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review http://www.kidney-international.org & 2007 International Society of Nephrology Podocytes in culture: past, present, and future SJ Shankland1, JW Pippin1, J Reiser2 and P Mundel3 1 Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington, USA; 2Nephrology Division, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA and 3Department of Medicine, Mount Sinai School of Medicine, New York, New Yor
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  Podocytes in culture: past, present, and future SJ Shankland 1 , JW Pippin 1 , J Reiser 2 and P Mundel 3 1 Division of Nephrology, Department of Medicine, University of Washington, Seattle, Washington, USA;  2 Nephrology Division,Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts, USA and   3 Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA Human genetic and  in vivo  animal studies have helped todefine the critical importance of podocytes for kidneyfunction in health and disease. However, as in any otherresearch area, by default these approaches do not allow formechanistic studies. Such mechanistic studies require theavailability of cells grown  ex vivo  (i.e., in culture) with theability to directly study mechanistic events and controlthe environment such that specific hypotheses can be tested.A seminal breakthrough came about a decade ago with thedocumentation of differentiation in culture of primary rat andhuman podocytes and the subsequent development of conditionally immortalized differentiated podocyte cell linesthat allow deciphering the decisive steps of differentiationand function of ‘ in vivo ’ podocytes. Although this paper is notintended to provide a comprehensive review of podocytebiology, nor their role in proteinuric renal diseases orprogressive glomerulosclerosis, it will focus specifically onseveral aspects of podocytes in culture. In particular, we willdiscuss the scientific and research rationale and need forcultured podocytes, how podocyte cell-culture evolved, andhow cultured podocytes are currently being used to uncovernovel functions of podocytes that can then be validated in vivo  in animal or human studies. In addition, we provide adetailed description of how to properly culture andcharacterize podocytes to avoid potential pitfalls. Kidney International   (2007)  72,  26–36; doi:10.1038/sj.ki.5002291;published online 25 April 2007KEYWORDS: podocyte biology; cultivation of differentiated podocytes;differentiation markers; proteinuric kidney diseases RATIONALE FOR THE NEED OF PODOCYTES IN CULTURE The study of kidney disease is complex because the onset isoften undetected, diseases may be acute or chronic in nature,the genetic makeup of the host leads to variable clinicalsyndromes, and multiple organs are often involved simulta-neously. To study disease susceptibility, mechanisms, prog-nosis, and potential therapies, most authorities, including theauthors, would say that studying humans is ideal and, inmost instances, this is the gold standard. However, this is notalways possible and we would argue that this is an exceptionrather than a rule. Moreover, many diseases, in particularkidney diseases, are not that common in the generalpopulation, thus increasing the challenge of limiting studiesto man. There is no doubt that the research questions shouldprimarily stem from patients with kidney disease. However,studies verifying proof of principle are limited, if notimpossible in man.Thus, in renal and nonrenal research, the use of experimental models in animals has proved invaluable.Moreover, the genome era with the development of null ortransgenic mice, and more recently, with the ability to restrictor delete expression to a specific cell, has significantly advanced our understanding of many aspects of kidney disease. Nevertheless, animal models are often limitedbecause they do not always fully replicate their humancounterpart. For example, the current mouse models of diabetic nephropathy do not typically demonstrate thefeatures of the human disease such as Kimmelstiel–Wilsonnodules. Moreover, there are no mouse models of otherpodocyte diseases such as membranous nephropathy and thebackground mouse strain plays a critical role in diseaseinitiation and progression. 1 Similarly, while positionalcloning has proven invaluable and has uncovered mutationssuch as those in phospholipase C epsilon (PLCE1) underlyingnephrotic syndrome, PLCE1-null mice do not have any overt phenotype, 2 suggesting that animal models too may be limited. Yet, well-characterized animal models doprovide insights into disease, albeit often descriptive, andin many instances, provide a good starting point for thedecision to try a particular therapy in human disease. Mostsignificantly, however, despite the many merits of transgenic/null mice and animal models, they do not, by default,allow for mechanistic studies, calling for the use of cellsgrown  in vitro . review  http://www.kidney-international.org &  2007 International Society of Nephrology Received 2 March 2007; accepted 7 March 2007; published online25 April 2007Correspondence:  P Mundel, Division of Nephrology, Mount Sinai School of Medicine, One Gustave L. Levy Place, Box 1243, New York, NY 10029-6574,USA. E-mail: peter.mundel@mssm.edu 26  Kidney International   (2007)  72 , 26–36  WHERE DID CELL-CULTURE STUDIES BEGIN? The initial studies using cells from either man or animalsgrown in culture date back to the 1950, 3–5 including cellsderived from the kidney. 6 Although cells grown  ex vivo  (i.e.,in culture) do not fully replicate the  in vivo  environment,they have several major advantages. These include the ability to directly study mechanistic events, to control the environ-ment such that specific hypotheses can be tested, and thatmultiple experiments can be performed to validate the initialobservations. Although perhaps obvious, it is critical to usepodocytes in culture to study events related to podocytes.Thus, although informative, the use of other cells such ashuman embryonic kidney cell line, COS, and mouseembryonic fibroblasts (MEFs) to study podocytes is not fully representative and thus has the potential to be erroneous.Although these nonpodocyte cells might be useful to initially characterize novel protein:protein interactions, they are notpodocytes and therefore the functional relevance of thesenovel interactions need to be confirmed in podocytes togeneralize these findings to podocytes.There is a large body of literature using cells in culture incardiac, central and peripheral nervous system, liver, blood,immune, and other organ-system research. Each cell-culturesystem has unique properties and characteristics that differfrom others and podocytes are no exception. In this review we will provide comprehensive and compelling support thatthe study of podocytes in culture is a valid and invaluablemodel system, when used together with animal models andhuman studies, to uncover novel and mechanistic events inthese important cells. It is our belief that with each modelsystem (man, animal, cells), validation of the results need tobe confirmed with a different model system. THE PAST: HISTORY OF PODOCYTES IN CULTURE Cultivation of podocytes  in vitro  was first introduced in themid-1970s 7–9 and glomerular epithelial cells in culture from avariety of species have since been described in man, 7,8,10–12 pigs, 13 rats, 14–16 and mice. 17,18 The first step in culturingpodocytes is based on the isolation of encapsulated ordecapsulated glomeruli from kidney cortex. 9 A goodpreparation is considered when 95% of the cellularconstituents are glomeruli, with the remainder comprisingtubules. Thus, to further reduce tubular contaminations (andenhance the percentage of glomeruli in the preparation), aFicoll-gradient centrifugation step may be included. 19 After 4or 5 days of primary culture, a monolayer of cobblestone-likeepithelial cells is observed. However, since the beginning of these pioneering experiments substantial doubts about thesrcin and validity of the cells growing out from the isolatedglomeruli have been raised. 20–22 Subsequent studies showedthat this uncertainty could be ruled out by starting thegeneration of primary cultures specifically in manually selected decapsulated glomeruli that are devoid of parietalepithelium. 11,16 The presence of podocytes in culture wasfurther supported by the observation that differentiatedpodocytes can leave their position on the glomerularbasement membrane and migrate onto the surface of cultured kidney slices. 23 After the isolation of glomeruli, two alternative protocolshave been used that ultimately lead to similar results, thegeneration of primary cultures of podocytes. In the firstprotocol, isolated glomeruli are generally cultured for 4 or 5days before they are subcultured by passing trypsinizedglomerular outgrowths over sieves with 25 m m pore size toremove the remaining glomerular cores consisting mainly of mesangial and endothelial cells. 24 In the second protocol,isolated glomeruli are digested with collagenase for 30min,passed over a sieve with 25 m m pore size and the cells thatpass through the sieve are used for cultivation. 25 In bothprotocols, cells are routinely plated on type I collagen-coateddishes to promote cell proliferation. The proliferating cellsform a monolayer of cobblestone appearance. As they reachconfluence, the cells are subcultured after trypsinizationor collagenase treatment. The media used varied fromDulbecco’s modified Eagle’s medium or RPMI 1640 supple-mented with 5–20% fetal calf serum 13 or 3T3-fibroblast-conditioned medium 24 to hormonally defined serum-freemedia. 26 The cells obtained under these conditions havemostly been used as primary cultures or as early subculturesof primary outgrowths, 14,27,28 but in addition permanent andnonpermanent cell lines have been established. 10,11,15,17,18 Despite advancing the field, these cells were still not ideal andthe quest for the cell that replicates the  in vivo  counterpartwas only just beginning. CLUES TO UNLOCKING THE TRUE  IN VITRO  PODOCYTEPHENOTYPE: THE TALE OF TWO DIFFERENT PHENOTYPESOF CULTURED PODOCYTES As discussed above, early on the cobblestone-type cells werethe first to be used to investigate podocyte function inculture because these cells were able to survive under cell cultureconditions. However, in these early studies the unspecificcobblestone-appearance of the cells was used as the solecharacteristic to prove that the glomerular outgrowths werepodocyte-derived. 9 A breakthrough emerged in that theidentification of podocytes was advanced by the demonstra-tion of podocyte ‘markers’ including TN10 29 and podo-calyxin. 10,11 Of note, podocalyxin is not specific forpodocytes because it is also expressed by endothelial cells. 30 Thus, podocycalyxin alone is not sufficient to prove that cellsin culture are podocytes. Expression of intermediate filamentproteins (e.g., cytokeratin, vimentin, or desmin) and cell junction proteins (e.g., ZO-1) have also been used, but they too do not allow definitive proof of origin of culturedpodocytes, since they are also expressed in other cell types.Thus, their presence or absence in cultured podocytes shouldrather be seen in the light of mimicking  in vitro  thedevelopmental modulations of these proteins observedduring podocyte maturation  in vivo . 31–35 Another major problem associated with culturing podo-cytes had been the historical rapid dedifferentiation  in vitro that accompanies the loss of the specific cell architecture and Kidney International   (2007)  72 , 26–36  27 SJ Shankland   et al.: Podocytes in culture  review  results in the cobblestone morphology of ‘standard’ podo-cytes in culture. The latter share many features withimmature podocyte precursor cells during early glomerulardevelopment, including the lack of synaptopodin expression(see below). Their low degree of differentiation is alsoreflected by the reappearance of lymphohaemopoietic markerantigens 36 expressed  in vivo  only transiently during the early stages of podocyte development. 37 A second type of epithelial cells growing out from isolatedglomeruli shows a different phenotype (Figure 1b). These arelarge (up to 500 m m) arborized, often binucleated cells thatexhibited no proliferative activity. This obviously limitedpropagation and the ability to establish a cell line  in vitro . Anovel discovery was that in contrast to cobblestone cells,these cells expressed synaptopodin. 16,38 Synaptopodin is a key marker of a differentiated podocyte phenotype, because in vivo  the expression of synaptopodin is specific topostmitotic differentiated podocytes. 39 Arborized podocyteshad been reported to contribute between 10% of rat 16 andalmost 100% of cultured porcine glomerular epithelial cell, 13 both after roughly 10 days of culture. THE PRESENT: INDUCTION OF DIFFERENTIATION INCULTURED RAT AND HUMAN PODOCYTES In the mid-1990s, serious efforts were undertaken to developa podocyte culture model that would demonstrate  in vitro expression of synaptopodin, a key marker of differentiatedpodocytes  in vivo. 39 This effort was motivated by theobservation discussed above that the cultivation of podocytesunder then standard conditions leads to rapid dedifferentia-tion, including the loss of processes and synaptopodinexpression. 39 It was further motivated by the fact thatsynaptopodin is not expressed in other cells such asfibroblasts, suggesting that results obtained from studyingsynaptopodin in fibroblasts or other model cell lines wouldnot necessary reflect its true function in podocytes. Tooptimize the  in vitro  characteristics, we modified the cultureconditions for rat and human podocytes by simply avoidingrepeated subcultivation. 38 This led to profound phenotypicchanges in podocytes  in vitro , including the conversionof cells with the cobblestone phenotype into cells withthe characteristic arborized phenotype that more closely resemble  in vivo  podocytes. Both cobblestone andarborized cells srcinate from podocytes, as evidenced by the expression of a podocyte-specific O-acetylated ganglio-side 40 and WT-1. 41 The differentiation into arborized cellsleads to growth arrest and is reflected by the formation of processes and the expression of synaptopodin, which wasnever detected in cobblestones. Taken together, we achieved(partial) differentiation of cultured podocytes by avoidingrepeated subcultivation, resulting in a phenotype moreclosely reflecting  in vivo  podocytes. 38 Although theseresults were indeed satisfying, there was still room forimprovement. A MAJOR BREAKTHROUGH: CONDITIONALLY IMMORTALIZEDMURINE PODOCYTES RETAIN A DIFFERENTIATIONPOTENTIAL SIMILAR TO THEIR  IN VIVO  COUNTERPARTS The differentiation of primary human and rat podocytesresults in rapid growth arrest. 38 Although this reflects themature  in vivo  counterpart, it limits cell culture abilitiesbecause passaging cells that do not increase in number istechnically problematic. To circumvent this problem, we took advantage of the Immortomouse 42 and established condi-tionally immortalized mouse podocyte cell lines, which arehighly proliferative when cultured under permissive condi- 33 ° C (+INF-  )33 ° C (day 3)37 ° C (day 7)37 ° C (day 14)    L  o  w  m  a  g  n   i   f   i  c  a   t   i  o  n   (       ×    1   0   0   )   H   i  g   h  m  a  g  n   i   f   i  c  a   t   i  o  n   (       ×    2   0   0   ) a b Figure 1 | Light microscopic morphology of cultured podocyte.  ( a ) Podcytes grown under permissive conditions (at 33 1 C with 20U/ml INF- g )display a characteristic cobblestone morphology. The cells form an epithelial monolayer as they reach confluence. ( b ) Differentiating (3, 7) anddifferentiated (14) podocytes grown under nonpermissive conditions (days at 37 1 C, no INF- g ). Under nonpermissive conditions, the cellsdevelop interdigitating processes, which are only connected at sites of process interdigitations. On day 14, large, flat arborized cells withwell-developed prominent processes can be seen. 28  Kidney International   (2007)  72 , 26–36 review  SJ Shankland   et al.: Podocytes in culture  tions. The details are described elsewhere. 43 In brief,nonpermissive conditions render the majority of podocytesgrowth arrested within 6 days and induce many characte-ristics of differentiated podocytes (Table 1). Both proliferat-ing and differentiating podocytes express WT-1. Duringdifferentiation, an ordered array of actin fibers and micro-tubules started to extend into the forming cellular processes,reminiscent of podocyte processes  in vivo . 43 Similar toprimary cultures, 38 the cytoskeletal rearrangement andprocess formation were accompanied by the onset of synaptopodin expression. Moreover, electrophysiologicalstudies showed that differentiated murine podocytesrespond to bradykinin by changes in intracellular calciumconcentration.Taken together, these studies established for the first timethat conditionally immortalized murine podocytes  in vitro retain a differentiation potential similar to podocytes  invivo , 43 including the formation of slit-diaphragm-like cell–cellcontacts 44,45 (Figure 2). This breakthrough has since made itpossible to explore the molecular mechanisms underlyingpodocyte differentiation and function using an inducible  invitro  model. 43 Of note, despite published 46,47 and unpub-lished claims (Table 1), when properly cultured (see below),these cells express virtually all proteins described inpodocytes to date, including among others:  a 3 b 1 integrin, 48 a -actinin-4, 49–51 angiotensin 2 receptor 52 , B7-1, 53 CD2AP, 54,55 ILK, 56 myosin II, 51 Nck1/2, 57 nephrin, 50,53 P-cadherin, 44 Pod1, 58 podocalyxin, 58 podocin, 50,53 synapto-podin, 43 TRPC6, 59 transforming growth factor- b , 60 vascularendothelial growth factor, 61,62 and WT-1 58 (Table 2). HOW CULTURED PODOCYTES HAVE ADVANCED THE FIELDIN HEALTH AND DISEASE BEYOND  IN VIVO  STUDIES Podocytes in culture are now being widely used to study virtually all aspects of podocyte biology in health and disease(Table 3). These innovative studies by laboratories through-out the world include the regulation of the cytoskele-ton, 49,51,63–68 cell cycle, 69–71 survival, 72,73 signaling 56,58,74–79 cell:cell, 53,80 and cell:matrix adhesion 48,81 as well as podocytechannel physiology. 68,82–85 Table 3 also shows how podocytesin culture have been successfully used to gain mechanisticinsight into the role of podocytes in the pathogenesis of diabetic 62,86 and human immunodeficiency virus-associatednephropathy. 87–89 Here, we will highlight two recent exam-ples because they exemplify how podocytes in culture havehelped to explore the function of proteins in podocytes,which could have not come from  in vivo  studies, because theprotein is not expressed in healthy podocytes (B7-1 53 ) orbecause the protein is dispensable for healthy podocytes buthas an important protective role under pathologic (stressed)conditions (cyclin I 71 ).What we have learned from the B7-1 experience andcultured podocytes: for a variety of reasons beyond thescope of this review, we developed cultured podocytesfrom  a 3 integrin-deficient ( a 3  /  ) mice that bear a strongmorphological resemblance to podocytes in congenitalnephrotic syndrome and other pathological conditionswith foot process (FP) effacement. 90,91 These  a 3  /  podo-cytes allowed us to uncover an unanticipated novel rolefor costimulatory molecule B7-1 in podocytes as aninducible modifier of glomerular permselectivity. 53 Inparticular, this study was the first to demonstrate that B7-1is expressed in podocytes under a variety of stressedconditions, in culture, and  in vivo , both in experimentaland human proteinuric kidney diseases. We specifically showed that podocyte B7-1 reorganizes the actin cytoskeletonof podocytes and modulates slit diaphragm (SD) organiza-tion. Most significantly, the effects of B7-1 in podocytes werefound independent of T and B cells and represent a novelunique function of B7-1. 53 As discussed above, these resultmost probably would have not come from  in vivo  studies,because B7-1 is not expressed in healthy podocytes. 53 Similarly, cyclin I is constitutively expressed in normalpodocytes but its function remained unclear. Cyclin I  /  mice develop normally and have no renal phenotype.Surprisingly, cyclin I  /  podocytes in culture revealed anovel role for this enigmatic cyclin by enhancing podocytesurvival following injury. Once a role for cyclin I in podocytesurvival was uncovered in culture, its role was confirmed in adisease model  in vivo . 71 Table 1|Similarities between podocytes  in vitro  and  in vivo : if they look, smell, and act like a podocyte, they are probablypodocytes Anti-cell culture view Pro-cell culture view Refs. Cultured podocytes lack slitdiaphragmsThey express a slit diaphragm-like structure Reiser  et al  . 44 and Gao  et al. 45 Cultured podocytes lack expressionof podocyte-specific proteinsThey stably express the vast majority, if not all, of the podocyte-specificproteins described  in vivo See Table 2They represent a nonquiescentphenotypePCNA, Brdu, cyclins E, A, and M are absent in differentiated podocytesgrown at 37 1 C, consistent with a quiescent phenotypeMundel  et al  ., 43 Griffin  et al. , 70,71 and Hiromura  et al  . 95 FACS analysis further supports cell-cycle exitCultured podocytes are notexposed to hemodynamic effectsMechanical stretch is available if deemed necessary Endlich  et al  . 51 Full differentiation in metanephric kidney culture is possible in theabsence of hemodynamic effectsNagata  et al  . 96 FACS, fluorescence-activated cell sorting; PCNA, proliferating cell nuclear antigen. Kidney International   (2007)  72 , 26–36  29 SJ Shankland   et al.: Podocytes in culture  review
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