Biomedical subjects

Hermann-Josef Gröne

Publications and source records attributed to Hermann-Josef Gröne.

At least 19 recordsLinked to original sources

PubMed2003

Iron overload in adult Hfe-deficient mice independent of changes in the steady-state expression of the duodenal iron transporters DMT1 and Ireg1/ferroportin.

Patients suffering from hereditary hemochromatosis (HH) show progressive iron overload as a consequence of increased duodenal iron absorption. It has been hypothesized that mutations in the HH gene HFE cause misprogramming of the duodenal enterocytes towards a paradoxical iron-deficient state, resulting in increased iron transporter expression. Previous reports concerning gene expression levels of the duodenal iron transporters DMT1 and IREG1 in HH patients and animal models are controversial, however, and in many cases only mRNA expression levels were investigated. To analyze the duodenal expression of DMT1, Ireg1, Dcytb, and hephaestin and the association with iron overload in adult Hfe(-/-) mice, an Hfe(-/-) mouse line was generated. Duodenal DMT1 and Ireg1 protein levels, duodenal DMT1, Ireg1, Dcytb, hephaestin, and TfR1 mRNA levels, and hepatic hepcidin mRNA levels were quantified and the correlation to liver iron contents was calculated. We report that duodenal DMT1 and Ireg1 mRNA levels and DMT1 and Ireg1 protein levels remained unaffected by the Hfe deletion. Furthermore, duodenal hephaestin and TfR1 mRNA expression and hepatic hepcidin mRNA expression remained unaltered, while the duodenal mRNA expression of the brush border ferric reductase Dcytb was significantly increased in Hfe(-/-) mice. We found no correlation between the expression level of any of the analyzed transcripts and the liver iron content. In conclusion, the lack of correlation between DMT1 and Ireg1 protein expression and the liver iron content suggests that elevated duodenal iron transporter expression is not required for high liver iron overload. Hfe(-/-) mice do not necessarily display features of iron deficiency in the duodenum, indicated by an increase in mRNA and protein levels of DMT1 and Ireg1. Rather, the duodenal ferric reductase Dcytb may act as a possible mediator of iron overload in Hfe deficiency.

Animals↗

PubMed2003

Dkk1 and noggin cooperate in mammalian head induction.

Growth factor antagonists play important roles in mediating the inductive effects of the Spemann organizer in amphibian embryos and its equivalents in other vertebrates. Dual inhibition of Wnt and BMP signals has been proposed to confer head organizer activity. We tested the requirement of this coinhibition in Xenopus and mice. In Xenopus, simultaneous reduction of the BMP antagonists chordin and noggin, and the Wnt antagonist dickkopf1 (dkk1) leads to anterior truncations. In mice, compound mutants for dkk1 and noggin display severe head defects, with deletion of all head structures anterior to the mid-hindbrain boundary. These defects arise as a result of a failure in anterior specification at the gastrula stage. The results provide genetic evidence for the dual inhibition model and indicate that dkk1 and noggin functionally cooperate in the head organizer.

Animals↗

PubMed2003

Isotretinoin ameliorates renal damage in experimental acute renal allograft rejection.

BACKGROUND: Retinoic acids, derivatives of vitamin A, act through retinoid receptors that are expressed in renal and immunocompetent cells (B and T cells; monocytes and macrophages). In experimental models of glomerulonephritis and renal interstitial disease, retinoids were shown to reduce both glomerular and tubular damage and inflammation. We therefore examined whether retinoids reduce cellular rejection and renal damage in a model of acute renal allograft rejection. METHODS: Kidneys of Fisher rats (F344, RT11v1) were orthotopically grafted to Lewis rats (RT11). Animals were killed 7 or 14 days after transplantation. Rats undergoing transplantation were treated with isotretinoin (13 cis-retinoic acid) at a low dose of 2 mg/kg body weight per day (LD isotretinoin) or at a high dose of 20 mg/kg body weight per day (HD isotretinoin) or with vehicle. RESULTS: At day 14, albuminuria was reduced by approximately 70% (vehicle: 1.1+/-0.2 mg/24 hr vs. LD isotretinoin: 0.32+/-0.1 mg/24 hr; P<0.001). At days 7 and 14 serum creatinine levels were significantly higher in the vehicle-treated group than in the LD and HD isotretinoin-treated rats (P<0.05). Both LD and HD isotretinoin significantly reduced acute vascular injury compared with vehicle-treated rats (score at day 14: LD isotretinoin 20.1+/-5.1 vs. vehicle 57.8+/-9.9, P<0.01), acute glomerular injury (score: LD isotretinoin 6.8+/-1.0 vs. vehicle 10.6+/-0.9 P<0.05), and the number of glomerular monocytes and macrophages and cytotoxic T cells. Isotretinoin also significantly lessened tubulointerstitial damage, tubulointerstitial cell proliferation, and the number of cells infiltrating the tubulointerstitium. CONCLUSIONS: Isotretinoin significantly ameliorated functional, vascular, glomerular, and tubulointerstitial lesions in acute graft rejection. Although the current study did not definitely eliminate the possibility that isotretinoin only delayed the rejection process, retinoic acid derivatives may provide a new approach in the treatment of acute rejection injury.

Albuminuria↗

PubMed2003

CC chemokine ligand 5/RANTES chemokine antagonists aggravate glomerulonephritis despite reduction of glomerular leukocyte infiltration.

The chemokine CC chemokine ligand (CCL)5/RANTES as well as its respective receptor CCR5 mediate leukocyte infiltration during inflammation and are up-regulated early during the course of glomerulonephritis (GN). We tested the effects of the two CCL5/RANTES blocking analogs, Met-RANTES and amino-oxypentane-RANTES, on the course of horse apoferritin (HAF)-induced GN. HAF-injected control mice had proliferative GN with mesangial immune complex deposits of IgG and HAF. Daily i.p. injections of Met-RANTES or amino-oxypentane-RANTES markedly reduced glomerular cell proliferation and glomerular macrophage infiltration, which is usually associated with less glomerular injury and proteinuria in HAF-GN. Surprisingly, however, HAF-GN mice treated with both analogs showed worse disease with mesangiolysis, capillary obstruction, and nephrotic range albuminuria. These findings were associated with an enhancing effect of the CCL5/RANTES analogs on the macrophage activation state, characterized by a distinct morphology and increased inducible NO synthetase expression in vitro and in vivo, but a reduced uptake of apoptotic cells in vivo. The humoral response and the Th1/Th2 balance in HAF-GN and mesangial cell proliferation in vitro were not affected by the CCL5/RANTES analogs. We conclude that, despite blocking local leukocyte recruitment, chemokine analogs can aggravate some specific disease models, most likely due to interactions with systemic immune reactions, including the removal of apoptotic cells and inducible NO synthetase expression.

Animals↗

PubMed2003

Laser-controlled microdissection of tissues opens a window of new opportunities.

Gene expression analysis using total RNA of bulk tissue usually cannot assign specific messages to particular cell types. Cell-specific RNA expression profiling, though, may be crucial for a better understanding ofthe role of each distinct cell type within a physiological or pathophysiological setting. RNA profiling based on laser-controlled microdissection (LCM) of defined cells of a tissue now provides a useful tool for studying molecular crosstalk between different cell types within a tissue. The LCM technique allows for efficient isolation of single cells with no or very low contamination of surrounding tissue components, simultaneously leaving the intracellular structure and molecules intact. In this review, different issues of the LCM technique and the RNA amplification procedure for microarray analysis are discussed. An exemplary summary of results obtained from gene profiling of epithelial and stromal cells from human prostate tumors is presented, demonstrating the power of LCM-based molecular analysis. Finally, we discuss the potential use of the LCM technique i) to study the transcriptome of distinct cells from formalin-fixed and paraffin-embedded tissues in subcellular RNA profiling and ii) high resolution proteomic and metabolistic studies.

Dissection↗

PubMed2003

Bacterial CpG-DNA aggravates immune complex glomerulonephritis: role of TLR9-mediated expression of chemokines and chemokine receptors.

Immune complex glomerulonephritis (GN) often deteriorates during infection with viruses and bacteria that, in contrast to mammals, have DNA that contains many unmethylated CpG motifs. Balb/c mice with horse apoferritin-induced GN (HAF-GN) were treated with either saline, CpG-oligodeoxynucleotides (ODN), or control GpC-ODN. Only CpG-ODN exacerbated HAF-GN with an increase of glomerular macrophages, which was associated with massive albuminuria and increased renal MCP-1/CCL2, RANTES/CCL5, CCR1, CCR2, and CCR5 mRNA expression. CpG-ODN induced a Th1 response as indicated by serum anti-HAF IgG(2a) titers, mesangial IgG(2a) deposits, and splenocyte IFN-gamma secretion. Messenger RNA for the CpG-DNA receptor Toll-like reeptor 9 (TLR9) was present in kidneys with HAF-GN but not in normal kidneys. The source of TLR9 mRNA in HAF-GN could be infiltrating macrophages or intrinsic renal cells, e.g., mesangial cells; but, in vitro, only murine J774 macrophages expressed TLR9. In J774 cells, CpG-ODN induced the chemokines MCP-1/CCL2 and RANTES/CCL5 and the chemokine receptors CCR1 and CCR5. It is concluded that CpG-DNA can aggravate preexisting GN via a shift toward a Th1 response but also by a novel pathway involving TLR9-mediated chemokine and chemokine receptor expression by macrophages, which may contribute to the enhanced glomerular macrophage recruitment and activation. This mechanism may be relevant during infection-triggered exacerbation of human immune-complex GN and other immune-mediated diseases in general.

Animals↗

PubMed2003

PDGF-C expression in the developing and normal adult human kidney and in glomerular diseases.

PDGF-C is a new member of the PDGF-family and has recently been identified as a rat mesangial cell mitogen. Its expression and function in human kidneys is unknown. Localization of PDGF-C protein was analyzed by immunohistochemistry using a rabbit polyclonal antibody directed against the core-domain of PDGF-C in human fetal kidneys (n = 8), normal adult human kidneys (n = 9), and in renal biopsies of patients with IgA nephropathy (IgAN, n = 31), membranous nephropathy (MGN, n = 8), minimal change disease (MC, n = 7), and transplant glomerulopathy (TxG, n = 12). Additionally, PDGF-C mRNA was detected in microdissected glomeruli by real-time RT-PCR in cases of normal adult kidneys (n = 7), IgAN (n = 27), MGN (n = 11), and MC (n = 13). In the fetal kidney, PDGF-C localized to the developing mesangium, ureteric bud epithelium, and the undifferentiated mesenchyme. In the adult kidney, PDGF-C was constitutively expressed in parietal epithelial cells of Bowman's capsule, tubular epithelial cells (loops of Henle, distal tubules, collecting ducts), and in arterial endothelial cells. A marked upregulation of glomerular PDGF-C protein was seen in MGN and TxG with a prominent positivity of virtually all podocytes. In MC, PDGF-C localized to podocytes in a more focal distribution. In MGN, increased glomerular PDGF-C protein expression was due to increased mRNA synthesis as a 4.3-fold increase in PDGF-C mRNA was detected in microdissected glomeruli from MGN compared with normal. PDGF-C protein was additionally expressed in individual mesangial cells in TxG. Finally, upregulated PDGF-C protein expression was detected within sclerosing glomerular and fibrosing tubulointerstitial lesions in individual cases from all analyzed groups. We conclude that PDGF-C is constitutively expressed in the human kidney and is upregulated in podocytes and interstitial cells after injury/activation of these cells.

Adult↗

PubMed2003

Cell biological and biochemical characterization of drebrin complexes in mesangial cells and podocytes of renal glomeruli.

Drebrins are actin-binding proteins (ABP) initially identified in and thought to be specific for neuronal cells, where they appear to contribute to the formation of cell processes. Recent studies have also detected the isoform drebrin E2 in a wide range of non-neuronal cell types, notably in and near actin-rich lamellipodia and filopodia. The present study demonstrates drebrin enrichment in renal glomeruli. Immunohistochemistry and double-label confocal laser scanning microscopy have shown intense drebrin reactions in the mesangial cells of diverse mammalian species. In adult human and bovine kidneys, drebrin is, in addition, markedly enriched in the foot processes of podocytes, as also demonstrable by immunoelectron microscopy. By contrast, the podocytes of rodent glomeruli appear to contain significant drebrin concentrations only during early developmental stages. In differentiated murine podocytes cultured in vitro, however, drebrin is concentrated in the cell processes, where it partially codistributes with actin and other ABP. In biochemical analyses using protein extracts from renal cortices, large (approximately 20S) complexes ("drebrosomes") were found containing drebrin and actin. These findings confirm and extend our hypothesis that drebrin is involved in the regulation of actin dynamics also outside the nervous system. Clearly, drebrin has to be added to the ensemble of ABP regulating the actomyosin system and the dynamics of mesangial cells and foot processes in podocytes.

Animals↗

PubMed2003

Gene expression profiles of podocyte-associated molecules as diagnostic markers in acquired proteinuric diseases.

For identifying potential diagnostic markers of proteinuric glomerulopathies, glomerular mRNA levels of molecules relevant for podocyte function (alpha-actinin-4, glomerular epithelial protein 1, Wilms tumor antigen 1, synaptopodin, dystroglycan, nephrin, podoplanin, and podocin) were determined by quantitative real-time RT-PCR from microdissected glomeruli. Biopsies from 83 patients with acquired proteinuric diseases were analyzed (minimal change disease [MCD; n = 13], benign nephrosclerosis [n = 16], membranous glomerulopathy [n = 31], focal and segmental glomerulosclerosis [FSGS; n = 9], and controls [n = 14]). Gene expression levels normalized to two different housekeeping transcripts (glyceraldehyde-3-phosphate-dehydrogenase and 18 S rRNA) did not allow a separation between proteinuric disease categories. However, a significant positive correlation between alpha-actinin-4, glomerular epithelial protein 1, synaptopodin, dystroglycan, Wilms tumor antigen 1, and nephrin was found in all analyzed glomeruli, whereas podocin mRNA expression did not correlate. Because varying amounts of housekeeper cDNA per glomerulus can confound expression ratios relevant for a subpopulation of cells, an "in silico" microdissection was performed using a podocyte-specific cDNA as a reference gene. Expression ratio of podocin to synaptopodin, the two genes with the most disparate expression, allowed a robust separation of FSGS from MCD and nephrosclerosis. Segregation of FSGS from MCD via this ratio was confirmed in an independent population of formaldehyde-fixed archival biopsies (MCD, n = 5; FSGS, n = 4) after glomerular laser capture microdissection. In addition, the expression marker was able to predict steroid responsiveness in diagnostically challenging cases of MCD versus FSGS (n = 6). As the above approach can be performed as an add-on diagnostic tool, these molecular diagnostic parameters could give novel information for the management of proteinuric diseases.

Actinin↗

PubMed2003

Vascular endothelial growth factor in diabetic nephropathy.

BACKGROUND: Vascular endothelial growth factor (VEGF) increases endothelial permeability. VEGF is produced in podocytes and functional receptors are located on endothelial glomerular cells. The aim of the current study in diabetic patients with normal renal function to various degrees of proteinuric nephropathy was therefore to unravel a possible role of the most important isoform VEGF(165) for albuminuria and to investigate the impact of therapy with an inhibitor of the renin-angiotensin system on VEGF(165) secretion. SUBJECTS AND METHODS: A cross-sectional study in 72 patients (41 female, 31 male) with long-standing type 1 (n = 35, mean age 43.3 years, range 22-67) or type 2 (n = 37, mean age 66 years, range 53-83) diabetes mellitus was performed; in 19 patients the serum creatinine value was >1.5 mg/dl. Twenty-six healthy volunteers (17 female, 9 male, mean age 34.8 years, range 19-58) with normal renal function served as controls. Serum and urinary VEGF(165) was measured by ELISA. Urinary albumin was measured nephelometrically. Mann Whitney U tests were used for comparisons. RESULTS: In type 1 and type 2 diabetics mean urinary VEGF(165) concentration amounted to 112 +/- 88 (mean +/- SD) and 88 +/- 85 ng/l, respectively, compared to 101 +/- 60 ng/l in the normal volunteers (NS vs. diabetics). The respective mean urinary albumin concentrations were 443 +/- 1029, 394 +/- 749 and 20 +/- 33 mg/l (p < 0.01 vs. diabetics type 2). There was a correlation between urinary VEGF and albumin, but only in patients with type 2 diabetes (R = 0.497; n = 36; p = 0.002). Urinary VEGF(165) was similar in patients with (n = 40) and without ACE inhibitor/AT1 antagonist therapy (n = 32) and in normal volunteers, whereas serum VEGF(165) was higher in the treated type 1 diabetics. CONCLUSIONS: These results may suggest that VEGF(165) plays some role in the development of albuminuria in diabetic nephropathy due to type 2 but not type 1 diabetes.

Adult↗

PubMed2002

Anti-inflammatory effects of tetrahydrobiopterin on early rejection in renal allografts: modulation of inducible nitric oxide synthase.

Oxidative stress contributes to the development of early transplant failure. As nitric oxide synthases (NOS) can act as sources of superoxide, we investigated the effect of the NOS cofactor tetrahydrobiopterin (BH4) on oxyradical production and early rejection in a rat kidney transplantation model. Allograft transplantation (Brown Norway to Lewis) showed more renal superoxide production and monocyte infiltration when compared with isografts (Lewis to Lewis). Administration of the stable BH4 precursor sepiapterin had no effect on superoxide production in the isografts (51+/-10 vs. 69+/-17 cps/10 mg protein), but led to a marked decrease in superoxide production in the allografts (116+/-11 vs. 60+/-6 cps/10 mg protein; P<0.05) and was accompanied by a reduction in periarterial macrophage infiltration (3.3+/-0.7 vs. 1.3+/-0.3 cells/vessel; P<0.05) and an increase in NO production (78+/-22 vs. 173+/-12 AU/g kidney) (P<0.01). In vitro experiments confirm that iNOS can produce superoxide mainly from the heme domain, whereas BH4 administration can reverse this superoxide production in the presence of adequate anti-oxidant defense. Our findings support the hypothesis that BH4 can be used to modulate the function of the inflammatory iNOS isoform and suggest a potential therapeutic role for sepiapterin in early allograft rejection.

Animals↗

PubMed2002

Roles of SLC/CCL21 and CCR7 in human kidney for mesangial proliferation, migration, apoptosis, and tissue homeostasis.

The release of chemokines by intrinsic renal cells is an important mechanism for the regulation of leukocyte trafficking during renal inflammation. The expression of chemokine receptors by intrinsic renal cells such as mesangial cells (MC) suggests an expanded role for chemokine-chemokine receptor biology in local immunomodulation and potentially glomerular homeostasis. By immunohistochemistry we found the chemokine receptor CCR7 expressed in a mesangial pattern while the CCR7 ligand SLC/CCL21 showed a podocyte-specific expression. CCR7 expression was further characterized by RT-PCR, RNase protection assays, and FACS analysis of cultured human MC, and was found to be constitutively present. Real-time PCR of microdissected glomeruli confirmed the expression of SLC/CCL21. A functional role for CCR7 was demonstrated for human MC migration and proliferation. A protective effect of SLC/CCL21 was shown for MC survival in Fas Ab-induced apoptosis. Finally, "wound healing" was enhanced in the presence of SLC/CCL21 in an in vitro injury model. The constitutive glomerular expression of CCR7 and its ligand SLC/CCL21 in adjacent cell types of the human kidney suggests novel biological functions of this chemokine/chemokine receptor pair and a potential role in processes involved in glomerular homeostasis and regeneration.

Apoptosis↗

PubMed2002

Temporal Cre-mediated recombination exclusively in endothelial cells using Tie2 regulatory elements.

SUMMARY: The versatility of the bacteriophage Cre/LoxP system is dependent on the availability of a spectrum of tissue-specific Cre transgenic mice to address a host of biological questions. In this paper, we report on the generation of an inducible Tie2Cre transgenic mouse line that facilitates gene targeting exclusively in endothelial cells. The temporal manner of recombination is feasible through the use of a Cre-estrogen receptor fusion protein ER(T2) and was, in practical terms, achieved by feeding the animals the estrogen antagonist tamoxifen orally for 5 weeks. High efficiency of recombination was found in the vast majority of endothelial cell populations examined, as monitored by an EGFP reporter mouse line. Critically, no EGFP expression was observed in any uninduced mice. This inducible Cre line will be a very beneficial asset to investigating the role of endothelial specific genes in the adult mouse and to induce transgenes in the endothelium in an extremely efficient manner. genesis 33:191-197, 2002.

Animals↗

PubMed2002

Decrease and gain of gene expression are equally discriminatory markers for prostate carcinoma: a gene expression analysis on total and microdissected prostate tissue.

Information on over- and underexpressed genes in prostate cancer in comparison to adjacent normal tissue was sought by DNA microarray analysis. Approximately 12,600 mRNA sequences were analyzed from a total of 26 tissue samples (17 untreated prostate cancers, 9 normal adjacent to prostate cancer tissues) obtained by prostatectomy. Hierarchical clustering was performed. Expression levels of 63 genes were found significantly (at least 2.5-fold) increased, whereas expression of 153 genes was decreased (at least 2.5-fold) in prostate cancer versus adjacent normal tissue. In addition to previously described genes such as hepsin, overexpression of several genes was found that has not drawn attention before, such as the genes encoding the specific granule protein (SGP28), alpha-methyl-acyl-CoA racemase, low density lipoprotein (LDL)-phospholipase A2, and the anti-apoptotic gene PYCR1. The radiosensitivity gene ATDC and the genes encoding the DNA-binding protein inhibitor ID1 and the phospholipase inhibitor uteroglobin were significantly down-regulated in the cancer samples. DNA microarray data for eight genes were confirmed quantitatively in five normal and five cancer tissues by real-time reverse transcriptase-polymerase chain reaction with a high correlation between the two methods. Laser capture microdissection of epithelial and stromal compartments from cancer and histological normal specimens followed by an amplification protocol for low levels of RNA (<0.1 microg) allowed us to distinguish between gene expression profiles characteristic of epithelial cells and those typical of stroma. Most of the genes identified in the nonmicrodissected tumor material as up-regulated were indeed overexpressed in cancerous epithelium rather than in the stromal compartment. We conclude that development of prostate cancer is associated with down-regulation as well as up-regulation of genes that show complex differential regulation in epithelia and stroma. Some of the gene expression alterations identified in this study may prove useful in the development of novel diagnostic and therapeutic strategies.

Adenocarcinoma↗

PubMed2002

Absence of decorin adversely influences tubulointerstitial fibrosis of the obstructed kidney by enhanced apoptosis and increased inflammatory reaction.

Decorin, a small dermatan-sulfate proteoglycan, participates in extracellular matrix assembly and influences directly and indirectly cell behavior via interactions with signaling membrane receptors and transforming growth factor (TGF)-beta. We have therefore compared the development of tubulointerstitial kidney fibrosis in wild-type (WT) and decorin-/- mice in the model of unilateral ureteral obstruction. Without obstruction, kidneys from decorin-/- mice did not differ in any aspect from their WT counterparts. However, already 12 hours after obstruction decorin-/- animals showed lower levels of p27(KIP1) and soon thereafter a more pronounced up-regulation and activation of initiator and effector caspases followed by enhanced apoptosis of tubular epithelial cells. Later, a higher increase of TGF-beta1 became apparent. After 7 days, there was an up to 15-fold transient up-regulation of the related proteoglycan biglycan, which was mainly caused by the appearance of biglycan-expressing mononuclear cells. Other small proteoglycans showed no similar response. Because of enhanced degradation of type I collagen, end-stage kidneys from decorin-/- animals were more atrophic than WT kidneys. These data suggest that decorin exerts beneficial effects on tubulointerstitial fibrosis, primarily by influencing the expression of a key cyclin-dependent kinase inhibitor and by limiting the degree of apoptosis, mononuclear cell infiltration, tubular atrophy, and expression of TGF-beta1.

Animals↗

PubMed2002

Immunohistochemical detection of hypochlorite-modified proteins in glomeruli of human membranous glomerulonephritis.

A proposed analogy between atherosclerosis and glomerulosclerosis suggests that factors that contribute to the development of atherosclerosis, ie, oxidatively modified (lipo)proteins, may also participate in glomerular injury. Although the nature of the in vivo oxidants has not been clearly identified, increasing evidence suggested the myeloperoxidase (MPO)-H(2)O(2)-halide system to be responsible for the damage observed in leukocyte-dependent glomerulonephritis. MPO, a heme protein secreted by activated phagocytes, may generate modified/oxidized proteins in vivo via intermediate formation of hypochlorous acid (HOCl)/hypochlorite. HOCl, a reactive oxygen species and powerful oxidant, can convert (lipo)proteins into atherogenic forms in vitro and in vivo. Here we demonstrate the presence of HOCl-modified proteins in glomeruli of patients with membranous glomerulonephritis using monoclonal antibodies that do not cross-react with other oxidative modifications. Immunostaining for HOCl-modified epitopes in human minimal change glomerulopathy revealed glomeruli that were unreactive, although the number of MPO-positive cells/glomerulus was slightly increased in comparison to controls. In contrast to minimal change glomerulopathy, a pronounced infiltration of mononuclear cells/glomerulus in membranoproliferative glomerulonephritis is in line with pronounced staining for HOCl-modified epitopes. Immunostaining was detected in intracapillary cells and immune complex deposits within the glomerular basement membrane. In human membranous glomerulonephritis (Stages I to III), staining for HOCl-modified proteins was localized at the basement membrane and podocytes. Staining of serial sections revealed colocalization of HOCl-modified epitopes and MPO in glomerular peripheral basement membranes. Subsequently, tubulointerstitial staining for HOCl-modified epitopes was observed in foam cells at the border of the cytoplasm and in damaged tubular epithelia in focal advanced chronic lesions. Our results indicate that oxidative modification of the basement membrane structure by phagocyte-derived HOCl may be of importance for glomerular defects. The observed colocalization of HOCl-modified proteins and MPO in podocytes and adjacent basement membranes strengthens the assumption that the MPO-H(2)O(2)-halide system contributes to glomerular dysfunction in patients with membranous glomerulonephritis.

Antibodies, Monoclonal↗

PubMed2002

Laser microdissection and gene expression analysis on formaldehyde-fixed archival tissue.

BACKGROUND: Analysis of renal biopsies is currently based on histological recognition of typical structural patterns and immunohistological detection of protein expression alterations. Both can be performed using formaldehyde as the tissue fixative. As a consequence of recent advances in molecular medicine, mRNA expression analysis may offer an attractive option to obtain functionally relevant information. However, quantification of mRNA expression in human renal biopsies thus far has not been possible in formaldehyde-fixed tissue. METHODS: The present study evaluated a recently reported mRNA extraction protocol. Using this approach gene expression analysis could be performed on formaldehyde-fixed archival renal tissues by laser microbeam microdissection, laser pressure catapulting and real time reverse transcription-polymerase chain reaction. RESULTS: For an initial feasibility study, the expression of two chemokines (IP-10 and RANTES) in renal transplant rejection was examined. Induction of protein expression in allografts undergoing rejection was demonstrated for both chemokines by immunohistochemistry. The mRNA expression alterations in the defined renal compartments of glomeruli, vessels and tubulointerstitium were quantified using laser microdissection from formaldehyde-fixed, paraffin-embedded or frozen tissue sections. A pronounced increase of mRNA expression compared to controls was demonstrated for IP-10 as well as RANTES with both tissue-processing protocols. CONCLUSIONS: Using formaldehyde as the tissue fixative, information on the disease process can now be obtained by histological, immunohistochemical and gene expression techniques. In the future this may allow the study of activated molecular programs in routine renal biopsies as well as archival tissue samples.

Adolescent↗

PubMed2002

Inducible nitric oxide synthase in renal transplantation.

The importance of the endothelial isoform of nitric oxide synthase (eNOS) has been well established. Endothelium-derived nitric oxide has been shown to be essential for vascular homeostasis and modulation of eNOS has thus become a target in prevention of cardiovascular disease. The role of the inducible form of nitric oxide synthase (iNOS) in vascular biology, however, is less clear. Classically, iNOS has been regarded as an enzyme that produces nmolar amounts of the nitric oxide radical, thereby leading to cellular damage. More recent data, however, have shown that the iNOS can be a superoxide, peroxynitrite as well as a nitric oxide-producing enzyme, while the biological effects of iNOS probably depend upon the sort of radical species released by the enzyme as well as the anti-oxidant capacity of the cellular microenvironment of the enzyme. This brief review discusses these aspects in relation to renal transplantation.

Acute Disease↗