The tight regulation of cell growth and division within an organ is essential for the development and maintenance of correct structure and function. Perturbations of renal growth occurring either developmentally or following injury to mature renal cells contribute to the abnormalities observed in a wide range of diseases. The changes in growth are increasingly recognized as an influence on the progression of the initial disease process, and the ultimate clinical outcome. Abnormal cell growth is classified according to the presence of an increase in cell number or cell size. Hyperplasia refers to abnormal growth resulting in an increased absolute number of cells, whereas hypertrophy refers to an increase in individual cell size. Both processes may be present in a given cell population and contribute to the increase in overall kidney size.
Introduction
The tight regulation of cell growth and division within an organ is essential for the development and maintenance of correct structure and function. Perturbations of renal growth occurring either developmentally or following injury to mature renal cells contribute to the abnormalities observed in a wide range of diseases. The changes in growth are increasingly recognized as an influence on the progression of the initial disease process, and the ultimate clinical outcome. Abnormal cell growth is classified according to the presence of an increase in cell number or cell size. Hyperplasia refers to abnormal growth resulting in an increased absolute number of cells, whereas hypertrophy refers to an increase in individual cell size. Both processes may be present in a given cell population and contribute to the increase in overall kidney size.
Of particular interest to clinical nephrologists and renal pathologists is the fact that the kidney has several different resident cell types. Within the glomerulus, the growth responses of the mesangial cell, podocyte, parietal epithelial cell, and endothelial cell differ. The tubulointerstitial cells and vascular smooth muscle cells also vary in their growth responses following injury. Thus, characterizing the mechanisms that regulate each cell’s growth response enables the potential development of specific therapies that will modify the response to injury. We recognize that renal cell hyperplasia and hypertrophy are regulated by numerous pathways, involving growth factors, signaling pathways, and transcription factors. However, the focus of this review is to update the reader on recent advances in the regulation of these growth processes at the level of the cell cycle. We will first describe cell cycle regulation by specific cell cycle proteins, and then discuss hyperplasia and hypertrophy for individual glomerular and tubular cell types.
Although highly metabolically active, under normal conditions the cells of the mature kidney are relatively quiescent with respect to cell cycle entry. Following injury to either the glomerulus or the tubules, cell cycle progression with proliferation is often an essential part of the reparative process. However, if unchecked, proliferation can lead to compromise of renal function. Similarly, renal hypertrophy may occur as a compensatory physiological response, but unregulated hypertrophy is maladaptive, and is one of the hallmarks of diabetic nephropathy.
Cell proliferation is ultimately regulated at the level of the cell cycle, which occurs within the nucleus. Within the kidney, the control of the cell cycle is particularly intriguing, given the contrasting responses of the various resident cell types to injury. For example, the mesangial cell is capable of marked proliferation, often accompanied by the deposition of extracellular matrix. In contrast, the podocyte has been considered a relatively inert cell, although this view has recently been challenged, and the reparative proliferation of glomerular endothelial cells following injury has also been described. Renal tubular cells readily undergo both proliferative and hypertrophic responses following injury. The last decade has seen a rapid expansion in our understanding of the molecular mechanisms underlying the cell cycle, and therapeutic options for its manipulation are becoming available. There is currently increasing awareness of the need to reduce the progression of renal diseases. Knowledge of the cell cycle and an understanding of how this can be influenced may be crucial to the prevention, control, and amelioration of a wide range of renal diseases.
Measurement of Cell Growth
Hyperplasia
During a hyperplastic response, the number of proliferating cells is increased. A number of methods are available for measuring this increase, both in vivo and in cell culture. The majority of these have as their basis the detection of increased DNA synthesis. This may be done by determining the presence of proteins known to be associated with DNA synthesis, such as proliferating cell nuclear antigen (PCNA) or Ki-67, or by exogenously labeling cells with a compound known to be incorporated into newly synthesized DNA, such as 3 H thymidine or bromodeoxyuridine (BrdU). In cell culture, a convenient and high-throughput method for determining cell number is the MTT assay, in which the yellow tetrazolium salt is reduced in metabolically active cells to form insoluble formazan crystals, which are solubilized by the addition of detergent. The color intensity may then be quantified spectrophotometrically, allowing quantification of changes in proliferation. A caveat for this method is that a decrease in cell viability will mimic a decrease in proliferation, and concomitant apoptosis should be excluded. Analysis by fluorescent activated cell sorting (FACS) is a valuable tool for the assessment of hyperplasia, because it also allows quantification of the number of cells in each phase of the cell cycle.
Hypertrophy
Cellular hypertrophy may be defined as an increase in cell size due to an increase in protein and RNA content without DNA replication, and this forms the basis for the majority of methods for detection of hypertrophy. Upon entry into G1, cells undergo a physiologic increase in protein synthesis prior to the DNA synthesis of S-phase. Thus, one mechanism underlying hypertrophy is cell cycle arrest at the G1/S checkpoint, so that while protein synthesis and hence content, increase, there is no subsequent increase in DNA. Hypertrophy may also occur independently of the cell cycle, due to an inhibition of protein synthesis, and this mechanism is considered to contribute to tubular cell hypertrophy. Measurement of leucine or proline incorporation and comparison to 3 H thymidine incorporation allow determination of cell protein/DNA content, and hence assessment of hypertrophy. FACS analysis is also useful and enables direct measurement of cell size. Defining the growth response to a given stimulus as either hyperplastic or hypertrophic is important, as each will result from different alterations in cell signaling pathways, with implications for possible interventions.
Cell Cycle and Cell Cycle Regulatory Proteins
Cell Cycle
The cell cycle is divided into distinct phases, each representing a different function, and each being regulated by specific proteins ( Figure 28.1 ). Quiescent cells are termed as in G0, and upon mitogenic stimuli enter the cell cycle at early G1. Cells pass through the restriction point in late G1, beyond which they are typically unresponsive to extracellular cues, and are committed to complete the cell cycle despite the withdrawal of mitogenic stimuli. DNA synthesis occurs in S-phase. Cells then progress through G2, in preparation for mitosis (M-phase). Ultimately, cell division follows during cytokinesis. Our current understanding suggests there are at least two checkpoints to ensure fidelity of DNA duplication, at G1/S and G2/M, where cell cycle progression may be arrested. The length of the cell cycle is cell-type-specific, but this variability is largely due to differences in the duration of G1. For mammalian cells, the typical duration of G1 is approximately 12 hours, S- and G2-phases 6 hours, and mitosis 30 minutes.
Cyclins and Cyclin-Dependent Kinases: Positive Regulators of the Cell Cycle
Overview
The progress of a somatic cell through the cell cycle is dependent on the sequential and coordinated activation of the cyclin-dependent kinases (Cdks) by their specific partners, called cyclins ( Figure 28.2 ). Cdks belong to the family of proline-directed serine/threonine kinases with a specific (K/R) (S*/T*) PX (K/H/R)-phosphorylation motif. Once active, Cdks phosphorylate downstream targets, ultimately to induce DNA synthesis. While the levels of the Cdk catalytic subunits remain constant throughout the cell cycle, they are only functional following the binding of their specific cyclin partners. In contrast, cyclins are unstable proteins that are sequentially expressed and subsequently degraded by ubiquitination throughout the cell cycle, which activate their partner Cdks by inducing conformational changes. Originally described for their fluctuation during the cell cycle, members of the cyclin family are now defined by the presence of a conserved 100 amino acid cyclin box, which binds their complementary Cdk. In addition, the binding of inhibitors and accessory proteins, subcellular localization, and both inhibitory and activating phosphorylations influence the functional activity of the Cdk–cyclin complex.
Jumpstarting the Cycle
The cell cycle is initiated by the mitogen-driven induction of cyclin D. Depending on the cell type, three forms of cyclin D have been described (D1, D2, and D3), which interact allosterically with Cdk4 and Cdk6. Receptor-activated Ras signaling pathways lead to accumulation of cyclin D by three mechanisms: gene transcription; assembly; and stabilization of the cyclin D–Cdk complex. The Ras-Raf-1-mitogen-activated, protein kinase kinase (MEK), extracellular signal-related protein kinase (ERK) pathway both induces cyclin D transcription and promotes assembly of cyclin D–Cdk. The rate of degradation of cyclin D is controlled by a separate Ras signaling pathway involving phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB/Akt), which inhibits the phosphorylation of cyclin D on threonine-286 (Thr-286) by glycogen synthase kinase 3β (GSKβ). Thr-286 phosphorylated cyclin D would otherwise be exported to the cytoplasm for ubiquitination and degradation. This requirement for mitogen signaling prevents the cell from autonomous cycling. Although ectopic expression of cyclin D is insufficient to drive cell cycle progression, constitutive activation of the cyclin D pathway can reduce the reliance of the cell on mitogenic stimulation, and lower the threshold for oncogenic transformation. The cyclin D–Cdk4/6 complex enters the cell nucleus and is phosphorylated by Cdk-activating kinase (CAK).
Once DNA replication begins, active cyclin D-dependent kinase activity is not required until mitosis is complete, and the cell re-enters the next G1 phase. In continuously dividing cells, cyclin D1 is exported to the cytoplasm during S-phase, and its turnover is accelerated. However, cyclin D1 synthesis stimulated by Ras is stabilized in G2 as described above, allowing reaccumulation before cells divide. Hence, in the presence of continuous mitogen stimulation, the second and subsequent cell cycles are shorter than the first. Withdrawal of mitogens results in a rapid decline in cyclin D kinase activity, and cell cycle exit.
Active cyclin D-dependent kinases phosphorylate the retinoblastoma protein (pRb), which in quiescent cells has a growth-inhibitory effect. In its hypophosphorylated state, pRb suppresses the transcription of several genes whose proteins are required for DNA synthesis, including the E2F transcription factors. Upon phosphorylation of pRb, the E2Fs are released from inhibition, leading to the transcription of cyclins E and A, and many genes whose products are required for DNA replication. Furthermore, cyclin D–Cdk4 complexes also phosphorylate Smad3, negatively regulating the functions of transcriptional proteins responsible for mediating the growth inhibitory effects of transforming growth factor β (TGFβ). Cyclin D-dependent kinases therefore affect the activity of at least two pathways that independently inhibit the expression of cell cycle promoting genes.
The activity of cyclin E–Cdk2 is maximal at the G1- to S-phase transition, when its function to further phosphorylate pRb releases the cell from mitogen dependency. In addition to preferentially phosphorylating pRb on different sites to the cyclin D-dependent kinases, which may modify the interaction with E2Fs, cyclin E–Cdk2 phosphorylates a second set of substrates involved in cell replication, thus affecting histone gene expression, and centrosome duplication. The timing of expression and wider range of substrates suggest a role for cyclin E–Cdk2 in coordinating G1 regulation and the core cell cycle machinery.
The abrupt decline in cyclin E–Cdk2 activity in early S-phase results from cyclin E degradation. Phosphorylation by GSK-3β and Cdk2 itself target cyclin E for ubiquitination by the SCF Fbw7 E3 ligase, leading to proteasomal destruction.
Low levels of cyclin A–Cdk2 activity are first detected in late G1-phase, increase as cells begin to replicate their DNA, and decline as cyclin A is degraded in early mitosis. The substrate specificity of cyclin A–Cdk2 is different from that of cyclin E–Cdk2. In S-phase, cyclin A–Cdk2 is thought to phosphorylate substrates that control the start of DNA replication from preassembled replication initiation complexes, and control the integration of the end of S-phase with the activation of the mitotic Cdks. The apparently central role of Cdk2 in coordinating cell cycle progression through S-phase and entry into mitosis has been challenged by the surprising observation that Cdk2 null mice are viable. The possibility that other Cdks compensate for the loss of Cdk2 is currently a focus of intense research.
The entry to mitosis is controlled by cyclin B–Cdc2. Cell cycle-regulated transcription of cyclin B begins at the end of S-phase. Phosphorylation on Thr161 by CAK parallels cyclin B binding to Cdc2. During G2, cyclin B–Cdc2 complexes are maintained in an inactive state by phosphorylation on two inhibitory sites, Thr14 and tyrosine 15 (Tyr15) ( Figure 28.3 ). Phosphorylation on Tyr15 is mediated by the nuclear Wee1 kinases, and that on Thr14 by the membrane-bound Myt1. In late G2 phase, both Thr14 and Tyr15 are dephosphorylated by Cdc25, thus activating cyclin B–Cdc2, and initiating mitosis. Inappropriate triggering of mitosis is also prevented by the translocation of cyclin B to the cytoplasm by the nuclear export factor CRM1 (exportin 1) during S- and G2-phases. Phosphorylation of cyclin B is thought to promote nuclear import at the G2/M transition. Cyclin B–Cdc2 phosphorylates numerous downstream targets responsible for the structural reorganization of the cell to enable mitosis.
Although what is described above represents the basic paradigm of the control of cell cycle progression in mammalian cells, recent studies of knockout mice have demonstrated that much fetal development can occur normally despite the absence of cyclins and Cdks formerly considered to be vital. Clearly, individual cyclins and Cdks are able to act more promiscuously than previously appreciated to enable compensation for the lack of a specific cell cycle protein.
Stopping the Cell Cycle: Cdk Inhibitors Act as Negative Regulators
In essence, Cdk inhibitors bind and inhibit target cyclin–Cdk complexes. Two classes of Cdk inhibitors have been described, the INK4 proteins and the Cip/Kip family . Within each family, individual proteins are named according to their molecular weight. INK4 proteins were originally named for their ability to in hibit Cd k4 . This family comprises four proteins, namely p16 INK4a , p15 INK4b , p18 INK4c , and p19 INK4d . Structurally these proteins are made up of multiple ankyrin repeats, and bind only to the catalytic subunits Cdk4 and Cdk6, thus inhibiting G1 progression. An alternate reading frame of the genetic locus encoding p16 INK4a also encodes a second structurally and functionally unrelated protein named p19 ARF in the mouse (p14 ARF in the human). Whereas p16 INK4a acts to stabilize Rb by inhibition of Cdk4/6, p19 ARF stabilizes p53 by binding its negative regulator, Mdm2. Data from knockout mice suggest that p19 ARF , rather than p16 INK4a , is responsible for the tumor suppressor function of this locus.
The second class of Cdk inhibitors is the Cip/Kip family, which includes p21 Cip1 , p27 Kip1 , and p57 Kip2 , which share a conserved N-terminal Cdk-binding domain. They are capable of binding a wider range of targets, and can variably affect the activities of cyclin D-, E-, A-, and B-dependent kinases. Although potent inhibitors of cyclin E- and A-dependent CDK2, and to a lesser extent Cdc2, the Cip/Kip proteins have recently also been characterized, paradoxically, as positive regulators of the cyclin D-dependent kinases.
The first member of the family to be identified was p21 Cip1 , and it is usually present at a low level in quiescent cells. As the cell enters the replicative cycle, p21 Cip1 levels rise, displace INK4 proteins from binding to Cdk 4/6, and promote the assembly of cyclin D-Cdk complexes. This stabilizes the active complex and additionally provides a nuclear localization signal (NLS). The transcription of p21 Cip1 is increased by both p53-dependent and -independent pathways, such as those mediated by TGFβ. The inhibitory role of p21 Cip1 becomes dominant later in the cell cycle, and levels are also increased in senescent cells.
In contrast to p21 Cip1 , the level of p27 Kip1 is usually high in quiescent cells, where its primary role is as an inhibitor of cell division. Whereas p21 Cip1 is a principal mediator of the p53-dependent G1 arrest that occurs following DNA damage, p27 Kip1 appears to be primarily responsible for mediating extracellular anti-proliferative signals. The levels and activity of p27 Kip1 are post-transcriptionally regulated by changes in the rates of translation, ubiquitination, and phosphorylation. As cyclin D levels rise in response to mitogens, both p21 Cip1 and p27 Kip1 are sequestered by cyclin D–Cdk complexes, and therefore are unable to inhibit Cdk2. Cyclin E–Cdk2 phosphorylates p27 Kip1 on Thr 187, proving a recognition motif for an E3 ligase that targets p27 Kip1 for ubiquitination and proteasomal degradation.
The most recently identified member of the family, p57 Kip2 , was cloned in 1995. While tissue expression of p21 Cip1 and p27 Kip1 is widespread, that of p57 Kip2 is restricted to placenta, muscle, heart, brain, lung, and kidney. In addition to the Cdk inhibitory domain and putative C terminal NLS, p57 Kip2 also has a proline-rich domain containing a consensus ERK phosphorylation site, and an acidic domain, the functions of which are not known. A role for p57 Kip2 in the cell cycle exit that accompanies terminal differentiation has been suggested.
Despite their structural similarities, knockout studies have demonstrated divergent roles for the three Cip/Kip Cdk inhibitors. While p21 Cip1 and p27 Kip1 are not essential for normal embryogenesis, lack of p57 Kip2 results in profound developmental abnormalities. Most p57 Kip2 null mice die shortly after birth and have severe cleft palates, abdominal wall and gastrointestinal tract defects, and abnormal skeletal ossification. Unlike adult p21 Cip1−/− mice, p27 Kip1−/− mice are larger than wild-type animals, and have hyperplasia of organs that usually express high levels of p27 Kip1 , such as the thymus, spleen, adrenal and pituitary glands, testes, and ovaries. In contrast, only 10% of p57 Kip2−/− mice survive the weaning period and are much smaller than wild-type. The kidneys of p57 Kip2−/− mice have medullary dysplasia, although glomerular development appears normal.
Hyperplasia: An Increase in Cell Number Due to Proliferation
Glomerular Hyperplasia
Mesangial Cell Proliferation
Mesangial cell proliferation characterizes many forms of both experimental and human glomerular disease, including IgA nephropathy, lupus nephritis, diabetic nephropathy, and other forms of membranoproliferative glomerulonephritis ( Figure 28.4 ). It is frequently associated with, and likely underlies, matrix expansion and subsequent glomerulosclerosis, the significance of which has been shown in a range of experimental models. This simple observation provides the impetus for understanding what switches mesangial proliferation on and what switches it off. Several growth factors and cytokines are mitogens for mesangial cells, including platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), interleukin 6, and the product of growth arrest-specific gene 6 (Gas6). Intervention to reduce mesangial proliferation also reduces matrix expansion, confirming the tight link between these two processes. This has been achieved in experimental models using complement depletion, heparin infusion, blocking the action PDGF and bFGF, and inhibiting their specific intracellular signaling pathways with phosphodiesterase inhibitors. Warfarin has been used with mixed results in the treatment of glomerular diseases since the 1970s, and was originally hypothesized to reduce fibrin deposition. However, low-dose warfarin may also be effective, suggesting a mechanism of action not directly related to anticoagulation. Gas6 is a vitamin K-dependent growth factor for mesangial cells, and its inhibition by warfarin is likely to underlie the reported benefits of this treatment in human disease. Careful research since the mid-1990s has delineated the role of individual cell cycle proteins in mesangial cell proliferation, and also its resolution by apoptosis ( Figure 28.5 ).
Role of CDK2 in Mesangial Cell Proliferation
Cdk2 protein and kinase activity increase in cultured mesangial cells in response to mitogenic growth factors. The Thy1 model of experimental mesangial proliferative glomerulonephritis, induced in rats by an antibody directed against the mesangial Thy1 antigen, has provided an opportunity to study the regulation and consequences of mesangial cell proliferation in vivo . The initial complement-dependent mesangiolysis is followed by a phase of marked mesangial proliferation, paralleled by an increase in extracellular matrix accumulation and a decline in renal function. This model is useful as not only may the fluctuations of cell cycle proteins during proliferation be defined, but also the effect of their manipulation. Mesangial cell proliferation is associated with an increase in cyclin D1 and A, and their partners Cdk4 and Cdk2. Cdk2 expression is absent in the normal rat glomerulus. Proliferation is associated with increased Cdk2 activity, measured by the histone H1 kinase assay on protein extracted from isolated glomeruli. Bokemeyer et al. identified activation of the map kinase ERK as an upstream regulator of Cdk2 activity in the Thy1 model. Inhibition of ERK was associated with decreased cell proliferation by 67%. Cdk2 protein levels are also increased in the remnant kidney model, a nonimmune glomerular disease associated with mesangial proliferation. Taken together, these studies show that in contrast to most nonrenal cells, Cdk2 protein is at low levels in quiescent mesangial cells, and its levels and activity increase following injury.
Cdk Inhibitors and Mesangial Cell Proliferation
The Cdk inhibitor p27 Kip1 is constitutively expressed in quiescent mesangial cells both in vitro and in vivo , whereas p21 Cip1 and p57 Kip2 are essentially absent. In cultured mesangial cells, proliferation induced by mitogenic growth factors reduces p27 Kip1 levels. Mesangial cells derived from p27 Kip1−/− mice have augmented proliferation in response to mitogens, and lowering p27 Kip1 levels with antisense oligonucleotides has a similar effect in rat mesangial cells.
Complement-induced injury in the Thy1 model is associated with a marked decrease in p27 Kip1 levels. However, there is de novo synthesis of p21 Cip1 in the resolution phase of the disease, coincident with a decrease in proliferation. To further explore the role of p27 Kip1 in inflammatory disease, we induced experimental glomerulonephritis in p27 Kip1−/− mice. Our results showed a marked increase in the onset and magnitude of glomerular cell proliferation and cellularity in nephritic p27 Kip1−/− mice compared to control nephritic p27 Kip1+/+ mice. Moreover, this was associated with increased extracellular matrix proteins and a decline in renal function. To demonstrate that this result was not specific to glomerular cells or immune-mediated injury, we also obstructed a ureter by ligation to induce nonimmune injury to tubuloepithelial cells. Our results showed that tubuloepithelial proliferation was increased in obstructed p27 Kip1−/− mice compared to obstructed p27 Kip1+/+ mice. Taken together, these studies were the first to show that in inflammatory diseases, renal cell proliferation is regulated by the CKI p27 Kip1 , supporting a role for p27 Kip1 in controlling the threshold at which proliferation occurs.
Little is known about the role of the Cdk inhibtiors p21 Cip1 and p57 Kip2 . In an immune-mediated model of MC disease, the absence p21 Cip1 was associated with increased focal segmental tuft necrosis, mesangiolysis, and mesangial hypercellularity.
Role of Cell Cycle Proteins in Resolution of Mesangial Hyperplasia: Apoptosis
Although a characteristic response to mesangial cell injury is proliferation, apoptosis is often simultaneously increased. Studies have shown that apoptosis is a vital mechanism required to normalize cell number in the reparative phase of injury. However, the cellular pathways linking these opposing responses remain unclear. Many cells undergoing apoptosis have entered the cell cycle, but rather than completing their replication, they are destined to leave by programmed cell death. This suggests a role for the cell cycle proteins in directing these alternative outcomes.
Evidence to support this hypothesis was the observation that the resolution phase of Thy1 mesangial proliferation in the rat is characterized by mesangial cell apoptosis, a process that peaks when the levels of p27 Kip1 are at their lowest. Considering glomerulonephritis or unilateral ureteric obstruction in p27 Kip1−/− mice as described above, in addition to the increase in either glomerular or tubuloepithelial cell proliferation following injury, there was a marked increase in apoptosis in the p27 Kip1−/− mice compared to wild-type disease controls. Moreover, apoptosis was also increased in p27 Kip1−/− mesangial cells in culture following growth factor deprivation or cycloheximide, and reconstituting p27 Kip1 levels by transfection-rescued cells from apoptosis. In wild-type rat mesangial cells, apoptosis was increased following treatment with anti-p27 Kip1 antisense oligonucleotides. These results showed for the first time that p27 Kip1 has a role beyond the regulation of proliferation, in that it also protects cells from apoptosis. This dual role of regulating the proliferative threshold and governing apoptosis makes p27 Kip1 a potent regulator of overall mesangial cell numbers. In contrast, p21 Cip1 showed no effect on MC apoptosis.
How does P27 KIP1 Protect Cells from Apoptosis?
A clue to a possible mechanism was the increase in Cdk2 activity in p27 Kip1−/− mesangial cells when deprived of growth factors. The increase was due specifically to cyclin A–Cdk2, and not cyclin E–Cdk2. Moreover, inhibition of cyclin A–Cdk2 activity by roscovitine or a dominant negative mutant reduced apoptosis in mesangial cells and fibroblasts. In apoptotic p27 Kip1−/− mesangial cells, Cdk2 was bound to cyclin A, without a preceding increase in cyclin E–Cdk2 activity. We suggest that, in the absence of p27 Kip1 , uncoupling of Cdk2 activity from the scheduled sequence of cell cycle protein expression may lead to an inappropriate and premature initiation of G 1 /S-phase transition, causing the cell to respond by undergoing apoptosis, rather than inappropriately progressing through an unscheduled cell cycle.
How Might Cdk2 Control Growth and Apoptotic Fate of Cells?
Apoptosis typically begins in the cytoplasm, whereas DNA synthesis and mitosis are nuclear events. Accordingly, we tested the hypothesis that the subcellular localization of Cdk2 determines if cells undergo apoptosis or proliferation. As expected, Cdk2 protein was cytoplasmic in quiescent, and nuclear in proliferating, mesangial cells. However, in proliferating cells injured by an apoptotic stimulus, Cdk2 localized to the cytoplasm, was no longer nuclear, and importantly, remained active. Our results also showed that cyclin A, and not cyclin E, co-localized to the cytoplasm with Cdk2 in apoptotic cells, to form an active cytoplasmic cyclin A–Cdk2 complex. The translocation of Cdk2 is not p53-dependent, and inhibiting the nuclear localization signal has no effect. That inhibiting Cdk2 decreased apoptosis provides further support for a critical role for cytoplasmic Cdk2 in triggering programmed cell death. Thus, the subcellular localization of active Cdk2 determines the fate of a cell: when nuclear, cells proliferate; when cytoplasmic, cells die by apoptosis. The mechanism by which nuclear Cdk2 is translocated to the cytoplasm remains to be elucidated. These studies provide the novel paradigm that specific cell cycle regulatory proteins have a role in glomerular disease beyond the regulation of proliferation.
Therapeutic Inhibition of Mesangial Proliferation at Cell Cycle Level
In vitro and animal studies have recently revealed the potential of several novel therapies to modulate glomerular cell proliferation: the purine analog roscovitine, which inhibits Cdk2 activity; retinoids, derived from vitamin A; and lipoxins, endogenously produced eicosanoids. Roscovitine and retinoids have also been used to beneficial effect in the treatment of podocyte diseases, discussed in the next sections.
Roscovitine
The significance of increased Cdk2 activity in mesangial proliferation was demonstrated by Pippin et al., using roscovitine to inhibit Cdk2 in rats with Thy1 mesangioproliferative gomerulonephritis. Given immediately after disease induction, roscovitine significantly reduced mesangial cell proliferation. Moreover, administering roscovitine to rats once mesangial proliferation was already established also reduced proliferation. This inhibition of Cdk2 activity was accompanied by a marked reduction in the accumulation of glomerular extracellular matrix proteins (collagen IV, laminin, and fibronectin), and an improvement in renal function compared to controls. These results suggest that inhibiting Cdk2 may be a potential therapeutic target in glomerular diseases characterized by proliferation.
Retinoids
Retinoic acid (RA) has an established role in kidney development. RA binds to specific nuclear receptors, and the RA receptor complex then binds to DNA–RA response elements to cause the transcription of target genes. RA is used therapeutically in acute promyelocytic leukemia to slow proliferation and promote differentiation. RA-induced cell cycle arrest in nonrenal cells has been reported to involve reduction in c-Myc, cyclin D1, and cyclin E levels, with upregulation of p21 Cip1 and p27 Kip1 . The treatment of rats with experimental Thy1 glomerulonephritis with RA reduced mesangial cell proliferation, glomerular lesions, and albuminuria. In addition to a direct antiproliferative action, RA has also been reported to modulate both the renin–angiotensin system and TGFβ signaling, in addition to anti-inflammatory and immune modulatory effects. The efficacy of RA in the treatment of glomerulonephritis is likely due to its pleiotropic effects on these numerous pathways.
Lipoxins
Lipoxins are endogenously produced eicosanoids with potent anti-inflammatory actions, and are generated during the resolution phase of an acute inflammatory insult. Lipoxin A 4 biosynthesis has been demonstrated in glomerulonephritis, and its effects include modulation of leukocyte trafficking and phagocytic clearance of apoptotic cells. In vitro , lipoxin A4 inhibits PDGF-induced activation of Akt/PKB in human mesangial cells, and modulates PDGF-induced decrements of p21 Cip1 and p27 Kip1 . PDGF-induced increases in Cdk2–cyclin E complex formation are also inhibited by lipoxin A 4 . Prolonged exposure of mesangial cells to PDGF is associated with autocrine TGFβ production, and this is ameliorated by lipoxin A 4 .
In vivo , lipoxins are rapidly metabolized. To enable study of these compounds in disease models, stable synthetic analogs have been developed that are modified at C-15, C-16, and/or C20. These compounds retain the biological activity and receptor-binding affinity of the native lipoxin. The effect of a lipoxin A 4 analog, 15-epi-16-(FPho)-LXA-Me, has been studied in the immediate phase of experimental anti-GBM nephritis in mice, and found to inhibit neutrophil infiltration and glomerular nitrotyrosine staining. Although animal studies with lipoxins are at an earlier stage than those with roscovitine or retinoids, their potential to augment the resolution phase of glomerulonephritis suggests that they may in the future have an important therapeutic role.
The Podocyte and Cell Cycle: Why Is Lack of Podocyte Proliferation Important?
The podocyte, or visceral glomerular epithelial cell, is a highly specialized, terminally differentiated cell overlying the outer aspect of the glomerular basement membrane. In contrast to the mesangial cell, numerous studies of both animal models and human disease have shown that aside from a few specific conditions, podocytes do not typically proliferate in vivo ( Figure 28.6 ). Indeed, following injury, podocyte numbers may become depleted, because following cell loss by detachment or apoptosis, the lack of proliferation prevents normalization of podocyte number. Although initially the remaining podocytes may undergo a degree of compensatory hypertrophy, the decrease in podocyte number will eventually result in areas of “denuded” basement membrane, which is thought to predispose to the formation of synechiae between the GBM and Bowman’s capsule, leading to the development of secondary focal glomerulosclerosis and subsequent decline in renal function. Podocytes provide a size- and charge-dependent barrier to protein leakage into the urine, and are therefore a critical component of the glomerular filtration apparatus. Several studies in diverse renal diseases have shown a close correlation between the onset and progression of proteinuria and reduced podocyte number. These events provide a compelling rationale to define the mechanisms underlying the lack of podocyte proliferation.
Mature Podocyte has Exited Cell Cycle and is Postmitotic
During glomerulogenesis, immature podocytes are capable of proliferation. However, during the critical S-shaped body stage of glomerular development, podocytes exit the cell cycle in order to become terminally differentiated and quiescent, which are necessary requirements for normal function. Thus, in podocytes, proliferation and differentiation are closely linked, akin to neurons and cardiac myocytes. In both mouse and human glomerulogenesis, immunostaining for p27 Kip1 and p57 Kip2 is absent in proliferating podocytes during the S-shaped body stage. On cessation of proliferation, there is strong expression of both Cdk inhibitors, so that p27 Kip1 and p57 Kip2 are constitutively expressed in mature podocytes. The Cdk inhibitors p21 Cip1 , p27 Kip1 , and p57 Kip2 alone are not required for normal glomerular development because, as described previously, the glomeruli from null mice are histologically normal. However, functional redundancy of p27 Kip1 and p57 Kip2 , at least within the podocyte, has been suggested by studies of E13.5 embryonic metanephroi from double p27 Kip1 /p57 Kip2−/− mice. Glomeruli from these mice have been reported to be significantly larger than those from wild-type or single mutants, due to an increase in podocyte number. Differentiation of podocytes was judged to be normal by electron microscopy and immunostaining for WT-1, suggesting a synergistic role for p27 Kip1 and p57 Kip2 in determining the final complement of podocytes.
Resistance of Podocytes to Proliferation: Role of Cell Cycle Regulatory Proteins
Studies have shown that the frequently observed lack of podocyte proliferation may be due to abnormalities in DNA synthesis or mitosis and cytokinesis ( Figure 28.7 ).
The passive Heymann nephritis (PHN) model, induced by the administration of an antibody reactive against the Fx1A antigen on the rat podocyte, has many similarities to human membranous nephropathy. In common with the Thy1 model of mesangial proliferative glomerulonephritis, PHN is complement (C5b-9)-dependent. However, in contrast to the observed mesangial cell proliferation in response to complement-mediated injury, there is no increase in podocyte number. Mitotic figures and an increase in ploidy are seen in the acute phase of disease, but over time the number of podocytes decreases. The comparison of patterns of expression of cell cycle proteins between the PHN and Thy-1 disease models has provided an opportunity to elucidate the role of cell cycle proteins in experimental podocyte disease.
Following C5b-9-induced injury in PHN rats, protein levels for cyclin A and Cdk2 rise, indicating engagement of the cell cycle. However, only a limited increase in DNA synthesis occurs, suggesting the presence of an inhibitor to cell cycle progression. Indeed, the levels of the Cdk inhibitors p21 Cip1 and p27 Kip1 increase specifically in podocytes following the induction of PHN. The increase in p21 Cip1 is attenuated by administering the mitogen bFGF to PHN rats, and this augments the increase in podocyte DNA synthesis and ploidy. Furthermore, upregulation of M-phase cell cycle proteins Cdc2 and cyclin B is also observed in PHN podocytes, suggesting that a disturbance in cytokinesis is ultimately responsible for the development of polynucleated cells and lack of podocyte proliferation in this experimental glomerular disease.
Cell culture studies have further explored the inability of podocytes to proliferate following C5b-9-induced injury, and support the hypothesis of a defect in completing mitosis. When cultured podocytes are exposed to sublytic C5b-9 attack, the cells engage the cell cycle. However, there is a delay or inhibition in entering mitosis, suggesting a block in the G 2 /M transition, and involvement of mechanisms that regulate this checkpoint. This response is typical of that following DNA damage, to which podocytes appear to be particularly susceptible. The occurrence of DNA damage following exposure to sublytic C5b-9 has subsequently been confirmed, together with increased checkpoint kinase-1 and -2 protein levels, thus arresting cells at G 2 /M. The mechanism by which DNA damage occurs in podocytes is not currently well-understood, but is thought to involve the generation of reactive oxygen radicals.
A key role for p21 Cip1 in limiting the proliferative response of podocytes has been demonstrated in studies using p21 Cip1 knockout mice. The administration of an antiglomerular antibody to induce experimental podocyte injury caused podocyte dedifferentiation and proliferation in p21 Cip1−/− mice compared to control mice receiving the same antibody. Glomerular extracellular matrix accumulation was also increased in p21 Cip1−/− mice, and was associated with a significant decrease in renal function. Additional in vitro data could link the pro-apoptotic effect of TGF-beta1 in podocytes to p21 Cip1 . Podocyte apoptosis induced by TGF-beta1 required sufficient p21 Cip1 levels.
Intravenous application of the podocyte toxin Adriamycin resembles the histological findings of focal segemental glomerulosclerosis (FSGS) in mice. Diseased p21 Cip1−/− mice presented with increased podocyte apotosis and decreased podocyte number, leading to aggravated glomerular disease including worse albuminuria, glomerulosclerosis, and increased BUN compared to control animals.
The resistance of podocytes to proliferation in the majority of animal models has been confirmed in human diseases, and similar underlying mechanisms have been observed. Normal quiescent podocytes express p27 Kip1 and p57 Kip2 , and immunostaining for these proteins is maintained in conditions without proliferation, namely minimal change diseases and membranous glomerulopathy. In contrast, expression of both these proteins is uniformly decreased in diseases characterized by podocyte proliferation, that is, cellular FSGS, collapsing glomerulopathy, and HIV-associated nephropathy. This is accompanied by the de novo expression of p21 Cip1 . The mechanisms by which the podocyte eludes the usual constraints on proliferation are discussed below.
Resistance of Podocytes to Proliferation: Role of Mechanical Stretch
An additional factor reported to influence the proliferative capacity of the podocyte is the presence of mechanical stress. Independent of the site of initial injury, a common pathway to progressive glomerulosclerosis is an increase in intraglomerular pressure, also known as glomerular hypertension. Lowering intraglomerular pressure reduces progression in a number of glomerular diseases, including diabetic nephropathy. Glomerular hypertension is associated with glomerular hypertrophy (see below), and the resultant mechanical stretch causes injury to all three glomerular cell types. Whereas applying mechanical stretch to cultured mesangial cells increases proliferation, the opposite response is seen in cultured podocytes. Stretching mouse podocytes in culture decreased the levels of cyclins D1, A, B1, and Cdc2, in association with an increase in the Cdk inhibitors. Stretch caused an early increase in p21 Cip1 , followed by an increase in p27 Kip1 at 24 hours and p57 Kip2 at 72 hours. In contrast to the growth arrest seen in wild-type cells exposed to stretch, p21 Cip1−/− podocytes exposed to stretch continued to proliferate, suggesting a role for p21 Cip1 in the inability of the podocyte to progress through the cell cycle in response to stretch. These studies show that in addition to being injurious to mesangial cells, mechanical stretch affects podocytes and reduces their proliferative potential by altering specific cell cycle proteins.
Podocyte Proliferation
Although the majority of both experimental animal models of podocyte injury and human podocyte diseases are not associated with proliferation, this has been reported to occur in a limited number of settings. The true ability of the mature podocyte to proliferate remains controversial, principally because glomerular cells believed to be proliferating podocytes frequently lack defining cell-type-specific markers. However, the use of transgenic animals has been invaluable in resolving the debate. A well-studied animal model of podocyte proliferation is crescentic glomerulonephritis in the mouse, which has been examined in detail by several groups. In this model, predictable proliferation of glomerular cells occurs, and early in the course of the disease this is not associated with rupture of Bowman’s capsule, nor with the presence of infiltrating cells. It is therefore a useful model for studying the contribution of intrinsic glomerular cells to crescent formation. Moeller et al. generated a mouse with constitutive expression of β galactosidase specifically in podocytes in vivo . Experimental crescentic nephritis was induced by injecting rabbit IgG ip, followed six days later by an intravenous injection of rabbit antimouse GBM antibody. The crescents contained numerous β-galactosidase-expressing cells, confirming their podocyte origin. Furthermore, expression of the nuclear proliferation marker Ki-67 by these cells demonstrated the capability of these podocyte-derived cells to undergo proliferation. Matsusaka et al. generated a transgenic mouse with podocyte-specific expression of human CD25. Injury was then induced using an immunotoxin, resulting in a proliferative glomerular lesion. However, immunohistochemistry indicated that the proliferating cells were of parietal epithelial origin, not podocytes. The disparity between these two results likely results from the different initiating injuries used, but illustrates the ongoing debate which was further fueled by recent data. Smeets et al. could show, by lineage tracing of either podocytes or parietal epithelial cells (PECs), that the majority of cells within extracapillary proliferative leasions were PECs, and only a minority were of podocyte origin. This held true in the nephrotoxic nephritis model of inflammatory crescentic glomerulonephritis, and in the Thy-1.1 transgenic mouse model of collapsing glomerulopathy. As described above, once podocytes proliferate they lose several of their defining characteristic proteins, such as WT-1, making identification difficult. However, there is broad agreement that the proliferating resident glomerular cells making up crescents are of epithelial origin, although the relative contributions of parietal cells and podocytes remains disputed.
In human disease, podocyte proliferation is considered to occur in collapsing glomerulopathy, cellular focal segmental glomerulosclerosis, and HIV-associated nephropathy. In these diseases, there is increased expression of cyclin A and the proliferation marker Ki-67, with a marked reduction in p27 Kip1 and p57 Kip2 . In contrast, all other diseases of podocytes in humans, including membranous nephropathy, minimal change disease, and focal segmental glomerulosclerosis, are not associated with a decrease in Cdk inhibitor levels, and typical markers of proliferation are absent. Interestingly, the rate of progression to end-stage renal failure is increased in glomerular diseases characterized by podocyte proliferation. The pathogenesis of HIV-associated nephropathy has been further detailed using transgenic mice expressing HIV-1 genes. The Tg26 mouse model develops murine HIVAN secondary o renal expression of HIV-1 mRNAs from the HIV-1 NL4-3 gag-pol proviral transgene. This model has enabled characterization of the cellular effects of HIV-1 using both the intact animal and conditionally immortalized podocytes in culture. HIV-1 induces loss of contact inhibition in podocytes, and expression of cyclin D1 and phosphorylation of pRb. The cells become dedifferentiated, with loss of specific podocyte-expressed proteins. Interestingly, there have been reports of reversibility of HIV nephropathy in both mice (discussed below) and human disease following treatment with highly active antiretroviral therapy, suggesting that once the virus is cleared the podocyte is capable of exiting the cell cycle and redifferentiating to its mature phenotype.
Atypical Effects of cell cycle proteins on Podocytes
The traditional view of cell cycle proteins focuses on the regulation of cellular proliferation. The view on cell cycle proteins has since been broadened by the discovery of atypical cyclin proteins and dependent kinases. These include cyclin C-Cdk8, cyclin H-cdk7, cyclin K-Cdk9, cyclin T-Cdk9 or cyclin I-Cdk5. We could show that cyclin I null mice are normal consistent with the notion that cyclin I is not required for cell differentation. Cyclin I is predominantly expressed in terminally differentiated cells, including neurons and podocytes. Lack of cyclin I rendered podocytes more susceptible to apoptosis. A potential mechanism included a decreased expression and protein stability of p21.
The Cdk partner of cyclin I remained elusive since its discovery. We could identify Cdk5 as the Cdk partner of cyclin I. Cdk5 itself is essential for normal development, as Cdk5 null mice die from developmental deficits, but little is known about its function in the kidney. Cdk5 is mainly activated by the non-cyclin proteins p35 and p39. The latter is not expressed in podocytes, but p35–Cdk5 has been shown to be present in podocytes. In 2004, Griffin et al. demonstrated an essential role for p35–Cdk5 in podocyte differentation, proliferation, and maintenance. Cdk5 levels decline in experimental disease associated with podocyte dedifferentation and proliferation, including anti-glomerular antibody disease and HIV-associated nephropathy. More recent in vivo and in vitro data support the model that Cdk5 is central to podocyte survival, and is dually regulated by cyclin I and p35. Downstream targets of Cdk5 were the pro-survival proteins Bcl-2 and Bcl-XL.
Therapeutic Inhibition of Podocyte Proliferation at Cell Cycle Level
As with glomerular diseases principally affecting the mesangial cell, both roscovitine and retinoids have shown promise as therapeutic agents in modulating the podocyte response to injury. In addition, recent data suggest a direct effect of glucocorticoids on podocytes at the cell cycle level.
Roscovitine
We hypothesized that inhibition of podocyte proliferation with the Cdk2 inhibitor roscovitine in a mouse model of crescentic glomerulonephritis would improve renal outcome, similar to that observed following inhibition of mesangial cell proliferation in Thy 1 nephritis described previously. Inhibition of Cdk2 activity was confirmed by a histone kinase assay, and podocyte DNA synthesis measured by incorporation of BrdU. Compared to control nephritic animals receiving the vehicle, in mice treated with roscovitine there was a significant decrease in BrdU at day 5 of nephritis. This was accompanied by less accumulation of laminin at day 14, and significantly improved renal function, suggesting a similar intervention may be beneficial in human diseases. Roscovitine has also been demonstrated to be beneficial in the treatment of Tg26 mice at doses that did not decrease HIV-1 transgene expression, suggesting an effect mediated by inhibition of cell cycle progression.
Retinoids
Retinoids are particularly attractive agents for the treatment of podocyte disease, as it has been demonstrated that following podocyte injury, both decrease podocyte proliferation and maintain the expression of markers of differentiation. The promoter region of the human nephrin gene (NPHS1) contains three putative retinoic acid-response elements, and shows enhancer activity in response to all-trans-retinoic acid (ATRA) in a dose-dependent manner. We have recently demonstrated that ATRA in vitro significantly retards podocyte proliferation, as measured by the MTT assay, while inducing process formation and increasing the expression of both nephrin and podocin. Similarly, in mice with antiglomerular antibody nephritis, treatment with ATRA both reduces podocyte proliferation and prevents the decreases in nephrin, podocin, and synaptopodin in experimental animals. This was accompanied by a reduction in proteinuria. The dual roles of retinoids to both inhibit proliferation while promoting differentiation underscore their potential value as therapeutic agents for human podocyte diseases.
Glucocorticoids
Glucocorticods are the clinical backbone to treat patients with glomerular diseases. Their mode of action is poorly-understood, particularly in patients with steroid-sensitive nephritic syndrome which lacks any signs of inflammation in the glomerulus. In vitro data in human podocytes revealed direct effects of dexamethasone on human podocytes. Incubation with steroids led to decreased expression of p21 and suppression of inflammatory chemokines (IL-6/IL-8), but did not induce apoptosis.
Parietal Epithelial Cell Proliferation
As outlined above, there is an ongoing debate on the cellular origin of extracapillary proliferative lesions in glomerular disease. The parietal epithelial cell (PEC) has long been neglected, but recently moved into the center of glomerular research. PECs share a common lineage with podocytes until the S-shaped stage of glomerulogenesis. Between the S-shaped body and capillary loop stages, each cell begins to express distinct genes, used as “cell-specific markers.” For example, Wilm’s tumor suppressor protein 1 (WT-1) expression is no longer detected in PECs, whereas podocytes maintain WT-1 and gain vimentin expression. In mice, PECs express CD10 in the capillary loop stage of development. Potential mechanisms underlying PEC proliferation result from studies of cell cycle proteins. In contrast to podocytes, expression of the Cdk inhibitors p21 Cip1 and p57 Kip2 were not detected in healthy PECs, whereas p27 Kip1 was expressed at low levels. In experimental and human forms of FSGS, PECs showed increased DNA-synthesis. The increased expression of Ki-67 and cyclin A were accompanied by decreased p27 Kip1 expression in the absence of p21 Cip1 and p57 Kip2 . Suzuki et al. recently showed in a mouse model of FSGS, that PEC proliferation was in part regulated by p21 Cip1 .
Glomerular Endothelial Cell Proliferation
The endothelial cells of mature glomeruli are quiescent, but retain the capacity to proliferate and form new capillaries following injury. The degree of proliferation appears to be dependent on the local balance of proangiogenic factors, such as vascular endothelial growth factor (VEGF), and antiangiogenic factors, such as thrombospondin-1. An inadequate proliferative response may lead to loss of the glomerular microvasculature, and contribute to glomerulosclerosis and progressive renal impairment. The beneficial effects of VEGF administration have been demonstrated in animal models of acute glomerulonephritis, suggesting that amelioration of human diseases may be achieved by augmenting the reparative potential of the glomerular endothelial cells. However, the underlying role of individual cyclins, Cdk, and Cdk inhibitors in these cells remains unknown.
Tubular Cell Hyperplasia
Renal tubular hyperplasia is most frequently encountered during the reparative phase following an acute injury, such as ischemia or toxin exposure. Tubular injury results in cell loss by either necrosis or apoptosis, and therefore there is a requirement for the remaining cells to spread and migrate to cover the exposed basement membrane. These cells then dedifferentiate and proliferate to restore cell number, and finally differentiate again to restore the functional integrity of the nephron. Interestingly, the tubular repair recapitulates organogenesis in patterns of gene expression, including vimentin, neural cell adhesion molecule, growth factors such as IGF-1, fibroblast growth factor, and hepatocyte growth factor; and matrix molecules such as osteopontin. This dedifferentiated phenotype is likely to be important for the spreading and proliferative properties of the viable tubular epithelial cells as they cover the denuded basement membrane to replace lost cells. However, the factors controlling the reversion to a less-differentiated phenotype, and the subsequent re-establishment of the mature phenotype, remain poorly-understood.
As might be expected, the proliferation observed following a transient ischemic injury is associated with an increase in the mRNA and protein expression of cyclins D1, D3, and B, mRNA expression of cyclin A, and protein expression of Cdks 2 and 4. Both cyclin-D and -E kinase activities are increased. Thus, the proliferative response is due to increased expression of both the regulatory and catalytic subunits of the G1 kinases, and an increase in their activity.
The Cdk inhibitors appear to have a critical role in limiting the proliferative response. Following acute renal injury induced by ischemia, ureteral obstruction or cisplatin, p21 Cip1 protein expression is increased in the thick ascending limb of the loop of Henle, and in the distal convoluted tubule. Induction of the same injury in p21 Cip1−/− mice was associated with increased proliferation, as assessed by BrdU incorporation and PCNA staining. The p21 Cip−/− mice had a more rapid onset of acute renal failure, developed more severe morphological damage, and had a higher mortality, emphasizing the requirement for proliferation following injury to be at a controlled and appropriate level.
Hypertrophy
Hypertrophy has been described in most segments of the nephron, but proximal tubular hypertrophy is observed most frequently. Hypertrophy of the proximal tubule has been described in compensatory renal growth, chronic metabolic acidosis and chronic hypokalemia, diabetes mellitus, and during protein-loading. In compensatory renal growth, there is also hypertrophy of the glomerulus and collecting tubules; in diabetes, hypertrophy of glomerular cells is well-described, and with prolonged protein-loading hypertrophy of the initial segments of the thick ascending limb occurs.
As mentioned previously, cellular hypertrophy may result from either cell cycle-dependent or -independent mechanisms. Within the glomerulus, an increase in cell size is predominantly due to cell cycle re-entry without progression, whereas in the tubules there appears to be a more significant contribution from the inhibition of protein synthesis.
Glomerular Hypertrophy
Glomerular cell hypertrophy occurs during many forms of chronic renal disease, and may herald the development of glomerulosclerosis. Glomerular diseases associated with glomerular hypertrophy include diabetic nephropathy, minimal change nephropathy, focal segmental glomerulosclerosis, and a reduction in renal mass. However, there are clear differences in the prognosis of glomerular hypertrophy, depending on the underlying disease. Following uninephrectomy, the hypertrophy is compensatory, and is not typically associated with the development of glomerulosclerosis. In contrast, the hypertrophy of diabetic nephropathy antecedes and probably underlies the development of glomerulosclerosis. Diabetic nephropathy is the leading cause of end-stage renal disease in Western countries, and is discussed further below. Pathological glomerular hypertrophy leads to progressive glomerulosclerosis and scarring by a number of different mechanisms. The increased metabolic rate of cells undergoing hypertrophy results in enhanced mitochondrial oxygen consumption, which may lead to tissue injury due to the generation of reactive oxygen radicals and the subsequent peroxidation of proteins and lipids. The hypertrophy is often initiated by growth factors that also stimulate the cells to increase production of extracellular matrix components, such as type IV collagen.
Diabetic Nephropathy
Diabetes mellitus is now the most frequent cause of end-stage renal failure in Western countries, and the pathogenesis of diabetic nephropathy is discussed in detail elsewhere in this book. The focus of this chapter is on the disordered cell growth seen in association with diabetic renal involvement. The hallmarks of human diabetic nephropathy are similar in both type I and type II diabetes, and consist of mesangial cell hypertrophy and podocyte loss, with accumulation of extracellular matrix in the mesangium and tubulointerstitium, resulting in glomerulosclerosis and tubulointerstitial fibrosis. Research has concentrated principally on the ability of hyperglycemia and TGF-β to induce mesangial cell hypertrophy, and more recently the roles of RAGE and hyperinsulinemia have also been studied.
Diabetes and Mesangial Cells
HYPERGLYCEMIA In vitro culture of mesangial cells in high glucose media causes cell cycle entry and a biphasic growth response. Following initial proliferation, the cells arrest in G1-phase, and there is progressive hypertrophy. Both kidney and glomerular hypertrophy induced by hyperglycemia are associated with an early and sustained increase in expression of cyclin D1 and activation of Cdk4. An arrested cell cycle suggests a role for the Cdk inhibitors in mediating hypertrophy, and indeed high glucose increases the levels of both p21 Cip1 and p27 Kip1 in cultured mesangial cells. This is mediated by a number of factors, including glucose itself, TGF-β, which then acts in an autocrine fashion, and CTGF. High glucose directly stimulates the transcription of p21 Cip1 , and activates MAP kinases, which prolong the half-life of p27 Kip1 by phosphorylation on serine residues. Lowering p21 Cip1 or p27 Kip1 levels with antisense oligonucleotides reduces the hypertrophic effects of high glucose. Moreover, hypertrophy is not induced by high glucose in p21 Cip1−/− (unpublished observations) and p27 Kip1−/− mesangial cells in vitro . Indeed, high glucose increases proliferation in p27 Kip1−/− mesangial cells, whereas it arrests cell cycle progression in p27 Kip1+/+ mesangial cells. Reconstituting p27 Kip1 levels in p27 Kip1−/− mesangial cells by transfection restores the hypertrophic phenotype. These studies show a compelling role for the Cdk inhibitors p21 Cip1 and p27 Kip1 in mediating the hypertrophy induced by high glucose.
These in vitro findings have been confirmed in experimental models of both type I and type II diabetic nephropathy. Considering type I diabetes, the glomerular protein levels of p21 Cip1 are increased in the streptozotocin (STZ)-induced model in the mouse, and both p21 Cip1 and p27 Kip1 levels are increased in the glomeruli of diabetic BBdp rats. A similar increase was noted in glomeruli of db/db mice and the Zucker diabetic fatty rat, models of type II diabetic nephropathy. Diabetic p21 Cip1−/− mice are protected from glomerular hypertrophy. Diabetic p27 Kip1−/− mice have only mild mesangial expansion and no glomerular or renal hypertrophy compared to control diabetic p27 Kip1+/+ mice, despite upregulation of glomerular TGF-β. These results support a critical role for the Cdk inhibitors p21 Cip1 and p27 Kip1 in mediating the glomerular hypertrophy seen not only in association with diabetes, but also as described in the following, a reduction in nephron number.
TRANSFORMING GROWTH FACTOR β The cytokine TGF-β has been shown in numerous settings to be a key mediator of progressive fibrosis in renal disease. TGF-β also decreases proliferation in mesangial cells, an effect that appears to be independent of p21 Cip1 and p27 Kip1 , and induces cell hypertrophy. To determine the role of Cdk inhibitors in mediating the hypertrophic effects of TGF-β, mesangial cells derived from single and double null mice were studied. Compared to wild-type mice, hypertrophy was significantly reduced in double p21 Cip1 /p27 Kip1− / − mesangial cells. A less marked reduction in hypertrophy was seen in the single p21 Cip1− / − and p27 Kip1− / − cells. These results show that although p21 Cip1 and p27 Kip1 each contribute to the hypertrophic action of TGF-β, the presence of both is required for maximal effect.
The expression of Cdk inhibitors has also been explored in response to CTGF, considered to be a principle mediator of the downstream effects of TGF-β. Abdel Wahab et al. demonstrated that CTGF is a hypertrophic factor for human mesangial cells, and that this hypertrophy is associated with the induction of p15 INK4b , p21 Cip1 , and p27 Kip1 , with the maintenance of pRb in a hypophosphorylated state.
Diabetes and Podocytes
Morphometric analyses have demonstrated that the podocyte undergoes hypertrophy early in the course of both animal models of diabetic nephropathy and in human disease. This hypertrophy may be in direct response to the metabolic changes associated with diabetes or compensatory, consequent to the loss of podocytes that is known to occur.
At the level of the cell cycle, mRNA and protein expression of p27 Kip1 is increased in both cultured podocytes exposed to high glucose, and in glomeruli isolated from streptozotocin-induced diabetic rats. The p27 Kip1 gene appears to be haplo-insufficient, as diabetic p27 Kip1+/− mice exhibited an intermediate degree of functional and structural renal injury. High glucose also significantly increased angiotensin II levels both in cell lysates and media compared with normal glucose, and exogenous angiotensin II increased p27 Kip1 mRNA and protein expression. Exposure of cultured cells and treatment of diabetic rats with an angiotensin II receptor antagonist (ARB) inhibited the increase in p27 Kip1 . Glomerular hypertrophy was also significantly prevented by ARB treatment. It appears likely that similar cell cycle mechanisms drive both mesangial cell and podocyte hypertrophy in diabetic nephropathy, suggesting that podocytes in this setting are also capable of engaging the cell cycle.
Compensatory Glomerular Hypertrophy
A reduction in nephron number results in compensatory hypertrophy in the remaining viable nephron. Uninephrectomy does not alter the protein expression of cyclins D1 or D2, nor of Cdk2 or Cdk4, when total renal lysates are studied at day 7. However, there may be a differential effect in specific renal compartments, and an increase in tubular cyclin E–Cdk2 activity was demonstrated during compensatory hypertrophy following uninephrectomy. Prior to hypertrophy, severe renal ablation produced by 5/6 nephrectomy resulted in an early proliferative glomerular response, associated with an increase in cyclin E expression and phosphorylation of pRb.
A role for the Cdk inhibitors is now emerging in the pathogenesis of glomerular hypertrophy. Following partial renal ablation, p21 Cip1−/− mice develop glomerular hyperplasia rather than hypertrophy and increased intraglomerular pressure, with protection from the development of progressive renal failure. Increased intraglomerular pressure is a final pathway toward glomerulosclerosis in systemic hypertension, diabetes, and focal segmental glomerulosclerosis (FSGS). This glomerular hypertension causes stress-tension, or stretch, on resident glomerular cells, with differing consequences for the different cell types. Exposure of mesangial cells to cyclical mechanical stretch results in cell cycle entry and proliferation. In contrast, mechanical stretch reduces cell cycle progression and induces podocyte hypertrophy in vitro . This is unchanged in p27 Kip1−/− cells, but hypertrophy was not induced in p21 Cip1 and double p21 Cip1 /p27 Kip1−/− podocytes, indicating a requirement for p21 Cip1 . Stretch-induced hypertrophy required cell cycle entry, and was prevented by specifically blocking Erk1/2 or Akt. However, it is not clear whether podocyte hypertrophy represents a beneficial adaptive response to raised intraglomerular pressure or whether this change in podocyte phenotype is accompanied by a disturbance in function that is detrimental to the glomerulus.
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