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J Am Soc Nephrol 14:819-822, 2003
© 2003 American Society of Nephrology

EDITORIALS

p27Kip1: The "Rosebud" of Diabetic Nephropathy?

Gunter Wolf* and Stuart J. Shankland{dagger}

*Department of Medicine, Division of Nephrology and Osteology, University of Hamburg, Hamburg, Germany; and {dagger}Department of Medicine, Division of Nephrology, University of Washington, Seattle, Washington.

Correspondence to Dr. Stuart J. Shankland, Associate Professor of Medicine, Director, Nephrology Fellowship Program, Division of Nephrology, Box 356521, University of Washington Medical Center, 1959 NE Pacific Avenue, Seattle, WA 98195-6521. Phone: 206-543-3792; Fax: 206-685-8661; E-mail: stuartjs{at}u.washington.edu

"Rosebud" was the last enigmatic word of dying protagonist Charles Foster Kane in Orson Welles’ 1941 masterpiece Citizen Kane, considered by many cineastes as the best movie ever made. The search for its meaning is the film’s theme. The investigative reporter thinks that by discovering what or who "rosebud" was will possibly provide a simple secret to Kane’s mysterious, complex life. We are facing an anticipated epidemic of ends-tage renal disease in the next decade, which will largely be due to diabetic nephropathy. This reflects our inability to prevent and control the disease; therefore, anything that has promise to contribute to controlling diabetic nephropathy is a cause for considerable excitement.

What is p27Kip1, and is it the missing link in understanding the pathogenesis and potential treatment of diabetic nephropathy? This question is posed by Awazu et al. in this issue of JASN. In attempting to answer this question, we must first understand the complicated mechanisms of cell cycle regulation, which have been a major focus of many laboratories over the past two decades, culminating in awarding the Nobel prize for medicine in 2001 to Hartwell and Hunt. Depending on the cell type and underlying specific cell injury (1), renal cells respond by undergoing proliferation, apoptosis, de-differentiation, or hypertrophy (2,3).

Morphometric studies on renal specimens from patients with type 1 and 2 diabetes show characteristic glomerular hypertrophy, particularly of mesangial cells, but also of endothelial cells, early in diabetic nephropathy (1,4,5). Hypertrophy is biochemically defined as an increase in protein and RNA content, but without DNA replication (6). Morphometrically, this is accompanied by an increase in cell size. A more detailed analysis of the evolution of diabetic nephropathy that can only be performed in animal models shows a biphasic mesangial growth response, with early proliferation and subsequent hypertrophy (7). These growth responses to the diabetic environment are ultimately governed at the level of the nucleus (5), providing a rationale for the study of cell cycle regulatory proteins in diabetic nephropathy, as was undertaken by Awazu et al. in this issue of JASN (8).

It has been known for more than a century that the growth of a cell has two main phases: interphase and mitosis. Figure 1 shows that interphase is further divided into G1, S, and G2 phases (9). Nondividing cells enter the G1 phase from a quiescent G0 phase, pass through G1 into the S phase, where DNA replication takes place, and enter mitosis after progressing through G2.



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  		Figure 1. Phases of the cell cycle. Sequential transition through each phase of the cell cycle results in proliferation. In contrast, when cells engage G1 phase but arrest at G1/S, they undergo hypertrophy.

 
Once in G1, a cell can undergo three fates. First, the classic proliferation pathway requires that cells progress from G1, though S and M phases, followed by cytokinesis (cell division) (Figure 1). Second, upon G1 entry, cells can exit G1 or any phase of the cell cycle to undergo apoptosis. Third, and the focus of this editorial, is that after G1 entry, there is arrest at the G1/S transition, which prevents DNA synthesis. However, under these circumstances, because the protein content increases in G1 phase (in anticipation of the cell dividing into two daughter cells), a block at G1/S phase prevents DNA synthesis, resulting an increase in the protein:DNA ratio, which causes an increase in cell size (hypertrophy) (Figure 1).

Cell Cycle Regulatory Proteins Govern Cell Growth
What regulates the phases of the cell cycle, and how does this relate to diabetic nephropathy? Transitions between the different phases of the cell cycle are governed by positive (cyclins and cyclin-dependent kinases [CDK]) and negative (CDK-inhibitors) cell cycle regulatory proteins (Figure 2). Cyclins bind to and activate specific CDK in each phase of the cell cycle. In early G1, D-type cyclins (D1, 2, 3) associate with CDK4 and CDK6, and cyclin E associates with CDK2 in late G1. Cyclin A binds to CDK2 at the G1/S phase boundary, and it is essential for DNA synthesis. Finally, cyclins B1 and B2 form complexes with cdc2 (formerly CDK1) during mitosis. The activated heterodimer cyclin-CDK complexes exhibit kinase activity and in turn phosphorylate other target proteins that are necessary for cell cycle progression (10).



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  		Figure 2. Cell cycle regulatory proteins. Progression through each phase of the cell cycle requires that cyclins bind to and activate a partner cyclin dependent kinase (CDK). Specific CDK-inhibitors (p21, p27, p57) inactivate cyclin-CDK complexes in different phases of the cell cycle; in so doing, they prevent cell cycle progression.

 
Although cyclin-CDK may have a role in diabetic nephropathy, Awazu et al. (8) and other investigators, Terada, Safirstein, Wolf, Shankland, Preisig, and Mason (11–17), have focused on specific CDK-inhibitors in the pathogenesis of renal cell hypertrophy. The rationale for this is that, although there is cell cycle entry in diabetes, cell cycle progression is halted. CDK-inhibitors are relatively small molecules that bind to cyclin-CDK complexes, inhibit their activity, and thus halt cell cycle progression (Figure 2). Two main families of CDK-inhibitors exist (18): the INK4 family inhibit only D-type cyclin-CDK complexes in G1; the Cip/Kip (p21Cip1 and p27Kip1) family are more promiscuous and inhibit CDK2, CDK4, and CDK6 in most phases of the cell cycle. An increase in CDK-inhibitors arrests cells in the G1 phase of cell cycle, and a decrease in CDK-inhibitors is required for cycle progression and proliferation.

CDK-Inhibitors and Diabetic Nephropathy
Why study CDK-inhibitors in diabetic hypertrophy? The answer begins by examining the cell cycle kinetics during diabetic nephropathy. Cultured mesangial cells exposed to high glucose (450 mg/dl) undergo a biphasic growth response, being pro-proliferation early (within 24 h), followed by anti-proliferation due to G1 arrest (19). Studies have shown that G1 phase arrest is necessary for glucose-induced cellular hypertrophy and also for the increase in extracellular matrix proteins. Earlier studies focused on transforming growth factor-{beta} (TGF-{beta}) and showed that glucose-induced G1 phase arrest is mediated in part by the autocrine synthesis and activation of the antiproliferative and hypertrophic cytokine (19,20). However, there is a growing body of literature showing that specific CDK-inhibitors are also critical determinants of diabetic nephropathy (see below) and that these are both dependent and independent of TGF-{beta}.

Exposing cultured mesangial cells to high glucose increases the CDK-inhibitors, p21Cip and p27Kip1, but does not influence mRNA abundance (13). The glomerular (predominantly mesangial) expression of p21Cip and p27Kip1 also increases in experimental type 1 and 2 diabetic nephropathy (14, 21). Treatment with an angiotensin-converting enzyme (ACE) inhibitor reduces p27Kip1 expression and prevents renal hypertrophy of diabetic rats (22). The increase in p27Kip1 requires protein kinase C activation and is also partly dependent on the induction of TGF-{beta} (13). More recently, studies have shown that high glucose increases p27Kip1 protein expression through posttranscriptional mechanisms involving MAP kinases that directly phosphorylate this protein (23). Mason and colleagues showed that glucose-induced connective tissue growth factor increases expression of the CDK-inhibitors p15, p21Cip1, and p27Kip1 in mesangial cells undergoing hypertrophy (24). Taken together, these studies demonstrate that the levels of specific CDK-inhibitors, especially p27, increase in cultured cells exposed to high glucose.

What does the increase in p27Kip1 mean? The increase in p27Kip1 abundance due to high glucose concentrations causes this CDK-inhibitor to associate with and inhibit CDK2. This leads to cell cycle arrest at G1/S and prevents DNA synthesis (13). In contrast to wild-type mesangial cells, when p27Kip1knockout (-/-) mesangial cells are exposed to high glucose, they do not undergo G1 arrest (25). However, reconstituting p27Kip1 in p27 -/- cells inhibits cells at G1/S in the presence of high glucose (25). These in vitro results clearly demonstrate that p27Kip1 is required for glucose-induced cell cycle arrest.

A definitive and functional role for p27Kip1 in G1-phase arrest and glucose-induced hypertrophy comes from studies utilizing p27Kip1 -/- mesangial cells. In contrast to wildtype (+/+) mesangial cells, high glucose fails to induce hypertrophy in p27Kip1 -/- mesangial cells (25). However, reconstituting p27Kip1 in p27Kip1 -/- cells with an inducible vector system restored the hypertrophic phenotype induced by high glucose. These studies show that p27 is required for glucose-induced hypertrophy in cultured mesangial cells.

The role for CDK-inhibitor p27 in diabetic hypertrophy in vivo has been enigmatic until the current study by Awazu et al. in the current issue of JASN (8). The authors induced experimental type 1 diabetes in p27Kip1 +/+ and -/- mice by streptozotocin injection and showed that blood glucose and BP were comparable between diabetic p27Kip1 +/+ and -/- mice. However, in contrast to the increase in the kidney weight to body weight ratio, glomerular volume, and mesangial expansion in diabetic p27Kip1+/+ animals at 12 wk, these measures of hypertrophy did not increase in diabetic p27 -/- animals (8). Moreover, albuminuria only developed in diabetic p27Kip1 +/+ animals and was not detected in diabetic p27 -/- mice. Furthermore, despite a similar increase in glomerular TGF-{beta} expression in diabetic p27Kip1 +/+ and -/- mice, the glomerular protein expression of fibronectin increased only in diabetic p27Kip1 wild-type mice.

The studies described above by Awazu et al. (8) focused on established glomerular hypertrophy. Is there a role for the CDK-inhibitor p27 in early experimental diabetic hypertrophy? Studies have shown that at 4 wk after the development of hyperglycemia, glomerulosclerosis, tubulointerstitial sclerosis, and vascular sclerosis, indices were significantly less in diabetic p27Kip1 -/- mice compared with the diabetic wild-type (26). Most interestingly, diabetic heterozygotic p27Kip1+/- animals exhibited scores between those of p27Kip1 +/+ and -/- mice, suggesting that these animals are haplotype insufficient (26).

Is p27Kip1 the whole story, or do other CDK-inhibitors also have a role in mediating renal cell hypertrophy? Mice deficient for the CDK-inhibitor p21Cip1 are partially protected from diabetic glomerular hypertrophy (15), and the lack of p21 also attenuates glomerular hypertrophy after a significant reduction in renal mass (12). Recent studies have also shown that although TGF-{beta} inhibits proliferation in double p21Cip1/p27Kip1 -/- mesangial cells (16), the hypertrophic growth effects of TGF-{beta} were significantly reduced in the absence of both p21Cip1 and p27Kip1. This finding indicates that more than one CDK-inhibitor is likely pivotal in mediating the maximal hypertrophic response (16).

Taken together, there is now a growing literature showing that the CDK-inhibitor p27Kip1 has a critical role beyond simply regulating proliferation. The study by Awazu et al. (8) shows that p27Kip1 also regulates hypertrophy (8). Studies have also shown that p27Kip1 safeguards against apoptosis (27, 28). What research directions should we be focusing on in the future? These are numerous and include testing the possibility that targeted reductions of p27Kip1 could potentially protect patients from diabetic nephropathy. We need to understand why p27Kip1 increases predominantly in mesangial cells and not in other renal cell types, how p27Kip1 expression is regulated post-translationally in diabetes, and if p27Kip1 mediates other forms of renal hypertrophy.

We all know the sled named Rosebud that had been tossed into a furnace is not the answer for Kane’s life. It is rather the emblem of the security, hope, and innocence of childhood, which a man can spend his life seeking to regain. Similarly, knowing what p27Kip1 is does not necessarily explain how its control ameliorates diabetic nephropathy. It will certainly be a fascinating story to decipher the exact role of this cell cycle protein in the evolution of diabetic nephropathy and to determine how its manipulation can bring benefit to humans akin to those described in Awazu’s study in mice.

References

  1. Mogensen CE, Andersen MJ: Increased kidney size and glomerular filtration rate in early juvenile diabetes. Diabetes 22: 706–712, 1973[Medline]
  2. Shankland SJ, Wolf G: Cell cycle regulatory proteins in renal disease: Role in hypertrophy, proliferation, and apoptosis. Am J Physiol 278: F515–F529, 2000
  3. Ekholm SV, Reed SI: Regulation of G1 cyclin-dependent kinases in mammalian cell cycle. Curr Opin Cell Biol 12: 676–684, 2000[CrossRef][Medline]
  4. Osterby R, Gundersen HJ: Glomerular size and structure in diabetes mellitus. I. Early abnormalities. Diabetologia 11: 225–229, 1975[CrossRef][Medline]
  5. Wolf G, Ziyadeh FN: Molecular mechanisms of diabetic renal hypertrophy. Kidney Int 56: 393–405, 1999[CrossRef][Medline]
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  7. Young BA, Johnson RJ, Alpers CE, Eng E, Gordon K, Floege J, et al: Cellular events in the evolution of experimental diabetic nephropathy. Kidney Int 47: 935–944, 1995[Medline]
  8. Awazu M, Omori S, Ishikura K, Hida M, Fujita H: The lack of cyclin kinase inhibitor p27(Kip1) ameliorates progression of diabetic nephropathy. J Am Soc Nephrol 14: 699–708, 2003[Abstract/Free Full Text]
  9. Shankland SJ: Cell cycle control and renal disease. Kidney Int 52: 294–308, 1997[Medline]
  10. Lees E: Cyclin dependent kinase regulation. Curr Opin Cell Biol 7: 773–780, 1995[CrossRef][Medline]
  11. Terada Y, Inoshita S, Nakashima O, Tamamori M, Ito H, Kuwahara M, et al: Cell cycle inhibitors (p27(Kip1) and p21(Cip1)) cause hypertrophy in LLC-PK1 cells. Kidney Int 56: 494–501, 1999[CrossRef][Medline]
  12. Megyesi J, Price PM, Tamayo E, Safirstein RL. The lack of a functional p21WAF1/CIP1 gene ameliorates progression to chronic renal failure. Proc Natl Acad Sci USA 96: 10830–10835, 1999[Abstract/Free Full Text]
  13. Wolf G, Schroeder R, Ziyadeh FN, Thaiss F, Zahner G, Stahl RAK: High glucose stimulates expression of p27Kip1 in cultured mouse mesangial cells: relationship to hypertrophy. Am J Physiol 42: F348–F356, 1997
  14. Wolf G, Schroeder R, Thaiss F, Ziyadeh FN, Helmchen U, Stahl RAK: Glomerular expression of p27Kip1 in diabetic db/db mouse: Role of hyperglycemia. Kidney Int 53: 869–879, 1998[CrossRef][Medline]
  15. Al-Douahji M, Brugarolis J, Brown PAJ, Stehman-Breen CO, Alpers CE, Shankland SJ: The cyclin kinase inhibitor p21 WAF/CIP1 is required for glomerular hypertrophy in experimental diabetic nephropathy. Kidney Int 56: 1691–1699, 1999[CrossRef][Medline]
  16. Monkawa T, Hiromura K, Wolf G, Shankland SJ: The hypertrophic effect of transforming growth factor-beta is reduced in the absence of cyclin-dependent kinase-inhibitors p21 and p27. J Am Soc Nephrol 13: 1172–1178, 2002[Abstract/Free Full Text]
  17. Franch HA, Shay JW, Alpers RJ, Preisig PA: Involvement of pRB family in TGFbeta-dependent epithelial cell hypertrophy. J Cell Biol 129: 245–254, 1995[Abstract/Free Full Text]
  18. Sherr CJ, Roberts JM: CDK inhibitors: Positive and negative regulators of G 1-phase progression. Genes Dev 13: 1501–1512, 1999[Free Full Text]
  19. Wolf G, Sharma K, Chen Y, Ericksen M, Ziyadeh FN. High glucose-induced proliferation in mesangial cells is reversed by autocrine TGF-{beta}. Kidney Int 42: 647–656, 1992[Medline]
  20. Ziyadeh FN, Sharma K, Ericksen M, Wolf G: Stimulation of collagen gene expression and protein synthesis in murine mesangial cells by high glucose is mediated by autocrine activation of transforming growth factor-beta. J Clin Invest 93: 536–542, 1994
  21. Kuan CJ, Al-Douahji M, Shankland SJ. The cyclin kinase inhibitor p21WAF1, CIP1 is increased in experimental diabetic nephropathy: Potential role in glomerular hypertrophy. J Am Soc Nephrol 9: 986–993, 1998[Abstract]
  22. Wolf G, Wenzel U, Ziyadeh FN, Stahl RAK. Angiotensin converting-enzyme inhibitor treatment reduces glomerul p16INK4 and p27Kip1 expression in diabetic BBdp rats. Diabetologia 42: 1425–1432, 1999[CrossRef][Medline]
  23. Wolf G, Hannken T, Zahner G, Shankland SJ, Stahl RAK: p44/42 MAP kinase directly phosphorylates the CDK-inhibitor p27(Kip1) (p27): Role in hypertrophy [Abstract]. J Am Soc Nephrol 12: 623A, 2001
  24. Wahab NA, Weston BS, Roberts T, Mason RM: Connective tissue growth factor and regulation of the mesangial cell cycle: Role in cellular hypertrophy. J Am Soc Nephrol 13: 2437–2445, 2002[Abstract/Free Full Text]
  25. Wolf G, Schroeder R, Zahner G, Stahl RAK, Shankland SJ. High glucose-induced hypertrophy of mesangial cells requires p27 Kip1, an inhibitor of cyclin-dependent kinases. Am J Pathol 158: 1091–1100, 2001[Abstract/Free Full Text]
  26. Wolf G, Schanze A, Wenzel U, Shankland SJ, Stahl RAK, Amann K: p27(Kip1) (p27) knockout mice are protected of diabetic nephropathy [Abstract]. J Am Soc Nephrol 13: 164A, 2002
  27. Ophascharoensuk V, Fero ML, Hughes J, Roberts JM, Shankland SJ: The cyclin-dependent kinase inhibitor p27Kip1 safeguards against inflammatory injury. Nat Med 4: 575–580, 1998[CrossRef][Medline]
  28. Hiromura K, Pippin JW, Fero ML, Roberts JM, Shankland SJ: Modulation of apoptosis by the cyclin-dependent kinase inhibitor p27Kip1. J Clin Invest 103: 597–604, 1999[Medline]



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