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Targeting the podocyte to treat glomerular kidney disease Mark A. Lal1, Kenneth W. Young2 and Uwe Andag2 1 2
CVMD iMED, AstraZeneca, Mo¨lndal, Sweden Evotec AG, Germany
The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics. The kidneys The kidneys are a remarkable set of paired organs that receive 20% of cardiac output and that filter some 180 l blood daily, which is subsequently modified to give a final urine volume of approximately 1.5 l. This phenomenal physiological capacity is carried out by the concerted actions of the glomerular filter and renal tubular systems that together represent the single functional unit of the kidney known as the nephron. In performing this outstanding feat, the kidneys participate in establishing wholebody ion homeostasis, acid-base balance and blood pressure regulation. It should therefore come as no surprise that loss of kidney function and a compromised ability to carry out such fundamental processes is associated with considerable risks to overall health [1].
Chronic kidney disease Chronic kidney disease (CKD) is a progressive malady defined by reduced glomerular filtration rate, increased urinary albumin excretion or both, and is a major global public health concern with an extremely high unmet medical need. CKD is estimated to occur in 8–16% of the worldwide population and results in a substantially reduced life expectancy [1]. Diabetes itself is recognized as the primary cause of kidney failure in almost half of all new CKD cases and, given the current global diabetes pandemic, the prevalence of renal complications will continue to grow. Corresponding author: Lal, M.A. ([email protected]) 1359-6446/ß 2015 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2015.06.003
There is a strong relationship between CKD and cardiovascular disease which itself is twice as common in CKD patients as compared with the general population [2]. Accordingly, the current clinical paradigm for treating CKD patients is primarily focused on reducing cardiovascular risk and is, not surprisingly, inadequate to prevent patients from continuing their inexorable loss of renal function because such strategies do not directly address the principal mechanisms accounting for renal disease itself. Despite overall advances in renal replacement therapy and dialysis for CKD patients ultimately succumbing to end-stage renal failure, these last resort options are insufficient in terms of limited organ availability and high mortality and/or cost. The potential identification and implementation of novel, kidney-specific treatment strategies therefore represents a significant untapped opportunity to improve the prognosis of these patients. The future development of such kidney-centric therapeutics will require the identification of suitable renal targets and can only be achieved through a comprehensive understanding of disease pathophysiology, the underlying molecular mechanisms of disease initiation and progression, and the implementation of translatable models and technologies [3].
The glomerular filtration barrier The vast majority of all cases of CKD have their origin in the glomerulus [4]. Clinically, although kidney disease progression shows the strongest correlation with the degree of fibrosis in the
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FIGURE 1
Ultrastructural images of the glomerular capillary and filtration barrier. (a,b) Scanning electron micrographs of the exterior surface of the glomerular capillary depicting the intricate pattern in which podocyte foot processes interdigitate, surround and enwrap the outer capillary surface. (c) Transmission electron micrograph of the glomerular filtration barrier in cross section. The * indicates the slit diaphragm. Size marker 1 mm (a) and 200 nm (b,c). Abbreviations: FP, foot process; GBM, glomerular basement membrane; EC, endothelial cell; RBC, red blood cell; CL, capillary lumen; US, urinary space.
tubules and interstitium, it is lesions within the highly specialized microvascular unit that initiate disease. The primary function of the glomerulus is to filter blood selectively across the capillary wall and to elaborate an ultrafiltrate that is secondarily modified by the renal tubule system. The filtration capacity of the glomerulus is defined by the functional properties of the glomerular filtration barrier (GFB), a trilaminar molecular sieve comprising endothelial cells and visceral podocytes that medially elaborate the constituents of the glomerular basement membrane (GBM) that lies between them (Fig. 1). Although each individual component of the GFB is necessary for its concerted function, the podocyte is largely recognized as the final determinant of size-selective filtration and the ultimate barrier to albumin [4]. This is of particular clinical relevance because even small increases in albuminuria confer clinical risk and, at increasing levels of proteinuria, life expectancy is substantially reduced [5].
discovery of nephrin, the identification of a host of additional human genetic mutations in various proteins implicit in podocyte function has highlighted the central involvement of this cell type in the etiology of various inherited diseases of the glomerulus [7] (Table 1). Collectively, glomerular diseases can be classified as a related spectrum of podocytopathies where abnormalities in podocyte biology (i.e. dysfunction, injury and loss) are shared among them. They encompass purely genetic forms of podocyte disease TABLE 1
Selection of genes directly implicated in the development of podocyte-dependent human glomerular diseases and the subcellular localization and function of their corresponding proteins Subcellular location and function
Gene
Protein
Slit diaphragm
NPHS1 NPHS2 CD2AP PLCE1 TRPC6 ANLN
Nephrin Podocin CD2-associated protein Phospholipase Ce1 Transient receptor potential channel 6 Anillin
Actin cytoskeleton
ACTN4 MYO1E INF2 ARHGDIA ARHGAP24
a-Actinin-4 Myosin 1E Inverted formin-2 Rho GDP-dissociation inhibitor 1 Rho-GTPase-activating protein 24
GBM-associated
LAMB2 ITGA3 ITGB4 COL4A3,4,5 CD151
Laminin-a2 Integrin-a3 Integrin-b4 Collagen IV CD151
Nucleus
WT1 LMX1B
Wilms tumor protein LIM homeobox transcription factor 1b ATP-driven annealing helicase Nuclear RNA export factor 5
The podocyte The podocyte is a terminally differentiated, highly specialized cell type with a remarkable morphology exquisitely designed to match its function. The podocyte establishes the integrity and function of the GFB and slit diaphragm, maintains capillary structure and resists intraglomerular blood pressure, contributes to the formation and modulation of the glomerular basement membrane and also determines endothelial cell homeostasis [6]. Ultrastructurally, the podocyte is like no other cell. From its voluminous cell body exposed in the urinary space extend primary and secondary processes that arborize into slender foot processes that interdigitate with those of neighboring podocytes to enwrap the outer surface of the glomerular capillary firmly (Fig. 1). One of the most unique features of the podocytes and their foot processes is the slit diaphragm, a modified cell–cell adherens junction that spans the length of their interaction. The slit diaphragm, visible upon electron microscopy as a bridge connecting juxtaposed foot processes, comprises a host of unique structural proteins, the founding member of which is nephrin [4]. It is the initial identification of nephrin and genetic mutations therein as the cause of the massive proteinuria that characterizes patients with congenital nephrotic syndrome of the Finnish type (CNSF) that has placed the podocyte at the center of current activities aimed at understanding the molecular and cellular determinants of proteinuria. Since the 2
SMARCAL1 NXF5 Mitochondria
COQ2 COQ6 PDSS2 ADCK4
Polyprenyltransferase Ubiquinone biosynthesis monooxygenase Decaprenyl diphosphate synthase subunit 2 AarF-domain-containing protein kinase 4
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TABLE 2
Primary pathway
Target examples
Nuclear signaling
Glucocorticoid receptor, vitamin D receptor, Notch intracellular domain, all-trans retinoic acid, peroxisome proliferator-activated receptors gamma
Soluble factors
Angiopoietin-like-4, soluble urokinase receptor
Actin cytoskeleton
Transient receptor potential cation channel 5 and 6, RhoGTPase modulators, phospholipase C epsilon 1
Kinases
Calcineurin, glycogen synthase kinase 3b, Janus kinase, protein kinase C
Slit diaphragm signaling
Nephrin, podocin, CD2-associated protein
Redox signaling
Nuclear factor (erythroid-derived 2)-like 2, nitric oxide synthase, NADPH oxidase
Podocyte–GBM interaction
a3b1 Integrin, integrin-linked kinase, laminin-521, Cd151
Podocyte–endothelial crosstalk
Vascular endothelial growth factor, endothelin, angiopoietins
Mitochondrial function
Coenzyme Q10, prohibitin-2
Energy metabolism
Mammalian target of rapamycin (mTOR), AMP-activated protein kinase, autophagy proteins (ATGs)
Receptor signaling
Angiotensin receptor, insulin receptor, melanocortin 1 receptor, epidermal growth factor receptor, B7-1
Target examples are primarily chosen where in vitro and in vivo experimental data exist. The functions of most targets are difficult to define singularly and probably impact multiple pathways.
[such as CNSF, Alport syndrome and some forms of focal and segmental glomerular sclerosis (FSGS) and membranous nephropathy] and also include nonhereditary glomerular diseases that occur secondary to conditions such as hypertension and diabetic nephropathy [8]. Taken together, glomerular diseases account for the vast majority of all end-stage kidney disease cases with diabetic nephropathy representing the single largest cause. One of the earliest cellular lesions observed in the various podocytopathies that lead to glomerulosclerosis is a loss of podocytes or, more specifically, a reduction in the number of podocytes per glomerulus. Because podocytes are terminally differentiated and have a limited capacity for repair or regeneration, glomerular function is particularly sensitive to situations in which there is a mismatch between podocyte number and the glomerular filtration surface area. Experimental animal models show that a loss of 120 000 compounds using protection from palmitate and highglucose-induced podocyte apoptosis as a primary readout. Protection against effects on the actin cytoskeleton was used as a secondary orthogonal assay. To this end, we have identified a number of targets, some previously recognized as important regulators of podocyte biology [e.g. mammalian target of rapamycin (mTOR), glucocorticoid receptor, all-trans retinoic acid, among others]. Interestingly, a number of compounds that had the capability to attenuate palmitate and high-glucose-driven podocyte apoptosis in the screen were identified as glycogen synthase kinase (GSK)3b inhibitors. 1-Azakenpaullone (1-AZK), a selective GSK3b inhibitor identified by the podocyte screening approach, was further analyzed for its potency to protect ex vivo isolated porcine glomeruli against palmitate and high-glucose injury. 1-AZK significantly increased cell viability of glomeruli exposed to palmitate and high glucose, highlighting again the protective effect of GSK3b inhibitors in this injury setting (Fig. 3). Recently, GSK3b inhibitors have been demonstrated to exert protective effects on injured podocytes in culture as well as in mouse models of kidney disease including diabetic nephropathy [37,38]. These findings suggest that pharmacologically targeting GSK3b could represent a therapeutic strategy to protect podocytes against injury. The second, often more complex phase, of phenotypic screening is the need to identify the target or targets of hits found in the screening phase. Here, the process involves expansion of the chemical matter around the initial hit chemotype to understand the basic structure-related properties for the molecule. Next, chemical proteomics is employed to identify cellular binding partners for the hits and ultimately these targets need to be confirmed using the screening assay set up [39,40]. Together, Evotec and AstraZeneca have successfully progressed podocyte hit compounds to target identification and are currently developing compounds around these novel targets.
Tools to evaluate podocyte function Although the ability to culture podocytes has tremendously enhanced our understanding of the function of individual proteins
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FIGURE 2
Podocyte high-content screening. Conditionally immortalized human podocytes were screened using a combination of palmitate and high glucose to mimic conditions observed in diabetic nephropathy. Small-molecule inhibitors of phenotypic changes were identified using the OperaTM high-content imaging platform. The primary screen was run using activation of caspase 3/7 as a marker of podocyte apoptosis. Hit compounds were also tested in an orthogonal assay which used Alexa-labeled phalloidin to detect changes in the actin cytoskeleton.
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Glycogen synthase kinase (GSK)3b inhibition protects against podocyte injury. Representative images of the degree of podocyte apoptosis measured as caspase 3/7 activation (green) under control (a) and 200 mM palmitate with 25 mM glucose (b) conditions. Podocyte nuclei are counterstained red. (c) 1-Azakenpaullone (1-AZK) dose-dependently reduces caspase 3/7 activation in human podocytes challenged with 200 mM palmitate and 25 mM glucose for 48 h. Podocyte apoptosis was quantified and normalized to confluence level by measuring caspase 3/7 activity and confluence using IncuCyte live cell imaging (n = 4). (d) Overall cell viability, quantified by AlamarBlueW assay (n = 3), of glomeruli freshly isolated from Go¨ttingen minipig via graded sieving technique and cultured in 11 mM glucose ( ) or 400 mM palmitate with 25 mM glucose (+) with indicated 1-AZK concentrations for three days.
in this cell type at a biochemical level, such methods and the conclusions drawn from their results need to be integrated within the complex framework of podocyte function in vivo as a requisite component of the GFB. This is important to consider because the podocyte in vitro is an imperfect representation of its in vivo counterpart at a molecular and functional level [25–27,41]. The isolated podocyte certainly mimics certain facets of podocyte biology observed in vivo, but there is a need for additional unbiased and integrated strategies that can facilitate our understanding of
the mechanistic determinants of podocyte function in its native microenvironment. A number of novel and exciting strategies have been developed to fill the void between cell culture and whole-animal, mammalian studies of podocyte biology. The concept of the podocyte as a dynamic, motile cell type in vitro has been around for some time, but the corollary of this phenomenon in vivo and its putative physiological significance in human health and disease is a subject of active debate. Through the use of transgenic animals (i.e. mice and zebrafish) expressing
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fluorescently labeled podocytes and advanced microscopic imaging techniques it is possible to study and interrogate the occurrence of podocyte motility in vivo and its relevance to glomerular disease [42,43]. The conclusions drawn from such studies are not in complete harmony and continue to fuel the debate over podocyte plasticity and the cellular nature of their origin. Regardless, such strategies are valuable tools that will be important to evaluating the efficacy of novel therapeutic approaches designed to exploit podocyte dynamics and the potential to control podocyte regeneration from progenitor cell niches. Another novel and very interesting imaging-based approach for the study of glomerular function is a recently published technique based on repetitive noninvasive in vivo imaging of GFB integrity in isolated glomeruli transplanted into the anterior chamber of the mouse eye [44]. Podocytes from such glomeruli maintained their differentiation and displayed interdigitating podocyte foot processes for up to six months after transplantation and are amenable to repeated imaging. Transplanting healthy or diseased human glomeruli into mouse eyes might provide a means to image functioning human glomeruli and acutely examine the effect of therapeutics on glomerular function. The fruit fly has also recently seen its debut as a model organism to study podocyte biology [45]. Despite gross anatomical differences in the excretory system of humans and fruit flies, the Drosophila pericardial nephrocyte bears a remarkable evolutionary conservation of molecular components and functional properties with the mammalian podocyte. To exploit this observation, and to harness the power of Drosophila genetics, a reporter system of nephrocyte filtration function has been combined with a large RNA interference genetic screen that enables the scanning of the entire genome for genes specifically required for pericardial nephrocyte function [46]. An initial genetic screen of about 1000 genes resulted in the identification of about 7% that were essential for nephrocyte function, notable among them the mammalian homologs of nephrin and podocin [46]. In the near future, the complete repertoire of genes required for nephrocyte-specific function will be defined and this will be an invaluable source of unbiased information ready to be translated to higher animal species. The zebrafish, as mentioned above, has taken its place as an exceptional model for studying podocyte biology and glomerular function. The ability to knockdown individual genes and subsequently to follow functional parameters of filtration capacity has been an important tool in the armamentarium of the podocyte biologist to accelerate the study of podocyte gene function [47]. Reverse genetic screens can be readily used to investigate the potential role of novel podocyte proteins. Additionally, novel transgenic zebrafish models that incorporate inducible podocyte injury with a fluorescent tracer for proteinuria make it possible to use this functional model for the screening of therapeutic candidates that could improve overall podocyte health and GFB integrity [48]. Another strategy not yet fully realized for the kidney, but demonstrated in principal for other organ systems including that of the lung and liver, is the microfluidic organ-on-a-chip which aims to facilitate the in vitro organomimetic modeling of complex human physiology [49,50]. Similar to recapitulating the alveolar– capillary interface of the lung in a mechanical environment that mirrors physiological function, the GFB of the kidney would 6
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appear to be a particularly well-suited functional unit for such a strategy. One can envisage the culturing of human podocytes and glomerular endothelial cells on either side of a structural support in a self-contained unit where separated perfusion chambers bath cells in appropriate media and biomechanical properties such as hydrostatic pressure, fluid flow and shear stress can be modulated to simulate in vivo properties. Such a biomimetic device could be what is required to promote the differentiation of cultured podocytes to develop foot processes and slit diaphragms as is functionally required for GFB function. Interestingly, it has also been demonstrated through the cellular repopulation of decellularized kidneys that the native kidney extracellular matrix, including that of the GBM, provides cellular cues that might promote the potential differentiation of podocytes in the glomerulus [51]. The future development of a human GFB biomimetic represents a significant hurdle but holds great promise as a means to study podocyte function and to evaluate drug pharmacology and toxicity.
Podocyte delivery and targeting Briefly, cell-specific targeting of therapeutic agents to podocytes of the glomerulus might potentially be an attractive method to increase their efficacy and/or minimize their side-effects. Size and charge characteristics could be exploited to facilitate the capability of drug–carrier conjugates to reach and interact specifically with the correct target cells. Within the kidney, the podocyte is an attractive target cell because of its unique properties. Large molecules that accumulate in the GBM might not need to be filtered to reach the podocyte because of the robust endocytic capacity of this cell type [52]. This raises the possibility of using tissue-specific homing strategies to deliver large proteins, antibodies and siRNAs into cells in a biologically active form [53,54]. Ultrasound-microbubble-mediated delivery of gene therapies to the kidney is an interesting approach that has been used to achieve drug targeting to the kidney [55]. Efficacy of ultrasound-microbubble-mediated gene delivery has been demonstrated in rat models of renal fibrosis but upregulation of the transgene of interest was noted in all kidney tissues [56]. The ability to combine such ultrasound microbubble technology with cell-specific targeting should be possible for the podocyte as has been demonstrated for achieving endothelium-specific transgene expression of a shRNA [57].
Concluding remarks The number of randomized clinical trials addressing renal disease continues to be outpaced by most other specialties and the failure of a number of recent kidney clinical trials suggests a need for new thinking and new strategies to address CKD [5,58]. Delineation of the crucial molecular species and signaling pathways underlying podocyte function will undoubtedly lead to the identification of cell-specific targets that can be exploited for treating various glomerulopathies and CKD. Realizing this goal will require a holistic approach where the short-comings of any single strategy in isolation will be overcome only by integrating their individual strengths. With this in mind, the future for developing podocytespecific therapeutics looks bright.
Conflicts of interest The authors declare no conflicts of interest.
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Acknowledgements The authors would like to thank the members of the AstraZeneca–Evotec collaboration team involved in exploring compounds and targets with disease-modifying potential for
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the treatment of kidney disease. We are grateful to Tanja Koepp and Patrick Starremans for providing us with podocyte apoptosis and glomerulus cell viability data, respectively.
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References
Targeting the podocyte to treat glomerular kidney disease Mark A. Lal1, Kenneth W. Young2 and Uwe Andag2 1 2
CVMD iMED, AstraZeneca, Mo¨lndal, Sweden Evotec AG, Germany
The majority of chronic kidney disease (CKD) cases have their origin in the glomerulus, the microvascular unit of the nephron that serves as a filter tasked with forming primary urine. This selective filtration process is determined to a large extent by the functional capacity of the podocyte, a highly differentiated cell type that enwraps the outer aspect of the glomerular capillary wall. In this short review, we describe the biology of the podocyte, its central role in the etiology of various glomerulopathies and highlight current and future opportunities to exploit the unique properties of this cell type for developing kidney-specific therapeutics. The kidneys The kidneys are a remarkable set of paired organs that receive 20% of cardiac output and that filter some 180 l blood daily, which is subsequently modified to give a final urine volume of approximately 1.5 l. This phenomenal physiological capacity is carried out by the concerted actions of the glomerular filter and renal tubular systems that together represent the single functional unit of the kidney known as the nephron. In performing this outstanding feat, the kidneys participate in establishing wholebody ion homeostasis, acid-base balance and blood pressure regulation. It should therefore come as no surprise that loss of kidney function and a compromised ability to carry out such fundamental processes is associated with considerable risks to overall health [1].
Chronic kidney disease Chronic kidney disease (CKD) is a progressive malady defined by reduced glomerular filtration rate, increased urinary albumin excretion or both, and is a major global public health concern with an extremely high unmet medical need. CKD is estimated to occur in 8–16% of the worldwide population and results in a substantially reduced life expectancy [1]. Diabetes itself is recognized as the primary cause of kidney failure in almost half of all new CKD cases and, given the current global diabetes pandemic, the prevalence of renal complications will continue to grow. Corresponding author: Lal, M.A. ([email protected]) 1359-6446/ß 2015 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.drudis.2015.06.003
There is a strong relationship between CKD and cardiovascular disease which itself is twice as common in CKD patients as compared with the general population [2]. Accordingly, the current clinical paradigm for treating CKD patients is primarily focused on reducing cardiovascular risk and is, not surprisingly, inadequate to prevent patients from continuing their inexorable loss of renal function because such strategies do not directly address the principal mechanisms accounting for renal disease itself. Despite overall advances in renal replacement therapy and dialysis for CKD patients ultimately succumbing to end-stage renal failure, these last resort options are insufficient in terms of limited organ availability and high mortality and/or cost. The potential identification and implementation of novel, kidney-specific treatment strategies therefore represents a significant untapped opportunity to improve the prognosis of these patients. The future development of such kidney-centric therapeutics will require the identification of suitable renal targets and can only be achieved through a comprehensive understanding of disease pathophysiology, the underlying molecular mechanisms of disease initiation and progression, and the implementation of translatable models and technologies [3].
The glomerular filtration barrier The vast majority of all cases of CKD have their origin in the glomerulus [4]. Clinically, although kidney disease progression shows the strongest correlation with the degree of fibrosis in the
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FIGURE 1
Ultrastructural images of the glomerular capillary and filtration barrier. (a,b) Scanning electron micrographs of the exterior surface of the glomerular capillary depicting the intricate pattern in which podocyte foot processes interdigitate, surround and enwrap the outer capillary surface. (c) Transmission electron micrograph of the glomerular filtration barrier in cross section. The * indicates the slit diaphragm. Size marker 1 mm (a) and 200 nm (b,c). Abbreviations: FP, foot process; GBM, glomerular basement membrane; EC, endothelial cell; RBC, red blood cell; CL, capillary lumen; US, urinary space.
tubules and interstitium, it is lesions within the highly specialized microvascular unit that initiate disease. The primary function of the glomerulus is to filter blood selectively across the capillary wall and to elaborate an ultrafiltrate that is secondarily modified by the renal tubule system. The filtration capacity of the glomerulus is defined by the functional properties of the glomerular filtration barrier (GFB), a trilaminar molecular sieve comprising endothelial cells and visceral podocytes that medially elaborate the constituents of the glomerular basement membrane (GBM) that lies between them (Fig. 1). Although each individual component of the GFB is necessary for its concerted function, the podocyte is largely recognized as the final determinant of size-selective filtration and the ultimate barrier to albumin [4]. This is of particular clinical relevance because even small increases in albuminuria confer clinical risk and, at increasing levels of proteinuria, life expectancy is substantially reduced [5].
discovery of nephrin, the identification of a host of additional human genetic mutations in various proteins implicit in podocyte function has highlighted the central involvement of this cell type in the etiology of various inherited diseases of the glomerulus [7] (Table 1). Collectively, glomerular diseases can be classified as a related spectrum of podocytopathies where abnormalities in podocyte biology (i.e. dysfunction, injury and loss) are shared among them. They encompass purely genetic forms of podocyte disease TABLE 1
Selection of genes directly implicated in the development of podocyte-dependent human glomerular diseases and the subcellular localization and function of their corresponding proteins Subcellular location and function
Gene
Protein
Slit diaphragm
NPHS1 NPHS2 CD2AP PLCE1 TRPC6 ANLN
Nephrin Podocin CD2-associated protein Phospholipase Ce1 Transient receptor potential channel 6 Anillin
Actin cytoskeleton
ACTN4 MYO1E INF2 ARHGDIA ARHGAP24
a-Actinin-4 Myosin 1E Inverted formin-2 Rho GDP-dissociation inhibitor 1 Rho-GTPase-activating protein 24
GBM-associated
LAMB2 ITGA3 ITGB4 COL4A3,4,5 CD151
Laminin-a2 Integrin-a3 Integrin-b4 Collagen IV CD151
Nucleus
WT1 LMX1B
Wilms tumor protein LIM homeobox transcription factor 1b ATP-driven annealing helicase Nuclear RNA export factor 5
The podocyte The podocyte is a terminally differentiated, highly specialized cell type with a remarkable morphology exquisitely designed to match its function. The podocyte establishes the integrity and function of the GFB and slit diaphragm, maintains capillary structure and resists intraglomerular blood pressure, contributes to the formation and modulation of the glomerular basement membrane and also determines endothelial cell homeostasis [6]. Ultrastructurally, the podocyte is like no other cell. From its voluminous cell body exposed in the urinary space extend primary and secondary processes that arborize into slender foot processes that interdigitate with those of neighboring podocytes to enwrap the outer surface of the glomerular capillary firmly (Fig. 1). One of the most unique features of the podocytes and their foot processes is the slit diaphragm, a modified cell–cell adherens junction that spans the length of their interaction. The slit diaphragm, visible upon electron microscopy as a bridge connecting juxtaposed foot processes, comprises a host of unique structural proteins, the founding member of which is nephrin [4]. It is the initial identification of nephrin and genetic mutations therein as the cause of the massive proteinuria that characterizes patients with congenital nephrotic syndrome of the Finnish type (CNSF) that has placed the podocyte at the center of current activities aimed at understanding the molecular and cellular determinants of proteinuria. Since the 2
SMARCAL1 NXF5 Mitochondria
COQ2 COQ6 PDSS2 ADCK4
Polyprenyltransferase Ubiquinone biosynthesis monooxygenase Decaprenyl diphosphate synthase subunit 2 AarF-domain-containing protein kinase 4
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TABLE 2
Primary pathway
Target examples
Nuclear signaling
Glucocorticoid receptor, vitamin D receptor, Notch intracellular domain, all-trans retinoic acid, peroxisome proliferator-activated receptors gamma
Soluble factors
Angiopoietin-like-4, soluble urokinase receptor
Actin cytoskeleton
Transient receptor potential cation channel 5 and 6, RhoGTPase modulators, phospholipase C epsilon 1
Kinases
Calcineurin, glycogen synthase kinase 3b, Janus kinase, protein kinase C
Slit diaphragm signaling
Nephrin, podocin, CD2-associated protein
Redox signaling
Nuclear factor (erythroid-derived 2)-like 2, nitric oxide synthase, NADPH oxidase
Podocyte–GBM interaction
a3b1 Integrin, integrin-linked kinase, laminin-521, Cd151
Podocyte–endothelial crosstalk
Vascular endothelial growth factor, endothelin, angiopoietins
Mitochondrial function
Coenzyme Q10, prohibitin-2
Energy metabolism
Mammalian target of rapamycin (mTOR), AMP-activated protein kinase, autophagy proteins (ATGs)
Receptor signaling
Angiotensin receptor, insulin receptor, melanocortin 1 receptor, epidermal growth factor receptor, B7-1
Target examples are primarily chosen where in vitro and in vivo experimental data exist. The functions of most targets are difficult to define singularly and probably impact multiple pathways.
[such as CNSF, Alport syndrome and some forms of focal and segmental glomerular sclerosis (FSGS) and membranous nephropathy] and also include nonhereditary glomerular diseases that occur secondary to conditions such as hypertension and diabetic nephropathy [8]. Taken together, glomerular diseases account for the vast majority of all end-stage kidney disease cases with diabetic nephropathy representing the single largest cause. One of the earliest cellular lesions observed in the various podocytopathies that lead to glomerulosclerosis is a loss of podocytes or, more specifically, a reduction in the number of podocytes per glomerulus. Because podocytes are terminally differentiated and have a limited capacity for repair or regeneration, glomerular function is particularly sensitive to situations in which there is a mismatch between podocyte number and the glomerular filtration surface area. Experimental animal models show that a loss of 120 000 compounds using protection from palmitate and highglucose-induced podocyte apoptosis as a primary readout. Protection against effects on the actin cytoskeleton was used as a secondary orthogonal assay. To this end, we have identified a number of targets, some previously recognized as important regulators of podocyte biology [e.g. mammalian target of rapamycin (mTOR), glucocorticoid receptor, all-trans retinoic acid, among others]. Interestingly, a number of compounds that had the capability to attenuate palmitate and high-glucose-driven podocyte apoptosis in the screen were identified as glycogen synthase kinase (GSK)3b inhibitors. 1-Azakenpaullone (1-AZK), a selective GSK3b inhibitor identified by the podocyte screening approach, was further analyzed for its potency to protect ex vivo isolated porcine glomeruli against palmitate and high-glucose injury. 1-AZK significantly increased cell viability of glomeruli exposed to palmitate and high glucose, highlighting again the protective effect of GSK3b inhibitors in this injury setting (Fig. 3). Recently, GSK3b inhibitors have been demonstrated to exert protective effects on injured podocytes in culture as well as in mouse models of kidney disease including diabetic nephropathy [37,38]. These findings suggest that pharmacologically targeting GSK3b could represent a therapeutic strategy to protect podocytes against injury. The second, often more complex phase, of phenotypic screening is the need to identify the target or targets of hits found in the screening phase. Here, the process involves expansion of the chemical matter around the initial hit chemotype to understand the basic structure-related properties for the molecule. Next, chemical proteomics is employed to identify cellular binding partners for the hits and ultimately these targets need to be confirmed using the screening assay set up [39,40]. Together, Evotec and AstraZeneca have successfully progressed podocyte hit compounds to target identification and are currently developing compounds around these novel targets.
Tools to evaluate podocyte function Although the ability to culture podocytes has tremendously enhanced our understanding of the function of individual proteins
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FIGURE 2
Podocyte high-content screening. Conditionally immortalized human podocytes were screened using a combination of palmitate and high glucose to mimic conditions observed in diabetic nephropathy. Small-molecule inhibitors of phenotypic changes were identified using the OperaTM high-content imaging platform. The primary screen was run using activation of caspase 3/7 as a marker of podocyte apoptosis. Hit compounds were also tested in an orthogonal assay which used Alexa-labeled phalloidin to detect changes in the actin cytoskeleton.
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Glycogen synthase kinase (GSK)3b inhibition protects against podocyte injury. Representative images of the degree of podocyte apoptosis measured as caspase 3/7 activation (green) under control (a) and 200 mM palmitate with 25 mM glucose (b) conditions. Podocyte nuclei are counterstained red. (c) 1-Azakenpaullone (1-AZK) dose-dependently reduces caspase 3/7 activation in human podocytes challenged with 200 mM palmitate and 25 mM glucose for 48 h. Podocyte apoptosis was quantified and normalized to confluence level by measuring caspase 3/7 activity and confluence using IncuCyte live cell imaging (n = 4). (d) Overall cell viability, quantified by AlamarBlueW assay (n = 3), of glomeruli freshly isolated from Go¨ttingen minipig via graded sieving technique and cultured in 11 mM glucose ( ) or 400 mM palmitate with 25 mM glucose (+) with indicated 1-AZK concentrations for three days.
in this cell type at a biochemical level, such methods and the conclusions drawn from their results need to be integrated within the complex framework of podocyte function in vivo as a requisite component of the GFB. This is important to consider because the podocyte in vitro is an imperfect representation of its in vivo counterpart at a molecular and functional level [25–27,41]. The isolated podocyte certainly mimics certain facets of podocyte biology observed in vivo, but there is a need for additional unbiased and integrated strategies that can facilitate our understanding of
the mechanistic determinants of podocyte function in its native microenvironment. A number of novel and exciting strategies have been developed to fill the void between cell culture and whole-animal, mammalian studies of podocyte biology. The concept of the podocyte as a dynamic, motile cell type in vitro has been around for some time, but the corollary of this phenomenon in vivo and its putative physiological significance in human health and disease is a subject of active debate. Through the use of transgenic animals (i.e. mice and zebrafish) expressing
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fluorescently labeled podocytes and advanced microscopic imaging techniques it is possible to study and interrogate the occurrence of podocyte motility in vivo and its relevance to glomerular disease [42,43]. The conclusions drawn from such studies are not in complete harmony and continue to fuel the debate over podocyte plasticity and the cellular nature of their origin. Regardless, such strategies are valuable tools that will be important to evaluating the efficacy of novel therapeutic approaches designed to exploit podocyte dynamics and the potential to control podocyte regeneration from progenitor cell niches. Another novel and very interesting imaging-based approach for the study of glomerular function is a recently published technique based on repetitive noninvasive in vivo imaging of GFB integrity in isolated glomeruli transplanted into the anterior chamber of the mouse eye [44]. Podocytes from such glomeruli maintained their differentiation and displayed interdigitating podocyte foot processes for up to six months after transplantation and are amenable to repeated imaging. Transplanting healthy or diseased human glomeruli into mouse eyes might provide a means to image functioning human glomeruli and acutely examine the effect of therapeutics on glomerular function. The fruit fly has also recently seen its debut as a model organism to study podocyte biology [45]. Despite gross anatomical differences in the excretory system of humans and fruit flies, the Drosophila pericardial nephrocyte bears a remarkable evolutionary conservation of molecular components and functional properties with the mammalian podocyte. To exploit this observation, and to harness the power of Drosophila genetics, a reporter system of nephrocyte filtration function has been combined with a large RNA interference genetic screen that enables the scanning of the entire genome for genes specifically required for pericardial nephrocyte function [46]. An initial genetic screen of about 1000 genes resulted in the identification of about 7% that were essential for nephrocyte function, notable among them the mammalian homologs of nephrin and podocin [46]. In the near future, the complete repertoire of genes required for nephrocyte-specific function will be defined and this will be an invaluable source of unbiased information ready to be translated to higher animal species. The zebrafish, as mentioned above, has taken its place as an exceptional model for studying podocyte biology and glomerular function. The ability to knockdown individual genes and subsequently to follow functional parameters of filtration capacity has been an important tool in the armamentarium of the podocyte biologist to accelerate the study of podocyte gene function [47]. Reverse genetic screens can be readily used to investigate the potential role of novel podocyte proteins. Additionally, novel transgenic zebrafish models that incorporate inducible podocyte injury with a fluorescent tracer for proteinuria make it possible to use this functional model for the screening of therapeutic candidates that could improve overall podocyte health and GFB integrity [48]. Another strategy not yet fully realized for the kidney, but demonstrated in principal for other organ systems including that of the lung and liver, is the microfluidic organ-on-a-chip which aims to facilitate the in vitro organomimetic modeling of complex human physiology [49,50]. Similar to recapitulating the alveolar– capillary interface of the lung in a mechanical environment that mirrors physiological function, the GFB of the kidney would 6
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appear to be a particularly well-suited functional unit for such a strategy. One can envisage the culturing of human podocytes and glomerular endothelial cells on either side of a structural support in a self-contained unit where separated perfusion chambers bath cells in appropriate media and biomechanical properties such as hydrostatic pressure, fluid flow and shear stress can be modulated to simulate in vivo properties. Such a biomimetic device could be what is required to promote the differentiation of cultured podocytes to develop foot processes and slit diaphragms as is functionally required for GFB function. Interestingly, it has also been demonstrated through the cellular repopulation of decellularized kidneys that the native kidney extracellular matrix, including that of the GBM, provides cellular cues that might promote the potential differentiation of podocytes in the glomerulus [51]. The future development of a human GFB biomimetic represents a significant hurdle but holds great promise as a means to study podocyte function and to evaluate drug pharmacology and toxicity.
Podocyte delivery and targeting Briefly, cell-specific targeting of therapeutic agents to podocytes of the glomerulus might potentially be an attractive method to increase their efficacy and/or minimize their side-effects. Size and charge characteristics could be exploited to facilitate the capability of drug–carrier conjugates to reach and interact specifically with the correct target cells. Within the kidney, the podocyte is an attractive target cell because of its unique properties. Large molecules that accumulate in the GBM might not need to be filtered to reach the podocyte because of the robust endocytic capacity of this cell type [52]. This raises the possibility of using tissue-specific homing strategies to deliver large proteins, antibodies and siRNAs into cells in a biologically active form [53,54]. Ultrasound-microbubble-mediated delivery of gene therapies to the kidney is an interesting approach that has been used to achieve drug targeting to the kidney [55]. Efficacy of ultrasound-microbubble-mediated gene delivery has been demonstrated in rat models of renal fibrosis but upregulation of the transgene of interest was noted in all kidney tissues [56]. The ability to combine such ultrasound microbubble technology with cell-specific targeting should be possible for the podocyte as has been demonstrated for achieving endothelium-specific transgene expression of a shRNA [57].
Concluding remarks The number of randomized clinical trials addressing renal disease continues to be outpaced by most other specialties and the failure of a number of recent kidney clinical trials suggests a need for new thinking and new strategies to address CKD [5,58]. Delineation of the crucial molecular species and signaling pathways underlying podocyte function will undoubtedly lead to the identification of cell-specific targets that can be exploited for treating various glomerulopathies and CKD. Realizing this goal will require a holistic approach where the short-comings of any single strategy in isolation will be overcome only by integrating their individual strengths. With this in mind, the future for developing podocytespecific therapeutics looks bright.
Conflicts of interest The authors declare no conflicts of interest.
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Acknowledgements The authors would like to thank the members of the AstraZeneca–Evotec collaboration team involved in exploring compounds and targets with disease-modifying potential for
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the treatment of kidney disease. We are grateful to Tanja Koepp and Patrick Starremans for providing us with podocyte apoptosis and glomerulus cell viability data, respectively.
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30 Hayashi, K. et al. (2014) KLF4-dependent epigenetic remodeling modulates podocyte phenotypes and attenuates proteinuria. J. Clin. Invest. 124, 2523–2537 31 Rinschen, M.M. et al. (2014) Phosphoproteomic analysis reveals regulatory mechanisms at the kidney filtration barrier. J. Am. Soc. Nephrol. 25, 1509–1522 32 Ju, W. et al. (2013) Defining cell-type specificity at the transcriptional level in human disease. Genome Res. 23, 1862–1873 33 Shankland, S.J. et al. (2007) Podocytes in culture: past, present, and future. Kidney Int. 72, 26–36 34 Saleem, M.A. et al. (2002) A conditionally immortalized human podocyte cell line demonstrating nephrin and podocin expression. J. Am. Soc. Nephrol. 13, 630–638 35 Lee, H.W. et al. (2015) A podocyte-based automated screening assay identifies protective small molecules. J. Am. Soc. Nephrol. pii:ASN.2014090859 36 Korn, K. and Krausz, E. (2007) Cell-based high-content screening of small-molecule libraries. Curr. Opin. Chem. Biol. 11, 503–510 37 Paeng, J. et al. (2014) Enhanced glycogen synthase kinase-3beta activity mediates podocyte apoptosis under diabetic conditions. Apoptosis 19, 1678–1690 38 Xu, W. et al. (2015) Glycogen synthase kinase 3beta orchestrates microtubule remodeling in compensatory glomerular adaptation to podocyte depletion. J. Biol. Chem. 290, 1348–1363 39 Brehmer, D. et al. (2005) Cellular targets of gefitinib. Cancer Res. 65, 379–382 40 Raida, M. (2011) Drug target deconvolution by chemical proteomics. Curr. Opin. Chem. Biol. 15, 570–575 41 Warsow, G. et al. (2013) PodNet, a protein-protein interaction network of the podocyte. Kidney Int. 84, 104–115 42 Endlich, N. et al. (2014) Two-photon microscopy reveals stationary podocytes in living zebrafish larvae. J. Am. Soc. Nephrol. 25, 681–686 43 Hackl, M.J. et al. (2013) Tracking the fate of glomerular epithelial cells in vivo using serial multiphoton imaging in new mouse models with fluorescent lineage tags. Nat. Med. 19, 1661–1666 44 Kistler, A.D. et al. (2014) In vivo imaging of kidney glomeruli transplanted into the anterior chamber of the mouse eye. Sci. Rep. 4, 3872 45 Weavers, H. et al. (2009) The insect nephrocyte is a podocyte-like cell with a filtration slit diaphragm. Nature 457, 322–326 46 Zhang, F. et al. (2013) An in vivo functional analysis system for renal gene discovery in Drosophila pericardial nephrocytes. J. Am. Soc. Nephrol. 24, 191–197 47 Ebarasi, L. et al. (2011) Zebrafish: a model system for the study of vertebrate renal development, function, and pathophysiology. Curr. Opin. Nephrol. Hypertens. 20, 416–424 48 Zhou, W. and Hildebrandt, F. (2012) Inducible podocyte injury and proteinuria in transgenic zebrafish. J. Am. Soc. Nephrol. 23, 1039–1047 49 Bhatia, S.N. and Ingber, D.E. (2014) Microfluidic organs-on-chips. Nat. Biotechnol. 32, 760–772 50 Huh, D. et al. (2010) Reconstituting organ-level lung functions on a chip. Science 328, 1662–1668 51 Song, J.J. et al. (2013) Regeneration and experimental orthotopic transplantation of a bioengineered kidney. Nat. Med. 19, 646–651 52 Akilesh, S. et al. (2008) Podocytes use FcRn to clear IgG from the glomerular basement membrane. Proc. Natl. Acad. Sci. U. S. A. 105, 967–972 53 Chiang, W.C. et al. (2010) Establishment of protein delivery systems targeting podocytes. PLoS ONE 5, e11837 54 Hauser, P.V. et al. (2010) Novel siRNA delivery system to target podocytes in vivo. PLoS ONE 5, e9463 55 Dolman, M.E. et al. (2010) Drug targeting to the kidney: advances in the active targeting of therapeutics to proximal tubular cells. Adv. Drug Deliv. Rev. 62, 1344– 1357 56 Lan, H.Y. et al. (2003) Inhibition of renal fibrosis by gene transfer of inducible Smad7 using ultrasound-microbubble system in rat UUO model. J. Am. Soc. Nephrol. 14, 1535–1548 57 Huang, L. et al. (2014) AKI after conditional and kidney-specific knockdown of stanniocalcin-1. J. Am. Soc. Nephrol. 25, 2303–2315 58 Fernandez-Fernandez, B. et al. (2014) Therapeutic approaches to diabetic nephropathy—beyond the RAS. Nat. Rev. Nephrol. 10, 325–346
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References
