CELL CYCLE REGULATION OF RENAL HYPERTROPHY
Gunter Wolf, M.D.
University of Hamburg, University Hospital Eppendorf
Department of Medicine. Division of Nephrology and Osteology
Compensatory renal hypertrophy is a process to compensate for the loss of functioning renal tissue during chronic renal injury. In addition, renal hypertrophy is one of the earliest structural changes of diabetic nephropathy (1). For example, ultrasound investigations documented in humans an increase in overall renal size at the time of diagnosis of type 1 diabetes (2). Although one cannot be certain about the duration of the diabetic milieu in an individual patient before diagnosis, an increase in renal size is nevertheless one of the earliest organ alterations observed during the course of diabetes. Active growth of nephrons is largely responsible for the increase in renal size, but hemodynamic mechanisms such as hyperfiltration and hyperperfusion leading to kidney hyperemia as well as osmotic changes additionally contribute to enlargement of the kidneys (3-7). Although renal hypertrophy occurs in several pathophysiological as well as in physiological situations such as pregnancy, the current contribution will focus on molecular mechanisms of hypertrophy induced by the diabetic environment.
A controversial discussion has been going on for years whether changes in glomerular hemodynamics or adaptive growth changes of the kidney are more important for the progression of diabetic nephropathy (7-9). This issue, however, seems to have been resolved because hemodynamic changes and renal growth are intimately connected and may represent just different faces of the same coin. Hemodynamic changes such as mechanical stretch or alterations in laminar flow induce the synthesis of specific growth factors, activate distinct signal transduction pathways, and stimulate growth and extracellular matrix production in glomerular cells (10-12). Many of these changes are amplified in hyperglycemic situations (1,12). Proteinuria, which is increased in glomerular hyperdynamic states, fundamentally influences activation, growth, and matrix synthesis of tubular cells (13,14. On the other hand, induction of renal growth, mediated by the diabetic environment, may also eventually alter glomerular hemodynamics, for example by an increase in capillary filtration surface (1). Lastly, vasoactive hormones such as angiotensin II (ANG II) or endothelin all exert profound growth stimulatory actions on many renal cells, particularly in the presence of high glucose (15-18).
Expansion of the glomerular mesangium occurs within a few years of the onset of insulin-dependent diabetes mellitus and correlates closely with the deterioration in glomerular function (1). Although it can be debated whether initial adaptive renal growth, as observed in the diabetic state, is causally linked to the irreversible later changes such as glomerulosclerosis and tubulointerstitial fibrosis, there is accumulating evidence suggesting that this is indeed the case (1). Since the growth response of every cell is dictated by the mechanisms of cell cycle regulation, an understanding of these molecular processes is essential for the development of novel therapeutic strategies to prevent the development of diabetic nephropathy.
Renal growth in diabetes
The particular growth response of a distinct renal cell (proliferation versus hypertrophy) during the diabetic state depends on its intrinsic genetic program, which is specific for the cell type, and the presence of growth factors in the local environment (1). Despite the fact that all cells have the same cell cycle machinery, a distinct growth factor may cause different growth responses in the kidney depending on the specific cell type. For example, transforming growth factor-beta (TGF-b), a key player in the development of diabetic nephropathy, induces hypertrophy of mesangial and tubular cells, but stimulates proliferation of tubulointerstitial fibroblasts (5, 19-22). In addition, ANG II exerts proliferative effects on some renal cells (mesangium cells, fibroblasts, distal tubular cells), but mediates hypertrophy of proximal tubules (15,23-25).
In addition to an increase in protein synthesis, a cell cycle-independent decrease in proteinase activity leading to an attenuated turnover of structural and extracellular matrix proteins likely contributes to diabetic hypertrophy of the kidney (26-30). Inhibition of such proteases may be caused by multiple factors including high glucose, advanced glycated endproducts (AGEs), ANG II, and TGF-b (30-32).
Cell cycle regulation
The growth of each cell is determined by different phases called the cell cycle. The period between two mitoses defines the somatic cell cycle and is also called the interphase. Renal cells, under normal conditions, have a very low turnover rate of less than 1% (32,33). These dormant cells can be considered to rest in a G0-phase and have left the active processes of the cell cycle . After stimulation with a growth factor or cytokine, dormant cells actively enter the G1-phase in which cells increase their size and stimulate protein and mRNA synthesis in order to prepare for DNA replication. Immediate early genes, encoding various transcription factors, are also activated in the early G1-phase (33). If conditions to pass the restriction point are satisfied, cells will enter the S-phase after a short lag. During S-phase, the double helix unfolds, DNA strands are separated, stabilized, and DNA is replicated by polymerases. At the end of the S-phase, the total content of DNA doubles to the fully replicated value of 4n. Cells prepare for mitosis in the G2-phase then enter mitosis (M-phase) which itself is composed of several distinct steps. After cell division, cells may start another cell cycle with reentering the G1-phase or may, alternatively, withdraw from the active cell cycle into the G0-phase.
The progression through the cell cycle and transition between different phases is controlled by a series of protein kinases (for review see 33-39). These active protein kinases are holoenzymes composed of two subunits: cyclins and their partner cyclin-dependent kinases (CDK). Some cyclin/CDK complexes act only in specific cell phases whereas others are more promiscuously distributed (37). Cyclins have very short half-lives (<60 minute) and their protein levels fluctuate throughout the cell cycle with increasing synthesis and subsequent degradation by the ubiquitin-proteasome complex (36). The transcription and synthesis of various cyclins may be negatively or positively controlled through specific growth factors. In contrast, CDKs are constitutively expressed, and their activity depends on binding to cyclins and phosphorylation. Cyclins accumulate during specific phases of the cell cycles and bind, after reaching a critical concentration, to their putative CDK partners. Subsequent to complex phosphorylation and dephosphorylation steps whose upstream effectors are only incompletely characterized, the cyclin/CDKs heterodimers are activated and they exert kinase activity (37). For example, a critical substrate for cyclin D/CDK4,6 complexes which control G1-S-phase transit, is the protein product of the retinoblastoma gene (pRB). Upon phosphorylation by active cyclin D/CDK4,6 kinases, hyperphosphorylated pRB releases the transcription factor E2F that binds to the promoter regions of multiple target genes which are essential for further cell cycle progression (33).
The kinase activity of cyclin/CDK complexes is also negatively regulated by small proteins called CDK-inhibitors (CKI). Two families of CKIs can be grouped according to structural homology (36). Although common sense may suggest that an overall increase in CKIs directly inactivates cyclin/CDK complexes by simple binding to those complexes and thus interfering with their kinase activity, the situation is likely much more complex. For example, the CKI p21Cip1 can inhibit cyclin/CDK complexes, but may alternatively function as assembly factor cyclin D/CDK4 heterodimers (39). CKIs can be also redistributed between different cyclin/CDK complexes (33). A decrease in synthesis of a specific cyclin may lead to release of CKIs which then could bind and inhibit other cyclin/CDKs complexes (36). Finally, recent evidence suggests that peptide fragments of CKIs, released after ubiquitin-mediated proteolysis, could actually act as downstream activators of cyclin/CDK complexes facilitating cell cycle progression (33).
Cell cycle events in diabetic nephropathy
High glucose in vitro as well as the diabetic milieu in vivo results in a biphasic growth response (5,20). Initially, there is a limited degree of proliferation, followed by cell cycle arrest and hypertrophy (5,20). Mesangial cells exposed to high glucose actively enter the cell cycle as demonstrated by expression of immediate early gene such as c-fos, c-jun, and Egr-1 (40-42). After one or two complete rounds of cell cycle progression with completion of mitosis, cells are arrested in the G1-phase and they undergo hypertrophy (5). This change in phenotype is mediated by TGF-b because a neutralizing anti-TGF-b antibody prevented the late inhibitory effects of high glucose on hypertrophy of mesangial cells (5).
Cell cycle regulation of mesangial cells in diabetes
To gain a better insight into the molecular mechanisms surrounding this high glucose-mediated G1-phase arrest, we and others investigated the regulation of CKIs. Incubation of mouse mesangial cells, in the absence of other factors for 48-96 hours, in medium with high glucose stimulated p27Kip1 protein expression but did not influence mRNA abundance (43). These effects were independent of the osmolarity of the medium. High glucose-stimulated expression of p27Kip1 involved the activation of protein kinase C and was partly dependent on induction of TGF-b (44). p27Kip1 protein, induced by high glucose, mainly bound to CDK2 but not to CDK4 (43). p27Kip1 antisense, but not missense, oligonucleotides inhibited high glucose-stimulated total protein synthesis and converted the hypertrophy into a proliferative phenotype suggesting G1-phase exit (43) We extended these studies to diabetic db/db mice, a model of type 2 diabetes (44). Glomerular p27Kip1 protein, but not mRNA expression, was strongly enhanced in diabetic db/db mice compared with non-diabetic db/+ littermates (44). Immunohistochemical studies revealed that this stimulated expression was due to an increase in mesangial and endothelial staining for p27Kip1 (44). Primary cultures of mesangial cells from db/+ and db/db revealed a low p27Kip1 expression when cultured in normal glucose-containing medium (44). However, increasing the glucose concentration of the medium p27Kip1 expression induced in both cell lines and arrested the cells in the G1-phase. Glomerular p27Kip1 protein also increased in the murine streptozotocin type 1 diabetes (44). These data clearly indicate that the stimulated mesangial expression of p27Kip1 in db/db mice compared to normoglycemic littermates is due to high glucose and is not caused by additionally genetic effects (44). Moreover, ANG II also induces p27Kip1 expression in renal cells (60). This mechanism may additional contribute to p27Kip1 expression in diabetic nephropathy in vivo because the intrarenal renin-angiotensin system is activated during diabetes (45-46). We have obtained preliminary evidence that mitogen-activated protein (MAP)-kinases directly phosphorylates p27Kip1 and increases its stability. Since MAP kinases are activated in cultured mesangial cells exposed to high glucose and in glomeruli from diabetic rats (47-49), it is possible that phosphorylation of p27Kip1 partly contributes to the induced expression observed in the diabetic environment. To further investigate a functional role of p27Kip1 in high glucose-mediated mesangial hypertrophy, we compared the growth behavior of mesangial cells cultured from p27Kip1 +/+ and -/- mice. In contrast to p27Kip1 wild-type cells, high glucose did not increase hypertrophy in p27Kip1 -/- mesangial cells (50). Reconstitution of p27Kip1 expression in -/- cells using an inducible expression system lead to G1-phase arrest (50). However, mesangial hypertrophy was only observed when p27Kip1 was induced in high glucose suggesting that p27Kip1-mediated G1-phase arrest is a necessary prerequisite, but alone was not sufficient for the development of hypertrophy (50).
In accordance with this observation, Kuna, Al-Douahji, and Shankland studied the expression of p21Cip1 in experimental diabetic nephropathy (51). They found a progressive increase in mesangial cells that stained positive for p21Cip1 at day 3 and 9 after induction of diabetes with streptozotocin compared with normoglycemic control mice (51). This increase in p21Cip1 was associated with glomerular hypertrophy. Furthermore, cellular hypertrophy, induced in cultured mesangial cells exposed to high ambient glucose concentration, was also associated with an increase in p21Cip1 protein expression, whereas the levels of p57Kip2, another member of the CIP/KIP family of CKIs, did not change (52).
It is well known that angiotensin-converting enzyme (ACE) inhibitor treatment prevents glomerular hypertrophy in diabetes mellitus (53,54). We tested recently the effects of enalapril on glomerular expression of CKIs in BBdp rats, an autoimmune model of type I diabetes (55). Glomerular expression of p16INK4, p21Cip1, and p27Kip1 were all stimulated in BBdp rats compared with non-diabetic BBdr animals (55). Enalapril treatment for three weeks reduced the glomerular expression of p16INK4 and p27Kip1 but not of p21Cip1 (55).The ACE inhibitor treatment also prevented renal hypertrophy, but had, in the used dose, no effect on systolic blood pressure or glucose concentration (55). These data demonstrate that ACE inhibitor treatment attenuates glomerular hypertrophy in diabetes by interfering with the expression of selected CKI (55). Although it remains unclear whether these effects are due to ANG II itself or are caused by normalization of glomerular hemodynamics, these findings provide strong evidence that modulation of renal cell cycle events is feasible in diabetes mellitus.
An unifying cell cycle regulatory model for mesangial and tubular cell hypertrophy in diabetic nephropathy is proposed as follows. Cells subjected to the diabetic environment actively enter the G1-phase and may complete one or two mitoses. Then cells become growth arrested in late G1-phase and undergo hypertrophy. The induction of CKIs such as p21Cip1 and p27Kip1 is pivotal for this arrest. High glucose and other factors such as ANG II and AGEs induce TGF-b. TGF-b in turn stimulates the expression of p21Cip1 and p27Kip1 . Other CKIs (p16INK4, p57Kip2) may play additional roles. The high glucose-mediated induction of p27Kip1 is to some extent independent of TGF-b. High glucose activates MAP kinases which, in turn, phosphorylate and may further stabilize p27Kip1. Both CDK-inhibitors, likely in concert, mediate G1-phase arrest by binding to and inhibiting G1-phase cyclin/CDK complexes. Moreover, it has been shown that TGF-b leads to a downregulation of cyclin D as well as induction of p16INK4 with a potential liberation of p27Kip1 that could now bind to cyclin E and further reinforce the G1-phase arrest.
Address All Correspondence To: Gunter Wolf, M.D.
University of Hamburg, University Hospital Eppendorf Department of Medicine. Division of Nephrology and Osteology Pavilion 61. Martinistraße 52
D-20246 Hamburg. Germany
Address All Correspondence To:
Gunter Wolf, M.D.
University of Hamburg, University Hospital Eppendorf
Department of Medicine. Division of Nephrology and Osteology
Pavilion 61. Martinistraße 52
D-20246 Hamburg. Germany