Podocyte Injury Model Research Articles

Advanced glycation end products induce the apoptosis of and inflammation in mouse podocytes through CXCL9-mediated JAK2/STAT3 pathway activation.

Diabetic nephropathy (DN) is a serious and one of the most common microvascular complications of diabetes. There is accumulating evidence to indicate that advanced glycation end products (AGEs), senescent macroprotein derivatives formed at an accelerated rate under conditions of diabetes, play a role in DN. In this study, we found that the serum and urine levels of C-X-C motif chemokine ligand 9 (CXCL9) were significantly elevated in patients with DN compared with healthy controls. Based on an in vitro model of mouse podocyte injury, AGEs decreased the proliferation of podocytes and increased the expression of CXCL9 and C-X-C motif chemokine receptor 3 (CXCR3), and promoted the activation of signal transducer and activator of transcription 3 (STAT3). The knockdown of CXCL9 by the transfection of mouse podoyctes with specific siRNA significantly increased the proliferation and decreased the apoptosis of the podoyctes. Moreover, the levels of inflammatory factors, such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 were also decreased in the podoyctes transfected with siRNA-CXCL9, accompanied by the increased expression of nephrin and podocin, and decreased levels of Bax/Bcl-2 and activated caspase-3. The knockdown of CXCL9 also led to the inactivation of the Janus kinase 2 (JAK2)/STAT3 pathway. Importantly, the use of the JAK2 inhibitor, AG490, and valsartan (angiotensin II receptor antagonist) attenuated the injury induced to mouse podoyctes by AGEs. On the whole, and to the best of our knowledge, this study demonstrates for the first time that AGEs exert pro-apoptotic and pro-inflammatory effects in mouse podoyctes through the CXCL9-mediated activation of the JAK2/STAT3 pathway. Thus, our data provide a potential therapeutic target for DN.

Acute podocyte injury is not a stimulus for podocytes to migrate along the glomerular basement membrane in zebrafish larvae

Podocytes have a unique 3D structure of major and interdigitating foot processes which is the prerequisite for renal blood filtration. Loss of podocytes leads to chronic kidney disease ending in end stage renal disease. Until now, the question if podocytes can be replaced by immigration of cells along the glomerular basement membrane (GBM) is under debate. We recently showed that in contrast to former theories, podocytes are stationary in the zebrafish pronephros and neither migrate nor change their branching pattern of major processes over 23 hours. However, it was still unclear whether podocytes are able to migrate during acute injury. To investigate this, we applied the nitroreductase/metronidazole zebrafish model of podocyte injury to in vivo two-photon microscopy. The application of metronidazole led to retractions of major processes associated with a reduced expression of podocyte-specific proteins and a formation of subpodocyte pseudocyst. Electron microscopy showed that broad areas of the capillaries became denuded. By 4D in vivo observation of single podocytes, we could show that the remaining podocytes did not walk along GBM during 24 h. This in vivo study reveals that podocytes are very stationary cells making regenerative processes by podocyte walking along the GBM very unlikely.

Combination of cilostazol and probucol protected podocytes from lipopolysaccharide-induced injury by both anti-inflammatory and anti-oxidative mechanisms.

Podocytes are essential for maintaining kidney glomerular functions. Injuries to podocyte are closely related to the pathological process of proteinuria. However, a treatment for podocyte injury has still not been established. Cilostazol (CSZ) and probucol (PBC) have been shown to possess renoprotective effects. Therefore, we evaluated these drugs in a lipopolysaccharide (LPS)-induced podocyte injury model. 7-week-old female C57BL/6J mice were fed a normal diet or a diet containing 0.3% CSZ, 0.5% PBC, or both for 10 days. Then, mice were intraperitoneally injected with 13μgg-1 body weight LPS. Both CSZ and PBC decreased LPS-induced albuminuria and co-administration was found to be most effective. These treatments ameliorated the upregulation of monocyte chemoattractant protein 1. In cultured podocytes, CSZ suppressed LPS-induced activation of nuclear factor-kappa B (NF-κB) and phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). PBC reduced LPS-induced activation of NF-κB and reactive oxygen species production. Furthermore, PBC decreased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase4 expression. Our findings suggest that CSZ and PBC are able to inhibit podocyte-injury through different mechanisms, indicating that a combination of these two old drugs is a good treatment option to protect podocytes from injury.

The costimulatory receptor B7-1 is not induced in injured podocytes

Recent research on podocytes has proposed B7-1 as an important player in podocyte biology and as a potential new therapeutic target. B7-1 was upregulated in injured podocytes and described as a biomarker to identify patients who may benefit from abatacept, a B7-1 blocker. However, after this initial enthusiasm, several reports have not confirmed the efficiency of abatacept at inducing proteinuria remission in patients. In order to resolve these discrepancies, we explored the role of B7-1 in the injured podocyte. Both primary cultured and immortalized podocytes were exposed to lipopolysaccharides, but this failed to induce B7-1 expression at the mRNA and protein levels. Importantly, TLR-4 engagement confirmed lipopolysaccharide efficacy. We then evaluated B7-1 expression in several mouse models of podocyte injury including treatment with lipopolysaccharide or Adriamycin, a lupus prone model (NZB/W F1) and subtotal nephrectomy. Using 3 commercially available anti-B7-1 antibodies and appropriate controls, we could not find B7-1 expression in podocytes, whereas some infiltrating cells were positive. Thus, our findings do not support a role for B7-1 in podocyte biology. Hence, further studies are mandatory before treating proteinuric patients with B7-1 blockers.

A-306989, an inhibitor of adenosine kinase, is renoprotective in rodent models of podocyte, basement membrane, and obstructive injury

Adenosine (ADO) is an important regulatory purine nucleoside that accumulates at sites of inflammation and tissue injury including in diseases associated with renal pathology. Endogenous levels of ADO may be increased by inhibiting the ADO-metabolizing enzyme, ADO kinase (AK). AK inhibitors have demonstrated protection in rodent models of diabetic nephropathy. To further investigate AK inhibition as a potential mechanism for renal protection, A-306989, a potent non-nucleoside AK inhibitor, was examined in both in vitro and in vivo assays of renal injury. A-306989 prevented podocyte damage (disruption of actin cytoskeleton) and increased podocyte survival following puromycin aminonucleoside (PAN) application in both mouse and human conditionally immortalized podocytes. Prophylactic oral administration of A-306989 (1.5, 5 and 15mg/kg) reduced proteinuria in a dose-dependent manner and repressed pro-inflammatory/fibrotic gene up-regulation; A-306989 was also efficacious when administered two days following the PAN-insult. A-306989 (10 and 30mg/kg) also significantly reduced proteinuria and macrophage infiltration in a rat model of glomerulonephritis. Finally, A-306989 (15 and 50mg/kg) reduced the expression levels of pro-inflammatory/fibrotic genes, and reduced macrophage infiltration (50mg/kg), but did not affect the deposition of interstitial collagen in fibrotic kidneys from mice with unilateral ureter obstruction. A-306989 also had beneficial actions on “quality of life” measures including improving body weight loss. Thus, these data indicate that enhancement of endogenous ADO levels by A-306989 can positively modulate renal pathology and mimic some of the previously reported beneficial actions of ADO A2A receptor agonists.

Nephrin Suppresses Hippo Signaling through the Adaptor Proteins Nck and WTIP

Podocytes are key components of the kidney blood filtration barrier, and their ability to withstand hemodynamic strain is proposed to be closely tied to their unique and flexible cytoarchitecture. However, the mechanisms that control such mechanotransduction are poorly understood. We have previously established that tyrosine phosphorylation of the transmembrane protein nephrin promotes recruitment of the Nck1/2 cytoskeletal adaptor proteins and downstream actin remodeling. We now reveal that Nck integrates nephrin with the Hippo kinase cascade through association with the adaptor protein WTIP. Using mutational analysis, we show that Nck sequesters WTIP and its binding partner Lats1 to phosphorylated nephrin, resulting in decreased phospho-activation of Lats1. We further demonstrate that, coincident with nephrin dephosphorylation in a transient model of podocyte injury in mice, Lats1 is rapidly activated, and this precedes significant down-regulation of the transcription regulator Yap. Moreover, we show reduced levels of Yap protein in mice with chronic disruption of nephrin phospho-signaling. Together, these findings support the existence of a dynamic molecular link between nephrin signaling and the canonical Hippo pathway in podocytes, which may facilitate the conversion of mechanical cues to biochemical signals promoting podocyte viability.

Melatonin protects podocytes from angiotensin II-induced injury in an invitro diabetic nephropathy model.

Diabetic nephropathy (DN) is a severe renal disorder characterized by podocyte damage and accumulation of extracellular matrix leading to further glomerulosclerosis, possibly via the activation of inflammatory signaling and the generation of oxidative stress. Melatonin has been considered a robust anti‑oxidant, and is able to attenuate DN in several animal models. In the present study, cell viability was examined by CCK-8 assay. Cell apoptosis, intracellular reactive oxygen species and mitochondrial membrane potential (MMP) levels were measured by flow cytometry. Western blot analysis was performed to detect the expression of Nephrin, Bcell lymphoma‑2 (Bcl‑2)‑associatedXprotein (Bax), Bcl-2, Caspase-3, Janus kinase (JAK)2 and Signal transducer and activator of transcription (STAT)3 in podocytes. Based on an invitro podocyte injury model, the present study found that the application of melatonin significantly reduced AngII‑induced apoptosis and increased the proliferative rate of cells, as evidenced by decreased expression levels of apoptotic proteins, including Caspase‑3 and Bax, and a change in the ratio of Bax/Bcl‑2. Further investigation demonstrated that a reduction in oxidative stress and the recovery of mitochondrial function were involved in this protective effect. In addition, the Jak/STAT signaling pathway was inhibited, indicating that cytokine‑mediated inflammation was also targeted by melatonin. The present study demonstrated for the first time, to the best our knowledge, that melatonin exerted an anti-apoptotic effect in AngII-mediated podocyte injury, and indicates a possible mechanism to explain the protective effect of melatonin in DN.

Thrombospondin 1 Deficiency Ameliorates the Development of Adriamycin-Induced Proteinuric Kidney Disease

Accumulating evidence suggests that thrombospondin 1 (TSP1) is an important player in diabetic nephropathy. However, the role of TSP1 in podocyte injury and the development of non-diabetic proteinuric kidney disease is largely unknown. In the current study, by using a well-established podocyte injury model (adriamycin-induced nephropathy mouse model), we examined the contribution of TSP1 to the development of proteinuric kidney disease. We found that TSP1 was up-regulated in the glomeruli, notably in podocytes, in adriamycin injected mice before the onset of proteinuria. ADR treatment also stimulated TSP1 expression in cultured human podocytes in vitro. Moreover, increased TSP1 mediated ADR-induced podocyte apoptosis and actin cytoskeleton disorganization. This TSP1’s effect was through a CD36-dependent mechanism and involved in the stimulation of p38MAPK pathway. Importantly, in vivo data demonstrated that TSP1 deficiency protected mice from ADR induced podocyte loss and foot process effacement. ADR induced proteinuria, glomerulosclerosis, renal macrophage infiltration and inflammation was also attenuated in TSP1 deficient mice. Taken together, these studies provide new evidence that TSP1 contributes to the development of non-diabetic proteinuric kidney disease by stimulating podocyte injury and the progression of renal inflammation.

Calpain-Mediated Cleavage of Calcineurin in Puromycin Aminonucleoside-Induced Podocyte Injury.

The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus are widely used in the treatment of proteinuria diseases. As the direct target of these drugs, calcineurin has previously been demonstrated to play a role in proteinuria diseases. However, aside from its immune-related effects, the local status of calcineurin in renal inherent cells has not been fully explored in the settings of proteinuria disease and podocyte injury. In this study, calcineurin activity and protein expression in the well-known puromycin aminonucleoside (PAN)-induced podocyte injury model were examined. Interestingly, we found that calcineurin activity was abnormally increased in PAN-treated podocytes, whereas the expression of the full-length 60-kDa calcineurin protein was decreased. This result suggests that there may be another activated form of calcineurin that is independent of the full-length phosphatase. To investigate whether calpain is involved in regulating calcineurin, we exposed PAN-treated podocytes to both pharmacological inhibitors of calpain and specific siRNAs against calpain. Calpain blockade reduced the enhanced calcineurin activity and restored the down-regulated expression of 60-kDa calcineurin. In addition, purified calpain protein was incubated with podocyte extracts, and a 45-kDa fragment of calcineurin was identified; this finding was confirmed in PAN-induced podocyte injury and calpain inhibition experiments. We conclude that calcineurin activity is abnormally increased during PAN-induced podocyte injury, whereas the expression of the full-length 60-kDa calcineurin protein is down-regulated due to over-activated calpain that cleaves calcineurin to form a 45-kDa fragment.

Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury.

In most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury.

Nephrin Tyrosine Phosphorylation Is Required to Stabilize and Restore Podocyte Foot Process Architecture.

Podocytes are specialized epithelial cells of the kidney blood filtration barrier that contribute to permselectivity via a series of interdigitating actin-rich foot processes. Positioned between adjacent projections is a unique cell junction known as the slit diaphragm, which is physically connected to the actin cytoskeleton via the transmembrane protein nephrin. Evidence indicates that tyrosine phosphorylation of the intracellular tail of nephrin initiates signaling events, including recruitment of cytoplasmic adaptor proteins Nck1 and Nck2 that regulate actin cytoskeletal dynamics. Nephrin tyrosine phosphorylation is altered in human and experimental renal diseases characterized by pathologic foot process remodeling, prompting the hypothesis that phosphonephrin signaling directly influences podocyte morphology. To explore this possibility, we generated and analyzed knockin mice with mutations that disrupt nephrin tyrosine phosphorylation and Nck1/2 binding (nephrin(Y3F/Y3F) mice). Homozygous nephrin(Y3F/Y3F) mice developed progressive proteinuria accompanied by structural changes in the filtration barrier, including podocyte foot process effacement, irregular thickening of the glomerular basement membrane, and dilated capillary loops, with a similar but later onset phenotype in heterozygous animals. Furthermore, compared with wild-type mice, nephrin(Y3F/Y3F) mice displayed delayed recovery in podocyte injury models. Profiling of nephrin tyrosine phosphorylation dynamics in wild-type mice subjected to podocyte injury indicated site-specific differences in phosphorylation at baseline, injury, and recovery, which correlated with loss of nephrin-Nck1/2 association during foot process effacement. Our results define an essential requirement for nephrin tyrosine phosphorylation in stabilizing podocyte morphology and suggest a model in which dynamic changes in phosphotyrosine-based signaling confer plasticity to the podocyte actin cytoskeleton.

GADD45B mediates podocyte injury in zebrafish by activating the ROS-GADD45B-p38 pathway.

GADD45 gene has been implicated in cell cycle arrest, cell survival or apoptosis in a cell type specific and context-dependent manner. Members of GADD45 gene family have been found differentially expressed in several podocyte injury models, but their roles in podocytes are unclear. Using an in vivo zebrafish model of inducible podocyte injury that we have previously established, we found that zebrafish orthologs of gadd45b were induced upon the induction of podocyte injury. Podocyte-specific overexpression of zebrafish gadd45b exacerbated edema, proteinuria and foot-process effacement, whereas knockdown of gadd45b by morpholino-oligos in zebrafish larvae ameliorated podocyte injury. We then explored the role of GADD45B induction in podocyte injury using in vitro podocyte culture. We confirmed that GADD45B was significantly upregulated during the early phase of podocyte injury in cultured human podocytes and that podocyte apoptosis induced by TGF-β and puromycin aminonucleoside (PAN) was aggravated by GADD45B overexpression but ameliorated by shRNA-mediated GADD45B knockdown. We also showed that ROS inhibitor NAC suppressed PAN-induced GADD45B expression and subsequent activation of p38 MAPK pathway in podocytes and that inhibition of GADD45B diminished PAN-induced p38 MAPK activation. Taken together, our findings demonstrated that GADD45B has an important role in podocyte injury and may be a therapeutic target for the management of podocyte injury in glomerular diseases.

The mTORC2/Akt/NFκB Pathway-Mediated Activation of TRPC6 Participates in Adriamycin-Induced Podocyte Apoptosis

Background/Aims: Although increased expression and gain function of transient receptor potential cation channel 6 (TRPC6) has been associated with the pathogenesis of some proteinuric glomerular diseases, it remains elusive how TRPC6 participates in the process of podocyte damage. Methods: The potential signaling responsible for TRPC6 activation was investigated using immunoblot assays in an in vitro podocyte injury model induced by Adriamycin (ADR). Podocyte apoptosis was measured using FITC-conjugated Annexin V and Propidium Iodide staining. The channel activity of TRPC6 was assessed using the Ca²⁺ influx assay. Results: Increase of TRPC6 expression was detected in ADR-treated podocytes, and TRPC6 knockdown significantly decreased ADR-induced podocytes apoptosis. Following ADR treatment, phospho-mTOR^Ser2481 and phospho-Akt^Ser473 was significantly increased in a time-dependent manner, whereas phospho-mTOR^Ser2448 and phospho-p70S6K^Thr389 showed no change. ADR-induced apoptosis was prevented by ku0063794 (a dual mTOR complexes inhibitor), not by rapamycin (a specific mTORC1 inhibitor). Furthermore, nuclear translocation of NFκB/p65 was detected in ADR-treated podocytes, which was prevented by an Akt inhibitor triciribine. Of note, NFκB inhibitor PDTC prevented ADR-induced increase of TRPC6, and decreased ADR-induced apoptosis. We found that Akt activation and NFκB nuclear translocation was significantly inhibited by knockdown of mTORC2 protein Rictor, not by mTORC1 protein Raptor. In comparison with control, the Ca²⁺ influx was significantly increased in ADR-treated podocytes, which was remarkably prevented by TRPC6 knockdown. ADR-induced increase of TRPC6 channel activity was dramatically prevented by ku0063794, but not by rapamycin. Additionally, knockdown of Rictor, not Raptor, prevented ADR-induced increase of the Ca²⁺ influx. Moreover, the application of NFκB inhibitor PDTC also prevented the Ca²⁺ influx in ADR-treated podocytes. Conclusions: Our findings revealed that the mTORC2/Akt/NFκB pathway-mediated activation of TRPC6 participates in ADR-induced podocyte apoptosis.

Biallelic Mutations in Nuclear Pore Complex Subunit NUP107 Cause Early-Childhood-Onset Steroid-Resistant Nephrotic Syndrome

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133kDa) and NUP107 incorporation into NPCs invitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties ofthe podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a "podocyte-injury model" as the pathomechanism for SRNS due to biallelic NUP107 mutations.

MiR-135 family members mediate podocyte injury through the activation of Wnt/β-catenin signaling.

The upregulation of Wnt/β-catenin signaling occurs in virtually all types of kidney disease and is associated with podocyte injury. However, the precise mechanisms involved in the development of kidney disease remain to be elucidated. MicroRNAs (miRNAs or miRs) are a class of short non-coding RNAs and they have been shown to be regulators of gene expression, mainly by binding to the untranslated region (UTR) of mRNAs. The aim of the present study was to determine the role of the 2 members of the miR-135 family (miR-135a and miR-135b) in podocyte injury and to elucidate the mechanisms responsible for the damage to podocytes. The results revealed that miR-135a and miR-135b were upregulated in models of podocyte injury and in glomeruli isolated from patients with focal segmental glomerulosclerosis (FSGS). The ectopic expression of miR-135a and miR-135b led to severe podocyte injury and the disorder of the podocyte cytoskeleton. Our findings demonstrated that miR-135a and miR-135b activated Wnt/β-catenin signaling and induced the nuclear translocation of β-catenin. Using luciferase reporter assays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, glycogen synthase kinase 3β (GSK3β) was identified as a target gene of miR-135a and miR-135b. To the best of our knowledge, this is the first study to demonstrate that members of the miR-135 family (specifically miR-135a and miR-135b) regulate the expression of GSK3β, thus playing a role in the development of podocyte injury and the disorder of the podocyte cytoskeleton. This is an important finding as it may contribute to the development of novel therapeutics for podocyte injury-associated glomerulopathies.

A vital role for Angptl3 in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro.

BackgroundPodocyte detachment and apoptosis are two risk factors causing podocyte loss, F-actin rearrangement is involved in detachment and apoptosis. However, the nature of events that promote detachment and apoptosis of podocytes and whether detachment occurred simultaneously with apoptosis are still unclear. Previously, it was found that angiopoietin-like3 (Angptl3) induces F-actin rearrangement in podocytes. In this study we investigate whether Angptl3 influences podocyte loss (detachment and apoptosis) and the process through which Angptl3 exactly influenced the podocyte loss.MethodsIn conditionally immortalized mice podocytes, recombinant mice Angptl3 protein (rm-Angptl3) was used to mimic Angptl3 overexpression model and transfection with small interfering RNA (siRNA) to knockdown the expression of Angptl3. Both flow cytometry analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay were used to detect apoptosis. Rearrangement of F-actin was assessed using confocal microscopy. Western blot assay was used to measure levels of Angptl3, integrin α3β1, integrin-linked kinase (ILK), p53, caspase 3, and phosphorylation of integrin β1.ResultsIn a puromycin aminonucleoside (PAN)-induced podocyte injury model, rm-Angptl3 accelerated the loss of podocytes, both detachment and apoptosis occurred, and F-actin rearrangement is involved in the process. However, knockdown of Angptl3 by siRNA markedly ameliorated these injuries. Observed effects were partially correlated with the altered integrin α3β1, ILK and p53, rather than caspase 3.ConclusionsAngptl3 is a novel factor involved in the PAN-induced podocyte loss by affecting detachment and apoptosis in vitro. This study helps to deepen the understanding of the mechanisms of podocyte loss and lays the foundation for developing a new successful therapy for podocyte injury via lower expression of Angptl3.

Transgenic Strategies to Study Podocyte Loss and Regeneration

Podocyte death and regeneration are major topics in kidney research but remain controversial. Data obtained in humans demonstrate the existence of cells sited along Bowman's capsule that behave as podocyte progenitors in vitro and in in vivo mouse models of podocyte injury xenotrasplanted with this human-derived population. However, this podocyte reservoir still remains elusive in murine models, where it could be more easily studied. Transgenic models can be a powerful tool to identify this population and to better understand its dynamics and hierarchies in both physiological and pathological conditions. Indeed, exploiting transgenic approaches allows detecting, at the single cell level, movements, cell death, and replacement. Moreover, through lineage tracing it is now possible to identify specific population increase and to point out clonal expansions during or after the regenerative processes. However, applying transgenic strategies to study glomerular regeneration requires the search of markers to unequivocally identify this progenitor population. Achieving this aim would lead to a deep comprehension of the biological processes that underlie glomerular regeneration and clarify how different cell pools interface during this phase. Here we discuss strategies that have been used and new approaches in transgenic models finalized to study podocyte loss and subsequent replacement.

Smad3/Nox4-mediated mitochondrial dysfunction plays a crucial role in puromycin aminonucleoside-induced podocyte damage

Podocyte depletion due to apoptosis is the key hallmark of proteinuric kidney disease progression. Recently, several studies reported that mitochondrial (mt) dysfunction is involved in podocyte injury, while the underlying molecular mechanisms remain elusive. This study investigated the potential proximal signaling related to in vitro and in vivo mitochondrial dysfunction in a puromycin aminonucleoside (PA)-induced podocyte injury model. PA time- and dose-dependently resulted in cultured mouse podocyte damage, presenting with an increase of apoptotic cells and induction of activated caspase3/9. PA also caused mitochondrial damage and dysfunction based on the downregulation of the mtDNA level, decrease of transcriptional factors mtTfa and Nrf-1, decrease of CoxI, II and IV, and reduction of the oxygen consumption level and mitochondrial membrane potential level as well as excessive production of cellular ROS. Additionally, antioxidant MnSOD and catalase levels were decreased in mitochondrial fractions, and reduction of complex I and IV activity was also observed in PA-stimulated podocytes. Furthermore, an obvious translocation of p-Smad3 from the cytosol to nuclei and induction of mitochondrial Nox4 were detected following PA application. The PA-induced shift of cytochrome c was observed from mitochondria to the cytoplasm. Induction of Nox4 by PA administration was significantly repressed by Smad3-shRNA, while Nox4-shRNA showed no effect on PA-induced p-Smad3 activation. Notably, both Smad3 and Nox4 silencing significantly prevented the reduction of the mtDNA level, restored mitochondrial function, and decreased cellular apoptosis in PA-stimulated podocytes. A similar mitochondrial dysfunction was obtained in a PA-injected nephropathy rat, which was effectively inhibited by treatment with the antiproteinuric drug prednisone. In addition, Dab2 knockdown decreased albumin uptake and influx whereas it showed no effect on cellular apoptosis in PA-stimulated podocytes. In conclusion, our findings demonstrated that Smad3–Nox4 axis-mediated mitochondrial dysfunction is involved in PA-induced podocyte damage likely via increasing ROS generation and activating the cytochrome c–caspase9–caspase3 apoptotic signaling pathway. Dab2 may be required for the increased permeability of podocytes following injury.

Protein Tyrosine Phosphatase 1B Inhibition Protects against Podocyte Injury and Proteinuria

Protein tyrosine phosphatase 1B (PTP1B) is a ubiquitously expressed nonreceptor protein-tyrosine phosphatase that regulates various cellular functions, including migration. Recent studies suggest that an increased migratory phenotype of podocytes may be responsible for proteinuria and foot process effacement. The current study addresses the role of PTP1B in podocyte injury and proteinuria. PTP1B was markedly up-regulated in the glomerulus, notably in podocytes, in three rodent models of podocyte injury. Podocyte-specific ablation of the PTP1B gene ameliorated proteinuria induced by lipopolysaccharide and Adriamycin (doxorubicin). The use of a specific PTP1B inhibitor also protected against lipopolysaccharide-induced proteinuria. In contrast, podocyte-specific PTP1B transgenic male mice developed spontaneous proteinuria and foot process effacement. In cultured mouse podocytes, PTP1B knockdown and/or pretreatment with the PTP1B inhibitor blunted lipopolysaccharide-induced cell migration, activation of Src-family kinases (SFKs), and phosphorylation of focal adhesion kinase at Y397 (pFAK(Y397)), the latter being crucial for cell migration. Lipopolysaccharide-injected mice showed increased glomerular expression of active SFKs and pFAK(Y397), both of which were inhibited by podocyte-specific PTP1B knockout and the PTP1B inhibitor. Moreover, podocyte-specific PTP1B transgenic mice showed increased glomerular expression of active SFKs and pFAK(Y397). In summary, PTP1B up-regulation in podocytes induces a migratory response by activating SFKs and FAK, leading to foot process effacement and proteinuria. Pharmacological inhibition of PTP1B may have therapeutic potential in the treatment of proteinuric diseases.

Crk1/2 and CrkL form a hetero-oligomer and functionally complement each other during podocyte morphogenesis

Activation of the slit diaphragm protein Nephrin induces actin cytoskeletal remodeling resulting in lamellipodia formation in podocytes in vitro in a phosphatidylinositol-3 kinase, focal adhesion kinase, Cas, and Crk1/2-dependent fashion. In mice, podocyte-specific deletion of Crk1/2 prevents or attenuates foot process effacement in two models of podocyte injury. This suggests that cellular mechanisms governing lamellipodial protrusion in vitro are similar to those in vivo during foot process effacement. Since Crk1/2 null mice develop and aged normally, we tested whether the Crk1/2 paralog, CrkL, functionally complements Crk1/2 in a podocyte-specific context. Podocyte-specific CrkL null mice, like podocyte-specific Crk1/2 null mice, developed and aged normally but were protected from protamine sulfate-induced foot process effacement. Simultaneous podocyte-specific deletion of Crk1/2 and CrkL resulted in albuminuria detected by six weeks post-partum and associated with altered podocyte process architecture. Nephrin-induced lamellipodia formation in podocytes in vitro was CrkL-dependent. CrkL formed a heterooligomer with Crk2 and, like Crk2, was recruited to tyrosine phosphorylated Nephrin. Thus, Crk1/2 and CrkL are physically-linked, functionally complement each other during podocyte foot process spreading, and together are required for developing typical foot process architecture.