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Molecular Mechanisms of Diabetic Mesangial Cell Hypertrophy: A Proliferation of Novel Factors

Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany.

Correspondence to Dr. Gunter Wolf, Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, University Hospital Eppendorf, Pavilion 26, Martinistr. 52, D-20246 Hamburg, Germany. Phone: 49-40-42803-5011; Fax: 49-40-42803-5186; E-mail: Wolf{at}uke.uni-hamburg.de

The French clinician Pierre-Francois-Olivier Rayer (1793–1867) wrote more than 150 years ago: "In diabetes, one finds hypertrophy of the renal cortex...the vessels are enlarged and the Malphigian corpuscles are much more prominent" (1). The underlying mechanisms of this correct description of diabetic glomerular hypertrophy, based exclusively on macroscopic studies, have puzzled and challenged nephrophiles ever since. Extensive morphometric studies on renal specimens from patients with diabetes mellitus type 1, performed almost thirty years ago, provided convincing evidence that glomerular, mainly mesangial cell, hypertrophy and basement membrane thickening are among the earliest pathologic alterations found in diabetic nephropathy (2,3). However, the issue is not a simple one and requires a meaningful definition of cellular hypertrophy and tools to measure it. Mesangial expansion associated with early diabetic nephropathy is certainly a combination of increased deposition of extracellular matrix components and stimulation of mesangial cell growth (4). Cell culture studies and an enormous deluge of novel information regarding cell cycle regulation in the last decade have provided important insights into mechanisms of mesangial cell hypertrophy on a molecular level (5).

An organ can increase in overall size due to an increase in cell number caused by stimulated proliferation or decreased apoptosis or an increase in individual cell size called hypertrophy (5). Cell hypertrophy could be defined as cell enlargement due to an increase in RNA and protein content without concomitant changes in DNA synthesis (4). This definition includes active changes in cellular metabolism and growth regulation, indicating that hypertrophy is more than passive cell swelling. Hypertrophy could be cell cycle–dependent or cell cycle–independent (5). Cell cycle independent hypertrophy may be caused by reduced protein degradation due to an inhibition of various proteases (6). The concept that mesangial cell hypertrophy is rather an active cell cycle–dependent process comes from studies in which murine mesangial cells were exposed to high D-glucose concentrations (7). Raising the medium glucose concentrations from 100 to 450 mg/dl has a biphasic effect on cell proliferation. It stimulates an early limited proliferation after 24 h (7). However, prolonged exposure to high glucose inhibits mesangial cell proliferation and induces hypertrophy (7). A similar biphasic growth response of mesangial cells has also been observed in experimentally induced diabetes in vivo (8). Cell cycle analysis revealed that prolonged incubation in high glucose arrests cells in the G₁-phase of the cell cycle (7). This G₁-phase arrest is partly mediated by autocrine synthesis and activation of transforming growth factor– (TGF-) (7). In exploring potential molecular mechanisms of this cell cycle arrest, it was discovered that high glucose induces ,partly but not exclusively through TGF-, expression of p21^Cip1 and p27^Kip1, two cyclin-dependent kinase (CDK) inhibitors (9,10). These CDK inhibitors bind to cyclin/CDK complexes, inhibit their activity, and thereby prevent G₁/S-phase transit (5). Recent evidence indicates that high glucose activates MAP kinases, which in turn directly phosphorylate p27^Kip1on serine residues and prolong the half-life of this CDK inhibitor (11). In contrast, high glucose directly stimulates transcription of p21^Cip1 (12). Mesangial cell expression of p21^Cip1 and p27^Kip1 also occurs in different models of type 1 and 2 diabetes in vivo and could be modulated by angiotensin-converting enzyme (ACE) inhibitor treatment (12,13). The functional role of p27^Kip1 in mediating hypertrophy emanates from studies using p27^Kip1 knockout (-/-) mesangial cells (14). In contrast to p27^Kip1 +/+ mesangial cells, high glucose fails to induce hypertrophy in p27^Kip1 -/- mesangial cells. However, reconstituting p27^Kip1 expression in p27^Kip1 -/- cells with an inducible vector system restores the hypertrophic phenotype induced by high glucose (14). These results clearly demonstrate that p27^Kip1 is required for glucose-induced cell cycle arrest and hypertrophy.

In their article published in this issue of JASN, Wahab et al. (15) bring a new twist to this complex story by demonstrating that connective tissue growth factor (CTGF) mediates mesangial cell hypertrophy, presumably by induction of CDK inhibitors. CTGF was originally isolated from human umbilical vein endothelial cells in a search for peptides related to platelet-derived growth factor (16). It is a cysteine-rich peptide containing 349 amino acids and belongs to a family of growth factors called CNN (members include cef10, nov, wisp-1) that share certain conserved domains (17). CTGF is produced by a wide variety of different cells, including fibroblasts, endothelial cells, chondrocytes, and smooth muscle cells (18). In the kidney, CTGF is expressed in glomerular endothelial, mesangial, and tubular cells (18). Early data using fibroblasts indicate that TGF-1 is a strong inducer of CTGF, and the CTGF promoter contains a TGF- response element (19). CTGF expression is upregulated in several extrarenal fibrotic diseases, and it is thought that CTGF actually mediates many of the profibrotic effects of TGF- (18). In addition, CTGF is a mitogen for many cells. A suppressive subtractive hybridization screen to identify novel genes expressed in mesangial cells exposed to high glucose revealed CTGF as strongly upregulated (20). This high glucose–induced CTGF expression is TGF-1–dependent and requires signal transduction pathways involving protein kinase C (20). Glomerular CTGF transcript and protein expression are increased in various models of diabetic nephropathy (21,22). In situ hybridization of human kidney biopsies revealed a strong mesangial increase in CTGF mRNA expression in diabetic nephropathy, but CTGF transcripts are also strongly upregulated in other mesangial lesions primarily associated with proliferation such as IgA nephropathy (23). Cell culture studies demonstrated that high glucose–induced mesangial synthesis of extracellular matrix components such as fibronectin depends, at least to some extent, on TGF-1–mediated expression of CTGF (21). How could this pleiotropic factor contribute to mesangial cell hypertrophy?

Wahab et al. (15) found that exogenous CTGF (80 ng/ml) stimulates growth-arrested cultured human mesangial cells to reenter the cell cycle. This reentrance in the cell cycle was associated with increased protein expression of cyclin D. However, these CTGF-treated mesangial cells do not progress into the S-phase, where DNA replication takes place, but are arrested in the G₁-phase and undergo hypertrophy. Simultaneously, the CDK inhibitors p15^INK4, p21^Cip1, and p27^Kip1 are induced by CTGF (15). Although not formally tested in the present study, these findings suggest that the induced CDK inhibitors may bind to cyclin/CDK complexes, inhibit their activity, and eventually cause G₁-phase arrest with concomitant hypertrophy. The observation that CTGF, in contrast to mitogens, fails to stimulate phosphorylation of the retinoblastoma protein (Rb), a critical substrate for cyclin D/CDK4, supports this hypothesis because hypophosphorylated Rb does not release the transcription factor E2F that mediates cell cycle progression. CTGF also phosphorylates MAP kinases Erk 1,2, and this pathway is supposed to induce expression of CDK inhibitors (15). All these effects of CTGF in mediating mesangial cell hypertrophy are reminiscent of TGF-; in fact, the authors tested whether CTGF might be a signal intermediate of TGF-. Inhibiting CTGF expression by antisense technology, Wahab et al. (15) convincingly demonstrated that TGF-–mediated induction of p15^INK4, p21^Cip1, and p27^Kip1 partly depends on autocrine synthesis of CTGF. Thus, a signal transduction pathway postulating how high glucose induces hypertrophy of mesangial cells is shown in Figure 1.

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Overview of potential mechanisms mediating mesangial cell hypertrophy in diabetes mellitus. High glucose is an important inducer of transforming growth factor- (TGF-). Other factors activated in diabetes mellitus, such as advanced glycation end products (AGEs), Amadori glucose adducts, and angiotensin II, also stimulate TGF- expression. TGF-, in turn, upregulates connective tissue factor (CTGF), which is also directly induced by high glucose. As shown in the article by Wahab et al., CTGF activates MAP kinases. MAP kinases are additionally activated by high glucose, presumably through reactive oxygen species, and TGF-. Activation of MAP kinases leads to cyclin D transcription and in parallel to induction of CDK inhibitors through transcriptional (p21Cip1) and posttranscriptional (p27Kip1) mechanisms. Resting mesangial cells reenter the cell cycle from G0, but do not progress through G1/S-phase because of the concomitant induction of CDK inhibitors that associate with and inhibit active cyclin/CDK complexes. The consequence is G1-phase arrest leading to hypertrophy. However, recent studies suggest that TGF-–mediated induction of CDK inhibitors stimulates mesangial cell hypertrophy without cell cycle arrest by a currently unknown mechanism (24). Finally, there is also evidence that non–cell cycle-dependent mechanisms such as inhibition of protein turnover contribute to high glucose-mediated hypertrophy (left arrow).

In summary, the study by Wahab et al. (15) characterizes CTGF as a novel important factor involved in the complex process of mesangial cell hypertrophy. It is not difficult to predict that there will be a further proliferation of knowledge regarding the mechanisms of mesangial cell hypertrophy in the future. And the rapid progress in this area brings us ever closer to understanding one of the most characteristic and long-recognized features of diabetic nephropathy, and presumably thereby to controlling it.

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