PODOCYTE INJURY CAUSED BY GENETIC CHANGES
The molecules implicated in genetically mediated podocyte damage can be grouped according to their location and function. Not surprisingly, the first uncovered mutations were of genes encoding for proteins located at the slit diaphragm, that is, NPHS1, NPHS2, NPHS3, CD2AP, and TRPC6.
Interestingly, recent studies on NPHS2, the gene-encoding podocin, have revealed additional information, which is relevant to genetic counseling and patient health because pathogenicity of an NPHS2-mutant allele seems to depend on the presence of a second transassociated mutation [4▪▪]. The authors showed that podocin undergoes altered heterodimerization and mislocalization only in the presence of both mutant alleles, with a final dominant-negative effect, whereas mutation of a single allele behaves as recessive.
Cytoskeletal-related genes (SMARCL1, ACTN4, MYH9, Myo1E, ARHGAP24, INF2) have also emerged as important determinants of nephrotic syndrome, confirming the profound association between podocyte structure and function. A recent addition to this group came with the identification of two deleterious mutations of ANLN, the gene encoding the actin-binding protein anillin in two families with autosomal dominant focal segmental glomerulosclerosis (FSGS). Anillin has been shown to interact with Rho GTPase, F-actin, and myosin II. When anillin is knocked down, active Rho (Rho-GTP), F-actin, and myosin II are consequently altered at the intercellular junctions .
The identification of mutations in organelle-related genes, mostly responsible for syndromic forms of nephrotic syndrome, served to drive attention on the relevance of mitochondrial (MTTL1, COQ6, COQ2, PDSS2) and lysosomal (SCARB2) functions in podocytes.
Cong et al.[6▪▪] identified a homozygous missense mutation in the TTC21B gene in seven families with FSGS and rapid progression to end-stage renal failure. TTC21B is a ciliary gene, previously found associated with nephronophthisis , and novel data now show that the TTC21B gene product intraflagellar transport protein 139 is present at the base of the primary cilium in immature podocytes from human fetal kidney and in an undifferentiated podocyte cell line, whereas it is located along microtubules in mature cells. Intraflagellar transport protein 139 knockdown in podocytes led to cilia defects, altered migration, and cytoskeletal changes. Transfection of knockout (KO) podocytes with the mutant protein partially rescued the phenotype, indicating a hypomorphic effect [6▪▪]. Interestingly, kidney tissue from patients carrying the mutation displayed thickening of the tubular basement membrane, which may account for tubular damage and progression to renal failure.
More recently, WDR73 mutations were identified in two families affected by Galloway–Mowat syndrome [8▪], a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. The WDR73 product, a WD40-repeat-containing protein of previously unknown function, seems to be involved in the formation of spindle poles and microtubule asters during mitosis. In the kidney, podocyte expression is clearly observed during development, but it is lost in mature glomeruli, indirectly confirming the association with the mitotic cycle.
INFLAMMATORY, TOXIC, AND METABOLIC INJURY: THE LINK BETWEEN INFLAMMASOMES AND AUTOPHAGY
The large majority of glomerular diseases are characterized by deposition of immunoglobulins or complement components, or both of these, which initiate inflammatory pathways that lead to progressive glomerular and tubulointerstitial damage. Even in metabolic disorders, such as diabetic nephropathy, the role of inflammatory mechanisms is emerging as a prominent element in disease progression . In recent years, the attention of researchers investigating podocyte injury during inflammatory and metabolic diseases has been attracted by two major pathways, that is, the nucleotide-oligomerization domain-like receptor 3 (NLRP3) inflammasome and autophagy.
The inflammasome is a group of multimeric protein complexes that consist of first, sensor molecules, the best studied of which is the pattern recognition receptor NLRP3, second, adaptor proteins, the most common being apoptosis-associated speck-like protein, and third, caspase 1. When NLRP3 is complexed with pro-caspase-1, it leads to the formation of active caspase-1, which cleaves prointerleukin 1β and prointerleukin 18 into their active forms .
Inflammasome formation can be induced either by exogenous molecules, such as those deriving from infective or toxic events, or by mislocalization of endogenous molecules, which occurs during cell damage, autoimmunity, and metabolic imbalances.
Proper activation of the inflammasome is an important first-line defense that belongs to innate immunity, but aberrant inflammasome activation has now been proven to contribute to the pathogenesis of numerous diseases, including autoimmune diseases, such as systemic lupus erythematosus .
As demonstrated by Zhang et al., murine podocytes can express all the key components of the inflammasome (i.e. the NLRP3 receptor, the adaptor protein apoptosis-associated speck-like protein, and caspase 1), whose activation contributes to glomerulosclerosis in a model of hyperhomocysteinemia. Xia et al. observed all inflammasome components and interleukin 1β production in glomeruli of wild-type mice with hyperhomocysteinemia, but not in those lacking NLRP3, and lack of inflammasome formation in these animals corresponded to lower glomerular damage, more preserved glomerular expression of nephrin and podocin, and lower proteinuria.
Shahzad et al. observed inflammasome components and activation in endothelial cells and podocytes in in-vitro and in-vivo models of diabetes and in renal biopsies of diabetic nephropathy. Abolishing NLRP3 or caspase-1 expression selectively in bone marrow-derived cells failed to protect mice against diabetic nephropathy, and transplantation of wild-type bone marrow in NLRP3-KO diabetic animals did not increase glomerular damage. The authors also showed that administration of interleukin-1 receptor (IL-1R) antagonists prevented or even reversed diabetic nephropathy in mice. Activation of the NLRP3 inflammasome in these diabetic models appeared to be due to mitochondrial reactive oxygen species because inhibiting mitochondrial reactive oxygen species production prevented glomerular inflammasome activation and nephropathy.
Autophagy is a conserved intracellular catabolic pathway and a key process for maintaining intracellular homeostasis . Inflammatory responses have been shown to affect autophagy, but a clear understanding of the complex relationship between the immune system, autophagy, and glomerular diseases is still lacking.
Interestingly, a series of studies has found a mutual relationship between autophagy and the inflammasome, showing on one hand that autophagy negatively regulates inflammasome activation, but on the other hand that induction of autophagy depends on the presence of specific inflammasome sensors [16,17], and that autophagy plays a key role in biogenesis and secretion of interleukin 1β . In addition, it has been shown that inflammasomes themselves are ultimately degraded by autophagosomes via the selective autophagic receptor protein 62 .
So far, most studies concerning glomerular diseases have demonstrated a protective role of autophagy. Healthy podocytes, as reported by Hartleben et al., seem to exhibit a particularly high level of constitutive autophagy. These authors showed that podocyte-specific deletion of autophagy-related protein 5 makes the animals more susceptible to glomerular damage and with age causes the appearance of proteinuria and glomerular damage, with the accumulation of oxidized and ubiquitinated proteins and endoplasmic reticulum stress.
More recent works seem to confirm that podocytes need autophagy to maintain their functions. According to Zeng et al., the glomeruli of patients with minimal change disease have more Beclin1-mediated autophagic activity than those with FSGS, and progression from minimal change to FSGS is accompanied by decreasing autophagy. Beclin1-KO mice, or animals treated with autophagy inhibitors, experience more severe damage in the context of puromycin aminonucleoside-induced podocyte injury, whereas induction of autophagy by the mTOR inhibitor rapamycin reduces podocyte damage.
Kawakami et al. induced mutations of the autophagy genes autophagy-related protein 5 or autophagy-related protein 7 during mouse nephrogenesis. This was enough to cause a progressive podocyte and tubular disease that reached renal failure by 6 months. Both podocytes and tubular cells displayed vacuolization, abnormal mitochondria, and evidence of endoplasmic reticulum stress, which were detectable biochemically and by electron microscopy before renal lesions could be observed by light microscopy.
Improved knowledge of the molecular mechanisms occurring during podocyte injury rapidly leads to the identification of numerous therapeutic targets potentially useful to achieve podocyte repair, and translation of experimental data into clinical practice will constitute a major challenge in the near future. Timing of any therapeutic intervention constitutes a relevant issue, particularly considering that in humans podocyte damage remains silent until proteinuria is detected.
It is worth noting that a number of drugs already in use for the treatment of glomerular diseases have shown the ability to directly act on the podocyte because of podocyte expression of drug receptors or enzymatic targets [23–25]. Precise podocyte delivery of novel and old drugs can now be envisaged as a result of major developments in nanotechnology and nanomedicine, taking into account that the use of nanocarriers and engineered biomolecules for targeted therapies has already entered human application in other fields of medicine [26,27]. Recently, size-controlled inorganic nanomaterials, such as gold nanoparticles and quantum dots, have been investigated in vitro and in vivo for glomerular  and podocyte  targeting.
Specific cell delivery of drugs would have obvious advantages in terms of dose reduction, improvement of the drug half-life, and avoidance of side-effects, which could be significant in cases where a precise molecular target is relevant for other cell types. This concept has been proved by the appearance of glomerular injury and proteinuria in patients affected by cancer and treated with humanized antibodies against vascular endothelial growth factor .
As another example, it has been shown that amelioration of podocyte damage can be reached experimentally by acting on the Notch pathway, either by blocking Notch1, as recently reviewed by Kato and Susztak , or by activating Notch2, as described by Tanaka et al.. Several compounds have been produced that are influencing the Notch pathways and human trials are ongoing in the field of oncology (https://clinicaltrials.gov). However, these interventions in the kidney will necessitate specific cell delivery, as it has been found that glomeruli display a different Notch expression and pathway activation as compared with the tubulointerstitium .
One of the most attractive possibilities for driving podocyte repair, action on the integrins differentially expressed or activated/inactivated during podocyte damage, could potentially result in amelioration of cell attachment to the glomerular basement membrane and reequilibration of cell motility . Abatacept, a drug recently utilized in steroid-resistant nephrotic syndrome, seems to act on podocyte de-novo expression of the costimulatory molecule B7-1, which in turn results in changes of integrin beta1 activation and overall amelioration of podocyte adhesion and stability . Interestingly, integrin beta 1 activation can also result from activated Rap1, a small G protein with 53% amino acid identity to Ras . Reduced Rap1 activation, as it occurs during human and experimental podocyte damage, can be due to increased activity of Rap1GAP, a GTPase-activating protein that accelerates hydrolysis of bound GTP to GDP, blocking the activity of small G proteins .
Finally, numerous studies address the possibility of exploiting the regenerative potential of podocytes. Among different podocyte progenitors [38–42], most studies have focused on parietal epithelial cells, that is the cells forming the Bowman's capsule. At least a subpopulation of parietal cells has demonstrated the ability to become podocytes in vitro, and lineage tracing studies have shown the participation of parietal epithelial cells in the development of the glomerular tuft .
However, the actual possibility that parietal epithelial cells contribute to repairing podocyte damage is still controversial and different results have been obtained, most likely because of the different experimental models utilized, the timing of analysis, and the severity of disease [44,45,46▪,47▪]. Instead, there seems to be more consensus on the participation of parietal epithelial cells in glomerulosclerosis and extracapillary proliferation [48–51].
The use of mesenchymal stem cells has also been proposed to repair podocyte injury. Mesenchymal stem cells seem not to repopulate the glomerulus, but rather to act by secreting immunomodulatory factors that can help disease resolution .
An additional, as yet under-developed, possibility is the derivation of podocytes from induced pluripotent stem cells . This technology has several interesting research and therapeutic implications because of the possibility of obtaining induced pluripotent stem cells from patients’ accessible cells, such as fibroblasts or cells from the urinary sediment.
In summary, research is making rapid progress in uncovering the molecular pathways implicated in different types of podocyte injury.
These results offer multiple possibilities for the better treatment of podocyte damage, including possible regeneration, rising the hope of abating the number of patients reaching terminal renal failure.
The Renal Research Laboratory of Milan Policlinic belongs to the ‘Italian Network to Fight FSGS’ organized and supported by ‘Fondazione la Nuova Speranza ONLUS – Lotta alla Glomerulosclerosi Focale’, Rho (MI).
Financial support and sponsorship
This work was supported by Associazione Bambino Nefropatico ABN ONLUS, Milano, Italy.
Conflicts of interest
There are no conflicts of interest.
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Keywords:Copyright © 2015 Wolters Kluwer Health, Inc. All rights reserved.
anillin; autophagy; inflammasome; nanomedicine; parietal cells; podocin