Increased expression of heparanase in overt diabetic nephropathy.

In overt diabetic nephropathy (DNP), an increase in the permeability of the glomerular basement membrane (GBM) has been associated with a loss of negatively charged heparan sulfates (HS) in the GBM. Heparanase (HPSE), an endo-beta(1-4)-D-glucuronidase, can cleave HS and could be a potential candidate for the degradation of glomerular HS, leading to the development of proteinuria. We analyzed whether changes in HS expression are associated with HPSE expression in overt DNP. Immunofluorescence staining was performed to analyze HS, HPSE, and agrin core protein expression in kidney biopsies from patients with overt DNP and from rats and mice with streptozotocin (STZ)-induced diabetes. We also investigated the effect of transgenic HPSE overexpression in mice on glomerular HS and agrin expression. We demonstrate that the loss of GBM HS (-50%) and tubular HS (-60%) is associated with a four-fold increased HPSE expression in overt DNP. In addition, glomerular HPSE expression is upregulated in rats (messenger RNA (mRNA) 2.5-fold, protein three-fold) and mice (mRNA seven-fold, protein 1.5-fold) with STZ-induced diabetes. Furthermore, transgenic HPSE overexpression results in disappearance of HS, whereas expression of the core protein agrin remains unaltered. Our observations suggest that HPSE is involved in the pathogenesis of proteinuria in overt DNP by degradation of HS.

The role of oxidative stress in the ochratoxin A-mediated toxicity in proximal tubular cells.

Balkan endemic nephropathy (BEN), a disease characterized by progressive renal fibrosis in human patients, has been associated with exposure to ochratoxin A (OTA). This mycotoxin is a frequent contaminant of human and animal food products, and is toxic to all animal species tested. OTA predominantly affects the kidney and is known to accumulate in the proximal tubule (PT). The induction of oxidative stress is implicated in the toxicity of this mycotoxin. In the present study, primary rat PT cells and LLC-PK(1) cells, which express characteristics of the PT, were used to investigate the OTA-mediated oxidative stress response. OTA exposure of these cells resulted in a concentration-dependent elevation of reactive oxygen species (ROS) levels, depletion of cellular glutathione (GSH) levels and an increase in the formation of 8-oxoguanine. The OTA-induced ROS response was significantly reduced following treatment with alpha-tocopherol (TOCO). However, this chain-braking anti-oxidant did not reduce the cytotoxicity of OTA and was unable to prevent the depletion of total GSH levels in OTA-exposed cells. In contrast, pre-incubation of the cell with N-acetyl-L-cysteine (NAC) completely prevented the OTA-induced increase in ROS levels as well as the formation of 8-oxoguanine and completely protected against the cytotoxicity of OTA. In addition, NAC treatment also limited the GSH depletion in OTA-exposed PT- and LLC-PK(1) cells. From these data, we conclude that oxidative stress contributes to the tubular toxicity of OTA. Subsequently, cellular GSH levels play a pivotal role in limiting the short-term toxicity of this mycotoxin in renal tubular cells.

Structural requirements for O-glycosylation of the mouse hepatitis virus membrane protein.

The mouse hepatitis virus (MHV) membrane (M) protein contains only O-linked oligosaccharides. We have used this protein as a model to study the structural requirements for O-glycosylation. We show that MHV M is modified by the addition of a single oligosaccharide side chain at the cluster of 4 hydroxylamino acids present at its extreme amino terminus and identified Thr at position 5 as the functional acceptor site. The hydroxylamino acid cluster, which is quite conserved among O-glycosylated coronavirus M proteins, is not in itself sufficient for O-glycosylation. Downstream amino acids are required to introduce a functional O-glycosylation site into a foreign protein. In a mutagenic analysis O-glycosylation was found to be sensitive to some particular changes but no unique sequence motif for O-glycosylation could be identified. Expression of mutant M proteins in cells revealed that substitution of any 1 residue was tolerated, conceivably due to the occurrence of multiple UDP-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (GalNAc transferases). Indeed, MHV M served as a substrate for GalNac-T1, -T2, and -T3, as was demonstrated using an in situ glycosylation assay based on the co-expression of endoplasmic reticulum-retained forms of the GalNAc transferases with endoplasmic reticulum-resident MHV M mutants. The GalNAc transferases were found to have largely overlapping, but distinct substrate specificities. The requirement for a threonine as acceptor rather than a serine residue and the requirement for a proline residue three positions downstream of the acceptor site were found to be distinctive features.