Limits...
CXCL16 and oxLDL are induced in the onset of diabetic nephropathy.

Gutwein P, Abdel-Bakky MS, Doberstein K, Schramme A, Beckmann J, Schaefer L, Amann K, Doller A, Kämpfer-Kolb N, Abdel-Aziz AA, El Sayed el SM, Pfeilschifter J - J. Cell. Mol. Med. (2009)

Bottom Line: Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN.Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes.In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

View Article: PubMed Central - PubMed

Affiliation: Pharmazentrum Frankfurt/ZAFES, University Hospital, Goethe University Frankfurt, Frankfurt, Germany. p.gutwein@med.uni-frankfurt.de

ABSTRACT
Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated alpha(3)-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored alpha(3) integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

Show MeSH

Related in: MedlinePlus

Effect of HG on cellular CXCL16 and ADAM10 expression in human podocytes. (A) Immunofluorescence staining of CXCL16 and ADAM10 expression in human podocytes treated for 24 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. The cells were stained with DAPI to visualize nuclei (blue), and with Alexa Fluor 488 and Cy3 coupled secondary antibodies to visualize the distribution of ADAM10 (green) and CXCL16 (red) proteins. (B) Immunofluorescence results of CXCL16 and ADAM10 expression after 24 hrs glucose treatment using the BZ-Analyzer software (Keyence). Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG concentration. (C) Podocytes were incubated 72 hrs with 5 or 30 mM glucose containing media. Cells were than lysed and ADAM10 expression was determined by Western blot analysis. β-actin was used to demonstrate equal sample loading. (D) CXCL16 protein expression was measured with a CXCL16 specific ELISA 24 hrs after the treatment of podocytes with 5 mM glucose or 30 mM glucose. Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG containing media. (E) mRNA from podocytes was isolated at times indicated, transcribed in the cDNA as described in material and methods and real-time PCR was performed. (F) Release of CXCL16 was measured in podocytes incubated for 24, 72 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. Supernatants were prepared as described in material and methods and soluble CXCL16 was measured by CXCL16 specific ELISA. (G-I) ADAM10 expression was down-regulated in human podocytes by ADAM10 specific siRNA (controlled by ADAM10 western blot, depicted above the graph) and cellular (G) and soluble CXCL16 (I) was measured by a CXCL16 specific ELISA. A10 = ADAM10, sc = scrambled.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4516529&req=5

fig04: Effect of HG on cellular CXCL16 and ADAM10 expression in human podocytes. (A) Immunofluorescence staining of CXCL16 and ADAM10 expression in human podocytes treated for 24 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. The cells were stained with DAPI to visualize nuclei (blue), and with Alexa Fluor 488 and Cy3 coupled secondary antibodies to visualize the distribution of ADAM10 (green) and CXCL16 (red) proteins. (B) Immunofluorescence results of CXCL16 and ADAM10 expression after 24 hrs glucose treatment using the BZ-Analyzer software (Keyence). Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG concentration. (C) Podocytes were incubated 72 hrs with 5 or 30 mM glucose containing media. Cells were than lysed and ADAM10 expression was determined by Western blot analysis. β-actin was used to demonstrate equal sample loading. (D) CXCL16 protein expression was measured with a CXCL16 specific ELISA 24 hrs after the treatment of podocytes with 5 mM glucose or 30 mM glucose. Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG containing media. (E) mRNA from podocytes was isolated at times indicated, transcribed in the cDNA as described in material and methods and real-time PCR was performed. (F) Release of CXCL16 was measured in podocytes incubated for 24, 72 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. Supernatants were prepared as described in material and methods and soluble CXCL16 was measured by CXCL16 specific ELISA. (G-I) ADAM10 expression was down-regulated in human podocytes by ADAM10 specific siRNA (controlled by ADAM10 western blot, depicted above the graph) and cellular (G) and soluble CXCL16 (I) was measured by a CXCL16 specific ELISA. A10 = ADAM10, sc = scrambled.

Mentions: Hyperglycaemia is an important determinant in the pathogenesis of diabetic microvascular complications in both type 1 and type 2 diabetes [42]. To analyse the influence of hyperglycaemia on the expression of CXCL16 and ADAM10, human podocytes were cultured in LG (=5 mM) or HG (=30 mM) containing media. As documented in Fig. 4A and B, HG treatment led to a significant induction of cellular CXCL16 in human podocytes 24 and 120 hrs after treatment. Importantly we also noticed a down-regulation of ADAM10 expression after HG treatment at both time-points (Fig. 4A, B and C). In addition, with a CXCL16 specific ELISA we confirmed that hyperglycaemic conditions increased cellular CXCL16 expression (Fig. 4D). To investigate if HG levels induce CXCL16 mRNA expression in podocytes, we performed real-time PCR. As shown in Fig. 4E, treatment of podocytes for 24 and 72 hrs with HG containing media significantly induced CXCL16 mRNA. Furthermore, we provide evidence that hyperglycaemic conditions reduces the release of CXCL16 from podocytes (Fig. 4F). To investigate, if the down-regulation of ADAM10 is involved in the reduced shedding of CXCL16 we performed siRNA experiments against ADAM10. As documented in Fig. 4G, blocking ADAM10 protein synthesis increased cellular CXCL16 and reduced the constitutive cleavage of CXCL16 (Fig. 4H).


CXCL16 and oxLDL are induced in the onset of diabetic nephropathy.

Gutwein P, Abdel-Bakky MS, Doberstein K, Schramme A, Beckmann J, Schaefer L, Amann K, Doller A, Kämpfer-Kolb N, Abdel-Aziz AA, El Sayed el SM, Pfeilschifter J - J. Cell. Mol. Med. (2009)

Effect of HG on cellular CXCL16 and ADAM10 expression in human podocytes. (A) Immunofluorescence staining of CXCL16 and ADAM10 expression in human podocytes treated for 24 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. The cells were stained with DAPI to visualize nuclei (blue), and with Alexa Fluor 488 and Cy3 coupled secondary antibodies to visualize the distribution of ADAM10 (green) and CXCL16 (red) proteins. (B) Immunofluorescence results of CXCL16 and ADAM10 expression after 24 hrs glucose treatment using the BZ-Analyzer software (Keyence). Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG concentration. (C) Podocytes were incubated 72 hrs with 5 or 30 mM glucose containing media. Cells were than lysed and ADAM10 expression was determined by Western blot analysis. β-actin was used to demonstrate equal sample loading. (D) CXCL16 protein expression was measured with a CXCL16 specific ELISA 24 hrs after the treatment of podocytes with 5 mM glucose or 30 mM glucose. Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG containing media. (E) mRNA from podocytes was isolated at times indicated, transcribed in the cDNA as described in material and methods and real-time PCR was performed. (F) Release of CXCL16 was measured in podocytes incubated for 24, 72 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. Supernatants were prepared as described in material and methods and soluble CXCL16 was measured by CXCL16 specific ELISA. (G-I) ADAM10 expression was down-regulated in human podocytes by ADAM10 specific siRNA (controlled by ADAM10 western blot, depicted above the graph) and cellular (G) and soluble CXCL16 (I) was measured by a CXCL16 specific ELISA. A10 = ADAM10, sc = scrambled.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4516529&req=5

fig04: Effect of HG on cellular CXCL16 and ADAM10 expression in human podocytes. (A) Immunofluorescence staining of CXCL16 and ADAM10 expression in human podocytes treated for 24 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. The cells were stained with DAPI to visualize nuclei (blue), and with Alexa Fluor 488 and Cy3 coupled secondary antibodies to visualize the distribution of ADAM10 (green) and CXCL16 (red) proteins. (B) Immunofluorescence results of CXCL16 and ADAM10 expression after 24 hrs glucose treatment using the BZ-Analyzer software (Keyence). Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG concentration. (C) Podocytes were incubated 72 hrs with 5 or 30 mM glucose containing media. Cells were than lysed and ADAM10 expression was determined by Western blot analysis. β-actin was used to demonstrate equal sample loading. (D) CXCL16 protein expression was measured with a CXCL16 specific ELISA 24 hrs after the treatment of podocytes with 5 mM glucose or 30 mM glucose. Data represent mean ± S.D. ***P < 0.001, **P < 0.01 considered statistically significant compared to cells treated with LG containing media. (E) mRNA from podocytes was isolated at times indicated, transcribed in the cDNA as described in material and methods and real-time PCR was performed. (F) Release of CXCL16 was measured in podocytes incubated for 24, 72 or 120 hrs with LG (5 mM) or HG (30 mM) containing media. Supernatants were prepared as described in material and methods and soluble CXCL16 was measured by CXCL16 specific ELISA. (G-I) ADAM10 expression was down-regulated in human podocytes by ADAM10 specific siRNA (controlled by ADAM10 western blot, depicted above the graph) and cellular (G) and soluble CXCL16 (I) was measured by a CXCL16 specific ELISA. A10 = ADAM10, sc = scrambled.
Mentions: Hyperglycaemia is an important determinant in the pathogenesis of diabetic microvascular complications in both type 1 and type 2 diabetes [42]. To analyse the influence of hyperglycaemia on the expression of CXCL16 and ADAM10, human podocytes were cultured in LG (=5 mM) or HG (=30 mM) containing media. As documented in Fig. 4A and B, HG treatment led to a significant induction of cellular CXCL16 in human podocytes 24 and 120 hrs after treatment. Importantly we also noticed a down-regulation of ADAM10 expression after HG treatment at both time-points (Fig. 4A, B and C). In addition, with a CXCL16 specific ELISA we confirmed that hyperglycaemic conditions increased cellular CXCL16 expression (Fig. 4D). To investigate if HG levels induce CXCL16 mRNA expression in podocytes, we performed real-time PCR. As shown in Fig. 4E, treatment of podocytes for 24 and 72 hrs with HG containing media significantly induced CXCL16 mRNA. Furthermore, we provide evidence that hyperglycaemic conditions reduces the release of CXCL16 from podocytes (Fig. 4F). To investigate, if the down-regulation of ADAM10 is involved in the reduced shedding of CXCL16 we performed siRNA experiments against ADAM10. As documented in Fig. 4G, blocking ADAM10 protein synthesis increased cellular CXCL16 and reduced the constitutive cleavage of CXCL16 (Fig. 4H).

Bottom Line: Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN.Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes.In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

View Article: PubMed Central - PubMed

Affiliation: Pharmazentrum Frankfurt/ZAFES, University Hospital, Goethe University Frankfurt, Frankfurt, Germany. p.gutwein@med.uni-frankfurt.de

ABSTRACT
Diabetic nephropathy (DN) is a major cause of end-stage renal failure worldwide. Oxidative stress has been reported to be a major culprit of the disease and increased oxidized low density lipoprotein (oxLDL) immune complexes were found in patients with DN. In this study we present evidence, that CXCL16 is the main receptor in human podocytes mediating the uptake of oxLDL. In contrast, in primary tubular cells CD36 was mainly involved in the uptake of oxLDL. We further demonstrate that oxLDL down-regulated alpha(3)-integrin expression and increased the production of fibronectin in human podocytes. In addition, oxLDL uptake induced the production of reactive oxygen species (ROS) in human podocytes. Inhibition of oxLDL uptake by CXCL16 blocking antibodies abrogated the fibronectin and ROS production and restored alpha(3) integrin expression in human podocytes. Furthermore we present evidence that hyperglycaemic conditions increased CXCL16 and reduced ADAM10 expression in podocytes. Importantly, in streptozotocin-induced diabetic mice an early induction of CXCL16 was accompanied by higher levels of oxLDL. Finally immunofluorescence analysis in biopsies of patients with DN revealed increased glomerular CXCL16 expression, which was paralleled by high levels of oxLDL. In summary, regulation of CXCL16, ADAM10 and oxLDL expression may be an early event in the onset of DN and therefore all three proteins may represent potential new targets for diagnosis and therapeutic intervention in DN.

Show MeSH
Related in: MedlinePlus