubMed1999

Re-expression of the developmental gene Pax-2 during experimental acute tubular necrosis in mice 1.

BACKGROUND: The transcription factor Pax-2 is known to play a key regulatory role during embryonic development of the nervous and excretory systems in mammals and flies. During mouse kidney development, Pax-2 is expressed in the undifferentiated mesenchyme in response to ureter induction and continues to be expressed in the developing comma- and s-shaped bodies. These structures harbor the immediate precursors of the proximal tubular epithelial cells. Pax-2 expression is down-regulated as the differentiation of the functional units of the nephron proceeds. In the adult mammalian kidney, the Pax-2 protein is detectable exclusively in the epithelium of the collecting ducts. We sought to test the hypothesis that tissue regeneration is characterized by re-expression of developmentally important regulatory genes such as Pax-2. METHODS: The expression pattern of Pax-2 in kidneys after experimentally-induced acute tubular necrosis caused by intraperitoneally injected folic acid in mice was tested by indirect immunofluorescence, Western blotting, reverse transcriptase-polymerase chain reaction, and in situ hybridization analysis. RESULTS: A transient, temporally and locally restricted re-expression of Pax-2 in regenerating proximal tubular epithelial cells was observed following kidney damage. CONCLUSIONS: These data indicate that during the regeneration processes, developmental paradigms may be recapitulated in order to restore mature kidney function.

Animals↗
PubMed1999

Altered gene expression and functions of mitochondria in human nephrotic syndrome.

The molecular basis of glomerular permselectivity remains largely unknown. The congenital nephrotic syndrome of the Finnish type (CNF) characterized by massive proteinuria already present but without extrarenal symptoms is a unique human disease model of pure proteinuria. In search of genes and pathophysiologic mechanisms associated with proteinuria, we used differential display-PCR to identify differences in gene expression between glomeruli from CNF and control kidneys. A distinctly underexpressed PCR product of the CNF kidneys showed over 98% identity with a mitochondrially encoded cytochrome c oxidase (COX I). Using a full-length COX I cDNA probe, we verified down-regulation of COX I mRNA to 1/4 of normal kidney values on Northern blots. In addition, transcripts of other mitochondrially encoded respiratory chain complexes showed a similar down-regulation whereas the respective nuclearly encoded complexes were expressed at comparable levels. Additional studies using histochemical, immunohistochemical, in situ hybridization, RT-PCR, and biochemical and electron microscopic methods all showed a mitochondrial involvement in the diseased kidneys but not in extrarenal blood vessels. As a secondary sign of mitochondrial dysfunction, excess lipid peroxidation products were found in glomerular structures in CNF samples. Our data suggest that mitochondrial dysfunction occurs in the kidneys of patients with CNF, with subsequent lipid peroxidation at the glomerular basement membrane. Our additional studies have revealed similar down-regulation of mitochondrial functions in experimental models of proteinuria. Thus, mitochondrial dysfunction may be a crucial pathophysiologic factor in this symptom.

Adolescent↗
PubMed1998

Analysis of mouse glomerular podocyte mRNA by single-cell reverse transcription-polymerase chain reaction.

Selective investigation of glomerular podocytes is not possible using conventional methods in vivo. Analysis of glomerular epithelium-derived cells in culture yields dubious results because of the rapid dedifferentiation of podocytes. We developed a modification of the polymerase chain reaction (PCR) method previously used to analyze cultured neurons. Podocytes harvested from freshly dissected glomeruli are ideal target cells for this modified, single cell reverse transcription-PCR method to reproducibly identify specific mRNA species from resident intact podocytes in vivo.

Actins↗
PubMed1998

Expression of glucose transporters in human peritoneal mesothelial cells.

Glucose containing solutions, the basis of peritoneal dialysis fluids, affect the proliferation and regeneration of peritoneal mesothelial cells (MsC). The aim of this study was to examine mechanisms of glucose transport into MsC, that is, the expression of facilitative glucose transporters (GLUT) and the Na(+)-dependent glucose transporter (SGLT1) in human primary MsC and a transfected MsC line. Since expression of both transporters is differentiation dependent, we investigated the effects of cell differentiation induced by culturing MsC on membranes or by addition of hexamethylene bisacetamide (HMBA; 6 mM), which enhances SGLT1 expression in LLC-PK1 cells. Levels of mRNA for GLUT1 through GLUT4 and SGLT1 were evaluated by reverse transcriptase-polymerase chain reaction (RT-PCR). The presence of the corresponding proteins was examined by Western blotting and localized by immunofluorescence. Active, Na(+)-dependent glucose transport was assessed by alpha-methyl-D-[14C]glucopyranoside (AMG) with and without the SGLT1-specific inhibitor phlorizin and by patch clamp experiments in NaCl or choline-chloride, For Na(+) dependent glucose uptake choline chloride instead of NaCl served as negative control. Facilitative transport was assessed using 2-fluoro-2-deoxy-[14C]-D-glucose (FDG) with and without the inhibitors cytochalasin B or phloretin. Primary and transfected MsC express GLUT1 and GLUT3 mRNA while no transcripts were found for GLUT2 and GLUT4. No SGLT1 transcript was detectable in subconfluent cells. Semiquantitative RT-PCR analysis documented that the addition of the differentiation inducer HMBA to confluent cultures or growth of MsC on membranes for seven days produced a down-regulation of mRNA for GLUT1, no change for GLUT3, and a substantial increase for SGLT1 mRNA. Under these conditions MsC express SGLT1 protein and possess a Na(+)-dependent glucose uptake as assessed by AMG. Phlorizin (1 mM) inhibits AMG uptake by 30 to 40%. In patch clamp experiments the addition of extracellular glucose depolarized the membrane potential only in the presence of sodium. These results indicate that differentiated MsC express GLUT1, GLUT3, and SGLT1. Further characterization of these transport mechanisms and their regulation may help to understand the cellular effects of glucose on MsC in peritoneal dialysis.

Base Sequence↗
PubMed1998

Detection of multiple vascular endothelial growth factor splice isoforms in single glomerular podocytes.

Glomerular podocytes are major determinants of filtration permselectivity in the glomerulus. Although the molecular mechanisms determining the characteristics of the glomerular filtration unit are incompletely understood, vascular endothelial growth factor (VEGF) has been implicated. To analyze this process in situ, we established a method that allows exploration of in vivo mRNA expression of podocytes using single-cell reverse transcriptase-polymerase chain reaction (RT-PCR). Microdissected mouse glomeruli were held in a patch-clamp apparatus, and single podocytes were harvested by aspiration. After lysis, the cells were reverse transcribed, and PCR was performed (45 cycles). The podocyte nature of the material was confirmed by detection of podocyte-specific mRNA (glomerular epithelial protein 1 and Wilms' tumor protein 1). Using specific oligonucleotide primers, VEGF was detected in mRNA obtained from renal cortex, single microdissected glomeruli, cultured murine podocytes, and single podocytes in situ. All cells examined expressed three VEGF isoforms (121, 165, and 189). These differ in their capacity for binding to extracellular matrix and could have different potencies regulating glomerular endothelial permeability. Our approach should allow a semiquantitative, isoform-specific evaluation of VEGF mRNA expression in podocytes during nephrogenesis and in glomerular disease.

Animals↗
PubMed1998

Single cell RT-PCR analysis of ClC-2 mRNA expression in ureteric bud tip.

Embryonic epithelia at the tip of the ureteric bud (UB) face the interspace between epithelial and mesenchymal cells and are fundamentally involved in reciprocal signaling during early nephrogenesis. To characterize their membrane conductive proteins, patch-clamp and single cell RT-PCR techniques were applied to embryonic rat UBs [embryonic day 17 (day E17)] microdissected from the outer cortex. Cells at the UB tip had a high whole cell conductance (14 +/- 2 nS/10 pF, n = 8). The main fractional conductance resembled that of Ca-activated Cl channels in nonepithelial cells, with its time-dependent activation at depolarizing and inactivation at hyperpolarizing voltages. A second Cl-selective current fraction, by contrast, activated slowly during strong hyperpolarization, suggestive of a ClC-2-mediated conductance. To determine the origin of this current, cytoplasm was harvested into the patch pipette, RNA was reverse transcribed, and cDNA encoding the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) housekeeper gene or the ClC-2 Cl channel was amplified by polymerase chain reaction (PCR). GAPDH and ClC-2 PCR products were identified in 23 and 8 (out of a total of 57) single cell cDNA samples, respectively. ClC-2 PCR products with two different lengths were obtained, which might be due to two alternatively spliced ClC-2 mRNA isoforms. This first and combined approach by patch-clamp and single cell RT-PCR techniques to embryonic epithelia indicates that 1) cells at the UB tip express a phenotype remarkably different from that of postembryonic collecting duct principal cells and that 2) ClC-2 is likely to have a key role in early nephrogenesis.

Animals↗
PubMed1996

Stability and leakiness: opposing challenges to the glomerulus.

The complex architecture of the glomerular tuft is stabilized by several mechanisms. The basic system consists of the GBM and the mesangium maintaining the branching pattern of the capillary network. Superimposed are the podocytes, which appear to take effect by two mechanisms. First, podocytes contribute to the stabilization of the capillary folding pattern by supporting the angles between neighboring capillaries. Second, podocyte foot processes fixed to the outer aspect of the GBM probably function as contractile patches counteracting the elastic distension of the GBM. Simultaneously, the pattern of foot process interdigitation underlies the elaboration of a filtration slit and is thus pivotal for the high hydraulic permeability and the specificity of the glomerular filter. The loss of this pattern-commonly termed "foot process effacement" or "foot process fusion"-is frequently found in pathological situations and results in a decrease in permeability and impairment in specificity. On the other hand, foot process effacement is associated with prominent hypertrophy of the contractile apparatus of podocytes, suggesting an increased ability to generate forces counteracting capillary expansion. Thus, foot process effacement appears as an adaptive change in podocyte phenotype giving priority to the support function of podocytes for the prize of reducing the specific permeability.

Animals↗
PubMed1996

Novel mouse embryonic renal marker gene products differentially expressed during kidney development.

Investigators approaching the problem of renal organogenesis have been hampered by a paucity of suitable molecular markers that specify distinct developmental phenotypes. To identify such markers, differential display-polymerase chain reaction (DD-PCR) was used to survey the temporal pattern of gene expression in mouse kidney at 11.5, 13.5, 15.5, and 17.5 days after conception and in the adult kidney. Twenty-two differentially expressed amplification products were identified, isolated, and sequenced. Seventeen clones showed no significant similarity with previously reported nucleotide sequences: two were similar to two housekeeping gene products, and three were similar to human or rat expressed sequence tags. To confirm the differential expression patterns observed by DD-PCR, semiquantitative reverse transcription-PCR was performed using sequence-specific oligonucleotide primers. Nineteen of 22 clones were differentially expressed during kidney development [mouse embryonic renal marker (MERM) sequences 1-19]. The value of MERMs as developmental markers was further assessed in mouse metanephric organ culture, where the pattern of MERM transcript expression mimicked that observed in vivo. Therefore, the DD-PCR method permitted development of a panel of marker sequences that can be used to characterize renal developmental processes and that may allow the identification of novel, functionally relevant gene products.

Animals↗
PubMed1996

A frequent pathway to glomerulosclerosis: deterioration of tuft architecture-podocyte damage-segmental sclerosis.

Lesions in glomerular architecture include mesangial expansion, capillary ballooning, capillary unfolding and microaneurysm formation. Such lesions appear to develop in response to mechanical overextension. A frequent pathway to segmental glomerulosclerosis starts from capillary ballooning and unfolding. Podocytes supporting those deranged capillaries are exposed to increased mechanical stress. This may lead to podocyte injury terminating in detachments from the GBM. Naked GBM areas at peripheral capillary loops allow the attachment of parietal cells to the GBM, i.e. the formation of a tuft adhesion to Bowman's capsule. An adhesion has a strong tendency to progress to segmental sclerosis.

Animals↗
PubMed1996

Regulation of endothelin production and secretion in cultured collecting duct cells by endogenous transforming growth factor-beta.

Confluent cultures of two renal collecting duct cell lines (M-1 and mIMCD-K2 cells derived from cortical and inner medullary collecting ducts, respectively) express endothelin1 (ET1), transforming growth factor-beta (TGF beta; both TGF beta 1 and TGF beta 2), and both types of the TGF beta receptor. Experiments were performed to test whether endogenous TGF beta may be a paracrine modulator of ET1 expression in these cells. Treatment of M-1 and mIMCD-K2 cells with TGF beta 2 antisense oligodeoxynucleotides (ODN) significantly reduced ET1 messenger RNA (mRNA) and ET secretion (as well as TGF beta 2 mRNA) in a concentration-dependent manner, whereas control ODN were without significant effects. To produce ET inhibition, antisense ODN had to be present in the basolateral medium, whereas its sole presence in the apical medium was without effect. In addition, a pan-specific TGF beta antibody caused a significant reduction of ET1 mRNA expression and ET1 secretion. M-1 cells were found to express high levels of the mRNA for plasminogen activator of both tissue and urokinase types. Addition of the nonspecific serine protease inhibitor aprotinin (50 micrograms/ml) to the medium for 24 h significantly reduced the secretion of ET1. These results suggest that secretion of endogenous TGF beta, at least in part activated by the plasminogen/plasmin system, participates in the regulation of ET1 synthesis and secretion by collecting duct cell lines.

Animals↗
PubMed1994

Podocyte damage is a critical step in the development of glomerulosclerosis in the uninephrectomised-desoxycorticosterone hypertensive rat.

The progressive renal disease model of chronic uninephrectomy-desoxycorticosterone-trimethylacetate (UNX-DOCA) hypertension is associated with mesangial proliferation as a major disease mechanism. A detailed structural analysis of the alterations in glomerular structure which accompany the development of sclerosis in this model has not been made. Male Munich-Wistar rats underwent UNX, received weekly injections of the aldosterone agonist DOCA and 1% sodium chloride as drinking solution and were compared with sham operated controls (CON). Thirty eight days after onset, UNX animals had an albuminuria of 183 +/- 180 mg/day versus 0.38 +/- 0.22 mg/day in CON. Kidneys were fixed by total body perfusion and renal tissue processed for light and electron-microscopy. Superficial and deep total glomerular volume increased from 2.18 +/- 0.15 (deep: 2.57 +/- 0.24) 10(6) microns 3 in CON to 3.98 +/- 0.81 (deep: 3.95 +/- 0.63) 10(6) microns 3 in UNX. In addition to overall tuft hypertrophy, structural analysis revealed severe destruction of tuft architecture with mesangial expansion and/or capillary ballooning, leading to local tuft enlargements. Podocytes overlying the expanded areas appeared unable to adapt to cover the increased tuft surfaces. They developed severe lesions in cell architecture leading to denudation of glomerular basement membrane (GBM)-areas. "Naked" GBM appears to represent a nidus for hyalinosis, thrombosis and synechia formation, which progresses to segmental sclerosis. In the UNX-DOCA model of chronic glomerular hypertension local mesangial expansion was frequently encountered but no evidence was found that mesangial proliferation and matrix production proceeded to sclerosis. The crucial damage to the glomerulus in this model would appear to be attributable to podocyte failure, with the resultant GBM denudation triggering synechia formation, hyalinosis and ultimately glomerulosclerosis.

Animals↗
PubMed1994

Renin and renin mRNA in proximal tubules of the rat kidney.

The present study was undertaken to assess the presence of renin enzymatic activity and renin mRNA in proximal tubules of rat kidneys, and to determine the effect of converting enzyme inhibition (CEI) on proximal tubule renin gene expression. Proximal convoluted tubules (PCT), proximal straight tubules (PST), outer medullary collecting ducts (OMCD), and glomeruli (Gloms) were isolated by microdissection. Renin activity was measured in sonicated segments by radioimmunoassay. Renin mRNA levels were assessed using a quantitative PCR. Renin activity in PCT averaged 51 +/- 15 microGU/mm compared to 405 +/- 120 microGU/glomerulus. No measurable renin activity was found in PST and OMCD. Renin activity in both glomeruli and tubules had the same pH optimum, between 7.0 and 7.5. Renin mRNA was consistently detectable in cDNA prepared from PCT and PST, although its abundance per mm tubule was about 1/500th that found in one glomerulus. Renin mRNA was not detectable in OMCD. Tubular renin PCR product identity was confirmed by restriction digestion. CEI administration increased glomerular renin activity and renin mRNA, but not proximal tubular renin. The absence of a stimulatory effect of CEI on proximal tubule renin gene expression suggests the operation of different intracellular signals in control of renin synthesis in the proximal tubule than in the vascular compartment.

Angiotensin-Converting Enzyme Inhibitors↗