Podocytes

Podocytes are highly specialized cells of the kidney glomerulus that wrap around capillaries and that neighbor cells of the Bowman’s capsule. When it comes to glomerular filtration, podocytes play an active role in preventing plasma proteins from entering the urinary ultrafiltrate by providing a barrier comprising filtration slits between foot processes, which in aggregate represent a dynamic network of cellular extensions. Foot processes interdigitate with foot processes from adjacent podocytes and form a network of narrow and rather uniform gaps. The fenestrated endothelial cells retain blood cells but permit passage of small solutes and an overlying basement membrane less permeable to macromolecules, in particular to albumin. The cytoskeletal dynamics and structural plasticity of podocytes as well as the signaling between each of these distinct layers are essential for an efficient glomerular filtration and thus for proper renal function. The genetic or acquired impairment of podocytes may lead to foot process effacement (podocyte fusion or retraction), a morphological hallmark of proteinuric renal diseases. Here, we briefly discuss aspects of a contemporary view of podocytes in glomerular filtration, the patterns of structural changes in podocytes associated with common glomerular diseases, and the current state of basic and clinical research.


Podocytes and glomerular filtration
Podocytes (or visceral epithelial cells) are terminally differentiated cells lining the outer surface of the glomerular capillaries. As a major component of the ultrafiltration apparatus, podocytes have a complex cellular architecture consisting of cell body, major processes that extend outward from their cell body, forming interdigitated foot processes (FPs) that enwrap the glomerular capillaries 1 . Major processes are tethered by microtubules and intermediate filaments while FPs contain actin-based cytoskeleton [2][3][4] . Podocyte FPs comprise a functioning slit diaphragm (SD) in between 5,6 , a meshwork of proteins actively participating in podocyte signaling 7-9 . In addition, FPs have a thick, negatively charged coat (glycocalyx) facing the urinary space 10 ; this accounts for negative surface charges throughout the glomerular filtration barrier, which generates an electrostatic repel between the neighboring FPs and helps maintain the unique cytoarchitecture of podocytes by enhancing the physical separation 11 . Podocytes form the glomerular filtration barrier together with the opposing monolayers of fenestrated endothelium in the vascular space 12 and glomerular basement membrane (GBM) in between 13,14 . This three-layer filtration barrier serves as a size-selective and charge-dependent molecular sieve facilitating the filtration of cationic molecules, electrolytes, and small and midsized solutes but restricting the passage of anionic molecules and macromolecules 15,16 . It is important to bear in mind that those layers should be arranged with decreasing selectivity, with the SD being the least selective filter; otherwise, retained plasma proteins would routinely accumulate behind the filtration slits of podocytes 17 . This elegant structure has to oppose hydrostatic pressure in the glomerular capillary, which is the natural driving force behind macromolecular filtration.
If podocytes are injured, mutated, or lost, the elaborate structure of podocytes is physically altered-a process termed 'foot process effacement', which is found in many proteinuric kidney diseases. In some cases, once FPs are effaced (flattened down and fused), the glomerular filtration barrier is no longer intact as evidently indicated by the massive leak of proteins out of the vasculature into the urine, known as proteinuria 18 . Proteinuria (also referred to as 'albuminuria' or 'microalbuminuria') is a clinically important sign of early renal dysfunction. In the following sections, we outline the response of podocytes to various stimuli or injury (or both) to better understand the mechanisms underlying podocyte FP effacement, proteinuria, and glomerular disease progression.

Major causes of podocyte injury
Podocyte function depends on a highly ordered cellular arrangement of filtration compartments and the correct signaling within this microenvironment. Therefore, podocytes are uniquely sensitive to a variety of agents interfering with their actin cytoskeleton, their apical membrane domain (i.e., the negative surface charge), SD complex that regulates podocyte actin reorganization, and GBM structure to which podocytes adhere [19][20][21] . The mechanisms leading to podocytopathies at the molecular level include genetic events (genetic mutations and deletions) associated with common complex diseases 22,23 .
The core structural component of podocyte FPs is a highly regulated actin cytoskeletal network, which was represented either by a dense bundle of actin filaments that extends along the length of FPs or by a relatively short and branched cortical network, which is located at the cell periphery and anchors elements of the SD. The initial response of podocytes to injury is the disruption of these structures and actin dysregulation, where actin and actin-binding proteins accumulate.
In experimental models aiming to study various aspects of cell and molecular biology of podocytes, it has been demonstrated that podocytes are the major targets of various soluble and cellular products, including toxins, reactive oxygen species (ROS), complements, and antibodies, as outlined in Table 1. A commonly used experimental model to induce glomerular proteinuria is puromycin aminonucleoside (PAN) injection into rats. Upon PAN treatment, podocytes undergo significant alterations ranging from FP effacement and cytoskeletal rearrangement to diminished levels of actin cytoskeleton-and SD-associated proteins 24-28 . Abnormal distribution of SD proteins 29 and increasing levels of tight junction proteins 6,30,31 are also reported. Rats develop proteinuria after 4 or 5 days. The early phase of proteinuria is related to the secretion of hyposialylated angiopoietin-like 4 (ANGPTL4) from podocytes, which binds avidly to the GBM and is sensitive to glucocorticoids 32 . Later stages may be mediated by direct oxidative mechanisms. Many pharmacological agents, including dexamethasone 33,34 , fluvastatin 35 , erythropoietin analog darbepoetin 36 , mizoribine 37 , sialic acid 38 , and nuclear factor kappa B (NF-κB) inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) 39 , have been shown to possess the ability to reverse the reorganized stress fiber and cortical actin fiber phenotype observed after PAN treatment.
Similar structural and functional abnormalities are observed in a rat model of adriamycin (ADR) [40][41][42][43][44] . However, podocyte cytoskeleton returns to almost normal appearance by day 20 in PAN-treated rats, whereas pathological and functional changes progress and proteinuria sustains during a similar period of time in ADR-induced nephropathy. Of note, most mouse strains are not susceptible to either of these reagents except BALB/c and BALB/cJ mice, which develop severe proteinuria and progressive renal failure following ADR administration 45,46 . Recently, a nuclear DNA repair protein Prkdc (protein kinase, DNA-activated, catalytic peptide) was discovered to participate in the maintenance of the mitochondrial genome and prevent ADR-induced nephropathy 47 . Simvastatin 48 and thiazolidinedione 49 also confer renoprotective phenotype in response to ADR.
Other toxins, which act on podocytes and have been used experimentally, include diphtheria toxin (DT) 50-52 secreted by Corynebacterium diphtheria, which causes acute loss of podocytes in inducible diphtheria toxin receptor (iDTR) mice; uremic toxin indoxyl sulfate 53 , which decreases the expression of podocyte differentiation and functional marker proteins; and hemolytic uremic Shiga toxin 54-56 , which mediates the release of inflammatory cytokines and vasoactive mediators while potently inhibiting protein synthesis.
These models represent irreversible glomerular damage with major (20% and above in vivo) podocyte depletion 50 , which leads to progressive renal failure. If podocyte loss is less than this threshold, then podocytes have the capacity to recover the normal structure  -4) and this pro-inflammatory response upregulates expression of the co-stimulatory molecule B7-1 57 and the urokinase-type plasminogen activator receptor (uPAR) 60 . LPS also induces the cytosolic variant of cathepsin L (CatL) enzyme 61 , indicating that CatL upregulation in podocytes is associated with the development of proteinuria in mice through a mechanism that involves the cleavage of large GTPase dynamin 61 , synaptopodin 62 , and CD2-associated protein (CD2AP) 63 . In a recent study, we reported that the modification of intracellular pH by glutamine uptake was a protective mechanism of cultured mouse podocytes against cytosolic CatL activity, which was markedly elevated under the disease state 64 . Treatment of cultured human podocytes with TLR-3 immunostimulant polyinosinic-polycytidylic acid (polyIC) induces CatL mRNA and simultaneously downregulates podocyte marker protein synaptopodin 65 , suggesting that polyIC may follow an injury pathway similar to that of LPS.
Another means of podocyte injury are subepithelial immune complexes developing as a result of circulating antibodies, which damage or activate podocytes through complement-dependent processes. A number of signaling pathways have been implicated in complement-mediated podocyte injury 66-69 , in which sublethal concentrations of complement produce a pronounced but reversible disruption of the actin cytoskeleton and associated focal contacts 70 . Other intracellular events include endoplasmic reticulum (ER) stress, production of ROS, and proteases. Focusing on immunologically induced glomerular injury, podocytes also respond to immunologic processes particularly targeting GBM. Passive administration of heterologous sera containing cross-reacting antibodies against the GBM results in vacuolization of podocytes, focal detachment of podocytes from GBM, and immediate onset of glomerulosclerosis with crescent formation 71-78 consistent with a crosstalk between podocytes and the immune system.
Distortion of glomerular charge selectivity by neutralization of the negative charges on podocytes and SDs with polycation protamine sulfate (PS) causes FPs to broaden in vivo 30,79-81 and stress fibers to disintegrate in vitro 82 in a calcium-dependent manner 83,84 . These physiological changes happen within 15 minutes following PS treatment and can be reversed by reperfusion with heparin for another 15 minutes 81,85,86 . PS is also responsible for the phosphorylation of SD protein nephrin 81,86 and focal adhesion complex protein Cas 81 . On the other hand, protamine had little or no effect on the sieving coefficient (also referred to as fractional clearance) of bovine serum albumin once added to neutralize GBM polyanions, a finding that downplays the contribution of GBM to the charge selectivity exhibited by the glomerular filtration barrier 87 . A similar structural alteration can be induced by polycation poly-L-lysine 85 , by removal of the sialic acid 11,88 , or by mutation in glucosamine (UDP-N-acetyl)-2-epimerase/N-acetylmannosamine kinase (GNE), the rate-limiting enzyme of sialic acid biosynthesis 89 . Patients with mutations in GNE, however, develop the rare muscle disease HIBM (hereditary inclusion body myopathy) and never get kidney disease 89 , highlighting the differences between mice and humans in this pathway in the kidney.  In addition to these molecules, factors, and genes implicating various means of podocyte injury, there are proteins or agents such as angiopoietin-like 3 (ANGPTL3) 271 , B7-1 (CD80) 57,272 , cytosolic CatL 61-64 , glucose 273-277 , glutamine 64 , insulin 227 , integrin β3 (ITGB3) 60,278 , tumor necrosis factor-α (TNF-α) 279-281 , transient receptor potential cation channel 5/6 (TRPC5 and TRPC6) 282 , and uPAR 60 that are involved in the regulation of podocyte cytoskeleton. In some cases, this cytoskeletal disaggregation and the associated activation of certain pathways (including tumor suppressor protein p53 and caspases) lead to podocyte loss (in vitro) and detachment from GBM (in vivo). Seminal studies have shown that apoptotic stimuli are mediated by albumin [283][284][285] , aldosterone [286][287][288] , angiopoietin-like3 (ANGPTL3) 289 , angiotensin II 290-294 , fatty acids 295-297 , glucose 232,294,298 , IGF-binding protein-3 (IGFBP-3) 299 , oxidized low-density lipoprotein (LDL) 300 , and TGF-β1 237, [301][302][303][304] . Angiotensin II 305 and glucose 306 might also induce podocyte autophagic processes as evidenced by the presence of the increased number of autophagosomes and autophagic genes such as LC3-2 and beclin-1. Under specific conditions, podocyte injury leads to a phenotypic conversion, where podocytes lose their epithelial features such as nephrin, P-cadherin, and ZO-1 while acquiring mesenchymal markers such as desmin, fibroblast-specific protein-1 (FSP-1), α-smooth muscle actin (α-SMA), vimentin, type I collagen, and fibronectin 307 . This process is referred to as podocyte's epithelial-mesenchymal transition (EMT) and is driven in some cases by endothelin-1 (ET-1) 263 , ILK 212 , PAN 262 , and TGF-β1 308 . When infected by human immunodeficiency virus 1 (HIV-1) NEF protein 309,310 or treated by TNF-α 311 , the podocyte gives a proliferative response marked by the loss of differentiation markers such as synaptopodin, WT-1, and GLEPP-1 and the subsequent expression of the proliferation markers such as podocyte G 1 cyclin, cyclin A, cyclin D1, and Ki-67. There is evidence that non-cytokine-soluble factors such as soluble urokinase-type plasminogen activator receptor (suPAR) 278 cause podocyte FP effacement and proteinuria via a β3 integrin-dependent mechanism but that circulating sialylated ANGPTL4 312 reduces proteinuria via an endothelial β5 integrin-dependent mechanism. By contrast, podocyte-secreted hypo-sialylated ANGPTL4 causes proteinuria via interactions with the GBM 32 .

Podocytes in glomerular disease pathology
Although the podocyte injury is not the only cause of major glomerular diseases, a stable podocyte architecture with interdigitating FPs connected by highly specialized filtration slits is essential for the maintenance and proper function of the glomerular filtration barrier. Both experimental and clinical studies have indicated a pivotal role of podocyte injury in the development and progression of glomerular diseases.
A number of different conditions and health risk factors can result in glomerular disease. Nephrotic syndrome or glomerulonephritis (i.e., malfunction of glomerular filter) may be a direct result of an infection or accumulation of toxic agents in kidneys, or (podocyteand GBM-associated) genetic defects, or may be due to a secondary insult such as a pre-existing disease occurring in the body 320-322 . This represents the conventional approach to classification of glomerular diseases, which generally meets the needs of nephrologists.
The most common cause of primary glomerular disease in adults is focal segmental glomerulosclerosis (FSGS), which is defined by the scarring (sclerosis) of some but not all of the glomeruli (focal) that involves only a section of the affected glomeruli (segmental) by light microscopy of a renal biopsy specimen. In most cases, distinguishing primary (idiopathic) FSGS from the genetic form of FSGS associated with mutations in essential podocyte proteins 323,324 or secondary FSGS (linked to a variety of conditions, including viral infections, drug toxicity, or previous glomerular injury) 325 is challenging; however, it has been proven that this heterogeneous lesion results from podocyte injury 20,50,326,327 . Once the integrity of podocyte FPs is lost, podocytes start to detach from underlying GBM at certain sites revealing bare areas of glomerular capillary surface. Later, these bare areas of GBM contact the Bowman's capsule and form synechia, which represents the earliest committed FSGS lesion. This sequence of pathological events eventually leads to the development of more lesions and progression to glomerulosclerosis 328 . Recurrence of FSGS in renal transplant recipients has given rise to the existence of permeability or circulating factor(s) acting on podocytes as the cause of primary FSGS 329 . To date, a few plasma factors have been proposed 330 but most of these have been found to be non-specific to FSGS serum/plasma 331 . Recently, suPAR was found to be associated with FSGS; for example, two thirds of patients with primary FSGS exhibited high levels of suPAR and those with the highest levels had a greater chance of recurrence after transplantation 278 . In support of this, our group found that higher suPAR levels at baseline are independently associated with faster decline in eGFR and suPAR in plasma can predict risk of developing chronic kidney disease (CKD) in healthy people up to five years before its onset 332 .
Contrary to FSGS, in which podocytes are lost in the areas of sclerosis, minimal change disease (MCD) is a reversible disorder with normal histology and does not cause podocyte depletion. Diffuse effacement of podocyte FPs (accompanied by condensation of the actin-based cytoskeleton but not associated with reduction of any key podocyte-specific protein except podocyte alpha-dystroglycan 333 ) and loss of GBM charge are among the classic features of MCD. All of these changes, and the development of selective proteinuria, are attributed to the secretion from podocytes of a form of ANGPTL4 that lacks sialic acid residues 32,334 .
An FSGS-related but morphologically distinct phenotype was observed when podocytes are infected with HIV 335,336 or induced by infections, drugs, autoimmune diseases, or organ transplants 337-339 . This phenotype is described as collapsing glomerulopathy (CG) and is characterized by extensive loss of mature podocyte markers, severe FP effacement, and focal detachment together with the collapse of the capillary loops. Importantly, podocytes re-enter the cell cycle, become capable of proliferating, and lead to the formation of crescents filling the Bowman's space, making CG structurally distinct from other forms of FSGS 214,309,340 . If left untreated, HIV-1-associated nephropathy progresses to end-stage renal disease within weeks to months, whereas the combined antiretroviral therapy, which blocks HIV-1 replication, limits podocyte hyperplasia and hypertrophy and brings podocytes back to differentiation state 341 . Studies using animal models have demonstrated that CG can be ameliorated by using cell-cycle inhibitors 342 or by activating transcription factors involved in podocyte differentiation 343 ; however, full recovery from CG is scarce 344 .
The immunoglobulin A (IgA) nephropathy (IgAN), which is the most prevalent primary chronic glomerular disease worldwide 345 , exhibits significant heterogeneity in terms of histopathologic features and clinical outcomes 346 . Emerging data suggest that mesangial deposition of IgA1 immune complexes leads to podocyte necrosis and detachment from the GBM 347,348 with the subsequent reduction in nephrin mRNA 349 .
Obesity-related hypertension and diabetes have become epidemic health problems worldwide and major risk factors for the development of CKD 350 . High glucose altered podocyte actin assembly in vitro 351 , high blood glucose (hyperglycemia) induced podocyte apoptosis via the ROS-dependent pathway in obese rodents 298 , and podocyte density and number decreased in patients with obesityrelated glomerulopathy 352 .

Targeting podocytes as renal-specific therapy
Human kidney has been considered a terminally differentiated organ with minimal cellular turnover and limited capacity for repair, suggesting that kidney injuries carrying severe consequences have limited treatment options. The goal of clinical nephrologists and renal researchers should be to identify the renal protection mechanism and to develop strategies for the treatment of kidney or various renal compartments of which kidney is composed. Podocytes are probably the most likely candidate cell population to be analyzed on a molecular level since these intricate cells are the most vulnerable component of the glomerular filtration network even during early stages of injury and serve as hallmarks of a state of glomerular disease 353 . Owing to their post-mitotic nature, podocytes have a limited capacity for cell division and do not regenerate in response to injury and loss 354 . This leads to rapid progression of glomerular diseases unless treated. Regardless of the diverse origins of glomerular diseases, podocytes are critical determinants of outcome for all glomerular diseases, which makes podocytes a unique model for monitoring and investigating disease progression 355 .
Therefore, there has been a pronounced shift toward podocyte proteins as therapeutic targets in the last decade 356 . Sialic acid and its precursors show efficacy in MCD 32 and diabetic nephropathy 357 . Mutant forms of human ANGPTL4 reduce proteinuria without causing hypertriglyceridemia in FSGS and diabetic nephropathy 312,357 . Calcineurin inhibitor cyclosporine A (CsA) stabilizes of the actin cytoskeleton and stress fibers in podocytes by blocking the calcineurin-mediated phosphorylation and CatL-facilitated degradation of synaptopodin 62 . As mentioned earlier, suPAR, which activates integrin αvβ3 independent of uPAR, has been suggested as an FSGS factor 278 . A specific inhibitor of integrin αvβ3, cyclo-RGDfV, ameliorates proteinuria in mouse models of nephrotic syndrome by directly targeting the upregulated integrin αvβ3 on podocytes 60 . CD20 antibody rituximab binds to sphingomyelin phosphodiesterase acid-like 3b (SMPDL-3b) and stabilizes the structure and function of podocytes treated with the sera of patients with recurrent FSGS 358 . Abatacept blocks the interaction of B7-1 (CD80) with cytoskeletal protein talin and thereby stabilizes β1-integrin activity and prevents podocyte motility 359 . However, these results have been subjected to criticism since uncertainties still remain, preventing us from being too optimistic about the general efficacy of abatacept 360-362 . The GTPase dynamin, which promotes endocytosis and regulates actin cytoskeleton 61,97 , is induced by smallmolecule Bis-T-23 as a potential therapeutic approach 363 . Bis-T-23 effectively promotes dynamin assembly into higher-order structures and increases actin polymerization in injured podocytes 361 .
Molecular analysis of podocytes will lead to a better understanding of disease mechanisms and therefore may enable the identification of targets for early-onset diagnostics and disease treatment. In this context, cell-based high-throughput drug screening assays quantifying the phenotypic changes in podocytes (specifically changes in morphology, F-actin cytoskeleton, focal adhesions, cell volume, and so on) offer great value for the discovery of chemotherapeutic agents. Recently, a podocyte cell-based phenotypic assay was developed and applied to identify novel podocyte-protective small molecules and establish specific drug delivery strategies 364 .
The possibilities of targeting podocytes and thereby affecting kidney disease and progression early in the course set high expectations and hopefully will provide a significant benefit to human health in the future.

Competing interests
Jochen Reiser has pending and issued patents on novel strategies for kidney therapeutics and stands to gain royalties from their commercialization. He is co-founder of TRISAQ (Miami, FL, USA), a biotechnology company in which he has financial interest, including stock. MA declares that he has no competing interests.

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