Renal TLR4 mRNA expression correlates with inflammatory marker MCP-1 and profibrotic molecule TGF-ß1 in patients with chronic kidney disease.

BACKGROUND: Toll-like receptor-4 (TLR4) has been implicated in the pathogenesis of different renal diseases in rodent models. However, in human kidney disease, TLR4 expression and regulation is not well understood. We hypothesized that renal TLR4 expression plays a role in chronic kidney disease (CKD) and is associated with proteinuria, kidney function, histological diagnosis, and inflammatory mediators. METHODS: We quantified TLR4 mRNA as well as expression of macrophage chemoattractant protein-1 (MCP-1), transforming growth factor-ß1 (TGF-ß1) and interleukin-6 (IL-6) in human kidney biopsies from 70 patients with CKD. We measured kidney function, urinary MCP-1 protein excretion, and the amount of chronic damage. Macrophage load was quantified by CD68 and vascularization by CD34 immunostaining. RESULTS: TLR4 expression correlated significantly with MCP-1 and TGF-ß1 expression. High TLR4 expression was associated with high IL-6 expression. TLR4 expression was significantly upregulated in patients with severe proteinuria, and in patients with chronic ischemic renal damage and IgA nephropathy, when compared to patients with thin glomerular basement membrane disease. TLR4 expression did not correlate with creatinine clearance, renal outcome, macrophage load or chronic damage. CONCLUSIONS: We show for the first time that renal TLR4 expression was significantly associated with the pro-inflammatory marker MCP-1 and the profibrotic molecule TGF-ß1 in kidney biopsies from patients with CKD, suggesting that increased expression of TLR4 is an important feature of CKD.

Renal PPARγ mRNA expression increases with impairment of renal function in patients with chronic kidney disease.

AIM: Peroxisome proliferator-activated receptor gamma (PPARγ) is generally accepted as renoprotective factor in type 2 diabetes mellitus, and PPARγ agonists have been reported to reduce albuminuria. However, little is known about renal PPARγ expression in chronic kidney disease, and especially human data are scarce. We hypothesized that renal PPARγ expression is associated with extent of proteinuria, kidney function, histological diagnosis and inflammatory mediators. Therefore, we investigated PPARγ mRNA expression in human kidney biopsies. METHODS: We quantified PPARγ mRNA as well as the expression of macrophage chemoattractant protein-1, transforming growth factor beta-1 and interleukin-6 in 64 human kidney biopsies from patients with chronic kidney disease and mild-to-marked proteinuria of diverse aetiology. We measured renal function, and macrophage invasion was quantified by CD68 and vascularization by CD34 immunostaining. RESULTS: PPARγ mRNA expression correlated inversely with renal function. Higher blood pressure levels were associated with higher PPARγ expression levels. PPARγ mRNA expression correlated significantly (P<0.001) with macrophage chemoattractant protein-1 mRNA expression and showed a negative trend with transforming growth factor beta-1 mRNA expression. No differences in PPARγ expression were detected with regard to extent of proteinuria, histological diagnosis, macrophage invasion, interleukin-6 expression, and age or body mass index. CONCLUSIONS: PPARγ expression increases with loss of renal function and may be an important factor in maintaining normal renal function serving as a key protective mechanism to renal injury.

IGF-1, IGFBP-3 and ALS in adult patients with chronic kidney disease.

BACKGROUND: Insulin-like growth factor I (IGF-1) is for the most part bound in a ternary complex with IGF-binding protein-3 (IGFBP-3) and acid-labile subunit (ALS). This ternary complex is a storage form of IGF-1 in blood and passes not through the renal glomerulus. Little information is available in regard to the components of the ternary complex in adult renal disease. OBJECTIVE: To investigate levels of serum IGF-1, IGFBP-3 and ALS in relation to renal function and extent of proteinuria. DESIGN AND PATIENTS: We measured IGF-1, IGFBP-3 and ALS concentrations in 137 patients who were investigated due to proteinuria and/or haematuria and/or renal impairment. The patients received renal biopsies and the histological diagnosis was documented. Urinary albumin excretion and relevant clinical parameter were evaluated. RESULTS: IGF-1 showed a highly positive correlation to IGFBP-3 and ALS, and the latter to IGFBP-3. IGF-1, IGFBP-3 and ALS decreased with increasing age. IGF-1 and IGFBP-3 showed no significant change depending on the creatinine clearance. However, ALS decreased with decreasing renal function. In patients with heavy proteinuria ALS levels, but not IGF-1 and IGFBP-3 levels, decreased significantly. Patients with chronic ischaemic renal damage and diabetic glomerulopathy showed higher IGF-1 and IGFBP-3 levels compared to patients with thin glomerular basement membrane disease despite their older age. CONCLUSIONS: IGF-1 and IGFBP-3 levels seem to be independent of renal function and severity of proteinuria. However, ALS levels are altered in renal failure and nephrotic syndrome, which may be due to increased renal loss or diminished hepatic production or both.

The hypertensiogenetic steroid 19-nor-progesterone does not influence cortisol inactivation by 11beta-hydroxysteroid dehydrogenase type 2.

19-nor-progesterone (19-nor-P) has the characteristics of a potent mineralocorticoid in adrenalectomized or salt-loaded rats and is capable of causing hypertension. In human placenta, progesterone is converted to 19-hydroxy-progesterone, a precursor of 19-nor-P. In some states of pregnancy hypertension, 19-nor-P may inhibit renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2), thus allowing cortisol to bind to the mineralocorticoid receptor (MR). Therefore, we investigated the ability of 19-nor-P to inhibit human 11beta-HSD2. Fetal kidney cells (HEK 293) were transfected with human 11beta-HSD2 and incubated with increasing concentrations of 19-nor-P, labelled and unlabelled cortisol. Steroids were extracted, separated by TLC, and radioactivity was measured using a TLC scanner. 19-nor-P treatment did not significantly reduce 11beta-HSD2 activity (430 to 300 pmol/mg protein/h) in the range of tested concentrations. In conclusion, 19-nor-P did not inhibit human 11beta-HSD2 and seems not to be involved in human hypertension. Nevertheless, 19-nor-P may be converted by extra-adrenal tissues into 19-nor-deoxycorticosterone (DOC) or 19-nor-corticosterone, which are potent mineralocorticoids and may be involved in the pathogenesis of hypertension during pregnancy.

Vasculitic IgA nephropathy: prognosis and outcome.

BACKGROUND: A vasculitic glomerulonephritis is seen in both IgA nephropathy and Henoch-Schonlein purpura. No study has examined prognostic factors in vasculitic IgA nephropathy. METHODS: All patients with vasculitic IgA presenting to our centre between January 1996 and May 2003 were retrospectively reviewed by analysis of clinical and pathological features at presentation and during follow-up. RESULTS: Of 363 patients with IgA nephropathy, 67 patients had vasculitic IgA nephropathy (33 Henoch-Schönlein purpura; 34 IgA). Median length of follow-up was 32.38 months (range 0-90). Median glomerular filtration rate at presentation was 45.56 ml/min (range 4-110), arterial blood pressure 104.67 mm Hg and proteinuria 1.19 g/24 h (range 0-14.8) falling during follow-up to 0.11 g (range 0-3.1). Median chronic damage at presentation in the biopsy was 10% (range 0-91). The majority (79%) were immunosuppressed with 84% patient survival and 85% renal survival at 60 months. Three factors significantly affected renal outcome: renal function; blood pressure at presentation and the amount of chronic damage in the biopsy. The effect of immunosuppression on outcome was difficult to comment on as treated and untreated groups were not comparable. CONCLUSIONS: Presenting renal function, blood pressure and chronic damage in the biopsy are important prognostic factors in vasculitic IgA nephropathy. Immunosuppression is advocated in some patients.

Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation.

To examine any potential role for 1,25-dihydroxyvitamin D (1,25(OH)2D) in inflammation associated with chronic kidney disease we measured vitamin D metabolites, markers of inflammation and gene expression in 174 patients with a variety of kidney diseases. Urinary MCP-1 protein and renal macrophage infiltration were each significantly but inversely correlated with serum 1,25(OH)2D levels. Logistic regression analysis with urinary MCP-1 as binary outcome showed that a 10-unit increase in serum 1,25(OH)2D or 25OHD resulted in lower renal inflammation. Analysis of 111 renal biopsies found that renal injury was not associated with a compensatory increase in mRNA for the vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), its catabolic counterpart 24-hydroxylase, or the vitamin D receptor. There was, however, a significant association between tissue MCP-1 and CYP27B1. Patients with acute renal inflammation had a significant increase in urinary and tissue MCP-1, macrophage infiltration, and macrophage and renal epithelial CYP27B1 expression but significantly lower levels of serum 1,25(OH)2D in comparison to patients with chronic ischemic disease despite similar levels of renal damage. In vitro, 1,25(OH)2D attenuated TNFalpha-induced MCP-1 expression by human proximal tubule cells. Our study indicates that renal inflammation is associated with decreased serum vitamin D metabolites and involves activation of the paracrine/autocrine vitamin D system.

The role of capillary density, macrophage infiltration and interstitial scarring in the pathogenesis of human chronic kidney disease.

To assess the relationship between interstitial capillary density and interstitial macrophages we prospectively measured these factors in situ in 110 patients with chronic kidney disease. Macrophage numbers and urinary MCP-1/CCL2 levels significantly correlated inversely with capillary density which itself significantly correlated inversely with chronic damage and predicted disease progression. In 54 patients with less than 20% chronic damage, there was a significant correlation between the urinary albumin to creatinine ratio and MCP-1/CCL2, and MCP-1/CCL2 and macrophages but not between MCP-1/CCL2 and capillary density. Conversely, in 56 patients with over 20% chronic damage there was no correlation between MCP-1/CCL2 and macrophages but there were significant inverse correlations between capillary density and both macrophages and chronic damage. The expression of VEGF mRNA significantly correlated with macrophage infiltration, capillary density and chronic scarring. In an ischemic-hypertensive subgroup there was upregulation of the hypoxia marker carbonic anhydrase IX and with over 20% chronic damage an increased macrophage to CCR2 ratio. Our study shows that proteinuria and MCP-1/CCL2 are important for macrophage recruitment in early disease. As renal scarring evolves, alternative pathways relating to progressive tissue ischemia secondary to obliteration of the interstitial capillary bed predominate.

Reduced cytokine induction and removal of complement products with synthetic hemodialysis membranes.

The increasing use of high-flux membranes for hemodialysis (HD) has raised concerns that these membranes may confer a higher risk of exposure to cytokine-inducing, bacterial substances (CIS) in the dialysate. Several studies, however, reported higher transfer of CIS through low-flux cellulosic than high-flux synthetic membranes. This surprising paradox was explained by adsorption of CIS to certain high-flux membranes. In order to investigate flux and membrane type independently, we studied two synthetic Polyflux (PF) membranes of the same type but with different flux properties and compared them to a cellulosic membrane (Cuprophan). Three different approaches were employed: (1) cytokine induction in whole blood during in vitro HD contaminated with bacterial filtrates, (2) removal of recombinant C5a, and (3) transfer of purified lipopolysaccharide (LPS). After 90 min recirculation of whole blood, the appearance of IL-6-inducing substances on the blood side was lowest with high-flux PF (1.1 +/- 0.2 ng/ml), slightly higher with low-flux PF (1.9 +/- 0.7 ng/ml) and highest with Cuprophan (4.1 +/- 1 ng/ml). Recombinant C5a added to plasma on the blood side was markedly removed by high-flux PF (by 83%), to a lesser degree and only in the presence of ultrafiltration with low-flux PF (by 54%) and not significantly with Cuprophan (by 11%). Significant transfer of purified LPS from the dialysate onto the blood side was only observed with the cellulosic membrane. We conclude that in contrast to cellulosic membranes, certain synthetic membranes do not permit transfer of LPS. Cytokine induction on the blood side is further reduced by the use of high-flux membranes due to removal of activated complement factors.

Increased expression of mineralocorticoid effector mechanisms in kidney biopsies of patients with heavy proteinuria.

BACKGROUND: Aldosterone has emerged as a deleterious hormone in the heart, with mineralocorticoid receptor (MR) blockade reducing mortality in patients with severe heart failure. There is also experimental evidence that aldosterone contributes to the development of nephrosclerosis and renal fibrosis in rodent models, but little is known of its role in clinical renal disease. METHODS AND RESULTS: We quantified MR, serum- and glucocorticoid-regulated kinase 1 (sgk1), and mRNA expression of inflammatory mediators such as macrophage chemoattractant protein-1 (MCP-1), transforming growth factor-beta1, and interleukin-6 in 95 human kidney biopsies in patients with renal failure and mild to marked proteinuria of diverse etiologic origins. We measured renal function, serum aldosterone, urinary MCP-1 protein excretion, and the amount of chronic renal damage. Macrophage invasion was quantified by CD68 and vascularization by CD34 immunostaining. Serum aldosterone correlated negatively with creatinine clearance (P<0.01) and positively with renal scarring (P<0.05) but did not correlate with MR mRNA expression or proteinuria. Patients with heavy albuminuria (>2 g/24 h; n=15) had the most renal scarring and the lowest endothelial CD34 staining. This group showed a significant 5-fold increase in MR, a 2.5-fold increase in sgk1 expression and a significant increase in inflammatory mediators (7-fold increase in MCP-1, 3-fold increase in transforming growth factor-beta1, and 2-fold increase in interleukin-6 mRNA). Urinary MCP-1 protein excretion and renal macrophage invasion were significantly increased in patients with heavy albuminuria. CONCLUSIONS: These studies support animal data linking aldosterone/MR activation to renal inflammation and proteinuria. Further studies are urgently required to assess the potential beneficial effects of MR antagonism in patients with renal disease.

Expression of renal 11beta-hydroxysteroid dehydrogenase type 2 is decreased in patients with impaired renal function.

OBJECTIVE: Renal 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) enables selective access of aldosterone to the mineralocorticoid receptor (MR). Impaired 11beta-HSD2 activity has been suggested in patients with hypertension as well as in patients with renal disease, where it may contribute to sodium retention, oedema and hypertension. To date, these studies have relied upon urinary cortisol (F) metabolite levels as surrogate markers of renal 11beta-HSD2 activity. METHODS: We have directly analysed renal 11beta-HSD2 mRNA expression in 95 patients undergoing kidney biopsy using TaqMan real-time PCR. Serum and 24-h urine samples were used to document underlying renal function and endocrine parameters. Urinary F and cortisone (E) metabolites were analysed using gas chromatography/mass spectrometry. RESULTS: Expression of 11beta-HSD2 did not correlate with blood pressure or urinary Na/K ratio, but a significant positive correlation with creatinine clearance was observed (r = 0.284; P < 0.01). Immunofluorescence and confocal laser microscopy confirmed decreased 11beta-HSD2 expression in patients with impaired renal function. For the first time, we showed that 11beta-HSD2 mRNA expression correlated negatively with the urinary free (UF) F/E (UFF/UFE) ratio (r = 0.276; P < 0.05) as well as with the urinary tetrahydrocortisol + 5alpha-tetrahydrocortisol/tetrahydrocortisone ((THF + alphaTHF)/THE) ratio (r = 0.256; P < 0.05). No difference in 11beta-HSD2 mRNA expression or in the UFF/UFE ratio was found between groups with no proteinuria, microalbuminuria, moderate or severe proteinuria. In contrast, the urinary (THF + alphaTHF)/THE ratio increased significantly (P < 0.05) in patients with severe albuminuria, suggesting increased hepatic 11beta-HSD1 in those patients. CONCLUSIONS: These data suggest that renal 11beta-HSD2 expression may be represented only marginally better, if at all, by the UFF/UFE than by the (THF + alphaTHF)/THE ratio. Reduced renal 11beta-HSD2 expression may lead to occupancy of the MR by glucocorticoids such as cortisol and may contribute to the increased sodium retention seen in patients with impaired renal function.