Reversal of Collapsing Glomerulopathy in Mice with the Cyclin-Dependent Kinase Inhibitor CYC202
Dana Gherardi*,
Vivette D’Agati,
Te-Hua Tearina Chu,
Anna Barnett||,
Athos Gianella-Borradori||,
Irwin H. Gelman and
Peter J. Nelson*
*Division of Nephrology, New York University School of Medicine, Department of Pathology, Columbia University College of Physicians and Surgeons, Shared Microarray Facility, Mount Sinai School of Medicine, New York, and Roswell Park Cancer Institute, Buffalo, New York; and ||Cyclacel Ltd., Dundee, United Kingdom.
Correspondence to Dr. Peter J. Nelson, Division of Nephrology, OBV-CD696, Department of Medicine, NYU School of Medicine, 550 First Avenue, New York, NY 10016. Phone: 212-263-7681; Fax: 212-263-7683; E-mail: nelsop02{at}popmail.med.nyu.edu
ABSTRACT. Collapsing glomerulopathy (CG) has become an importantcause of end-stage renal disease. Whether associated with HIV-1or other potential etiologies, the pathogenesis of CG convergesto induce aberrant proliferation of renal epithelium along theentire nephron. This raises the possibility that targeting cell-cycleprogression may be an effective therapeutic strategy for CG.Here, we ask whether the cyclin-dependent kinase (CDK) inhibitor,CYC202 (R-roscovitine), could attenuate or reverse existingrenal disease in Tg26 mice, a well characterized HIV-1 transgenicmouse model of CG. Tg26 mice were age and disease matched throughanalysis of urine (protein/creatinine) to generate 12 treatmentpairs covering a range of mild to severe CG. One mouse fromeach pair received either vehicle or 75 mg/kg of CYC202 every12 h for 20 d, a dose 20% above that needed to prevent the developmentof CG. After treatment, urinary, serologic, and histopathologicindices of nephrosis showed reversal of CG in 8 of 12 CYC202-treatedmice compared with progression of CG in 10 of 12 vehicle-treatedmice, demonstrating a significant therapeutic benefit from CYC202(P < 0.05). Pharmacokinetic profiles showed that concentrationsof CYC202 known to inhibit cell-cycle and transcriptional CDKin vitro were achieved in plasma at efficacious doses. However,amelioration of CG by CYC202 did not correlate with decreasesin kidney HIV-1 transgene expression, indicating that suppressionof HIV-1 transcription was not a prerequisite for the antiproliferativeactivity of CYC202. These results demonstrate a novel therapeuticstrategy for CG.
Within the last two decades, there has been an increased focuson collapsing glomerulopathy (CG) as an important cause of end-stagerenal disease. First delineated as a distinct clinicopathologicvariant of focal segmental glomerulosclerosis (FSGS) in the1980s, CG has emerged as a leading cause of renal failure inHIV-infected patients (1,2). Concurrently, CG has been increasinglyrecognized in non-HIV-infected patients without apparent cause(idiopathic), or in association with other immunodeficiencies,viral infections, autoimmune diseases, lymphoproliferative disorders,and drug exposures (1,2). Despite a poor understanding of howthese conditions may trigger CG, retrospective analyses haveshown that CG causes a surprisingly rapid loss of renal functionwhen compared with other variants of FSGS (3–6). Coupledwith the fact that CG is poorly responsive to standard therapiesfor FSGS (1,2), these epidemiologic and clinical observationshave engendered the need to evaluate therapeutic strategiesthat may attenuate or possibly reverse the progression of CGto end-stage renal disease.
Irrespective of the potential etiology, CG is marked by pathogenicproliferation of renal epithelium along the entire nephron.Underlying the acute loss of glomerular filtration barrier toplasma proteins, diseased glomeruli show mild to severe "pseudocresentic"podocyte hyperplasia with variably collapsed capillary loops(1,2). Detailed immunohistochemical analyses of cell-cycle proteinsin biopsy specimens have demonstrated that in CG, but not inother proteinuric lesions, podocytes lose constitutive expressionof endogenous cyclin kinase inhibitors such as p27kip1 and p57kip2even before appreciable capillary collapse, suggesting thatpodocyte proliferation is pathogenic for this unique glomerularphenotype (7–10). Similarly, aberrant cell-cycle engagementoccurs in renal epithelium along more distal nephron segments,leading to extensive microcystic tubular disease that can affectany segment of the renal tubule (1,2,10,11). Given the low mitoticindex in normal, mature renal parenchyma (12,13), this diffuseepithelial proliferation is a major disruption to the structure-functionrelationships required for physiologic nephron function, andsuggests that directly targeting cell-cycle progression maybe an effective therapeutic strategy for CG.
After extensive molecular, cellular, preclinical, and clinicalstudies, small molecule inhibitors of cyclin-dependent kinases(CDK) have become promising therapeutic agents (14,15). Categorizedon the basis of chemical structure, small molecule CDK inhibitors,otherwise termed pharmacologic cyclin kinase inhibitors (PCI),disrupt cellular ATP binding at the CDK catalytic site, therebyinhibiting CDK phosphorylation of target substrates (16). Thetherapeutic utility of PCI may be dictated by their bindingaffinities to the various CDK members (16). If a PCI inhibitsa cell-cycle CDK, such as CDK-1, –2, or –4, aberrantcell-cycle progression might be arrested at G1/S or G2/M checkpoints(14,15). Alternatively, if a PCI inhibits a transcriptionalCDK, such as CDK-7, –8, or –9, pathogenic processesthat require RNA polymerase II-mediated transcription can bedisrupted (14,15). This has led to intensive efforts to notonly identify highly selective PCI, but also to explore thetherapeutic use of PCI with a broad range of CDK selectivityin diseases where several CDK may be activated.
We recently investigated whether the combined antiproliferativeand antiviral activity of specific PCI may be exploited as atherapeutic strategy for CG triggered by HIV-1-encoded geneproducts. HIV-1 gene expression is critically dependent on CDK-9,–7, and/or –2 (17), and we and others have demonstratedthat roscovitine and flavopiridol (Table 1), small moleculesoriginally designed as cell-cycle PCI, potently suppress HIV-1transcription in infected lymphocytes and renal epithelium invitro (18–21). Because HIV-1 gene products can induceG1- to S-phase progression and dedifferentiation of renal parenchyma(18,22), this raised the possibility of targeting both a potentialetiology and the aberrant cell-cycle progression in CG. Indeed,we found that chronic treatment with flavopiridol, administeredto suppress HIV-1 transcription in mouse kidney, prevented thedevelopment of CG in Tg26 mice, a well characterized HIV-1 modelof CG (11,23–25), without alterations in global renalgene expression patterns or normal renal function (26). To extendthe clinical relevance of these observations and to test whetherother PCI show the same capacity, we ask here whether existingCG in Tg26 mice can be attenuated or reversed by chronic drugtreatment with R-roscovitine (CYC202), an effective cell-cyclePCI in rodent kidney in vivo that is well tolerated (27,28).Our results demonstrate a novel therapeutic strategy for CGand broaden the range of proliferative renal lesions that mayrespond to PCI.
Mice
All animal experiments were performed under Institutional AnimalCare and Use Committee-approved protocols. The Tg26 mouse line[TgN(pNL4-3d14)Lom], one of three HIV-1 NL4-3gag-pol transgeniclines developed and originally characterized for their renaldisease by the laboratories of Malcolm Martin and Albert Notkinsat the National Institutes of Health (23), was used for thisstudy. Triggered by HIV-1 transgene expression in the kidney(29), weaning-age heterozygous Tg26 mice progressively developseveral of the histopathologic and clinical hallmarks of humanCG, including collapsed glomerular capillaries, podocyte hyperplasia("pseudocrescents"), microcystic tubular disease, epithelialdedifferentiation, extracellular matrix deposition, proteinuria,hyperlipidemia, and progression to end-stage renal disease overmonths (11,23–26).
To define the threshold dose at which CYC202 could prevent CG,four prevention cohorts, each consisting of ten randomly selected21-d old heterozygous/nontransgenic sibling pairs, were enteredinto a 20-d drug treatment protocol to compare vehicle withincremental doses of CYC202 (described below). To determinewhether CYC202 could attenuate or reverse existing CG, Tg26heterozygotes between 35 and 45 d of age were disease-matchedthrough analysis of urine (protein/creatinine) (U[P/C]) beforeenrollment in the drug treatment protocol (described below).Twelve treatment pairs representing a range of mild to severeCG were generated. The beginning U[P/C] for these 12 treatmentpairs is as follows: 2.0/2.7; 5.9/2.8; 6.8/10.9; 13.9/11.3;15.3/14.6; 15.4/18.5; 19.4/21.0; 30.7/33.3; 41.1/34.3; 44.1/43.3;45.6/47.0; 61.8/74.8.
Drug Treatment Protocols
CYC202 (R-roscovitine; 6-benzylamino-2-[R-1-ethyl-2-hydroxyethylamino]-9-isopropylpurine)was supplied by Cyclacel Ltd., Dundee, UK. To determine at whatdose CYC202 could prevent CG in Tg26 mice, one mouse from eachsibling pair in the prevention cohorts received either CYC202(20 mg/kg, 40 mg/kg, or 60 mg/kg) or an equivalent volume ofvehicle (40:60 vol/vol DMSO:PBS, pH 7.4), respectively, by intraperitonealinjection every 12 h for 20 d. To determine whether CYC202 couldattenuate or reverse existing CG in Tg26 mice, one transgenicmouse from each treatment pair received either 75 mg/kg of CYC202or an equivalent volume of vehicle intraperitoneally every 12h for 20 d.
Collection and Analysis of Specimens
From day 17 to day 20 of the 20-d drug treatment protocols,urine was collected from each mouse and individually pooled.Three to 5 h after the final drug dose in each mouse, wholeblood and serum were collected by heart puncture, one kidneywas homogenized in TriZol reagent (Life Technologies, Gaithersburg,MA) for RNA extraction, and the contralateral kidney was fixedin 10% buffered formalin for histopathology. To screen for existingCG, urine was collected from each mouse and individually pooledover the 3 d before enrollment in the drug protocol. Serum concentrationsof albumin, blood urea nitrogen, total cholesterol and triglycerides,complete blood counts with differential, and urinary concentrationsof random protein and random creatinine were determined by theClinical Pathology Laboratory of the Center for ComparativeMedicine and Surgery at the Mount Sinai School of Medicine,New York.
Quantitative Histopathology
Quantitative histopathology of CG in each mouse at the conclusionof the drug treatment protocols was performed as described previously(26). The severity of CG was quantified in four full-lengthcoronal kidney sections, each representing a different level.The glomerular compartment was graded by determining the percentof glomeruli (in at least 100 total) with any glomerulosclerosisand/or podocyte hyperplasia. The tubulointerstitial compartmentwas graded by determining the percent area of cortical parenchymawith tubular microcysts and/or tubular atrophy with interstitialfibrosis. The mean of these percentages gave a final histopathologicgrade of CG, reported here as percentage of parenchyma, in eachmouse that could range from 0% (no glomerular or tubulointerstitialdisease) to 100% (all glomeruli and the entire cortical tubulointerstitialspace with disease). Sample identifications were blinded tothe pathologist before analysis.
Real-time RT-PCR
Real-time RT-PCR for the level of HIV-1 transcription in thekidneys of Tg26 mice was performed as described previously (26).Briefly, cDNA from 2 µg of whole kidney RNA was preparedwith the Omniscript RT Kit (Qiagen, Valencia, CA) and used forreal-time RT-PCR with SYBR Green PCR Core Reagents (AppliedBiosystems, Foster City, CA) on an iCycler (Bio-Rad Laboratories,Hercules, CA) to determine the relative kidney expression ofHIV-1 NL4-3 env (forward primer: 5'-TGTCCAAAGGTATCCTTTGAGCCAATTCC-3';reverse primer: 5'-AGTAGAAAAATTCCCCTCCACAATTA-3') versus glyceraldehyde-3-phosphatedehydrogenase (forward primer: 5'-ACCACAGTCCATGCCATCAC-3'; reverseprimer: 5'-TCCACCACCCTGTTGCTGTA-3'), a cellular gene not affectedby the concentrations of CYC202 used in this study (18). Threeseparate analyses were performed on each kidney RNA sample.
Microarray Gene Analysis
Microarray gene analysis for changes in renal gene expressionpatterns due to chronic CYC202 treatment (60 mg/kg every 12h for 20 d) was performed on Murine Genome U74Av2 Arrays (Affymetrix,Santa Clara, CA) by the Mount Sinai School of Medicine SharedMicroarray Facility as described previously (26). The analysiswas performed on two separate groups of mice (Table 2). Eachgroup consisted of one vehicle-treated non-transgenic mousewith no renal disease, one CYC202-treated non-transgenic mousewith no renal disease, and one CYC202-treated transgenic mouse,which responded to treatment. In each group, the CYC202-treatednon-transgenic and the CYC202-treated transgenic mice were eachcompared with the vehicle-treated non-transgenic mice to flaggenes that showed a twofold or greater change in expressionin both the non-transgenic and transgenic mice after chronicCYC202 treatment. Only genes that were flagged in the two separategroups of mice were considered to be indicative of an effectfrom chronic CYC202 treatment.
Pharmacokinetics
The pharmacokinetics of CYC202 in mice (CD-1, Harlan, UK) afterreceiving 20 mg/kg, 40 mg/kg, or 60 mg/kg of CYC202 were determinedby Cyclacel Ltd. Mice received CYC202 either by intraperitonealinjection (vehicle: 40:60 vol/vol DMSO:PBS) or by oral gavage(vehicle: 50 mM HCl in 0.9% NaCl) followed by heart puncturefor whole blood collection at 15, 30, 45, 60, 90, 120, 240,and 480 min after dosing. Plasma was immediately isolated fromwhole blood (10 min centrifugation at 3000 rpm) and flash frozenin liquid nitrogen. The frozen plasma samples were stored at–70°C until determination of CYC202 plasma concentrationson a LC-MS/MS mass spectrometer (Quattro Ultima, Waters/Micromass,UK). Three mice were studied at each time point.
Cell Cycle Kinomes
The comparison of whole kidney cell-cycle kinome profiles betweenone 40 d-old nontransgenic mouse (U[P/C] = 1.5, histopathologicalgrade of CG = 0%) and one diseased 40 d-old transgenic sibling(U[P/C] = 48, histopathological grade of CG = 30%) was performedon Kinetworks Cell Cycle Protein Screen immunoblots by KinexusBioinformatics (Vancouver, BC, Canada). As a result of rigorousvalidation of commercial antibodies, the mouse Kinetworks CellCycle Protein Screen provides unambiguous detection and relativequantitation of 30 mouse cell-cycle proteins (30). As per Kinetworkssample preparation protocols, whole kidney protein lysates fromeach mouse were prepared as follows: immediately after a rapidnephrectomy, one whole kidney was homogenized in ice cold KinetworksProtein Lysis Buffer and clarified by centrifugation for 20min at 4°C; after determination of the lysate protein concentrationby Bradford Reagent (Bio-Rad), lysates where boiled for 5 minin Kinetworks Analysis Buffer at a final concentration of 1µg/µl for subsequent analysis on Kinetworks CellCycle Protein Screens at Kinexus Bioinformatics.
Statistical Analyses
Unless indicated otherwise, data are expressed as mean ±SD. In the case of the treatment cohorts, CYC202 or vehicle-treatedtransgenic mice were defined as having reversed the progressionof existing CG with treatment if the U[P/C] at the end of the20-d drug treatment protocol was less than the U[P/C] at enrollmentonto the drug treatment protocol. For these data on drug efficacy,comparisons were made by two-tailed Fisher’s exact test.For all other data, comparisons were made by two-tailed t test.Significance was accepted at the 0.05 level of probability.
CDK Targets of CYC202 in CG
Of the more than 500 putative protein kinases in the mammaliankinome, it is clear that activation of even one can induce orfacilitate disease (31). This is important when consideringthe therapeutic use of kinase inhibitors such as PCI, wherespecificity, efficacy, and toxicity are largely determined notonly by the pharmacology of small molecules, but also by thepathogenic versus physiologic expression and activity of drugtargets in diseased versus normal tissue, respectively (32).Steady-state drug treatment of HIV-1-infected mouse podocytesin vitro showed that the IC50 for suppression of HIV-1 transcriptionby flavopiridol or R-roscovitine (CYC202) was approximatelyfourfold greater than the IC50 of each drug for CDK-9, but inthe case of CYC202, nearly 40-fold greater than the IC50 forCDK-2 (18) (Table 1). Thus, to understand how the dynamics ofdrug dosing may affect the CDK specificity of CYC202 in mousekidney in vivo, the pharmacokinetics of CYC202 in mice weredetermined for three separate incremental doses of CYC202 (Figure 1and Table 3). This analysis showed that irrespective of intraperitonealor oral administration, the exposure (area under the curve)of mouse kidney to CYC202 in the plasma at 20 mg/kg, 40 mg/kg,or 60 mg/kg remained the same because the mean retention timewas on average five times longer after oral dosing. However,the maximum concentration of CYC202 in plasma (Cmax) was significantlyhigher (3.5-fold) after intraperitoneally dosing, temporarilyexceeding the IC50 for suppression of HIV-1 transcription atall intraperitoneal doses, but only at the highest oral dose.
Figure 1. CYC202 bioavailability in the mouse. (A) Pharmacokinetic profiles of the plasma concentration of CYC202 in mice after a 20 mg/kg, 40 mg/kg, or 60 mg/kg intraperitoneal dose. Data points represent the average plasma concentration of CYC202 calculated from three mice. The arrow indicates the IC50 for suppression of HIV-1 transcription in mouse podocytes. (B) Pharmacokinetic profiles of the plasma concentration of CYC202 in mice after a 20 mg/kg, 40 mg/kg, or 60 mg/kg oral dose.
Table 3. Pharmacokinetic parameters of CYC202 in mice
Although these pharmacokinetics suggest that intraperitonealdosing in mice may more readily achieve therapeutic CYC202 plasmaconcentrations for HIV-induced CG, the availability of the desiredCDK targets of CYC202 for G1- to S- phase progression (CDK-2,–4, and –7) and HIV-1 transcription (CDK-9, –7,and –2) in Tg26 kidneys is not known. Because HIV-inducedCG involves aberrant cell-cycle progression and HIV-1 expressionin renal epithelium along the entire nephron (9,11,22,33), wecompared whole kidney cell-cycle kinomes of one 40-d-old nontransgenicmouse to a transgenic sibling with extensive CG (histopathologicgrade = 30% of parenchyma; U[P/C] = 48). Quantitation of therelative expression of CDK-2, –4, –7, and –9on mouse Kinetworks Cell Cycle Protein Screens showed that theseCDK are clearly expressed in the transgenic kidney with CG whencompared with the normal kidney (Figure 2). Although not statisticallyconclusive, these results are supported by previous reportsshowing upregulation of proliferating cell nuclear antigen anddownregulation of CDK-5 in CG (10,34). Given that in normal,mature kidney parenchyma, CDK are rarely engaged in cell-cycleprogression and are not active in HIV-1 transcription (12,13),these results suggest that CYC202 may target several CDK ofpotential importance to the pathogenesis of HIV-induced CG inTg26 mice.
Figure 2. Cell-cycle kinome profiles in kidneys of Tg26 mice. (A) Kinetworks Cell Cycle Protein Screen immunoblots of whole kidney protein extracts from one normal, nontransgenic and one diseased, transgenic sibling. Identification of protein bands for cyclin-dependent kinase (CDK)-7 (#1), proliferating cell nuclear antigen (#2), CDK-2 (#3), CDK-5 (#4), CDK-9 (#5), and CDK-4 (#6) are indicated. (B) Relative change in expression (%) in the diseased transgenic (Tg) kidney when compared with the nontransgenic (Non-Tg) kidney.
CYC202 Prevents CG
Renal disease in the HIV-1 transgenic mouse, Tg26, recapitulatesmany of the histopathologic and clinical aspects of human CG(11,23–26). We demonstrated recently that flavopiridol,a more potent PCI of CDK-9 when compared with other CDK (Table 1),prevented CG in Tg26 mice when therapy was commenced atweaning age, the point of disease onset (26). To determine thethreshold dose at which CYC202, a more potent PCI of CDK-2 whencompared with other CDK (Table 1), could also prevent CG, cohortsof 21-d-old Tg26 heterozygous and nontransgenic siblings weretreated with either vehicle or 20 mg/kg incremental doses ofCYC202 intraperitoneally every 12 h for 20 d, the identicaldosing regimen used in the flavopiridol study (26). Comparisonof U[P/C] levels and histopathological grade of CG between cohortsat the end of the drug treatment protocol showed that preventionof CG by CYC202 began to occur at a threshold dose of 60 mg/kg(Figure 3). As in the flavopiridol study (26), there was a trendtoward a decrease in leukocyte counts in both non-transgenicand transgenic mice at this efficacious dose without any significantabnormalities in growth, or gastrointestinal or other hematologicindices (Table 4). In contrast to the flavopiridol study (26),chronic CYC202 treatment did not reduce kidney HIV-1 transcriptlevels in the responsive cohort receiving 60 mg/kg when comparedwith the vehicle-treated cohort (percentage change in [HIV-1env/G3PDH] in the CYC202-treated transgenic mice at 20 mg/kgwas –2% ± 50%; at 40 mg/kg was –1% ±47%; and at 60 mg/kg was + 8% ± 62%). Interestingly,unlike the flavopiridol study where dosing maintained drug troughlevels above the IC50 for suppression of HIV-1 transcription(26), CYC202 plasma levels fall beneath the IC50 for suppressionof HIV-1 transcription within 5 to 6 h after a 60 mg/kg intraperitonealdose (Figure 1). However, whether pharmacokinetics or some othermechanism explains this difference in PCI specificity is notexplored here. Nonetheless, these results do suggest that continualsuppression of HIV-1 expression in Tg26 kidney was not requiredfor CYC202 to prevent CG.
Figure 3. Dose-dependent prevention of collapsing glomerulopathy (CG) by CYC202. (A) Final urine (protein/creatinine) (U[P/C]) levels in Tg26 cohorts (Tg = transgenic; Non-Tg = non-transgenic) after receiving vehicle or 20 mg/kg incremental doses of CYC202 intraperitoneally every 12 h for 20 d. Final U[P/C] in Tg mice are as follows: vehicle, 33.4 ± 39.5; 20 mg/kg, 31.4 ± 23.2; 40 mg/kg, 30.2 ± 13.8; 60 mg/kg, *4.8 ± 3.3. Horizontal bars indicate the average U[P/C] in the Tg mice. *P < 0.05 when compared with vehicle-treated Tg mice. (B) Final histopathologic grade of CG in the same treatment cohorts as in Panel A. Final histopathologic grade of CG in Tg mice are as follows: vehicle, 18.6 ± 16.5; 20 mg/kg, 19.4 ± 17.6; 40 mg/kg, 13.8 ± 15.3; 60 mg/kg, *4.6 ± 2.6. Horizontal bars indicate the average histopathologic grade in Tg mice. *P < 0.05 when compared with vehicle-treated Tg mice.
Table 4. Toxic effects of chronic CYC202 treatment in the prevention cohorts
CYC202 Reverses CG
Compared with patients with other variants of FSGS, patientswith CG have a significantly greater incidence of nephroticsyndrome at the time of diagnosis (1,2). Thus, to determinewhether CYC202 may not only prevent but also attenuate or reverseCG, we enrolled Tg26 mice with existing CG into the CYC202 treatmentprotocol. One mouse from each of 12 age- and disease-matchedtreatment pairs, representing a range of mild to severe CG,received either vehicle or 75 mg/kg CYC202 every 12 h for 20d, a dose 20% above the threshold needed to prevent CG. Comparisonof U[P/C] at the beginning and end of the drug treatment protocolin each mouse showed that 8 of 12 CYC202-treated mice had decreasedproteinuria after treatment, whereas 10 of 12 vehicle-treatedmice had increased proteinuria after treatment (Figure 4), demonstratinga significant therapeutic benefit from CYC202 in reversing theclinical progression of CG (P < 0.05). Furthermore, the rateof increase in U[P/C] in the remaining four CYC202-treated micewas nearly twofold less when compared with their vehicle-treatedcontrol match (percentage increase per day of 6.4 ± 1.3%versus 10.2 ± 3.8%, respectively). Analysis of the histopathologic(Figure 4 and Figure 5) and serologic (Table 5) indices of nephrosisafter drug treatment confirmed that the clinical progressionof existing CG in Tg26 mice was reversed or attenuated by CYC202.
Figure 4. Reversal and attenuation of existing collapsing glomerulopathy (CG) by CYC202. (A) Change in urine (protein/creatinine) (U[P/C]) levels during the 20-d drug treatment protocol in vehicle-treated transgenic mice covering a range of mild to severe CG at the beginning of the protocol (day 0). Solid lines indicate an increase in U[P/C] from day 0 to day 20. Dashed lines indicate a decrease in U[P/C] from day 0 to day 20. (B) Change in U[P/C] levels in the disease-matched transgenic mice that received CYC202. (C) Listing of the final histopathologic grade of CG after treatment (C = CYC202; V = vehicle) in matched pairs. Matched pairs are listed in descending order from the highest beginning (U[P/C]) to the lowest beginning U[P/C]. CYC202- and vehicle-treated mice that had an increase in U[P/C] during treatment are indicated.
Figure 5. Histopathology of existing collapsing glomerulopathy (CG) after 20 d of drug treatment. (A) Periodic acid Schiff-stained kidney section of the transgenic with a beginning urine (protein/creatinine) (U[P/C]) of 47, an end U[P/C] of 82.6, and a final histopathologic grade of CG of 52% of renal parenchyma after treatment with vehicle (original magnification, x100). (B) Periodic acid Schiff-stained kidney section of the disease-matched transgenic of Panel A with a beginning U[P/C] of 45.6, an end U[P/C] of 44.3, and a final histopathologic grade of CG of 12% of renal parenchyma after treatment with CYC202 (original magnification, x100). Residual focal fibrotic changes are seen in both the glomerular and tubulointerstitial compartments of this CYC202-treated transgenic mouse.
Table 5. Serologic indices of nephrosis in the treatment cohorts
Pleotropic Therapeutic Activity of CYC202
On the basis of their potential to inhibit transcriptional CDK,cell-cycle PCI have been postulated to exhibit nonselectivetherapeutic benefits through modulation of cellular transcription(35,36). By use of microarray gene analysis, we found that dosesof flavopiridol, which prevented CG in Tg26 mice, dysregulatedless than 3% of cellular renal genes, none of which were identifiedas genes directly involved in cell-cycle progression (26). Todetermine whether the expression of any cellular renal geneswould be altered by a similarly efficacious dose of CYC202 (60mg/kg every 12 h for 20 d), we repeated the same Affymetrixmicroarray analysis on CYC202-treated mice. This analysis showedthat only 0.3% (21 of 5400) of the genes expressed in kidneysof vehicle-treated mice were dysregulated by twofold or greaterwith CYC202 treatment (examples in Table 6). The expressionof the vast majority of these genes were induced, not suppressed,as a result of chronic CYC202 treatment, and none of these genesare known to directly control cell-cycle progression.
Table 6. Genes dysregulated by at least twofold in Tg26 kidney with chronic CYC202 treatment
Because our previous study in Tg26 mice showed that chronicflavopiridol treatment decreased normal lipid levels in non-transgenicmice and corrected hyperlipidemia in transgenic mice beyondthat expected from the amelioration of CG (26), we determinedif CYC202 also altered lipid metabolism in Tg26 mice. At theconclusion of the 20-d drug treatment protocol, the concentrationsof total cholesterol and triglycerides in whole serum were determinedfor each mouse in the prevention cohorts. With each incrementaldose of CYC202, the hyperlipidemia of CG progressively correctedto normal levels by the threshold dose for prevention of CG(Table 7). However, total cholesterol and triglycerides werealso suppressed by CYC202 in the nontransgenic mice, suggestingthat this correction of hyperlipidemia by CYC202, as with flavopiridol,is not entirely attributable to the amelioration of CG.
Clinical and epidemiologic studies suggest that CG from anycause poses a significant therapeutic challenge (3–6).However, no currently available therapies for CG (corticosteroids,cytotoxic agents, other immunomodulators, highly active antiretroviralregimens, angiotensin-converting enzyme inhibitors) are knownto directly target pathogenic proliferation of renal parenchyma.Here, we extend our previous preclinical study with flavopiridolto show that a second PCI, CYC202, not only prevents, but reversesand attenuates the clinical progression of CG in Tg26 mice.Coupled with the fact that CG can develop in HIV- and non-HIV-infectedpatients (1,2), our data strongly suggest for the first timethat CG from any potential etiology may benefit from PCI treatment.Thus, bearing in mind the need for their careful evaluationin patients with CG-associated illnesses, PCI are promisingadditions to the limited therapeutic options for CG and otherproliferative renal lesions.
Even though both flavopiridol and CYC202 can ameliorate renaldisease in Tg26 mice, differences in drug specificity may haveimportant therapeutic implications for CG associated with HIV-1infection. Consistent with their CDK inhibition profiles (Table 1),flavopiridol, but not roscovitine, has been shown to suppressCDK-9-mediated HIV-1 transcription and replication at severalfoldlower concentrations than that needed to inhibit cell-cycleprogression (20,21). Congruent with this, we showed that roscovitine,but not flavopiridol, significantly inhibited proliferationof HIV-infected podocytes in vitro despite equivalent suppressionof HIV-1 expression by both PCI (18). This divergence in specificitywas further evidenced in vivo, where prevention of CG by flavopiridol(26), but not by CYC202, correlated with the suppression ofHIV-1 expression in Tg26 kidneys, indicating that cell-cyclePCI antitranscriptional activity is not a prerequisite for drugefficacy in CG. Interestingly, HIV-1 expression in Tg26 kidneyis lost as renal disease develops (24–26), and in humanbiopsy samples, nephron sites of HIV-1 expression do not consistentlyoverlap sites of histopathologic disease (33), suggesting thatthe progression of CG may become independent of HIV-1 expression.Nonetheless, any use of PCI in HIV-infected patients must considertheir potential effect on the dynamics of HIV-1 replicationand the reconstitution of cellular-based immunity (17). Promisingly,unlike the apparent idiosyncratic decrease in Tg26 leukocyteswith flavopiridol (26) or CYC202 treatment, early clinical useof these PCI has not uncovered any significant adverse changein immune function in humans (14,15).
Results from this animal study with CYC202 and from the previousstudy with flavopiridol suggest that PCI may have pleiotropictherapeutic activity in nephrosis. Consistent with previousgene expression profiling of in vitro treated cells (35,36),we found that efficacious doses of flavopiridol dysregulatemore cellular genes in the kidney than efficacious doses ofCYC202 (26). Yet after examining the identity of these genes,we did not find clear evidence that either CYC202 or flavopiridolprevented CG by modulating the transcription of genes that directlycontrol cell-cycle progression. However, both CYC202 and flavopiridoldecreased serum levels of total cholesterol and triglyceridesin normal and diseased Tg26 mice that could not be entirelyattributed to the amelioration of CG (26). Given that flavopiridoland CYC202 fall into different chemical classes, these resultsraise the possibility that PCI in general may modulate a centralbut as yet undefined mechanism in lipid metabolism. This mayrepresent a previously unrecognized benefit from PCI in nephrosis.Interestingly, the anti-lipidemic 3-hydroxy-3-methyl glutaryl-CoAreductase inhibitors have been shown to inhibit CDK activationfrom small GTPases by preventing isoprenylation (37), but whethera similar reciprocal relationship exists between CDK inhibitionand lipid metabolism has not been established. Other drugs thatmay prove to be beneficial in CG induced by HIV-1 (38) alsodisplay pleiotropic therapeutic activity. For example, HIV-1protease inhibitors have demonstrated significant anti-inflammatory,anti-fibrotic, and antitumorigenic activity through modulationof host, not HIV-1, proteases (39,40). These examples highlightthe importance of investigating drug specificity, a considerationfor PCI as they are evaluated further in proliferative renaldiseases.
In conclusion, we have demonstrated in a second "proof-of-concept"preclinical study that targeting aberrant proliferation of renalparenchyma may be an effective therapeutic strategy for CG.We found that reversal and attenuation of the progression ofHIV-induced CG was clinically evident after 20 d of continuoustreatment of Tg26 mice with the cell-cycle PCI, CYC202. Longerperiods of observation and/or PCI treatment will be needed todetermine if responders, particularly those with only mild decreasesin U[P/C], continue to regress or enter remission with chronicchanges in renal histopathology and renal function (41). Ineither respect, this study supports the further evaluation ofPCI in CG and in other proliferative renal diseases.
Acknowledgments
We thank Leslie Bruggeman for the gift of Tg26 mice, Iain Stuartfor help with pharmacokinetic analyses, and Luis Schang andFatah Kashanchi for helpful discussions. This work was supportedin part by a National Kidney Foundation research award (P.J.N.)and by Cyclacel Ltd., and was presented in abstract form atthe 36th Annual Meeting of the American Society of Nephrology.
Footnotes
See related editorial, "Negatively Regulating the Cell CycleCan Be Positive," on pages 1361-1362.
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Received for publication October 8, 2003.
Accepted for publication February 10, 2004.
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Abstract
Introduction
gag-pol transgenic lines developed and originally characterized for their renal disease by the laboratories of Malcolm Martin and Albert Notkins at the National Institutes of Health (23), was used for this study. Triggered by HIV-1 transgene expression in the kidney (29), weaning-age heterozygous Tg26 mice progressively develop several of the histopathologic and clinical hallmarks of human CG, including collapsed glomerular capillaries, podocyte hyperplasia ("pseudocrescents"), microcystic tubular disease, epithelial dedifferentiation, extracellular matrix deposition, proteinuria, hyperlipidemia, and progression to end-stage renal disease over months (11,23–26). 
