Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells1

Albumin up-regulates the type II transforming growth factor-beta receptor in cultured proximal tubular cells.BackgroundClinical and experimental observations suggest that proteinuria is not merely a marker of chronic nephropathies, but may also be involved in the progression to end-stage renal failure. Filtered proteins are taken up by tubular cells, and overwhelming this system may lead to tubular synthesis of various proinflammatory and profibrogenic cytokines, including transforming growth factor-beta (TGF-β). TGF-β acts by first binding to specific receptors. We studied in an in vitro system using a well-defined mouse proximal tubular cell line (MCT cells) whether fatty acid-free bovine albumin modulates expression of specific receptors for TGF-β.MethodsMCT (and LLC-PK1) cells were challenged in serum-free medium with different concentrations of albumin. Activation of a local renin-angiotensin system was tested by real-time polymerase chain reaction (PCR) for renin and angiotensinogen transcripts and determination of secreted angiotensin II (Ang II) by enzyme-linked immunosorbent assay (ELISA). Some cells were also treated with the AT1 receptor antagonist losartan. TGF-β receptor types I and II mRNA levels were determined by Northern analysis whereas protein abundance was measured by Western blots. To test for a functional consequence of up-regulated TGF-β receptors, MCT cells were preincubated with albumin and subsequently treated with low-dose TGF-β that normally does not induce collagen type IV expression by itself. Downstream signaling events were detected by Western blots for phosphorylated Smad2. Scatchard assays with [125I]TGF-β1 were performed to estimate affinity and number of specific binding sites. Different length TGF-β type II promoter constructs linked to CAT reporter were transiently transected into MCT cells to determine transcriptional activity.ResultsIncubation of MCT cells with 0.5 to 10 mg/mL albumin leads to an increase in type II TGF-β receptor mRNA and protein expression without influencing type I receptors. An increase in type II TGF-β receptor protein expression was detected after 12 hours of albumin incubation and was still detectable after 48 hours. The albumin-mediated increase in type II TGF-β receptor mRNA was attenuated in the presence of 1 μmol/L losartan, suggesting involvement of a local renin-angiotensin system. MCT cells treated with albumin significantly increased expression of angiotensinogen and renin transcripts and also secreted more Ang II into the culture supernatant. Analysis of transcriptional activity showed that promoter segments containing activating protein (AP-1)-binding sites are necessary for albumin-induced transcription of the TGF-β type II receptor. Binding assays revealed that albumin treatment significantly increased the overall binding sites as well as the affinity for TGF-β. This effect had functional consequences because MCT cells pretreated with albumin reacted with a stronger TGF-β–mediated phosphorylation of down-stream Smad2 and also increased collagen IV expression compared with control cells.ConclusionOur findings indicate that albumin up-regulates ligand-binding TGF-β receptors on cultured proximal tubular cells. Albumin-induced activation of local Ang II production appears to be responsible for this effect. This may amplify the matrix-stimulatory actions of TGF-β on tubular cells and could be a novel mechanism for how proteinuria exhibits pathophysiologic effects on tubular cells ultimately leading to tubulointerstitial fibrosis

Immune-mediated mesangial cell injury—Biosynthesis and function of prostanoids

Immune-mediated mesangial cell injury—Biosynthesis and function of prostanoids. We studied the formation of cyclo-oxygenase products in a rat model of mesangial cell injury, in order to determine a possible role of prostaglandin E2 (PGE2), prostaglandin I2 (determined as 6-keto-PGF1α and thromboxane A2 (TxA2) in immune-mediated glomerular disease. Selective immune-mediated mesangial cell injury was induced by i.v. administration of a rabbit anti-rat thymocyte antiserum (ATS). Intravenous ATS leads to immune deposits in the mesangium followed by mesangiolysis and the infiltration of polymorphonuclear granulocytes and monocytes. Glomerular TxB2 formation two hours (292 ± 27 pg/mg/min) and 48 hours (396 ± 69 pg/mg/min) following antibody was significantly (P < 0.05) higher compared to animals receiving non-antibody rabbit IgG (TxB2: 2hr 143 ± 13; 48hr 171 ± 32 pg/mg/min). Treatment with cobra venom factor (CVF) and the reduction of glomerular monocyte infiltration inhibited the increase of glomerular TxB2 formation significantly. Depletion of granulocytes with a rabbit anti-rat granulocyte serum had no effect on glomerular prostanoid formation following ATS. Glomerular PGE2 and 6-keto PGF1α production was not altered following ATS. Inulin clearance in rats with immune-mediated mesangial cell injury was significantly (P < 0.001) lower at two hours (456 ± 24 µl/min/100g body wt) and 48 hours (433 ± 54 µl/min/lOO g body wt) compared to their corresponding control animals which were treated with non-antibody IgG (2 hr: 914 ± 51; 48 hr: 694 ± 79 µl/min/100g body wt). Pretreatment of rats with indomethacin (Indo) or with the thromboxane synthetase inhibitor UK 38485 prevented the decrease in inulin clearance following ATS at two hours (Indo: 800 ± 67; UK 38485: 923 ± 115) and at 48 hours (Indo: 697 ± 60; UK 38485: 654 ± 99). The data demonstrate that selective, immune-mediated mesangial cell injury in rats is associated with increased glomerular TxB2 formation. Complement and monocyte/macrophage depletion reduces TxB2 production. The fall in inulin clearance following ATS is ameliorated when the rats receive indomethacin or the Tx synthetase inhibitor UK 38485. Thus, elevated TxB2 formation might mediate the reduction in GFR in this model of glomerular immune injury

Chronic anti-Thy-1 nephritis is aggravated in the nonclipped but not in the clipped kidney of Goldblatt hypertensive rats

Chronic anti-Thy-1 nephritis is aggravated in the nonclipped but not in the clipped kidney of Goldblatt hypertensive rats.BackgroundWe have previously shown that renovascular hypertension does not inhibit healing of the acute Thy-1 nephritis. To test whether a chronic model of the Thy-1 nephritis is more susceptible to high blood pressure, the repetitive hit model was evaluated in rats with 2-kidney, 1-clip Goldblatt hypertension.MethodsSix weeks after initiation of 2-kidney, 1-clip hypertension, chronic Thy-1 glomerulonephritis was induced in hypertensive rats by four consecutive injections of rabbit antiserum in weekly intervals. Renal structure and function were examined two weeks after the last injection. Glomerular binding of rabbit IgG as well as expression of transforming growth factor-beta (TGF-β), α-smooth muscle actin (α-SMA) and cyclooxygenase (COX)-1 and -2 were evaluated by Western blotting.ResultsSimilar glomerular deposition of rabbit IgG was detected in normotensive rats and in both kidneys of Goldblatt hypertensive rats indicating similar delivery and binding of the heterologous antibody. Induction of the repetitive Thy-1 model significantly enhanced glomerular damage in the nonclipped kidney and increased albuminuria. Surprisingly, no glomerular damage developed in the clipped kidney of nephritic hypertensive rats. In contrast, increased glomerular volume and increased expression of TGF-β, α-SMA as well as COX-1 and COX-2 were found in normotensive nephritic rats and in both kidneys of nephritic hypertensive rats.ConclusionGlomerular and tubulointerstitial damage of the chronic Thy-1 model is dramatically enhanced in the nonclipped kidneys of Goldblatt hypertensive rats. In contrast, the clipped kidney is completely protected from this immunological injury despite similar activation of glomerular cells, induction of TGF-β, COX-1 and COX-2 and glomerular hypertrophy

Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia

Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia Early diabetic nephropathy is characterized by glomerular hypertrophy. Previous studies in vitro have demonstrated that mesangial cells exposed to high glucose are arrested in the G1-phase of the cell cycle and express increased levels of the cyclin-dependent kinase inhibitor p27Kip1. The present study was performed to investigate the renal expression of p27Kip1 in db/db mice, a model of diabetes mellitus type II. Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates. Immunohistochemical studies revealed that this stimulated expression was mainly restricted to the nuclei of mesangial cells and podocytes, but glomerular endothelial cells occasionally also stained positively. Quantification of p27Kip1 positive glomerular cells showed a significant increase of these cells in db/db mice compared with non-diabetic db/+ animals. Although tubular cells revealed a positive staining for p27Kip1 protein, there was no difference between db/+ and db/db mice. Immunoprecipitation experiments revealed that p27Kip1 protein associates with Cdk2 and Cdk4, but not with Cdk6. To test for the influence of hyperglycemia on cell cycle arrest and p27Kip1 expression, mesangial cells were isolated from db/+ and db/db mice. There was a similar basal proliferation when these cells were grown in normal glucose-containing medium (100 mg/dl). However, raising the glucose concentration to 275 to 450 mg/dl induced cell cycle arrest in db/+ as well as db/db mesangial cells. Increasing the medium osmolarity with D-mannitol failed to induce p27Kip1 expression in mesangial cells. Transfection of cells with p27Kip1 antisense, but not missense, phosphorothioate oligonucleotides facilitated cell cycle progression equally well in db/+ and db/db mesangial cells. Furthermore, p27Kip1 expression was comparable in both cell lines in normal glucose, but increased in high glucose medium. Our studies demonstrate that p27Kip1 expression is enhanced in diabetic db/db animals. This induction appears to be due to hyperglycemia. Expression of p27Kip1 may be important in cell cycle arrest and hypertrophy of mesangial cells during early diabetic nephropathy

Expression of the chemokines MCP-1/CCL2 and RANTES/CCL5 is differentially regulated by infiltrating inflammatory cells

Expression of the chemokines MCP-1/CCL2 and RANTES/CCL5 is differentially regulated by infiltrating inflammatory cells.BackgroundChemokines are involved in the regulation of the cellular renal infiltrate in glomerulonephritis; however, it is unclear to which degree resident glomerular cells or infiltrating leukocytes contribute to the formation of chemokines in glomerular inflammatory lesions. We therefore examined whether monocytes/macrophages play a role in the expression of the C-C chemokines MCP-1/CCL2 and RANTES/CCL5 in renal tissue in a lipopolysaccharide (LPS)-induced model of inflammation, where previously we have shown increased glomerular RANTES expression and glomerular infiltration of ED-1-positive cells.MethodsInflammatory lesions were induced by an intraperitoneal injection of LPS. The infiltration of monocytes into the glomerulus was reduced by two experimental approaches. First, rats were depleted of monocytes by the use of specific monocyte-antisera or by cytotoxic drugs. Second, the infiltration of monocytes into the kidney was reduced by using intercellular adhesion molecule-1 (ICAM-1) knockout mice.ResultsBoth experimental approaches demonstrated a significant reduction in the number of infiltrating monocytes/macrophages after lipopolysaccharide injection. This reduction in the infiltration of inflammatory cells was associated with significantly reduced RANTES/CCL5 mRNA expression. However, MCP-1/CCL2 mRNA expression was not inhibited after the LPS injection by monocyte/macrophage depletion. Also, the increase in nuclear factor-κB (NF-κB) binding activity after the LPS injection was not reduced in pretreated animals. The experiments therefore demonstrate that infiltrating monocytes/macrophages contribute to increased RANTES/CCL5 mRNA expression in inflammatory renal lesions, whereas MCP-1/CCL2 mRNA expression and NF-κB activation were not reduced by monocyte/macrophage depletion.ConclusionMCP-1/CCL2 released from renal tissue upon stimulation plays a major role in the regulation of monocyte/macrophage infiltration, which contributes significantly to increased renal RANTES/CCL5 expression. This cross-talk between resident renal cells and monocytes/macrophages is therefore likely to boost the number of infiltrating inflammatory cells

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C

Increased NAD(P)H oxidase-mediated superoxide production in renovascular hypertension: Evidence for an involvement of protein kinase C.BackgroundAngiotensin II infusion has been shown to cause hypertension and endothelial dysfunction and to increase superoxide (O-·2) production in vascular tissue, mainly via an activation of nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]-dependent oxidase, the most significant O-·2 source in endothelial and/or smooth muscle cells. With these studies, we sought to determine whether endothelial dysfunction in renovascular hypertension is secondary to an activation of these oxidases.MethodsEndothelial function in aortas from rats with two kidney-one clip (2K-1C) hypertension and age-matched controls was assessed using isometric tension studies in organ chambers. Changes in vascular O-·2 production were measured using lucigenin-enhanced chemiluminescence and electron spin resonance spectroscopy.ResultsIn hypertensive animals, relaxation to endothelium-dependent (acetylcholine) and endothelium-independent nitrovasodilators (nitroglycerin) was impaired. Constriction to a direct activator of protein kinase C (PKC) phorbol ester 12,13 dibutyrate (PDBu) was enhanced, and vascular O-·2 was significantly increased compared with controls. Vascular O-·2 was normalized by the PKC inhibitor calphostin C, by the inhibitor of flavin-dependent oxidases, diphenylene iodonium, and recombinant heparin-binding superoxide dismutase, whereas inhibitors of the xanthine oxidase (oxypurinol), nitric oxide synthase (NG-nitro-L-arginine) and mitochondrial NADH dehydrogenase (rotenone) were ineffective. Studies of vascular homogenates demonstrated that the major source of O-·2 was a NAD(P)H-dependent oxidase. Incubation of intact tissue with PDBu markedly increased O-·2, the increase being significantly stronger in vessels from hypertensive animals as compared with vessels from controls. Endothelial dysfunction was improved by preincubation of vascular tissue with superoxide dismutase and calphostin C.ConclusionsWe therefore conclude that renovascular hypertension in 2K-1C rats is associated with increased vascular O-·2 leading to impaired vasodilator responses to endogenous and exogenous nitrovasodilators. Increased vascular O-·2 is likely secondary to a PKC-mediated activation of a membrane-associated NAD(P)H-dependent oxidase