Izabella Z.A. Pawluczyk

Acidosis Downregulates Leptin Production from Cultured Adipocytes through a Glucose Transport-Dependent Post-transcriptional Mechanism

Metabolic acidosis, a common feature of uremia, has a well documented wasting effect on skeletal muscle. In contrast, the effect of metabolic acidosis on adipose tissue is unknown. Serum levels of the adipocyte hormone leptin have been shown to be lower in acidotic uremic rats when compared with uremic controls. This study investigated the effect of acidosis on leptin protein secretion and leptin gene expression. This was studied in vitro by means of 3T3-L1 cultured adipocytes. Leptin secretion was decreased at an acid pH of 7.1 compared with a control pH of 7.5 (1277 versus 1950 pg/well/48 h, P < 0.05). In contrast, acidosis did not affect leptin mRNA content. Glucose transport was reduced by 39% at pH 7.1 at 24 h, which was comparable in magnitude with the inhibition of leptin secretion at the same pH. The glucose transport inhibitors cytochalasin B (0.5 to 50 micro M) and phloretin (0.05 to 0.25 mM) mimicked the effect of acidosis and reduced leptin secretion in a dose-dependent fashion (P < 0.02). Dose-response curves for the inhibition of glucose uptake showed that decreasing glucose transport to the same extent as with acid was sufficient to drive down leptin secretion, independently of changes of leptin mRNA. Acid decreases leptin secretion from 3T3-L1 adipocytes through a post-transcriptional mechanism via changes in glucose transport. This starvation-like response may be physiologically important in conditions such as uremia to prevent excessive energy expenditure.

Macrophage-induced rat mesangial cell expression of the 24p3-like protein alpha-2-microglobulin-related protein.

During screening of a murine macrophage cDNA repertoire for factors potentially able to modulate glomerular cell responses to injury, we identified a gene coding for the murine protein 24p3 lipocalin. Immunostaining of normal rat kidney sections showed positive 24p3-like staining in distal tubules/collecting ducts and small muscular arteries. Although most glomeruli were negative, some did exhibit small numbers of positively stained cells. Cultured rat glomeruli and glomerular mesangial cells secreted the 24p3-like protein in response to macrophage-conditioned medium (MPCM) and the cytokine IL-1beta. MPCM derived from TGFbeta-pretreated macrophages enhanced mesangial cell 24p3 secretion. In contrast, addition of anti-IL-1beta neutralising antibody to MPCM or IL-1beta resulted in suppression of 24p3 secretion. Co-culture of mesangial cells with varying numbers of non-LPS-treated macrophages resulted in dose-dependent secretion of 24p3 into culture supernatants. Archival sections from polyvinyl alcohol-treated and cholesterol-fed rats showed positive glomerular staining for 24p3 in and around glomerular foam cells. Nucleotide sequencing of rat mesangial cell-derived 24p3 cDNA revealed it to be identical to rat alpha-2-microglobulin-related protein (alpha2microGRP), the rat homologue of murine 24p3. These data provide the first description of rat alpha2microGRP in the context of mesangial cell pathophysiology.

Expression of T cell receptor variable region families by bone marrow γδ T cells in patients with IgA nephropathy

IgA nephropathy (IgAN) is characterized by mesangial deposition of polymeric IgA (pIgA). Abnormalities of the IgA system include reduced mucosal and increased bone marrow (BM) pIgA production. γδ T cells are regulators of mucosal IgA production and oral tolerance. We have described previously a deficiency of γδ T cells expressing Vγ3 and Vδ3 from the duodenal mucosa in IgAN. Since pIgA production is displaced to the BM, we have now studied BM γδ T cells in IgAN.

Rat mesangial cells exhibit sex-specific profibrotic and proinflammatory phenotypes

BACKGROUND Chronic renal disease progresses more rapidly in males compared to females. This study investigated whether there were any inherent differences between male and female mesangial cells that could contribute to this phenomenon and whether these differences could be modulated by sex hormones. METHODS Experiments were carried out on cultured mesangial cells derived from adult male and female Wistar rat kidneys. Fibronectin, TNFalpha and IL-1beta levels were measured in control and macrophage-conditioned medium (MCM)-injured cells in the presence and absence of 17beta estradiol or testosterone. RESULTS Male mesangial cells expressed higher baseline fibronectin levels compared to female cells. Similarly, basal levels of the proinflammatory cytokines TNFalpha and IL-1beta were higher in male cells. Fibronectin and IL-1beta levels were enhanced proportionately between the sexes in response to MCM stimulation, whilst the increase in TNFalpha levels was greater in MCM-stimulated female cells. Treatment with 10(-8) M estradiol down-regulated baseline fibronectin levels in female mesangial cells but had no effect on basal levels in male cells. Estradiol had no effect on MCM-stimulated fibronectin levels in female mesangial cells but further increased stimulated levels in male cells. Testosterone had no effect on basal fibronectin levels of either sex but further enhanced MCM-stimulated fibronectin levels in mesangial cells of both sexes. Sex hormone treatment had no effect on cytokine levels in male mesangial cells. However, in female cells estradiol decreased TNFalpha levels and increased IL-1beta levels, while testosterone increased the levels of both cytokines. CONCLUSION These data would suggest that male mesangial cells inherently exhibit greater profibrotic and proinflammatory characteristics than female cells. The inherent gender phenotypes are further modulated by sex hormones. This sexual dimorphism in mesangial cells may play a contributory role in the faster rate of progression to end-stage renal disease in males.

Kallikrein gene 'knock-down' by small interfering RNA transfection induces a profibrotic phenotype in rat mesangial cells.

Background Emerging evidence suggests that kallikrein exerts renoprotective effects independent of its haemodynamic actions. The aim of the current investigation was to delineate the role of kallikrein in the regulation of fibrosis, by ‘knocking down’ its expression using specific small interfering RNAs (siRNA). Methods Rat mesangial cells were treated with 12, 60, 120 nmol/l kallikrein-specific siRNAs. The consequent cellular genotypes and phenotypes were analysed. Results Western blotting demonstrated that mesangial cells produced a kallikrein protein, which was of a different molecular weight to urinary kallikrein from rats of the same species. Treatment of cells with siRNA resulted in a dose-dependent decrease in kallikrein mRNA levels, which impacted on other components of the kallikrein–kinin system, dose-dependently reducing bradykinin B2 receptor mRNA expression. Kallikrein suppression resulted in significant increases in fibronectin and transforming growth factor-β protein levels in culture supernatants over control levels. Gelatin zymography demonstrated a siRNA dose-dependent decrease in active MMP-2 enzyme levels. Bradykinin, an effector molecule of the kallikrein system, is known to stimulate tissue plasminogen activator production. Paradoxically, however, tissue plasminogen activator protein levels were augmented with increasing kallikrein mRNA silencing. This was accompanied by a dose-dependent decrease in low-density lipoprotein receptor-related protein mRNA levels, indicating that increased tissue plasminogen activator levels were due to an attenuation of receptor-mediated protease clearance. Conclusion These data lend strong support to the hypothesis that kallikrein exerts antifibrotic, renoprotective effects that are independent of its classical haemodynamic actions.

Sialic acid attenuates puromycin aminonucleoside-induced desialylation and oxidative stress in human podocytes

Sialoglycoproteins make a significant contribution to the negative charge of the glomerular anionic glycocalyx-crucial for efficient functioning of the glomerular permselective barrier. Defects in sialylation have serious consequences on podocyte function leading to the development of proteinuria. The aim of the current study was to investigate potential mechanisms underlying puromycin aminonucleosisde (PAN)-induced desialylation and to ascertain whether they could be corrected by administration of free sialic acid. PAN treatment of podocytes resulted in a loss of sialic acid from podocyte proteins. This was accompanied by a reduction, in the expression of sialyltransferases and a decrease in the key enzyme of sialic acid biosynthesis N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). PAN treatment also attenuated expression of the antioxidant enzyme superoxide dismutase (mSOD) and concomitantly increased the generation of superoxide anions. Sialic acid supplementation rescued podocyte protein sialylation and partially restored expression of sialyltransferases. Sialic acid also restored mSOD mRNA expression and quenched the oxidative burst. These data suggest that PAN-induced aberrant sialylation occurs as a result of modulation of enzymes involved sialic acid metabolism some of which are affected by oxidative stress. These data suggest that sialic acid therapy not only reinstates functionally important negative charge but also acts a source of antioxidant activity.

Pharmacological Enhancement of the Kallikrein-Kinin System Promotes Anti-Fibrotic Responses in Human Mesangial Cells

The aim of the present study was to investigate whether pharmacological enhancement of the renal kallikrein-kinin system using the vasopeptidase inhibitor omapatrilat plays a direct role in modulating the fibrotic responses of human mesangial cells to injury. Treatment with 40µmol/L omapatrilat was able to reduce macrophage-conditioned medium (MPCM)-induced fibronectin levels without affecting mRNA expression. MPCM injury also suppressed kallikrein and low molecular weight kininogen mRNA. Omapatrilat was able to attenuate this suppression. Bradykinin levels in contrast were increased by MPCM and treatment with omapatrilat further augmented levels. Co-incubation with the bradykinin B2 receptor antagonist HOE 140 attenuated the omapatrilat-induced lowering of fibronectin. Moreover, inhibition of cGMP release had a similar effect. Paradoxically, RT-PCR and Southern blotting demonstrated that bradykinin B2 receptor mRNA levels were down regulated in response to omapatrilat. Western blotting supported this data. Supernatant levels of tissue plasminogen activator (tPA), a product of bradykinin stimulation, were decreased by omapatrilat while cell associated tPA levels were increased. Matrix metalloproteinase-9 (MMP-9) mRNA expression was up regulated by omapatrilat treament, although no difference in active zymogen levels was observed. In conclusion enhancement of kallikrein-kinin system appears to play a direct role in promoting anti-fibrotic responses in MPCM-injured human mesangial cells.

β1,4-galactosyltransferase 1 is a novel receptor for IgA in human mesangial cells

IgA nephropathy is characterized by mesangial deposition of IgA, mesangial cell proliferation, and extracellular matrix production. Mesangial cells bind IgA, but the identity of all potential receptors involved remains incomplete. The transferrin receptor (CD71) acts as a mesangial cell IgA receptor and its expression is upregulated in many forms of glomerulonephritis, including IgA nephropathy. CD71 is not expressed in healthy glomeruli and blocking CD71 does not completely abrogate mesangial cell IgA binding. Previously we showed that mesangial cells express a receptor that binds the Fc portion of IgA and now report that this receptor is an isoform of β-1,4-galactosyltransferase. A human mesangial cell cDNA library was screened for IgA binding proteins and β-1,4-galactosyltransferase identified. Cell surface expression of the long isoform of β-1,4-galactosyltransferase was shown by flow cytometry and confocal microscopy and confirmed by immunoblotting. Glomerular β-1,4-galactosyltransferase expression was increased in IgA nephropathy. IgA binding and IgA-induced mesangial cell phosphorylation of spleen tyrosine kinase and IL-6 synthesis were inhibited by a panel of β-1,4-galactosyltransferase-specific antibodies, suggesting IgA binds to the catalytic domain of β-1,4-galactosyltransferase. Thus, β-1,4-galactosyltransferase is a constitutively expressed mesangial cell IgA receptor with an important role in both mesangial IgA clearance and the initial response to IgA deposition.

Effect of angiotensin type 2 receptor over-expression on the rat mesangial cell fibrotic phenotype: effect of gender

Background and aim: The protective role of angiotensin type 2 receptors (AT2-Rs) is still controversial. As AT2-Rs are minimally expressed in adult tissues the aim of the current study was to over-express AT2-Rs in rat mesangial cells in order to ascertain their potential role in modulating renal scarring. Methods: Male and female mesangial cells were transiently transfected with AT2-R or control vector then ‘injured’ with macrophage-conditioned medium (MCM). Culture supernatants and extracted RNA were analysed for evidence of an anti-fibrotic phenotype. Results: Supernatant fibronectin levels in female mesangial cells treated with MCM were reduced in AT2-R transfected cells (p < 0.001) compared to controls. AT2-R transfected male cells showed a trend towards lower constitutive fibronectin levels. There was no effect of AT2-R transfection on TGF-β or TNF-α secretion; however, IL-1β levels were reduced in male cells treated with MCM. RT-PCR demonstrated that constitutive kallikrein mRNA levels were suppressed in both male and female AT2-R transfected cells. Bradykinin receptors (BkB2-R and BkB1-R) were unaffected in female cells although the BkB1-R was upregulated in male cells treated with MCM. Conclusion: This data provides a case for AT2 receptors playing a protective role in rat mesangial cells independent of the effects of blood pressure control.

The effect of albumin on podocytes: the role of the fatty acid moiety and the potential role of CD36 scavenger receptor.

Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified. Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA+FA) or depleted of them (HSA-FA). Receptor-mediated endocytosis of FITC-HSA+FA over 60 min was 5 times greater than that of FITC-HSA-FA. 24h exposure of podocytes to albumin up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA-FA. Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake. These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin.