Podocyte mitosis - a catastrophe.

Podocyte loss plays a key role in the progression of glomerular disorders towards glomerulosclerosis and chronic kidney disease. Podocytes form unique cytoplasmic extensions, foot processes, which attach to the outer surface of the glomerular basement membrane and interdigitate with neighboring podocytes to form the slit diaphragm. Maintaining these sophisticated structural elements requires an intricate actin cytoskeleton. Genetic, mechanic, and immunologic or toxic forms of podocyte injury can cause podocyte loss, which causes glomerular filtration barrier dysfunction, leading to proteinuria. Cell migration and cell division are two processes that require a rearrangement of the actin cytoskeleton; this rearrangement would disrupt the podocyte foot processes, therefore, podocytes have a limited capacity to divide or migrate. Indeed, all cells need to rearrange their actin cytoskeleton to assemble a correct mitotic spindle and to complete mitosis. Podocytes, even when being forced to bypass cell cycle checkpoints to initiate DNA synthesis and chromosome segregation, cannot complete cytokinesis efficiently and thus usually generate aneuploid podocytes. Such aneuploid podocytes rapidly detach and die, a process referred to as mitotic catastrophe. Thus, detached or dead podocytes cannot be adequately replaced by the proliferation of adjacent podocytes. However, even glomerular disorders with severe podocyte injury can undergo regression and remission, suggesting alternative mechanisms to compensate for podocyte loss, such as podocyte hypertrophy or podocyte regeneration from resident renal progenitor cells. Together, mitosis of the terminally differentiated podocyte rather accelerates podocyte loss and therefore glomerulosclerosis. Finding ways to enhance podocyte regeneration from other sources remains a challenge goal to improve the treatment of chronic kidney disease in the future.

Proteinuria in diabetic kidney disease: a mechanistic viewpoint.

Proteinuria is the hallmark of diabetic kidney disease (DKD) and is an independent risk factor for both renal disease progression, and cardiovascular disease. Although the characteristic pathological changes in DKD include thickening of the glomerular basement membrane and mesangial expansion, these changes per se do not readily explain how patients develop proteinuria. Recent advances in podocyte and glomerular endothelial cell biology have shifted our focus to also include these cells of the glomerular filtration barrier in the development of proteinuria in DKD. This review describes the pathophysiological mechanisms at a cellular level which explain why patients with DKD develop proteinuria.

Darbepoetin alfa protects podocytes from apoptosis in vitro and in vivo.

Detachment or apoptosis of podocytes leads to proteinuria and glomerulosclerosis. There are no current interventions for diabetic or non-diabetic glomerular diseases specifically preventing podocyte apoptosis. Binding of erythropoiesis stimulating proteins (ESPs) to receptors on non-hematopoietic cells has been shown to have anti-apoptotic effects in vitro, in vivo, and in preliminary human studies. Recently, erythropoietin receptors were identified on podocytes; therefore, we tested effects of darbepoetin alfa in preventing podocyte apoptosis. Cultured immortalized mouse podocytes were treated with low-dose ultraviolet-C (uv-C) irradiation to induce apoptosis in the absence or presence of darbepoetin alfa. Apoptosis was quantified by Hoechst staining and by caspase 3 cleavage assessed by Western blots. Pretreatment with darbepoetin alfa significantly reduced podocyte apoptosis with this effect involving intact Janus family protein kinase-2 (JAK2) and AKT signaling pathways. Additionally, darbepoetin alfa was found protective against transforming growth factor-beta1 but not puromycin aminonucleoside induced apoptosis. Mice with anti-glomerular antibody induced glomerulonephritis had significantly less proteinuria, glomerulosclerosis, and podocyte apoptosis when treated with darbepoetin alfa. Our studies show that treatment of progressive renal diseases characterized by podocyte apoptosis with ESPs may be beneficial in slowing progression of chronic kidney disease.

Podocyte protection by darbepoetin: preservation of the cytoskeleton and nephrin expression.

Podocyte injury is a significant contributor to proteinuria and glomerulosclerosis. Recent studies have shown a renoprotective effect of erythropoietin (EPO) during ischemic kidney disease. In this study, we examine mechanisms by which a long acting recombinant EPO analog, darbepoetin, may confer renoprotection in the puromycin aminonucleoside-induced model of nephrotic syndrome. Darbepoetin decreased the proteinuria of rats treated with puromycin. This protective effect was correlated with the immunohistochemical disappearance of the podocyte injury markers desmin and the immune costimulator molecule B7.1 with the reappearance of nephrin expression in the slit diaphragm. Podocyte foot process retraction and effacement along with actin filament rearrangement, determined by electron microscopy, were all reversed by darbepoetin treatment. The protective effects were confirmed in puromycin-induced nephrotic rats that had been hemodiluted to normal hematocrit levels. Furthermore, puromycin treatment of rat podocytes in culture caused actin cytoskeletal reorganization along with deranged nephrin distribution. All these effects in vitro were reversed by darbepoetin. Our study demonstrates that darbepoetin treatment ameliorates podocyte injury and decreases proteinuria by a direct effect on podocytes. This may be accomplished by maintenance of the actin cytoskeleton and nephrin expression.

Podocytes in culture: past, present, and future.

Human genetic and in vivo animal studies have helped to define the critical importance of podocytes for kidney function in health and disease. However, as in any other research area, by default these approaches do not allow for mechanistic studies. Such mechanistic studies require the availability of cells grown ex vivo (i.e., in culture) with the ability to directly study mechanistic events and control the environment such that specific hypotheses can be tested. A seminal breakthrough came about a decade ago with the documentation of differentiation in culture of primary rat and human podocytes and the subsequent development of conditionally immortalized differentiated podocyte cell lines that allow deciphering the decisive steps of differentiation and function of 'in vivo' podocytes. Although this paper is not intended to provide a comprehensive review of podocyte biology, nor their role in proteinuric renal diseases or progressive glomerulosclerosis, it will focus specifically on several aspects of podocytes in culture. In particular, we will discuss the scientific and research rationale and need for cultured podocytes, how podocyte cell-culture evolved, and how cultured podocytes are currently being used to uncover novel functions of podocytes that can then be validated in vivo in animal or human studies. In addition, we provide a detailed description of how to properly culture and characterize podocytes to avoid potential pitfalls.

Puromycin aminonucleoside induces oxidant-dependent DNA damage in podocytes in vitro and in vivo.

A decline in podocyte number correlates with progression to glomerulosclerosis. A mechanism underlying reduced podocyte number is the podocyte's relative inability to proliferate in response to injury. Injury by the podocyte toxin puromycin aminonucleoside (PA) is mediated via reactive oxygen species (ROS). The precise role of ROS in the pathogenesis of PA-induced glomerulosclerosis remains to be determined. We sought to examine whether PA-induced ROS caused podocyte DNA damage, possibly accounting for the podocyte's inability to proliferate in response to PA. In vitro, podocytes were exposed to PA, with or without the radical scavenger 1,3-dimethyl-2-thiourea (DMTU). In vivo, male Sprague-Dawley rats were divided into experimental groups (n = 6/group/time point): PA, PA with DMTU, and control, killed at days 1.5, 3, or 7. DNA damage was measured by DNA precipitation, apurinic/apyrimidinic site, Comet, and 8-hydroxydeoxyguanosine assays. Cell cycle checkpoint protein upregulation (by immunostaining and Western blotting), histopathology, and biochemical parameters were examined. DNA damage was increased in cultured podocytes that received PA, but not PA with DMTU. PA exposure activated specific cell cycle checkpoint proteins, with attenuation by DMTU. DNA repair enzymes were activated, providing evidence for attempted DNA repair. The PA-treated animals developed worse proteinuria and histopathologic disease and exhibited more DNA damage than the DMTU pretreated group. No significant apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling staining. A mechanism underlying the lack of podocyte proliferation following PA-induced injury in vitro and in vivo may be ROS-mediated DNA damage, with upregulation of specific cell cycle checkpoints leading to cell cycle arrest.

The podocyte's response to injury: role in proteinuria and glomerulosclerosis.

The terminally differentiated podocyte, also called glomerular visceral epithelial cell, are highly specialized cells. They function as a critical size and charge barrier to prevent proteinuria. Podocytes are injured in diabetic and non-diabetic renal diseases. The clinical signature of podocyte injury is proteinuria, with or without loss of renal function owing to glomerulosclerosis. There is an exciting and expanding literature showing that hereditary, congenital, or acquired abnormalities in the molecular anatomy of podocytes leads to proteinuria, and at times, glomerulosclerosis. The change in podocyte shape, called effacement, is not simply a passive process following injury, but is owing to a complex interplay of proteins that comprise the molecular anatomy of the different protein domains of podocytes. These will be discussed in this review. Recent studies have also highlighted that a reduction in podocyte number directly causes proteinuria and glomerulosclerosis. This is owing to several factors, including the relative inability for these cells to proliferate, detachment, and apoptosis. The mechanisms of these events are being elucidated, and are discussed in this review. It is the hope that by delineating the events following injury to podocytes, therapies might be developed to reduce the burden of proteinuric renal diseases.

Distinct functions for Ras GTPases in the control of proliferation and apoptosis in mouse and human mesangial cells.

In previous work, we have demonstrated that Ras GTPases regulate proliferation in a range of human renal cells. The present work compares human and mouse mesangial cell (HMC and MMC) responses to specific knockdown of Ras genes with antisense oligonucleotides (AS-oligos), and examines the role of the p21 (cip1) and p27 (kip1) cyclin-dependent kinase inhibitors in these responses in mouse cells. HMC and MMC were lipofectin transfected with ras-targeted AS-oligo at 200-400 nM for 18 h followed by growth of cells in 20% serum for 18-72 h. Cell proliferation was assessed with an MTS assay and bromodeoxyuridine (BrdU) uptake. Apoptosis was quantified using nuclear stain with Hoechst 33342 dye. In MMC, Ha-ras AS-oligo caused an increase in apoptosis from <2% to 10-15% of cells after 18 h in serum (P<0.01). Control, Ki-ras and N-ras AS-oligos had minimal effects on apoptosis. BrdU uptake studies showed that BrdU+ve MMC were increased by 20-40% (P<0.05) after Ha-ras AS-oligo at 24 h; other ras AS-oligos were inactive. HMC number was reduced by 40-80% (P<0.01) at 48-72 h by both Ha-ras and Ki-ras AS-oligos. These actions were associated with reductions in BrdU+ve cells. In HMC, the ras AS-oligos did not induce apoptosis. p21(-,-) MMC showed exaggerated apoptotic responses to Ha-Ras AS-oligo. In mouse cells, Ha-Ras expression appears necessary to prevent apoptotic cell death; Ras expression does not appear necessary for cells to progress through the cell cycle. In human cells, Ras does not appear necessary to prevent apoptosis but Ha-Ras and Ki-Ras appear to be required for cell cycle progression.

Erk 1,2 phosphorylates p27(Kip1): Functional evidence for a role in high glucose-induced hypertrophy of mesangial cells.

AIMS/HYPOTHESIS: Mesangial cell hypertrophy is one of the earliest morphological abnormalities of diabetic nephropathy. We have previously shown that high glucose induces p27(Kip1) by a post-transcriptional mechanism and that mesangial cell hypertrophy depends on G(1)-phase arrest mediated by this CDK-inhibitor. However, it remains poorly understood how high glucose stimulates p27(Kip1) expression in mesangial cells. METHODS: Mesangial cells were isolated from p27(Kip1) +/+ and -/- mice and characterized by light microscopy and immunohistochemistry. It was tested by Western blotting and autoradiography whether high glucose medium activates Erk 1,2 and whether this activation phosphorylates p27(Kip1). The three consensus phosphorylation sites of p27(Kip1) were mutated and these constructs were expressed in p27(Kip1) -/- mesangial cells. Hypertrophy was assessed by different methods. RESULTS: High glucose stimulates phosphorylation of MAP kinases Erk 1,2 in p27(Kip1 )+/+ and -/- mesangial cells. Activation of Erk 1,2 leads to phosphorylation of p27(Kip1 )in vitro and in vivo. Mutations of serine(10) or threonine(187) still supported high glucose-induced hypertrophy. In contrast, a mutation of serine(178) converted the hypertrophic response into a proliferative phenotype. Mutation of serine(178) leads to the attenuated expression of p27(Kip1) protein in the presence of high glucose. CONCLUSIONS/INTERPRETATION: Our study shows that high glucose stimulates Erk 1,2 that phosphorylate p27(Kip1) at serine(178) increasing its expression. This is an important molecular mechanism of high glucose-induced hypertrophy of mesangial cells.

The subcellular localization of cyclin dependent kinase 2 determines the fate of mesangial cells: role in apoptosis and proliferation.

Apoptosis is closely linked to proliferation. In this study we showed that inducing apoptosis in mouse mesangial cells with ultraviolet (UV) irradiation was associated with increased cyclin A-cyclin dependent kinase (CDK) 2 activity. Inhibiting CDK2 activity with Roscovitine or dominant negative mutant reduced apoptosis. Because apoptosis typically begins in the cytoplasm, we tested the hypothesis that the subcellular localization of CDK2 determines the proliferative or apoptotic fate of the cell. Our results showed that cyclin A-CDK2 was nuclear in proliferating cells. However, inducing apoptosis in proliferating cells with UV irradiation was associated with a decrease in nuclear cyclin A and CDK2 protein levels. This coincided with an increase in protein and kinase activity for cyclin A-CDK2 in the cytoplasm. Translocation of cyclin A-CDK2 also occurred in p53-/- mesangial cells. Finally, we showed that caspase-3 activity was significantly reduced by inhibiting CDK2 activity with Roscovitine. In summary, our results show that apoptosis is associated with an increase in cytoplasmic cyclin A-CDK2 activity, which is p53 independent and upstream of caspase-3. We propose that the subcellular localization of CDK2 determines the proliferative or apoptotic fate of the cell.

Podocyte expression of the CDK-inhibitor p57 during development and disease.

BACKGROUND: The mature podocyte is a terminally differentiated cell with a limited proliferative capacity. The precise cell cycle proteins necessary for establishing podocyte quiescence during development or permitting podocyte cell cycle re-entry in disease states have not been fully defined. Accordingly, we studied the role of the cyclin dependent kinase (CDK)-inhibitor p57Kip2 (p57) in modulating these processes. METHODS: The expression of p57 protein in relation to markers of DNA synthesis was examined in developing mouse kidneys, and in the passive Heymann nephritis (PHN) and anti-glomerular antibody models of glomerular disease by immunohistochemistry. The role of p57 in glomerulogenesis was explored by examining renal tissue from embryonic p57-/- mice, and the expression of p21, p27 and p57 protein and mRNA was examined in podocytes in vitro. RESULTS: The de novo expression of p57 during glomerulogenesis coincides with the cessation of podocyte proliferation, and the establishment of a mature phenotype, and p57 is expressed exclusively in podocytes in mature glomeruli. However, p57 knockout mice have normal glomerular podocyte development. In addition, mRNA but not protein levels of p57 increased upon differentiation of podocytes in vitro. There was a marked decrease in p57 expression in both animal models of podocyte injury. This was diffuse in PHN, whereas in the murine model, loss of expression of p57 occurred predominantly in proliferating podocytes, expressing proliferating cell nuclear antigen (PCNA). CONCLUSION: Despite the de novo expression of p57 protein coinciding with the cessation of primitive podocyte proliferation during glomerulogenesis, embryonic p57-/- mice glomeruli were histologically normal. Cultured podocytes did not require changes in p57 protein levels to undergo differentiation. These data suggest that p57 alone is not required for podocyte differentiation, and that other cell cycle regulators may play a role. Furthermore, although injury to mature podocytes in experimental glomerular disease is associated with a decrease in p57, the levels of all three members of the Cip/Kip family of CDK inhibitors appear to determine the capability of podocytes to proliferate.

Complement (C5b-9) induces DNA synthesis in rat mesangial cells in vitro.

BACKGROUND: The membrane attack complex C5b-9 causes injury in many forms of immune-mediated glomerular diseases characterized by mesangial cell (MC) proliferation and inhibiting C5b-9 decreases MC proliferation in vivo. Membrane insertion of sublytic quantities of the membrane attack complex of complement (C5b-9) is a potent stimulus for cell activation and the production of a variety of cytokines, growth factors, oxidants, matrix components, and other nephritogenic molecules. In vivo, a common response of MC to C5b-9--mediated injury is cell proliferation, an event closely linked to matrix expansion and sclerosis. In this study, we tested the hypothesis that C5b-9 might also serve as a mitogenic stimulus for MCs. METHODS: Rat MCs in vitro were exposed anti-Thy1 antibody and 2% normal PVG serum (a complement source) to induce sublytic C5b-9 attack and DNA synthesis and cell number were measured. Control MCs were exposed to antibody and C6-deficient PVG serum. RESULTS: Sublytic C5b-9--induced injury to MCs is sufficient to induce DNA synthesis. Furthermore, C5b-9 augmented DNA synthesis induced by platelet-derived growth factor (PDGF) and 5% fetal calf serum. C5b-9--induced DNA synthesis was reduced by inhibiting reactive oxygen species (ROS) with superoxide dismutase and catalase, but not by neutralizing the mitogenic growth factors PDGF and basic fibroblast growth factor (bFGF). CONCLUSIONS: This study demonstrates that C5b-9 may directly increase DNA synthesis in cultured MCs, which are mediated in part by the release of ROS, and that C5b-9 also augments DNA synthesis induced in MCs by other known mitogens.

High glucose-induced hypertrophy of mesangial cells requires p27(Kip1), an inhibitor of cyclin-dependent kinases.

Hypertrophy of mesangial cells is one of the earliest morphological alterations in the kidney after the onset of diabetes mellitus. We have previously shown that cultured mesangial cells exposed to high ambient glucose arrest in the G1 phase of the cell cycle and that this is associated with an increased expression of inhibitors of the cyclin-dependent kinase (CDK)-inhibitors p21(Cip) and p27(Kip1). To further investigate a potential role of p27Kip1 in the development of glucose-induced hypertrophy, mesangial cells from p27Kip1 wild-type (+/+) and knockout (-/-) mice were established. High glucose medium (450 mg/dl) increased p21(Cip1) protein in p27Kip1+/+ and -/- mesangial cells, and increased p27Kip1 protein levels in p27Kip1+/+ cells. In contrast to high glucose increasing de novo protein synthesis in p27Kip1+/+ cells, high glucose did not increase protein synthesis in p27Kip1-/- cells. High glucose also reduced DNA synthesis and caused cell cycle arrest in p27Kip1+/+ cells. In contrast, despite an increase in transforming growth factor (TGF)-beta mRNA and protein expression, DNA synthesis and cell cycle progression were increased by high glucose in p27Kip1-/- cells. Exogenous TGF-beta comparably induced fibronectin mRNA in p27Kip1+/+ and -/- cells suggesting intact TGF-beta receptor transduction. In addition, high glucose failed to increase the total protein/cell number ratio in p27Kip1-/- cells. However, in the presence of high glucose, reconstituting p27Kip1 expression by transient or stable transfection in p27Kip1-/- cells, using an inducible expression system, increased the de novo protein synthesis and restored G1-phase arrest. These results show that p27Kip1 is required for glucose-induced mesangial cell hypertrophy and cell cycle arrest.

Vascular endothelial growth factor accelerates renal recovery in experimental thrombotic microangiopathy.

BACKGROUND: Renal microvascular injury characterizes thrombotic microangiopathy (TMA). The possibility that angiogenic growth factors may accelerate recovery in TMA has not been studied. METHODS: TMA was induced in rats by the selective right renal artery perfusion of antiglomerular endothelial cell IgG (30 mg/kg). Twenty-four hours later, rats received vascular endothelial growth factor (VEGF121, 100 microg/kg/day) or vehicle (control) daily until day 14. To evaluate renal function, the unperfused left kidney was removed at day 14, and rats were sacrificed at day 17. RESULTS: The induction of TMA was associated with loss of glomerular and peritubular capillary endothelial cells and decreased arteriolar density at day 1. Some spontaneous capillary recovery was present by day 17; however, repair was incomplete, and severe tubulointerstitial damage occurred. The lack of complete microvascular recovery was associated with reduced VEGF immunostaining in the outer medulla. VEGF-treated rats had more glomeruli with intact endothelium, less glomerular ischemia (collapsed glomeruli), and greater peritubular capillary density with less peritubular capillary loss. This was associated with less tubulointerstitial fibrosis, less cortical atrophy, and improved renal function. CONCLUSIONS: VEGF accelerates renal recovery in this experimental model of TMA. These studies suggest that angiogenic growth factors may provide a new therapeutic strategy for diseases associated with endothelial cell injury.

Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.

BACKGROUND: Normal human podocytes are terminally differentiated and quiescent cells. It is not known why podocytes fail to proliferate in response to most forms of injury. Proliferation is regulated by cell cycle proteins and their inhibitors. The Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) in general prevent proliferation by inhibiting cyclin-CDK complexes. In the current study, we determined the expression and possible role of specific CDK inhibitors in podocyte proliferation in human disease characterized by podocyte injury. METHODS: Immunostaining was performed for the CDK inhibitors p21, p27, and p57 and the proliferation marker Ki-67 on renal biopsies from patients with minimal change disease (MCD; N = 6), membranous glomerulopathy (MGN; N = 19), cellular variant of focal segmental glomerulosclerosis (FSGS; N = 12), collapsing glomerulopathy (CG; N = 9), and HIV-associated nephropathy (HIVAN; N = 16). Adult nephrectomy specimens without evidence of glomerular disease served as controls (N = 9). RESULTS: Normal quiescent podocytes express p27 and p57, but not p21. In diseases without podocyte proliferation (MCD, MGN), p21, p27, and p57 expression did not change. In contrast, there was a uniform decrease in p27 and p57 immunostaining in diseases with podocyte proliferation (cellular FSGS, CG, and HIVAN). This was accompanied by the de novo expression of p21 in podocytes. CONCLUSIONS: Our results show that podocyte quiescence may require the presence of the CDK inhibitors p27 and p57. In human glomerular diseases, a decrease in p27 and p57 may be permissive for the altered proliferative podocyte phenotype. p21 may have a multifactorial role in podocyte cell cycle regulation.

C5b-9 membrane attack complex mediates endothelial cell apoptosis in experimental glomerulonephritis.

We studied the role of the C5b-9 membrane attack complex in two models of inflammatory glomerulonephritis (GN) initiated by acute glomerular endothelial injury in Piebold-viral-Glaxo (PVG) complement-sufficient rats (C+), C6-deficient rats (C6-), and rats systematically depleted of complement with cobra venom factor (CVF). GN was induced by performing a left nephrectomy and selectively perfusing the right kidney with either 1) the lectin concanavalin A (Con A) followed by complement-fixing anti-Con A (Con A GN) or 2) purified complement-fixing goat anti-rat glomerular endothelial cell (GEN) antibody [immune-mediated thrombotic microangiopathy (ITM)]. Comparable levels of GEN apoptosis were detected in C+ animals in both models. CVF administration reduced GEN apoptosis by 10- to 12-fold. GEN apoptosis was C5b-9 dependent because PVG C6- rats were protected from GEN loss. Furthermore, functional inhibition of the cell surface complement regulatory protein CD59 by renal perfusion with anti-CD59 antibody in ITM resulted in a 3.5-fold increase in GEN apoptosis. Last, in Con A GN, abrogation of GEN apoptosis preserved endothelial integrity and renal function. This study demonstrates the specific role of C5b-9 in the induction of GEN apoptosis in experimental inflammatory GN, a finding with implications for diseases associated with the presence of antiendothelial cell antibodies.

Cell cycle regulatory proteins in renal disease: role in hypertrophy, proliferation, and apoptosis.

The response to glomerular and tubulointerstitial cell injury in most forms of renal disease includes changes in cell number (proliferation and apoptosis) and cell size (hypertrophy). These events typically precede and may be responsible for the accumulation of extracellular matrix proteins that leads to a decrease in renal function. There is increasing evidence showing that positive (cyclins and cyclin-dependent kinases) and negative (cyclin-dependent kinase inhibitors) cell cycle regulatory proteins have a critical role in regulating these fundamental cellular responses to immune and nonimmune forms of injury. Data now show that altering specific cell cycle proteins affects renal cell proliferation and improves renal function. Equally exciting is the expanding body of literature showing novel biological roles for cell cycle proteins in the regulation of cell hypertrophy and apoptosis. With increasing understanding of the role for cell cycle regulatory proteins in renal disease comes the hope for potential therapeutic interventions.

Cell cycle regulatory proteins in glomerular disease.

Evidence is accumulating that directly responsible for the rate of progression of glomerular disease are specific positive (cyclins and cyclin-dependent kinases) and negative (cyclin-kinase inhibitors) cell cycle regulatory proteins. The challenge for nephrologists is to determine which ones are expressed in renal disease and their precise role in glomerular cell proliferation, hypertrophy and differentiation. Ultimately the goal is to find ever more appropriate therapeutic strategies to arrest or prevent progressive renal disease.

Cyclin kinase inhibitor p21CIP1/WAF1 limits interstitial cell proliferation following ureteric obstruction.

Tubulointerstitial renal injury induced by unilateral ureteric obstruction (UUO) is characterized by marked cell proliferation and apoptosis. Proliferation requires cell cycle transit that is positively regulated by cyclins and cyclin-dependent kinases (CDKs) and inhibited by the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs: p21, p27, and p57). We have shown that the absence of p27 results in markedly increased tubular epithelial cell proliferation and apoptosis following UUO (V. Ophascharoensuk, M. L. Fero, J. Hughes, J. M. Roberts, and S. J. Shankland. Nat. Med. 4: 575-580, 1998). Since p21 mRNA is upregulated following UUO, we hypothesized that p21 would also serve to limit cell proliferation and apoptosis. We performed UUO in p21 +/+ and p21 -/- mice. Cell proliferation [bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA)], apoptosis [terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method], interstitial myofibroblast accumulation (actin), macrophage infiltration (F4/80), and collagen I expression were quantified at days 3, 7, and 14. In contrast to p27 -/- mice, there was no difference in tubular epithelial cell proliferation or apoptosis between p21 -/- and p21 +/+ mice at any time point. However, interstitial cell proliferation at day 3 was significantly increased in p21 -/- mice [BrdU, 40.7 +/- 1.9 cells/high-power field (cells/hpf) vs. 28.8 +/- 2, P < 0.005], although, interestingly, no difference was seen in interstitial cell apoptosis. Actin/BrdU double staining demonstrated increased interstitial myofibroblast proliferation at day 3 in p21 -/- animals (10 +/- 0.12 vs. 5.8 +/- 0. 11 cells/hpf, P < 0.05), which was followed by increased myofibroblast accumulation at day 7 in p21 -/- mice. No differences were detected in interstitial macrophage infiltration, collagen I deposition or transforming growth factor-beta1 mRNA (in situ hybridization) expression. In conclusion p21, unlike p27, is not essential for the regulation of tubular epithelial cell proliferation and apoptosis following UUO, but p21 levels do serve to limit the magnitude of the early myofibroblast proliferation. This study demonstrates a differential role for the CKI p21 and p27 in this model.