Renal Hyperplasia and Hypertrophy

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

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.

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.