Effect of disease progression on the podocyte cell cycle in Alport Syndrome

Abstract

Progression of glomerulosclerosis is associated with loss of podocytes with subsequent glomerular tuft instability. It is thought that a diminished number of podocytes may be able to preserve tuft stability through cell hypertrophy associated with cell cycle reentry. At the same time, reentry into the cell cycle risks podocyte detachment if podocytes cross the G1/S checkpoint and undergo abortive cytokinesis. In order to study cell cycle dynamics during chronic kidney disease (CKD) development, we used a FUCCI model (fluorescence ubiquitination-based cell cycle indicator) of mice with X-linked Alport Syndrome. This model exhibits progressive CKD and expresses fluorescent reporters of cell cycle stage exclusively in podocytes. With the development of CKD, an increasing fraction of podocytes in vivo were found to be in G1 or later cell cycle stages. Podocytes in G1 and G2 were hypertrophic. Heterozygous female mice, with milder manifestations of CKD, showed G1 fraction numbers intermediate between wild-type and male Alport mice. Proteomic analysis of podocytes in different cell cycle phases showed differences in cytoskeleton reorganization and metabolic processes between G0 and G1 in disease. Additionally, in vitro experiments confirmed that damaged podocytes reentered the cell cycle comparable to podocytes in vivo. Importantly, we confirmed the upregulation of PDlim2, a highly expressed protein in podocytes in G1, in a patient with Alport Syndrome, confirming our proteomics data in the human setting. Thus, our data showed that in the Alport model of progressive CKD, podocyte cell cycle distribution is altered, suggesting that cell cycle manipulation approaches may have a role in the treatment of various progressive glomerular diseases characterized by podocytopenia. Progression of glomerulosclerosis is associated with loss of podocytes with subsequent glomerular tuft instability. It is thought that a diminished number of podocytes may be able to preserve tuft stability through cell hypertrophy associated with cell cycle reentry. At the same time, reentry into the cell cycle risks podocyte detachment if podocytes cross the G1/S checkpoint and undergo abortive cytokinesis. In order to study cell cycle dynamics during chronic kidney disease (CKD) development, we used a FUCCI model (fluorescence ubiquitination-based cell cycle indicator) of mice with X-linked Alport Syndrome. This model exhibits progressive CKD and expresses fluorescent reporters of cell cycle stage exclusively in podocytes. With the development of CKD, an increasing fraction of podocytes in vivo were found to be in G1 or later cell cycle stages. Podocytes in G1 and G2 were hypertrophic. Heterozygous female mice, with milder manifestations of CKD, showed G1 fraction numbers intermediate between wild-type and male Alport mice. Proteomic analysis of podocytes in different cell cycle phases showed differences in cytoskeleton reorganization and metabolic processes between G0 and G1 in disease. Additionally, in vitro experiments confirmed that damaged podocytes reentered the cell cycle comparable to podocytes in vivo. Importantly, we confirmed the upregulation of PDlim2, a highly expressed protein in podocytes in G1, in a patient with Alport Syndrome, confirming our proteomics data in the human setting. Thus, our data showed that in the Alport model of progressive CKD, podocyte cell cycle distribution is altered, suggesting that cell cycle manipulation approaches may have a role in the treatment of various progressive glomerular diseases characterized by podocytopenia. Translational StatementLoss of podocytes characterizes disease progression in many glomerular diseases. Cell hypertrophy offers adaptation to podocyte loss, but reentry into the cell cycle leads to podocyte detachment if podocytes progress beyond the G1 stage. Using a mouse model with a fluorescent reporter of the cell cycle, we established that podocyte cell cycle phase distribution in progressive chronic kidney disease shifts from predominantly in G0 to increasingly in G1, followed by S and G2 phases. These studies could lead to the discovery of new cell cycle–modifying drugs that allow progression to G1, but not beyond, thus promoting stable hypertrophy of podocytes and prolonging renal function. Loss of podocytes characterizes disease progression in many glomerular diseases. Cell hypertrophy offers adaptation to podocyte loss, but reentry into the cell cycle leads to podocyte detachment if podocytes progress beyond the G1 stage. Using a mouse model with a fluorescent reporter of the cell cycle, we established that podocyte cell cycle phase distribution in progressive chronic kidney disease shifts from predominantly in G0 to increasingly in G1, followed by S and G2 phases. These studies could lead to the discovery of new cell cycle–modifying drugs that allow progression to G1, but not beyond, thus promoting stable hypertrophy of podocytes and prolonging renal function. For more than 20 years, a unifying hypothesis explaining progression to kidney failure in many glomerular diseases is based on podocyte loss and its effect on glomerular tuft stability.1Kriz W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar In human disease and experimental models2Lemley K.V. Lafayette R.A. Safai M. et al.Podocytopenia and disease severity in IgA nephropathy.Kidney Int. 2002; 61: 1475-1485Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar,3Wharram B.L. Goyal M. Wiggins J.E. et al.Podocyte depletion causes glomerulosclerosis: diphtheria toxin-induced podocyte depletion in rats expressing human diphtheria toxin receptor transgene.J Am Soc Nephrol. 2005; 16: 2941-2952Crossref PubMed Scopus (560) Google Scholar when ∼20% to 40% of podocytes are lost, subsequent podocyte loss and glomerular sclerosis ensue. Podocyte loss and shedding from the glomerular tuft in many cases seem to be caused when podocytes undergo abortive cytokinesis after passing through the S stage of the cell cycle.4Lemley K.V. Glomerular pathology and the progression of chronic kidney disease.Am J Physiol Renal Physiol. 2016; 310: F1385-F1388Crossref PubMed Scopus (13) Google Scholar Because of their particular location and anatomy,5Kriz W. Lemley K.V. A potential role for mechanical forces in the detachment of podocytes and the progression of CKD.J Am Soc Nephrol. 2015; 26: 258-269Crossref PubMed Scopus (161) Google Scholar podocytes cannot undergo cytokinesis without detachment into Bowman’s space. Binucleate podocytes are indeed seen in kidney biopsies6Nagata M. Yamaguchi Y. Komatsu Y. Ito K. Mitosis and the presence of binucleate cells among glomerular podocytes in diseased human kidneys.Nephron. 1995; 70: 68-71Crossref PubMed Scopus (63) Google Scholar and in the urine of patients with progressive chronic kidney disease (CKD),7Vogelmann S.U. Nelson W.J. Myers B.D. Lemley K.V. Urinary excretion of viable podocytes in health and renal disease.Am J Physiol Renal Physiol. 2003; 285: F40-F48Crossref PubMed Scopus (269) Google Scholar providing evidence that entering cytokinesis precedes shedding of viable podocytes. To avoid reentry into the cell cycle, postnatal podocytes express high levels of cyclin-dependent kinase inhibitors,8Shankland S.J. Eitner F. Hudkins K.L. et al.Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.Kidney Int. 2000; 58: 674-683Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar such as p21 and p27. Because of their inability to undergo regenerative replication, the glomerulus can only adapt to podocyte loss by hypertrophy of the remnant podocytes, thus allowing more effective structural coverage of the glomerular tuft by a diminished number of podocytes.1Kriz W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar,9Wiggins J.E. Goyal M. Sanden S.K. et al.Podocyte hypertrophy, “adaptation,” and “decompensation” associated with glomerular enlargement and glomerulosclerosis in the aging rat: prevention by calorie restriction.J Am Soc Nephrol. 2005; 16: 2953-2966Crossref PubMed Scopus (236) Google Scholar In this article, we quantitatively assess the progressive reentry of podocytes into the cell cycle by using a novel mouse cell cycle reporter construct10Abe T. Sakaue-Sawano A. Kiyonari H. et al.Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter.Development. 2013; 140: 237-246Crossref PubMed Scopus (102) Google Scholar,11Mort R.L. Ford M.J. Sakaue-Sawano A. et al.Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.Cell Cycle. 2014; 13: 2681-2696Crossref PubMed Scopus (68) Google Scholar applied to a model of progressive glomerular disease, Alport syndrome (AS), characterized by defective α3α4α5(IV) collagen production by podocytes.12Kashtan C.E. Ding J. Garosi G. et al.Alport syndrome: a unified classification of genetic disorders of collagen IV α345: a position paper of the Alport Syndrome Classification Working Group.Kidney Int. 2018; 93: 1045-1051Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar We show an increased number of podocytes in the G1 phase of the cell cycle accompanied by an increase in cell hypertrophy during the disease progression in both male and female AS mice. Our in vitro experiments, together with proteomic data on podocytes in G1 and G0 phases, highlighted that in disease and during reentry into the cell cycle, podocytes undergo a global reorganization of their proteome with a dramatic modulation of protein abundance in metabolic and cytoskeleton In we that in the discovery of that allow progression to G1, but not beyond, thus promoting and glomerular and function. To study the podocyte cell cycle in a CKD we FUCCI (fluorescence cell cycle indicator) R.L. Ford M.J. Sakaue-Sawano A. et al.Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.Cell Cycle. 2014; 13: 2681-2696Crossref PubMed Scopus (68) Google Scholar with X-linked AS A. et of cells progression of renal Am Soc Nephrol. PubMed Scopus Google H. M. et cell in Alport PubMed Scopus Google model of X-linked Alport Am Soc Nephrol. PubMed Scopus Google Scholar male mice with a show of progress to kidney disease with a of to Heterozygous female mice with kidney of were and experiments were in to the of for the and of and and of urine and were to and in experimental and for analysis podocyte and and glomerular in AS from a male patient of and urine of pathology showed glomerular and with A from the of a male patient with renal cell used as a were by the of and after patient were used to the podocyte and FUCCI expression and podocyte using our established A. et of cells progression of renal Am Soc Nephrol. PubMed Scopus Google H. M. et cell in Alport PubMed Scopus Google model of X-linked Alport Am Soc Nephrol. PubMed Scopus Google Lemley K.V. et novel of 2013; PubMed Scopus Google Scholar in with a using and were used to a For were and with and mouse and and protein for human For podocytes were and with an with a and to were with a in with For were and for to podocyte expression in different phases of the cell used as a podocytes from FUCCI mice were using for a by of the podocyte podocytes were for in Lemley K.V. et novel of 2013; PubMed Scopus Google Y. K. N. et and of expression in podocytes and of as a of J Physiol Renal Physiol. PubMed Scopus Google Scholar and for to different of or in podocytes by 2018; PubMed Scopus (13) Google Scholar and cells were through cell To the podocyte number in tissue were and used to glomerular glomerular and the glomerular tuft by using of in were using with differences between were using analysis of for experiments and an for data for of the between the of are expressed as Podocytes different phases of the cell cycle and in AS and wild-type FUCCI mice were used to a the podocyte as and protein a used to and K. Y. et for proteome of PubMed Scopus Google Scholar data were using an and data have to the the with the data on data and analysis are in To study the cell cycle phase distribution in podocytes, we the mouse with a mouse expressing Cre the and the FUCCI reporters in podocytes in mouse FUCCI is based on fluorescent and to and specific cell cycle T. Sakaue-Sawano A. Kiyonari H. et al.Visualization of cell cycle in mouse embryos with Fucci2 reporter directed by Rosa26 promoter.Development. 2013; 140: 237-246Crossref PubMed Scopus (102) Google Scholar,11Mort R.L. Ford M.J. Sakaue-Sawano A. et al.Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.Cell Cycle. 2014; 13: 2681-2696Crossref PubMed Scopus (68) Google in G1 and cells the S is by from the S phase through the G1 phase are in the phase are and in the G1/S both are cells In G0 and G1 phases, both reporters are and cells have showed podocytes expressing podocytes expressed We from mice and showed the expression of both FUCCI in podocytes as reentered the cell cycle in vitro To the specific expression of the FUCCI in podocytes, we used a to podocytes in G0 and G1 not express FUCCI reporter in and of cells were for We podocytes in different cell cycle phases. in of podocytes are in G1 are in G1 phase in S phase and and in G2 phase In the or and confirming that the FUCCI are expressed exclusively in podocytes. 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PubMed Scopus Google Scholar and in F. et podocyte detachment and progressive podocyte loss from with in Alport Int. Full Text Full Text PDF PubMed Scopus Google Scholar Podocyte cell cycle reentry in kidney disease J.E. Goyal M. Sanden S.K. et al.Podocyte hypertrophy, “adaptation,” and “decompensation” associated with glomerular enlargement and glomerulosclerosis in the aging rat: prevention by calorie restriction.J Am Soc Nephrol. 2005; 16: 2953-2966Crossref PubMed Scopus (236) Google N. et podocyte hypertrophy glomerular in mice and PubMed Scopus Google Scholar FUCCI model to study the podocyte cell cycle in the to cells to their cell cycle studies have podocyte cell cycle reentry using in podocytes in M. A. et cycle podocytes to Cycle. 2016; PubMed Scopus Google Scholar or using podocyte such as F. et podocyte cell cycle and by PubMed Scopus Google M. et al.Podocyte loss in 2013; PubMed Scopus Google Scholar We for the podocyte cell cycle reentry with disease progression in the of podocytes in G1 increased in kidney disease with AS to mice. In male AS mice more than a of podocytes reentered the cell cycle, in male mice, of podocytes were in increased of G1 podocytes in AS associated with of CKD, such as and we showed that in have milder than male mice and the of cells in G1 than that in AS male mice of the same We a in S phase in kidney disease. This a loss of of cyclin-dependent kinase S.J. Eitner F. Hudkins K.L. et al.Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.Kidney Int. 2000; 58: 674-683Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar to the cell G1/S the of podocyte This in S not followed by a in G2 of an increased loss of podocytes in the urine during abortive cytokinesis after S Because an effective of podocytes is not the to podocyte loss is through cell hypertrophy, allowing a number of podocytes to more the W. Gretz N. Lemley K.V. Progression of glomerular diseases: is the podocyte the culprit?.Kidney Int. 1998; 54: 687-697Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar,9Wiggins J.E. Goyal M. Sanden S.K. et al.Podocyte hypertrophy, “adaptation,” and “decompensation” associated with glomerular enlargement and glomerulosclerosis in the aging rat: prevention by calorie restriction.J Am Soc Nephrol. 2005; 16: 2953-2966Crossref PubMed Scopus (236) Google Scholar is evidence of podocyte from S.J. podocytes be in 26: PubMed Scopus Google Scholar we the for podocyte in this as this not seem to be able to in disease or podocyte hypertrophy is after a number of podocytes are a of glomerular hypertrophy of glomerular of a unifying of glomerular Int. 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