Caveolin-1 deficiency protects against mesangial matrix expansion in a mouse model of type 1 diabetic nephropathy.

AIMS/HYPOTHESIS: Glomerular matrix protein accumulation, mediated largely by resident mesangial cells (MCs), is central to the pathogenesis of diabetic nephropathy. We previously showed that caveolin (CAV)-1/caveolae mediate matrix upregulation by MCs in response to high glucose and TGFß, two important pathogenic mediators of diabetic glomerular sclerosis. Here, we evaluated the in vivo role of CAV-1/caveolae in the development of diabetic nephropathy. METHODS: Diabetes was induced in Cav1-knockout (KO) mice and their wild-type (WT) counterparts by streptozotocin injection. After 10 months, kidneys were evaluated for the development of nephropathy, including glomerular sclerosis and upregulation of matrix proteins. Parallel experiments assessing glucose-induced matrix upregulation were carried out in MCs isolated from KO mice. RESULTS: KO diabetic mice developed hyperglycaemia and renal hypertrophy, but were protected from developing albuminuria and glomerular sclerosis compared with WT mice. KO mice were significantly protected from the upregulation of glomerular collagen I, fibronectin, connective tissue growth factor (CTGF) and TGFß. In vitro, glucose induced collagen I A1 promoter activation and collagen I, fibronectin and CTGF protein upregulation in WT but not KO MCs. Re-expression of Cav1 in KO cells restored this response. CONCLUSIONS/INTERPRETATION: Cav1 deletion rendered significant protection from glomerular matrix accumulation and albuminuria in a mouse model of type 1 diabetes. These studies provide a foundation for the development of renal-targeted interference with CAV-1/caveolae as a novel approach to the treatment of diabetic nephropathy.

High glucose-induced RhoA activation requires caveolae and PKCß1-mediated ROS generation.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We previously showed that RhoA activation by high glucose in mesangial cells (MC) leads to matrix upregulation (Peng F, Wu D, Gao B, Ingram AJ, Zhang B, Chorneyko K, McKenzie R, Krepinsky JC. Diabetes 57: 1683-1692, 2008). Here, we study the mechanism whereby RhoA is activated. In primary rat MC, RhoA activation required glucose entry and metabolism. Broad PKC inhibitors (PMA, bisindolylmaleimide, Gö6976), as well as specific PKCß blockade with an inhibitor and small interfering RNA (siRNA), prevented RhoA activation by glucose. PKCß inhibition also abrogated reactive oxygen species (ROS) generation by glucose. The ROS scavenger N-acetylcysteine (NAC) or NADPH oxidase inhibitors apocynin and DPI prevented glucose-induced RhoA activation. RhoA and some PKC isoforms localize to caveolae. Chemical disruption of these microdomains prevented RhoA and PKCß1 activation by glucose. In caveolin-1 knockout cells, glucose did not induce RhoA and PKCß1 activation; these responses were rescued by caveolin-1 reexpression. Furthermore, glucose-induced ROS generation was significantly attenuated by chemical disruption of caveolae and in knockout cells. Downstream of RhoA signaling, activator protein-1 (AP-1) activation was also inhibited by disrupting caveolae, was absent in caveolin-1 knockout MC and rescued by caveolin-1 reexpression. Finally, transforming growth factor (TGF)-ß1 upregulation, mediated by AP-1, was prevented by RhoA signaling inhibition and by disruption or absence of caveolae. In conclusion, RhoA activation by glucose is dependent on PKCß1-induced ROS generation, most likely through NADPH oxidase. The activation of PKCß1 and its downstream effects, including upregulation of TGF-ß1, requires caveolae. These microdomains are thus important mediators of the profibrogenic process associated with diabetic nephropathy.

HB-EGF release mediates glucose-induced activation of the epidermal growth factor receptor in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We showed that transactivation of the epidermal growth factor receptor (EGFR) is an important mediator of matrix upregulation in mesangial cells (MC) in response to high glucose (HG). Here, we study the mechanism of EGFR transactivation. In primary MC, EGFR transactivation by 1 h of HG (30 mM) was unaffected by inhibitors of protein kinase C, reactive oxygen species, or the angiotensin II AT1 receptor. However, general metalloprotease inhibition, as well as specific inhibitors of heparin-binding EGF-like growth factor (HB-EGF), prevented both EGFR and downstream Akt activation. HB-EGF was released into the medium by 30 min of HG, and this depended on metalloprotease activity. One of the metalloproteases shown to cleave proHB-EGF is ADAM17 (TACE). HG, but not an osmotic control, activated ADAM17, and its inhibition prevented EGFR and Akt activation and HB-EGF release into the medium. siRNA to either ADAM17 or HB-EGF prevented HG-induced EGFR transactivation. We previously showed that EGFR/Akt signaling increases transforming growth factor (TGF)-ß1 transcription through the transcription factor activator protein (AP)-1. HG-induced AP-1 activation, as assessed by EMSA, was abrogated by inhibitors of metalloproteases, HB-EGF and ADAM17. HB-EGF and ADAM17 siRNA also prevented AP-1 activation. Finally, these inhibitors and siRNA prevented TGF-ß1 upregulation by HG. Thus, HG-induced EGFR transactivation in MC is mediated by the release of HB-EGF, which requires activity of the metalloprotease ADAM17. The mechanism of ADAM17 activation awaits identification. Targeting upstream mediators of EGFR transactivation including HB-EGF or ADAM17 provides novel therapeutic targets for the treatment of diabetic nephropathy.

EGFR-PLCgamma1 signaling mediates high glucose-induced PKCbeta1-Akt activation and collagen I upregulation in mesangial cells.

Glomerular matrix accumulation is a hallmark of diabetic nephropathy. We have recently shown that epidermal growth factor receptor (EGFR) transactivation mediates high glucose (HG)-induced collagen I upregulation through PI3K-PKCbeta1-Akt signaling in mesangial cells (MC). Phospholipase Cgamma1 (PLCgamma1) interacts with activated growth factor receptors and activates classic PKC isoforms. We thus studied its role in HG-induced collagen I upregulation in MC. Primary rat MC were treated with HG (30 mM) or mannitol as osmotic control. Protein kinase activation was assessed by Western blotting and collagen I upregulation by Northern blotting. Diabetes was induced in rats by streptozotocin. HG treatment for 1 h led to PLCgamma1 membrane translocation and Y783 phosphorylation, both indicative of its activation. Mannitol was without effect. PLCgamma1 Y783 phosphorylation was also seen in cortex and glomeruli of diabetic rats. HG induced a physical association between EGFR and PLCgamma1 as identified by coimmunoprecipitation. PLCgamma1 activation required EGFR kinase activity since it was prevented by the EGFR inhibitor AG1478 or overexpression of kinase-inactive EGFR (K721A). Phosphoinositide-3-OH kinase inhibition also prevented PLCgamma1 activation. HG-induced Akt S473 phosphorylation, effected by PKCbeta1, was inhibited by the PLCgamma inhibitor U73122. PLCgamma1 inhibition or downregulation by small interference RNA also prevented HG-induced collagen I upregulation. Our results indicate that EGFR-PLCgamma1 signaling mediates HG-induced PKCbeta1-Akt activation and subsequent collagen I upregulation in MC. Inhibition of EGFR or PLCgamma1 may provide attractive therapeutic targets for the treatment of diabetic nephropathy.

Collagen I induction by high glucose levels is mediated by epidermal growth factor receptor and phosphoinositide 3-kinase/Akt signalling in mesangial cells.

AIMS/HYPOTHESIS: Glomerular matrix accumulation is a hallmark of diabetic nephropathy. Recent data have linked the serine/threonine kinase protein kinase B (Akt) to matrix modulation. Here, we studied its role in high glucose-induced collagen elaboration by mesangial cells. METHODS: Primary rat mesangial cells were treated with high glucose levels (30 mmol/l) or mannitol as osmotic control. Western blots, northern blots, ELISA and immunohistochemistry were used for assessment. Diabetes was induced in rats by streptozotocin. RESULTS: Phosphorylated Akt at S473 (pAktS473), corresponding to Akt activation, was seen in diabetic glomeruli. In mesangial cells, high glucose levels induced pAktS473 by 20 min. This was sustained to 72 h, while mannitol had no effect. Akt activation by kinase assay and phosphorylation on threonine 308 was also observed. Phosphoinositide 3-kinase (PI3K) inhibitors LY294002 (20 micromol/l) and wortmannin (100 nmol/l) prevented pAktS473. Collagen IA1 transcript and collagen I protein upregulation by high glucose levels were inhibited by PI3K blockade, as was collagen I secretion into the medium (ELISA). Dominant-negative Akt overexpression also inhibited high glucose-induced collagen IA1 transcript and collagen I protein production. Since signalling through the epidermal growth factor receptor (EGFR) can activate PI3K-Akt, we studied its activation by high glucose levels. EGFR was correspondingly activated by 10 min; mannitol had no effect. EGFR activation was also seen in glomeruli from diabetic rats and co-localised with collagen IA1 in diabetic glomeruli. Specific EGFR inhibition (AG1478, 5 micromol/l or dominant-negative EGFR) blocked high glucose-induced pAktS473, phosphorylation on threonine 308 and activation of the EGFR downstream target p44 extracellular signal-regulated kinase (Erk) mitogen-activated protein kinase. Finally, EGFR inhibition also blocked high glucose-induced collagen I upregulation at transcriptional and protein levels. CONCLUSIONS/INTERPRETATION: We conclude that EGFR-PI3K-Akt signalling mediates high glucose-induced collagen I upregulation in mesangial cells and that this pathway is activated in diabetic glomeruli. Targeting its components may provide a new therapeutic approach to diabetic kidney disease.