PubMed Health⌕ Search

Biomedical subjects

R Witzgall

Publications and source records attributed to R Witzgall.

31 records · Page 2Linked to original sources
PubMed1997

Renal osmotic stress-induced cotransporter: expression in the newborn, adult and post-ischemic rat kidney.

The renal osmotic stress-induced cotransporter (ROSIT), a new putative member of a family of organic solute transporters, is highly expressed in the kidney. Our in situ hybridization data now reveal that large amounts of ROSIT mRNA can be found in the S3 segment of the proximal tubule. In the developing kidney, ROSIT mRNA is expressed after the S-shaped body stage. Because the S3 segment is the major site of damage in the post-ischemic kidney, we evaluated alterations in ROSIT mRNA expression after ischemic acute tubular necrosis. Renal osmotic stress-induced cotransporter mRNA levels were already decreased eight hours post-ischemia. At seven days post-ischemia, ROSIT mRNA reappeared in a mosaic pattern in the regenerating S3 segment, being fully expressed three weeks after the insult except for focal areas. The exact localization of this putative osmolyte transporter in the kidney, together with that of other known osmolyte transporter will contribute to a better understanding of the mechanism of medullary osmolyte accumulation and its vectorial transport.

Age Factors↗
PubMed1997

Differentiation and cell polarity during renal cyst formation in the Han:SPRD (cy/+) rat, a model for ADPKD.

Despite the recent positional cloning of genes responsible for autosomal dominant polycystic kidney disease (ADPKD), the exact pathogenetic mechanisms underlying this disorder are still unclear. To learn more about cyst formation, we investigated cell differentiation and cell polarity in the Han:SPRD (cy/+) rat between 21 days and 60 wk of age. At early stages of cyst development, alkaline phosphatase, aquaporin-1, NaSi-1 cotransporter, and Na(+)-K(+)-adenosinetriphosphatase (Na(+)-K(+)-ATPase) were expressed normally. Clusterin mRNA was only sparsely expressed at the onset of cystic degeneration and increased thereafter, being highest in noncystic nephron segments. In cyst wall cells, clusterin on the one hand and alkaline phosphatase, aquaporin-1, NaSi-1-cotransporter, and Na(+)-K(+)-ATPase on the other were expressed in a mutually exclusive fashion. No change in cell polarity could be observed at any stage. Our data therefore argue against a change in cell polarity and against an early arrest in normal tubular development during cyst formation in the Han:SPRD (cy/+) rat model of ADPKD but favor the hypothesis that tubular epithelia develop in an orderly fashion and degenerate thereafter.

Aging↗
PubMed1997

Expression of the conjugate export pump encoded by the mrp2 gene in the apical membrane of kidney proximal tubules.

A novel ATP-dependent export pump for amphiphilic anionic conjugates, which has been cloned recently from liver, was identified in rat kidney and localized to the apical membrane domain of proximal tubule epithelia. This 190-kD membrane glycoprotein (Mrp2) has been described previously as the hepatocyte canalicular isoform of the multidrug resistance protein and as the canalicular multispecific organic anion transporter. Mrp2 was identified in kidney by reverse transcription PCR followed by sequencing of the amplified 786-bp fragment and by immunoblotting, using an antibody specifically reacting with the carboxy terminus of rat Mrp2. Double immunofluorescence and confocal laser-scanning microscopy showed the presence of Mrp2 in the brush-border membrane domain of segments S1, S2, and S3 of proximal tubule epithelia. Mrp2 was not detectable in other segments of the nephron. The onset of Mrp2 expression during development occurred in a very early stage of nephron development. Mrp2 represents the first cloned ATP-dependent export pump for amphiphilic organic anions identified in kidney and localized to the apical membrane domain of proximal tubule epithelia. Mrp2 may contribute to cellular detoxification and to the secretion of endogenous and xenobiotic anionic substances, most of which are conjugates, from the blood into urine.

Adenosine Triphosphate↗
PubMed1996

A novel member of the RING finger family, KRIP-1, associates with the KRAB-A transcriptional repressor domain of zinc finger proteins.

The Krüppel-associated box A (KRAB-A) domain is an evolutionarily conserved transcriptional repressor domain present in approximately one-third of zinc finger proteins of the Cys2-His2 type. Using the yeast two-hybrid system, we report the isolation of a cDNA encoding a novel murine protein, KRAB-A interacting protein 1 (KRIP-1) that physically interacts with the KRAB-A region. KRIP-1 is a member of the RBCC subfamily of the RING finger, or Cys3HisCys4, family of zinc binding proteins whose other members are known to play important roles in differentiation, oncogenesis, and signal transduction. The KRIP-1 protein has high homology to TIF1, a putative modulator of ligand-dependent activation function of nuclear receptors. A 3.5-kb mRNA for KRIP-1 is ubiquitously expressed among all adult mouse tissues studied. When a GAL4-KRIP-1 fusion protein is expressed in COS cells with a chloramphenicol acetyltransferase reporter construct with five GAL4 binding sites, there is dose-dependent repression of transcription. Thus, KRIP-1 interacts with the KRAB-A region of C2H2 zinc finger proteins and may mediate or modulate KRAB-A transcriptional repressor activity.

Amino Acid Sequence↗
PubMed1996

Cytosolic phospholipase A2 (PLA2), but not secretory PLA2, potentiates hydrogen peroxide cytotoxicity in kidney epithelial cells.

Phospholipase A2 (PLA2) and reactive oxygen species have been implicated both individually and synergistically in various forms of cellular injury. The form(s) of PLA2 important for cell injury and the implications of enhanced activity of the enzyme, however, have not been discerned. Previous studies reveal an increase in PLA2 activity associated with cell injury, but this association does not establish a causal relationship between the increase in activity and the injury. LLC-PK1 cell lines were created that express either the cytosolic PLA2 or a group II PLA2. The susceptibility of these cells to hydrogen peroxide toxicity was determined in order to evaluate the relative importance of these two forms of PLA2 in oxidant injury. Expression of cytosolic PLA2 in the LLC-cPLA2 cell line was associated with a 50-fold increase in PLA2 activity in the cytosolic fraction, an increase in agonist-stimulated arachidonate release, and immunodetection of the cytosolic PLA2 protein that was undetectable in control cells. Exposure to hydrogen peroxide or menadione, but not mercuric chloride, resulted in significantly greater lactate dehydrogenase release in LLC-cPLA2 cells when compared with control cells. Exogenous arachidonic acid (150 microM) did not enhance hydrogen peroxide-induced injury. The intracellular calcium chelator, 1,2-bis-(o-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid/tetra(acetoxymethyl) ester, protected the cells against injury, but the calcium ionophore, A23187, did not increase injury. Glycine conferred no protective effect against hydrogen peroxide toxicity. By contrast to these results with cytosolic PLA2-expressing cells, secretory PLA2 expression to very high levels did not increase susceptibility to hydrogen peroxide. Thus, cytosolic PLA2 may an be an important mediator of oxidant damage to renal epithelial cells.

Animals↗
PubMed1994

The Krüppel-associated box-A (KRAB-A) domain of zinc finger proteins mediates transcriptional repression.

We have previously reported the cloning, sequencing, and partial characterization of Kid-1, a zinc finger-encoding cDNA from the rat kidney. The Kid-1 protein and approximately one-third of all other zinc finger proteins contain a highly conserved region of approximately 75 amino acids at their NH2 terminus named Krüppel-associated box (KRAB), which is subdivided into A and B domains. The evolutionary conservation, wide distribution, and genomic organization of the KRAB domains suggest an important role of this region in the transcriptional regulatory function of zinc finger proteins. The functional significance of the KRAB domain was evaluated by studying transcriptional activities of yeast GAL4-rat Kid-1 fusion proteins containing various regions of the non-zinc-finger domain of Kid-1. Transcriptional repressor activity of GAL4-Kid-1 fusion proteins maps to the KRAB-A domain. The KRAB-A domain of another zinc finger protein, ZNF2, also has repressor activity. Site-directed mutagenesis of conserved amino acids in this motif results in decreased repressor activity. Thus, we have established a functional significance for the KRAB-A domain, a consensus sequence common in zinc finger proteins.

Amino Acid Sequence↗
PubMed1994

Genomic structure and chromosomal location of the rat gene encoding the zinc finger transcription factor Kid-1.

We have previously cloned and sequenced a novel zinc finger cDNA, Kid-1, from the rat. Because of its developmentally regulated expression pattern and its suppression after renal injury, as well as its kidney-predominant expression, we propose that Kid-1 is likely to play an important role in renal gene regulation. Kid-1 encodes a predicted protein with 13 zinc fingers at the carboxy end and Krüppel-associated box (KRAB) A and B regions at the amino terminus. Expression of a Kid-1-GAL4 chimeric protein results in strong transcriptional repression of cotransfected constructs containing GAL4 binding sites and a chloramphenicol acetyl transferase gene driven by either a minimal promoter or a SV40 enhancer. We now report the cloning, structural organization, and chromosomal localization of the Kid-1 gene. The Kid-1 gene is composed of four exons and three introns, closely reflecting the organization of the Kid-1 protein. The KRAB A and B regions are encoded by the second and third exons, respectively. The entire zinc finger region is encoded by the fourth exon. Using a combination of linkage analysis and somatic cell hybrid analysis, Kid-1 was mapped to rat chromosome (RNO) 10. Kid-1, Il3, and Sparc form a tight linkage group on RNO10. Regional sublocalization to RNO10q21.3-q22 was established by fluorescence in situ hybridization.

Amino Acid Sequence↗
PubMed1994

A mammalian expression vector for the expression of GAL4 fusion proteins with an epitope tag and histidine tail.

Expression of newly cloned cDNAs in mammalian cell lines is an essential tool for the functional analysis of the proteins encoded by these cDNAs. In many instances, however, evaluation of the protein is difficult because of the difficulty in purification of the expressed protein and/or the lack of specific antibodies which react with the proteins on Western blots or for immunocytochemistry or immunoprecipitation. A number of gene fusion systems have been employed in which a known peptide is fused to the expression product of interest and the fusion protein is purified using affinity chromatography and identified in extracts or by immunocytochemistry using antibodies directed against the affinity handle peptide. The DNA-binding domain of the yeast transcription factor GAL4 is widely used to construct fusion proteins with putative transcription factors to evaluate potential trans-acting domains. Because of the lack of commercially available anti-GAL4 antibodies, the further biochemical characterization of these fusion proteins has remained difficult. We describe the construction of two mammalian expression vectors, pMFH/GAL4 and pMFH2/GAL4 (where pMFH stands for pM2, Flag, Histidine tail), which encode the DNA-binding domain of the yeast transcription factor GAL4 with a Flag peptide (consisting of the 11-amino-acid leader peptide of the gene 10 product from bacteriophage T7) at the NH2-terminus and a tail of six histidines at the COOH-terminus. Unique restriction sites allow both the construction of fusion proteins with the GAL4 DNA-binding domain and the replacement of the GAL4 fragment with another insert.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Localization of proliferating cell nuclear antigen, vimentin, c-Fos, and clusterin in the postischemic kidney. Evidence for a heterogenous genetic response among nephron segments, and a large pool of mitotically active and dedifferentiated cells.

The mechanisms leading to the recovery of the kidney after ischemic acute renal failure are poorly understood. To explore the role played by mitogenesis and dedifferentiation in this repair process and to identify whether the genetic response of the nephron segments reflects the level of susceptibility to injury, the temporal and nephron segment expressions of various proteins implicated in mitogenesis, differentiation, and injury were determined. Proliferating cell nuclear antigen (PCNA), a marker for the G1-S transition in the cell cycle and hence mitogenesis, was detected primarily in the S3 segment of the proximal tubule, with maximal expression at 2 d postischemia. Vimentin, normally present in mesenchymal cells but not epithelial cells, and hence a marker for the state of differentiation, was prominently expressed in the S3 segment 2-5 d postischemia. In the S3 segments in the outer stripe of the medulla cells that stained positively for PCNA also stained positively for vimentin. Clusterin, a marker for cell injury, was expressed primarily in the S3 segment and in the distal tubule with distinct staining patterns in each segment. None of the cells that stained with clusterin antibodies were positively stained with PCNA or vimentin antibodies. Likewise, none of the PCNA or vimentin-positive cells expressed clusterin at detectable levels. Thus, in the S3 segment, where there is significant ischemic injury, surviving cells express markers indicating that they undergo mitogenesis and dedifferentiate in the postischemic period. While there is some expression of c-Fos in the S3 segment, c-Fos was expressed predominantly, at 1 and 3 h postischemia, in the nuclei of the distal nephron, particularly in the thick ascending limb. The data support the view that the mature renal S3 segment epithelial cell can be a progenitor cell.

Animals↗
PubMed1993

Kid-1, a putative renal transcription factor: regulation during ontogeny and in response to ischemia and toxic injury.

We have identified a new putative transcription factor from the rat kidney, termed Kid-1 (for kidney, ischemia and developmentally regulated gene 1). Kid-1 belongs to the C2H2 class of zinc finger genes. Its mRNA accumulates with age in postnatal renal development and is detected predominantly in the kidney. Kid-1 mRNA levels decline after renal injury secondary to ischemia or folic acid administration, two insults which result in epithelial cell dedifferentiation, followed by regenerative hyperplasia and differentiation. The low expression of Kid-1 early in postnatal development, and when renal tissue is recovering after injury, suggests that the gene product is involved in establishment of a differentiated phenotype and/or regulation of the proliferative response. The deduced protein contains 13 C2H2 zinc fingers at the COOH end in groups of 4 and 9 separated by a 32-amino-acid spacer. There are consensus sites for phosphorylation in the NH2 terminus non-zinc finger region as well as in the spacer region between zinc fingers 4 and 5. A region of the deduced protein shares extensive homology with a catalytic region of Raf kinases, a feature shared only with TFIIE among transcription factors. To determine whether Kid-1 can modulate transcription, a chimeric construct encoding the Kid-1 non-zinc finger region (sense or antisense) and the DNA-binding region of GAL4 was transfected into COS and LLC-PK1 cells together with a chloramphenicol acetyltransferase (CAT) reporter plasmid containing GAL4 binding sites, driven by either a minimal promoter or a simian virus 40 enhancer. CAT activity was markedly inhibited in cells transfected with the sense construct compared with the activity in cells transfected with the antisense construct. To our knowledge, this pattern of developmental regulation, kidney expression, and regulation of transcription is unique among the C2H2 class of zinc finger-containing DNA-binding proteins.

Amino Acid Sequence↗
PubMed1992

Soluble interleukin-2 receptors inhibit interleukin 2-dependent proliferation and cytotoxicity: explanation for diminished natural killer cell activity in cutaneous T-cell lymphomas in vivo?

In patients with cutaneous T-cell lymphomas (CTCL), soluble interleukin-2 receptor serum levels (sIL-2R) were determined by ELISA technique, and natural killer cell (NK) activity, by a 4-h chromium-51 release assay. Decrease of NK activity correlated with the augmentation of serum sIL-2R. After a 4-d stimulation with interleukin 2 CTCL patients' peripheral mononuclear cells (PMC) showed an increase of cytotoxic activity similar to that in healthy donors' PMC. Normal donors' PMC demonstrated a diminished IL-2-induced cytotoxic activity in 25% CTCL serum (sIL-2R of 3000, 7330, and 10700 U/ml, respectively) compared to control serum (sIL-2R of 400, 340, and 420 U/ml, respectively). IL-2-dependent proliferation of 2-d phytohemagglutinin (PHA) blasts was lower in CTCL serum than in control serum. sIL-2R was enriched from one CTCL patient's serum by IL-2 affinity chromatography. Transfection of the Tac gene into NIH/3T3 fibroblasts resulted in the production of a recombinant sIL-2R. The presence of enriched native or recombinant sIL-2R inhibited interleukin-2-dependent generation of cytotoxic activity and PHA blast proliferation. We suggest that elevated sIL-2R levels account for diminished NK activity by neutralizing interleukin 2 in CTCL patients.

Cytotoxicity, Immunologic↗
PubMedDate not supplied

The proximal tubule phenotype and its disruption in acute renal failure and polycystic kidney disease.

In light of recent developments in the fields of genetics, molecular, cell and developmental biology, the kidney is receiving increasing attention as a model system for organ development and human diseases. Gene disruption experiments have provided evidence for the essential role of a number of proteins in the earliest phase of nephron development, but very little is known about the identity of such proteins in more advanced stages. This minireview will focus on the proximal tubule and its role in the pathology of ischemic acute renal failure and polycystic kidney disease. Like all other nephron segments, the proximal tubule develops from the metanephrogenic mesenchyme. So far the only genetic model which affects the function of the proximal tubule is a strain of knockout mice with an inactivation of the HNF1 gene. After ischemic renal damage the proximal tubule responds with a different genetic program than the distal tubule. Evidence from human polycystic kidney disease and several animal models of polycystic kidney disease suggests that proximal tubules are affected differently by polycystic kidney disease than distal tubules and collecting ducts.

Acute Kidney Injury↗