Reduced proximal tubular expression of protein endocytic receptors in proteinuria is associated with urinary receptor shedding.

Background: Filtered proteins, including albumin, are reabsorbed in the proximal tubule (PT) mediated by megalin, cubilin and the neonatal Fc receptor (FcRn). Proteinuria is an important renal biomarker linked to poor prognosis but expression of these key receptors is not well studied. Methods: Megalin expression was determined at protein and messenger RNA (mRNA) levels in kidneys from proteinuric patients, and the expression of megalin, cubilin and FcRn was examined in the kidneys of mice with protein-overload proteinuria. The presence of receptors in the urine of proteinuric and control mice was also studied. Results: In nephrotic patients, megalin expression is reduced while mRNA is increased. In proteinuric mice megalin, cubilin and the neonatal FcRn protein are all reduced in PTs. Megalin and FcRn mRNA are increased in proteinuric mice, whereas that for cubilin was reduced. In proteinuric mice increased urinary excretion of each of these endocytic receptors was observed. Conclusions: It is concluded that in proteinuria, expression of all the key protein re-absorptive receptors is significantly reduced in the PT in association with increased turnover and urinary shedding.

CD36 mediates proximal tubular binding and uptake of albumin and is upregulated in proteinuric nephropathies.

Dysregulation of renal tubular protein handling in proteinuria contributes to the development of chronic kidney disease. We investigated the role of CD36 as a novel candidate mediator of albumin binding and endocytosis in the kidney proximal tubule using both in vitro and in vivo approaches, and in nephrotic patient renal biopsy samples. In CD36-transfected opossum kidney proximal tubular cells, both binding and uptake of albumin were substantially enhanced. A specific CD36 inhibitor abrogated this effect, but receptor-associated protein, which blocks megalin-mediated endocytosis of albumin, did not. Mouse proximal tubular cells expressed CD36 and this was absent in CD36 null animals, whereas expression of megalin was equal in these animals. Compared with wild-type mice, CD36 null mice demonstrated a significantly increased urinary protein-to-creatinine ratio and albumin-to-creatinine ratio. Proximal tubular cells expressed increased CD36 when exposed to elevated albumin concentrations in culture medium. Expression of CD36 was studied in renal biopsy tissue obtained from adult patients with heavy proteinuria due to minimal change disease, membranous nephropathy, or focal segmental glomerulosclerosis. Proximal tubular CD36 expression was markedly increased in proteinuric individuals. We conclude that CD36 is a novel mediator influencing binding and uptake of albumin in the proximal tubule that is upregulated in proteinuric renal diseases. CD36 may represent a potential therapeutic target in proteinuric nephropathy.

Statins but not thiazolidinediones attenuate albumin-mediated chemokine production by proximal tubular cells independently of endocytosis.

BACKGROUND: Proximal tubular epithelial cells (PTEC) secrete chemokines under proteinuric conditions. Both statins and thiazolidinediones (TZDs) possess pleiotropic anti-inflammatory effects. This study examined the ability of statins and TZDs and the natural peroxisome proliferator activated receptor-gamma (PPARgamma) agonist 15-deoxy-Delta(12,14)-prostaglandin J(2) (PGJ(2)) to attenuate the proteinuria-induced pro-inflammatory phenotype of PTEC. METHODS: Mouse PTEC were treated with statins, TZDs and PGJ(2 )and effects on uptake and binding of FITC-albumin determined. PTEC were incubated with fatty acid free bovine serum albumin with or without statins/TZDs/PGJ(2), and the release of MCP-1 and RANTES measured. RESULTS: Statins and TZDs significantly inhibited PTEC albumin endocytosis. PGJ(2 )had no effect. Incubation of PTEC with albumin significantly stimulated production of MCP-1 and RANTES. Co-treatment with statins and PGJ(2) significantly reduced albumin-stimulated chemokine production, an effect reversed by the addition of mevalonate and geranylgeranyl pyrophosphate. In contrast, TZDs had no effect on albumin-mediated chemokine production. CONCLUSION: Statins and PGJ(2), but not TZDs, prevent the development of a PTEC pro-inflammatory phenotype in response to albumin. Albumin endocytosis is not a prerequisite for PTEC chemokine production, and inhibition of albumin endocytosis alone is insufficient to attenuate chemokine production. These studies suggest a therapeutic role for statins and some PPARgamma ligands in proteinuric renal disease.

Mesangial matrix-activated monocytes express functional scavenger receptors and accumulate intracellular lipid.

BACKGROUND: Monocyte recruitment into the mesangium and foam cell formation are recognized features of glomerular injury. External signals encountered by infiltrating mononuclear cells may determine their behaviour and thereby potentially influence disease outcome. Having previously demonstrated that monocytes are activated by exposure to matrix secreted by mesangial cells, we set out to determine whether matrix activation of monocytes led to expression of a macrophage phenotype. METHODS: THP-1 mononuclear cells were incubated for up to 120 h (5 days) with 500 microg/ml solublized matrix extracted from cultured human mesangial cells or with phorbol myristate ester (PMA-positive control) or albumin (negative control). Expression of peroxisome proliferator activated receptor-gamma (PPAR-gamma) and of scavenger receptors was used as a marker of monocyte to macrophage differentiation. The presence of functional scavenger receptors was examined by assessing cellular uptake of Dil-labelled acetylated (Ac)-LDL by flow cytometry. Matrix-mediated LDL oxidation was assessed using agarose gel electrophoresis to determine mobility shifts. RESULTS: Matrix activation was associated with an increase in the expression of PPAR-gamma, scavenger receptor-B (CD36) and scavenger receptor-A mRNA with a corresponding increase in PPAR-gamma protein. Matrix-activated cells incubated with Ac-LDL demonstrated foam cell formation, whilst incubation with Dil-labelled Ac-LDL led to an increase in mean fluorescence intensity of 373 +/- 34.8% (P < 0.005) as compared to albumin (100%) and PMA (423 +/- 55.8%) (P < 0.005). This could be inhibited by the addition of excess unlabelled ligand, suggesting specific involvement of scavenger receptors. Incubation of LDL with mesangial matrix in the absence of mesangial cells or monocytes led to enhanced electrophoretic mobility of the recovered lipoprotein on agarose gel, an effect that could be inhibited by the addition of anti-oxidants. CONCLUSION: Exposure to mesangial cell matrix induces expression of monocyte characteristics associated with a macrophage phenotype and promotes oxidation of LDL, thereby converting this lipoprotein to a scavenger receptor ligand. These observations may help to explain foam cell formation in the mesangium in the context of glomerular disease.

Thiazolidinediones inhibit albumin uptake by proximal tubular cells through a mechanism independent of peroxisome proliferator activated receptor gamma.

BACKGROUND: Peroxisome proliferator activated receptor gamma (PPARgamma) is a ligand-activated transcriptional factor which exerts multiple effects on target cell function. A variety of PPARgamma ligands are known, including the antidiabetic thiazolidinediones (TZDs). There is evidence that suggests that these drugs may improve metabolic parameters, proteinuria, and blood pressure in type 2 diabetes. METHOD: We investigated the potentially beneficial effects of TZDs in opossum kidney proximal tubular cells, focussing particularly on protein handling. RESULTS: Three TZDs, ciglitazone, rosiglitazone, and troglitazone, all inhibited FITC-albumin uptake by cells in a dose-dependent manner in the absence of cell cytotoxicity or effects on binding. In contrast, the structurally unrelated PPARgamma ligand 15d-PGJ2 had no effect on albumin uptake. In cells overexpressing PPARgamma or treated with the PPARgamma antagonist GW9662, no alterations in the inhibitory effects of TZDs were observed. All TZDs reduced cholesterol synthesis, and supplementation of cells with non-sterol precursors of cholesterol, mevalonate, farnesol, and geranylgeranyl pyrophosphate, reversed the effects of TZDs. CONCLUSIONS: TZDs inhibit albumin uptake and cholesterol synthesis in proximal tubular cells independently of PPARgamma. Depletion of cholesterol precursors by TZDs is at least partially responsible for reduced albumin uptake. These results support a new role for TZDs to combat progressive proteinuric renal disease.

Ligand-independent activation of peroxisome proliferator-activated receptor-gamma by insulin and C-peptide in kidney proximal tubular cells: dependent on phosphatidylinositol 3-kinase activity.

Peroxisome proliferator-activated receptor gamma (PPARgamma) has key roles in the regulation of adipogenesis, inflammation, and lipid and glucose metabolism. C-peptide is believed to be inert and without appreciable biological functions. Recent studies suggest that C-peptide possesses multiple functions. The present study investigated the effects of insulin and C-peptide on PPARgamma transcriptional activity in opossum kidney proximal tubular cells. Both insulin and C-peptide induced a concentration-dependent stimulation of PPARgamma transcriptional activity. Both agents substantially augmented thiazolidinedione-stimulated PPARgamma transcriptional activity. Neither insulin nor C-peptide had any effect on the expression levels of PPARgamma. GW9662, a PPARgamma antagonist, blocked PPARgamma activation by thiazolidinediones but had no effect on either insulin- or C-peptide-stimulated PPARgamma transcriptional activity. Co-transfection of opossum kidney cells with dominant negative mitogen-activated protein kinase kinase significantly depressed basal PPARgamma transcriptional activity but had no effect on that induced by either insulin or C-peptide. Both insulin- and C-peptide-stimulated PPARgamma transcriptional activity were attenuated by wortmannin and by expression of a dominant negative phosphatidylinositol (PI) 3-kinase p85 regulatory subunit. In addition PI 3-kinase-dependent phosphorylation of PPARgamma was observed after stimulation by C-peptide or insulin. C-peptide effects but not insulin on PPARgamma transcriptional activity were abolished by pertussis toxin pretreatment. Finally both C-peptide and insulin positively control the expression of the PPARgamma-regulated CD36 scavenger receptor in human THP-1 monocytes. We concluded that insulin and C-peptide can stimulate PPARgamma activity in a ligand-independent fashion and that this effect is mediated by PI 3-kinase. These results support a new and potentially important physiological role for C-peptide in regulation of PPARgamma-related cell functions.

Differential effects of peroxisome proliferator activated receptor-gamma (PPAR gamma) ligands in proximal tubular cells: thiazolidinediones are partial PPAR gamma agonists.

UNLABELLED: Background. Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors with multiple effects on target cell function. PPAR gamma activity is regulated by extracellular signal-regulated protein kinase (ERK), mitogen-activated protein (MAP) kinase, and PPAR gamma ligands have varying effects on activity of ERK. Different PPAR gamma ligands have been shown to have both protective and detrimental effects in the kidney. Since transcriptional activation by different PPAR agonists is ligand- and depot-specific PPAR gamma, we have examined the effects of different agonists on PPAR activity in the proximal tubule. METHODS: Opossum kidney cells were used in all experiments, transiently transfected with a PPAR response element luciferase reporter and subject to stimulation with various PPAR ligands. The role of ERK and phosphorylation in PPAR gamma activation were studied, as were the effects of PPAR agonists on ERK activation and cell proliferation. RESULTS: Transcriptional activity of PPAR was not stimulated by PPAR alpha agonists, and only very modestly stimulated by a PPAR beta agonist. The PPAR gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and troglitazone stimulated significant transcriptional activation and phosphorylation of PPAR gamma. These effects were more marked with 15d-PGJ(2). Thiazolidinediones attenuated 15d-PGJ(2) evoked PPAR gamma activation and phosphorylation. ERK activity positively regulated PPAR activation. Only 15d-PGJ(2) stimulated ERK activity and cell proliferation, and these effects were also inhibited by thiazolidinediones. CONCLUSION: PPAR gamma agonists exert differential effects in proximal tubule cells with thiazolidinediones behaving as partial agonists.

Monocyte adhesion to mesangial matrix modulates cytokine and metalloproteinase production.

BACKGROUND: Monocytes migrate into the glomerular mesangium during acute inflammatory renal disease, differentiate into macrophages, and may play a key role in the development and progression of glomerular scarring. Treatment strategies that inhibit monocyte infiltration ameliorate glomerular injury in animal models. Mesangial matrix contains several potential monocyte-binding domains that may contribute to monocyte entrapment and modulate cell activation. METHODS: Adhesion of peripheral blood-derived monocytes to matrix synthesized by human mesangial cells and to individual matrix proteins was assessed by colorimetry of nuclear staining with crystal violet. Monoclonal antibodies were used to identify the cell-surface integrins and matrix ligands involved. Monocyte proliferation was assessed by 3H-thymidine incorporation and cytokine production using enzyme-linked immunosorbent assay (ELISA). Secretion of metalloproteinases and their inhibitors was determined by zymography and ELISA, respectively. RESULTS: Monocytes bound to matrix synthesized by mesangial cells. Prestimulation of mesangial cells with tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta (TGF-beta) enhanced matrix fibronectin content (P < 0.001) and monocyte binding (P < 0.001). Blocking antibodies to fibronectin, as well as to the integrins very late antigen-4 (VLA-4) and VLA-5, reduced monocyte adhesion to mesangial matrix by approximately 50%. Incubation of monocytes with matrix, fibronectin, laminin and collagen IV enhanced production of interleukin-1beta (IL-1beta), interleukin-6 (IL-6), TNF-alpha and metalloproteinase-9 (MMP-9) when compared to cells incubated in plastic wells. However, there was no apparent difference in proliferation rate and no change in production of metalloproteinase inhibitors. CONCLUSION: Monocyte activation within the glomerulus may be mediated by binding to mesangial matrix components, particularly fibronectin. Matrix-mediated activation enhances production of inflammatory cytokines and matrix-degrading enzymes.

Synthesis of 1,25-dihydroxyvitamin D(3) by human endothelial cells is regulated by inflammatory cytokines: a novel autocrine determinant of vascular cell adhesion.

In addition to its calciotropic function, the secosteroid 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) has potent nonclassical effects. In particular, local production of 1,25D(3) catalyzed by the enzyme 1alpha-hydroxylase (1alpha-OHase) may act as an autocrine/paracrine immunomodulatory mechanism. To investigate the significance of this in vascular tissue the expression and function of 1alpha-OHase in human endothelial cells was characterized. Immunohistochemical and in situ hybridization analyses show, for the first time, the presence of 1alpha-OHase mRNA and protein in endothelial cells from human renal arteries as well as postcapillary venules from lymphoid tissue. Reverse transcription-PCR and Western blot analyses confirmed the presence of 1alpha-OHase in primary cultures of human umbilical vein endothelial cells (HUVEC). Enzyme activity in HUVEC (318 +/- 56 fmoles 1,25(OH)(2)D(3)/hr/mg protein) increased after treatment with tumor necrosis factor-alpha (1054 +/- 166, P < 0.01), lipopolysaccharide (1381 +/- 88, P < 0.01), or forskolin (554 +/- 56, P < 0.05). Functional studies showed that exogenously added 1,25(OH)(2)D(3) or its precursor, 25-hydroxyvitamin D(3) (25(OH)D(3)), significantly decreased HUVEC proliferation after 72 h of treatment (33% and 11%, respectively). In addition, after 24 h treatment, both 1,25(OH)(2)D(3) and 25(OH)D(3) increased the adhesion of monocytic U937 cells to HUVEC (159% and 153%, respectively). These data indicate that human endothelia are able to produce active vitamin D. The rapid induction of endothelial 1alpha-OHase activity by inflammatory cytokines suggests a novel autocrine/paracrine role for the enzyme, possibly as a modulator of endothelial cell adhesion.