The Nephrotoxin Puromycin Aminonucleoside Induces Injury in Kidney Organoids Differentiated from Induced Pluripotent Stem Cells.

Kidney diseases, including acute kidney injury (AKI) and chronic kidney disease (CKD), which can progress to end stage renal disease (ESRD), are a worldwide health burden. Organ transplantation or kidney dialysis are the only effective available therapeutic tools. Therefore, in vitro models of kidney diseases and the development of prospective therapeutic options are urgently needed. Within the kidney, the glomeruli are involved in blood filtration and waste excretion and are easily affected by changing cellular conditions. Puromycin aminonucleoside (PAN) is a nephrotoxin, which can be employed to induce acute glomerular damage and to model glomerular disease. For this reason, we generated kidney organoids from three iPSC lines and treated these with PAN in order to induce kidney injury. Morphological observations revealed the disruption of glomerular and tubular structures within the kidney organoids upon PAN treatment, which were confirmed by transcriptome analyses. Subsequent analyses revealed an upregulation of immune response as well as inflammatory and cell-death-related processes. We conclude that the treatment of iPSC-derived kidney organoids with PAN induces kidney injury mediated by an intertwined network of inflammation, cytoskeletal re-arrangement, DNA damage, apoptosis and cell death. Furthermore, urine-stem-cell-derived kidney organoids can be used to model kidney-associated diseases and drug discovery.

Decreased Podocyte Vesicle Transcytosis and Albuminuria in APC C-Terminal Deficiency Mice with Puromycin-Induced Nephrotic Syndrome.

In minimal change nephrotic syndrome, podocyte vesicle transport is enhanced. Adenomatous polyposis coli (APC) anchors microtubules to cell membranes and plays an important role in vesicle transport. To clarify the role of APC in vesicle transport in podocytes, nephrotic syndrome was induced by puromycin amino nucleoside (PAN) injection in mice expressing APC1638T lacking the C-terminal of microtubule-binding site (APC1638T mouse); this was examined in renal tissue changes. The kidney size and glomerular area of APC1638T mice were reduced (p = 0.014); however, the number of podocytes was same between wild-type (WT) mice and APC1638T mice. The ultrastructure of podocyte foot process was normal by electron microscopy. When nephrotic syndrome was induced, the kidneys of WT+PAN mice became swollen with many hyaline casts, whereas these changes were inhibited in the kidneys of APC1638T+PAN mice. Electron microscopy showed foot process effacement in both groups; however, APC1638T+PAN mice had fewer vesicles in the basal area of podocytes than WT+PAN mice. Cytoplasmic dynein-1, a motor protein for vesicle transport, and α-tubulin were significantly reduced in APC1638T+PAN mice associated with suppressed urinary albumin excretion compared to WT+PAN mice. In conclusion, APC1638T mice showed reduced albuminuria associated with suppressed podocyte vesicle transport when minimal change nephrotic syndrome was induced.

The Use of High-Throughput Transcriptomics to Identify Pathways with Therapeutic Significance in Podocytes.

Podocytes have a unique structure that supports glomerular filtration function, and many glomerular diseases result in loss of this structure, leading to podocyte dysfunction and ESRD (end stage renal disease). These structural and functional changes involve a complex set of molecular and cellular mechanisms that remain poorly understood. To understand the molecular signature of podocyte injury, we performed transcriptome analysis of cultured human podocytes injured either with PAN (puromycin aminonucleoside) or doxorubicin/adriamycin (ADR). The pathway analysis through DE (differential expression) and gene-enrichment analysis of the injured podocytes showed Tumor protein p53 (P53) as one of the major signaling pathways that was significantly upregulated upon podocyte injury. Accordingly, P53 expression was also up-regulated in the glomeruli of nephrotoxic serum (NTS) and ADR-injured mice. To further confirm these observations, cultured podocytes were treated with the P53 inhibitor pifithrin-α, which showed significant protection from ADR-induced actin cytoskeleton damage. In conclusion, signaling pathways that are involved in podocyte pathogenesis and can be therapeutically targeted were identified by high-throughput transcriptomic analysis of injured podocytes.

MiR-27b regulates podocyte survival through targeting adenosine receptor 2B in podocytes from non-human primate.

MicroRNAs are a group of small non-coding RNAs that play key roles in almost every aspect of mammalian cell. In kidney, microRNAs are required for maintaining normal function of renal cells, disruption of which contributes to pathogenesis of renal diseases. In this study, we investigated the potential role of miRNAs as key regulators of podocyte survival by using a primary cell culture model from non-human primates (NHPs). Through microRNA profile comparison in glomeruli from mouse, rat and NHP, miR-27b was found to be among a list of glomeruli-enriched miRNA conserved across species. In NHP primary podocyte culture, significant downregulation of miR-27b was observed during treatment of puromycin aminonucleoside (PAN), a classic nephrotoxin. Overexpression of miR-27b enhanced PAN-induced apoptosis and cytoskeleton destruction in podocytes while its inhibition had a protective effect. Target identification analysis identified Adora2b as a potential direct target of miR-27b. Ectopic expression of miR-27b suppressed both Adora2b mRNA and protein expression, whereas inhibition of miR-27b increased the transcript and protein expression levels of Adora2B. Dual luciferase assay further confirmed Adora2b as a direct target of miR-27b. Furthermore, knockdown of Adora2b by siRNAs enhanced PAN-induced apoptosis, similar to the phenotypes we had observed with miR-27b overexpression. In addition, stimulating the adenosine signaling by an Adora2b agonist, NECA, improved podocyte survival upon PAN treatment. Taken together, our data identified a novel role of miR-27b-adora2b axis in primary podocyte survival upon injury and suggested a critical role of adenosine signaling pathway in podocyte protection.

Bone marrow-derived mesenchymal stem cells ameliorate nephrosis through repair of impaired podocytes.

PURPOSE: The purpose of this study was to investigate the effects of bone marrow-derived mesenchymal stem cells (BMSC) on podocytes of puromycin amino nuclear glucoside (PAN) -induced nephrosis in mice. METHODS: Mice were randomly divided into Control, PAN and BMSC groups. Mice were injected with PAN (0.5 mg/g weight) via the tail vein. The 24-h urinary protein was obtained after modelling, and urinary protein excretion was determined. The blood and kidney specimens were isolated after the tenth day of modelling. Blood samples were collected for measuring serum creatinine (SCr) and blood urea nitrogen (BUN). A sample of kidney was taken for observing pathological changes through hematoxylin-eosin staining and electron microscopy, and the rest of the kidney was used for detecting the protein and mRNA expression of nephrin, CD2AP, synaptopodin, TRPC6 by real-time quantitative PCR, Western-blot and immunohistochemistry. RESULTS: After PAN injection, podocyte foot process fusion was detected by electron microscopy, and the 24 h urinary protein excretion increased compared with control mice on days 3, 7 and 10 post-PAN injection (P.

Estrogen-related receptor (ERR) γ protects against puromycin aminonucleoside-induced podocyte apoptosis by targeting PI3K/Akt signaling.

Accumulating evidence has shown that podocyte apoptosis is of vital importance for the development of glomerulosclerosis and progressive loss of renal function. However, the molecular mechanisms leading to podocyte apoptosis are still elusive. In this study, we investigated the role of estrogen-related receptor (ERR) γ in podocyte apoptosis, as well as the underlying mechanisms. Treatment of PAN caused a dose- and time-dependent podocyte apoptosis in line with a significant downregulation of ERRγ. Interestingly, the occurrence of ERRγ downregulation appeared earlier than the onset of cell apoptosis, suggesting a potential that ERRγ reduction triggered apoptotic response in podocyte. To test this hypothesis, ERRγ siRNA was administered to the podocytes. Strikingly, ERRγ silencing resulted in a significant cell apoptosis accompanied with increased injury markers of B7-1 and cathepsin L and decreased podocyte protein nephrin. In contrast, overexpression of ERRγ remarkably attenuated PAN-induced cell apoptosis. Moreover, ERRγ overexpression stimulated PI3K/Akt signaling pathway evidenced by increased expression of PI3K subunits p85α and p110α and phosphorylated Akt. Importantly, a specific PI3K inhibitor LY294002 entirely reversed the anti-apoptotic effect of ERRγ following PAN treatment. Finally, we observed a striking downregulation of ERRγ in PAN-treated rat kidneys, suggesting that our cell model replicated the in vivo condition. Taken together, these data highly suggested that ERRγ played a novel role in modulating podocyte apoptosis by targeting PI3K/Akt signaling pathway.

Original Research: Potential of urinary nephrin as a biomarker reflecting podocyte dysfunction in various kidney disease models.

Urinary nephrin is a potential non-invasive biomarker of disease. To date, however, most studies of urinary nephrin have been conducted in animal models of diabetic nephropathy, and correlations between urinary nephrin-to-creatinine ratio and other parameters have yet to be evaluated in animal models or patients of kidney disease with podocyte dysfunction. We hypothesized that urinary nephrin-to-creatinine ratio can be up-regulated and is negatively correlated with renal nephrin mRNA levels in animal models of kidney disease, and that increased urinary nephrin-to-creatinine ratio levels are attenuated following administration of glucocorticoids. In the present study, renal nephrin mRNA, urinary nephrin-to-creatinine ratio, urinary protein-to-creatinine ratio, and creatinine clearance ratio were measured in animal models of adriamycin nephropathy, puromycin aminonucleoside nephropathy, anti-glomerular basement membrane glomerulonephritis, and 5/6 nephrectomy. The effects of prednisolone on urinary nephrin-to-creatinine ratio and other parameters in puromycin aminonucleoside (single injection) nephropathy rats were also investigated. In all models tested, urinary nephrin-to-creatinine ratio and urinary protein-to-creatinine ratio increased, while renal nephrin mRNA and creatinine clearance ratio decreased. Urinary nephrin-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA in almost all models, as well as a significant positive correlation with urinary protein-to-creatinine ratio and a significant negative correlation with creatinine clearance ratio. Urinary protein-to-creatinine ratio exhibited a significant negative correlation with renal nephrin mRNA. Following the administration of prednisolone to puromycin aminonucleoside (single injection) nephropathy rats, urinary nephrin-to-creatinine ratio was significantly suppressed and exhibited a significant positive correlation with urinary protein-to-creatinine ratio. In addition, the decrease in number of glomerular Wilms tumor antigen-1-positive cells was attenuated, and urinary nephrin-to-creatinine ratio exhibited a significant negative correlation in these cells. In conclusion, these results suggest that urinary nephrin-to-creatinine ratio level is a useful and reliable biomarker for predicting the amelioration of podocyte dysfunction by candidate drugs in various kidney disease models with podocyte dysfunction. This suggestion will also be validated in a clinical setting in future studies.

The Expression Profile of Complement Components in Podocytes.

Podocytes are critical for maintaining the glomerular filtration barrier and are injured in many renal diseases, especially proteinuric kidney diseases. Recently, reports suggested that podocytes are among the renal cells that synthesize complement components that mediate glomerular diseases. Nevertheless, the profile and extent of complement component expression in podocytes remain unclear. This study examined the expression profile of complement in podocytes under physiological conditions and in abnormal podocytes induced by multiple stimuli. In total, 23/32 complement component components were detected in podocyte by conventional RT-PCR. Both primary cultured podocytes and immortalized podocytes expressed the complement factors C1q, C1r, C2, C3, C7, MASP, CFI, DAF, CD59, C4bp, CD46, Protein S, CR2, C1qR, C3aR, C5aR, and Crry (17/32), whereas C4, CFB, CFD, C5, C6, C8, C9, MBL1, and MBL2 (9/32) complement factors were not expressed. C3, Crry, and C1q-binding protein were detected by tandem mass spectrometry. Podocyte complement gene expression was affected by several factors (puromycin aminonucleoside (PAN), angiotensin II (Ang II), interleukin-6 (IL-6), and transforming growth factor-ß (TGF-ß)). Representative complement components were detected using fluorescence confocal microscopy. In conclusion, primary podocytes express various complement components at the mRNA and protein levels. The complement gene expressions were affected by several podocyte injury factors.

Syndecan-4 ectodomain evokes mobilization of podocyte TRPC6 channels and their associated pathways: An essential role for integrin signaling.

PodocyteTRPC6 channels have been implicated in glomerular diseases. Syndecan-4 (Sdc4) is a membrane proteoglycan that can be cleaved to release a soluble ectodomain capable of paracrine and autocrine signaling. We have confirmed that overexpression of Sdc4 core protein increases surface abundance of TRPC6 channels in cultured podocytes, whereas Sdc4 knockdown has the opposite effect. Exposure to soluble Sdc4 ectodomain also increased the surface abundance of TRPC6, and increased cationic currents evoked by a diacylglycerol analog in podocytes. Sdc4 ectodomain increased generation of reactive oxygen species (ROS), reduced activation of RhoA, increased activation of Rac1, increased nuclear abundance of NFATc1, and increased total ß3-integrin. The effects of Sdc4 ectodomain on cell-surface TRPC6 were blocked by the ROS quencher TEMPOL, and by the Rac1 inhibitor NSC-23766, but were not blocked by inhibition of calcineurin-NFATc1 signaling. The Sdc4 core protein co-immunoprecipitates with ß3-integrin in cultured podocytes. Moreover, effects of Sdc4 ectodomain on TRPC6, ROS generation, Rac1 and RhoA modulation, and NFATc1 activation were blocked by cilengitide, a selective inhibitor of outside-in signaling through αv-containing integrins. Exposure to TNF, or serum from three patients with recurrent FSGS in relapse, increased shedding of podocyte Sdc4 ectodomains into the surrounding medium. This was also observed after treating podocytes with the metalloproteinase ADAM17 or after overexpression of the Sdc4 core protein. Increased concentrations of Sdc4 ectodomain were detected in urine of rats during acute puromycin aminonucleoside nephrosis. Locally generated Sdc4 may play a role in regulating TRPC6 channels, and may contribute to glomerular pathology.

Podocin is translocated to cytoplasm in puromycin aminonucleoside nephrosis rats and in poor-prognosis patients with IgA nephropathy.

Podocytes serve as the final barrier to urinary protein loss through a highly specialized structure called a slit membrane and maintain foot process and glomerular basement membranes. Podocyte injury results in progressive glomerular damage and accelerates sclerotic changes, although the exact mechanism of podocyte injury is still obscure. We focus on the staining gap (podocin gap) defined as the staining difference between podocin and synaptopodin, which are normally located in the foot process. In puromycin aminonucleoside nephrosis rats, the podocin gap is significantly increased (p < 0.05) and podocin is translocated to the cytoplasm on days 7 and 14 but not on day 28. Surprisingly, the gap is also significantly increased (p < 0.05) in human kidney biopsy specimens of poor-prognosis IgA nephropathy patients. This suggests that the podocin gap could be a useful marker for classifying the prognosis of IgA nephropathy and indicating the translocation of podocin to the cytoplasm. Next, we find more evidence of podocin trafficking in podocytes where podocin merges with Rab5 in puromycin aminonucleoside nephrosis rats at day 14. In immunoelectron microscopy, the podocin positive area was significantly translocated from the foot process areas to the cytoplasm (p< 0.05) on days 7 and 14 in puromycin aminonucleoside nephrosis rats. Interestingly, podocin is also translocated to the cytoplasm in poor-prognosis human IgA nephropathy. In this paper, we demonstrate that the translocation of podocin by endocytosis could be a key traffic event of critical podocyte injury and that the podocin gap could indicate the prognosis of IgA nephropathy.

CXCL10 induces the recruitment of monocyte-derived macrophages into kidney, which aggravate puromycin aminonucleoside nephrosis.

The mechanism responsible for trafficking of monocyte-derived macrophages into kidney in the puromycin aminonucleoside model of nephrotic syndrome in rats (PAN-NS), and the significance of this infiltration, remain largely unknown. CXCL10, a chemokine secreted in many T helper type 1 (Th1) inflammatory diseases, exhibits important roles in trafficking of monocytes and activated T cells. We hypothesized that induction of circulating interferon (IFN)-γ and glomerular tumour necrosis factor (TNF)-α during PAN-NS would stimulate the release of CXCL10 by podocytes, leading to infiltration of activated immune cells and greater glomerular injury. We found that serum IFN-γ, glomerular Cxcl10 mRNA and intra- and peri-glomerular macrophage infiltration were induced strongly during the late acute phase of PAN-NS in Wistar rats, but not in nude (Foxn1(rnu/rnu) ) rats lacking functional effector T lymphocytes. Wistar rats also developed significantly greater proteinuria than nude rats, which could be abolished by macrophage depletion. Stimulation of cultured podocytes with both IFN-γ and TNF-α markedly induced the expression of Cxcl10 mRNA and CXCL10 secretion. Together, these data support our hypothesis that increased circulating IFN-γ and glomerular TNF-α induce synergistically the production and secretion of CXCL10 by podocytes, attracting activated macrophages into kidney tissue. The study also suggests that IFN-γ, secreted from Th1 lymphocytes, may prime proinflammatory macrophages that consequently aggravate renal injury.

Fluvastatin attenuated the effect of expression of ß1 integrin in PAN-treated podocytes by inhibiting reactive oxygen species.

It is well accepted that ß1 integrin plays a key role in maintaining normal podocytes form and functions; however, its mechanism of the potential protective effect remains unclear. Furthermore, the investigation and understanding of the non-lipid-dependent renal protection of Statins in addition to well-known lipid-lowering effect may provide the therapeutic utility and ultimately improve clinical outcome for patients with renal diseases. In the present study, we investigated the effect and mechanism of fluvastatin (FLV) on the expression of ß1 integrin in puromycin aminonucleoside (PAN)-treated podocytes in vitro. Cultured human podocytes were treated with PAN, and/or different concentrations of FLV (1 × 10(-8)-1 × 10(-5 )mol/l), superoxide dismutase (SOD), or H2O2, respectively. The expression of ß1 integrin and reactive oxygen species (ROS) in human podocytes under each experimental condition was evaluated by western blot, RT-PCR, and 2'7'-dichlorofluorescein 3'6'-diacetate, respectively. The viability of podocytes was also assessed by MTT colorimetry in the present study. The expression of ß1 integrin was significantly decreased, and the synthesis of ROS was significantly increased in podocytes following either PAN or H2O2 treatment (p < 0.05). The up-regulation of ß1 integrin and down-regulation of ROS were also observed in PAN-treated podocytes following lower concentrations of FLV or SOD treatment (p < 0.05, respectively). The cytotoxicity data derived from MTT assay revealed that lower podocyte viability was found in the presence of higher concentrations of FLV, PAN, or H2O2. Lower concentration of FLV or SOD can protect podocytes from being impaired by PAN treatment. FLV attenuated the podocyte injury induced by PAN and increased the production of ß1 integrin in human podocytes in vitro. This underlying mechanism of FLV may be through inhibiting the activity of ROS in human podocytes.

Trehalose, an mTOR independent autophagy inducer, alleviates human podocyte injury after puromycin aminonucleoside treatment.

Glomerular diseases are commonly characterized by podocyte injury including apoptosis, actin cytoskeleton rearrangement and detachment. However, the strategies for preventing podocyte damage remain insufficient. Recently autophagy has been regarded as a vital cytoprotective mechanism for keeping podocyte homeostasis. Thus, it is reasonable to utilize this mechanism to attenuate podocyte injury. Trehalose, a natural disaccharide, is an mTOR independent autophagy inducer. It is unclear whether trehalose alleviates podocyte injury. Therefore, we investigated the efficacy of trehalose in puromycin aminonucleoside (PAN)-treated podocytes which mimic cell damage in minimal change nephrotic syndrome in vitro. Human conditional immortalized podocytes were treated with trehalose with or without PAN. Autophagy was investigated by immunofluorescence staining for LC3 puncta and Western blotting for LC3, Atg5, p-AMPK, p-mTOR and its substrates. Podocyte apoptosis and necrosis were evaluated by flow cytometry and by measuring lactate dehydrogenase activity respectively. We also performed migration assay to examine podocyte recovery. It was shown that trehalose induced podocyte autophagy in an mTOR independent manner and without reactive oxygen species involvement. Podocyte apoptosis significantly decreased after trehalose treatment, while the inhibition of trehalose-induced autophagy abolished its protective effect. Additionally, the disrupted actin cytoskeleton of podocytes was partially reversed by trehalose, accompanying with less lamellipodias and diminished motility. These results suggested that trehalose induced autophagy in human podocytes and showed cytoprotective effects in PAN-treated podocytes.

The role of survivin in podocyte injury induced by puromycin aminonucleoside.

OBJECTIVE: Survivin is a member of the inhibitor of apoptosis protein family, which uniquely promotes mitosis and regulates apoptosis in cancer cells. Recent studies have demonstrated that survivin also expresses in several normal adult cells. In the present study, we aimed to investigate the function of survivin in the terminally differentiated epithelial cells, podocytes. METHODS: Survivin expression and location were detected by Quantitative Real-Time PCR, western blot and fluorescence confocal microscopy methods in normal and injured mouse podocytes. Cyto-protection function of survivin was also studied in cultured podocyte injured by puromycin aminonucleoside (PAN), transfected with survivin siRNA to down-regulate survivin expression, or with survivin plasmid to transiently over-express survivin. RESULTS: In podocytes, PAN stimulated expressions of survivin and the apoptosis related molecule caspase 3. Knockdown of survivin expression by siRNA increased the activation of caspase 3, induced podocyte apoptosis and remarkable rearrangement of actin cytoskeleton. Moreover, over-expression of survivin inhibited PAN-induced podocyte apoptosis and cytoskeleton rearrangement. CONCLUSION: Our data provides the evidence that survivin plays an important role in protecting podocytes from apoptosis induced by PAN. The mechanism of survivin related anti-apoptosis may, at least partially, be through the activation of caspase 3.

cAMP signaling prevents podocyte apoptosis via activation of protein kinase A and mitochondrial fusion.

Our previous in vitro studies suggested that cyclic AMP (cAMP) signaling prevents adriamycin (ADR) and puromycin aminonucleoside (PAN)-induced apoptosis in podocytes. As cAMP is an important second messenger and plays a key role in cell proliferation, differentiation and cytoskeleton formation via protein kinase A (PKA) or exchange protein directly activated by cAMP (Epac) pathways, we sought to determine the role of PKA or Epac signaling in cAMP-mediated protection of podocytes. In the ADR nephrosis model, we found that forskolin, a selective activator of adenylate cyclase, attenuated albuminuria and improved the expression of podocyte marker WT-1. When podocytes were treated with pCPT-cAMP (a selective cAMP/PKA activator), PKA activation was increased in a time-dependent manner and prevented PAN-induced podocyte loss and caspase 3 activation, as well as a reduction in mitochondrial membrane potential. We found that PAN and ADR resulted in a decrease in Mfn1 expression and mitochondrial fission in podocytes. pCPT-cAMP restored Mfn1 expression in puromycin or ADR-treated podocytes and induced Drp1 phosphorylation, as well as mitochondrial fusion. Treating podocytes with arachidonic acid resulted in mitochondrial fission, podocyte loss and cleaved caspase 3 production. Arachidonic acid abolished the protective effects of pCPT-cAMP on PAN-treated podocytes. Mdivi, a mitochondrial division inhibitor, prevented PAN-induced cleaved caspase 3 production in podocytes. We conclude that activation of cAMP alleviated murine podocyte caused by ADR. PKA signaling resulted in mitochondrial fusion in podocytes, which at least partially mediated the effects of cAMP.

Slit diaphragm protein Neph1 and its signaling: a novel therapeutic target for protection of podocytes against glomerular injury.

Podocytes are specialized epithelial cells that are critical components of the glomerular filtration barrier, and their dysfunction leads to proteinuria and renal failure. Therefore, preserving podocyte function is therapeutically significant. In this study, we identified Neph1 signaling as a therapeutic target that upon inhibition prevented podocyte damage from a glomerular injury-inducing agent puromycin aminonucleoside (PAN). To specifically inhibit Neph1 signaling, we used a protein transduction approach, where the cytoplasmic domain of Neph1 (Neph1CD) tagged with a protein transduction domain trans-activator of transcription was transduced in cultured podocytes prior to treatment with PAN. The PAN-induced Neph1 phosphorylation was significantly reduced in Neph1CD-transduced cells; in addition, these cells were resistant to PAN-induced cytoskeletal damage. The biochemical analysis using subfractionation studies showed that unlike control cells Neph1 was retained in the lipid raft fractions in the transduced cells following treatment with PAN, indicating that transduction of Neph1CD in podocytes prevented PAN-induced mislocalization of Neph1. In accordance, the immunofluorescence analysis further suggested that Neph1CD-transduced cells had increased ability to retain endogenous Neph1 at the membrane in response to PAN-induced injury. Similar results were obtained when angiotensin was used as an injury-inducing agent. Consistent with these observations, maintaining high levels of Neph1 at the membrane using a podocyte cell line overexpressing chimeric Neph1 increased the ability of podocytes to resist PAN-induced injury and PAN-induced albumin leakage. Using a zebrafish in vivo PAN and adriamycin injury models, we further demonstrated the ability of transduced Neph1CD to preserve glomerular function. Collectively, these results support the conclusion that inhibiting Neph1 signaling is therapeutically significant in preventing podocyte damage from glomerular injury.

Downregulation of microRNA-30 facilitates podocyte injury and is prevented by glucocorticoids.

MicroRNAs (miRNAs) are essential for podocyte homeostasis, and the miR-30 family may be responsible for this action. However, the exact roles and clinical relevance of miR-30s remain unknown. In this study, we examined the expression of the miR-30 family in the podocytes of patients with FSGS and found that all members are downregulated. Treating cultured human podocytes with TGF-ß, LPS, or puromycin aminonucleoside (PAN) also downregulated the miR-30 family. Podocyte cytoskeletal damage and apoptosis caused by treatment with TGF-ß or PAN were ameliorated by exogenous miR-30 expression and aggravated by miR-30 knockdown. Moreover, we found that miR-30s exert their protective roles by direct inhibition of Notch1 and p53, which mediate podocyte injury. In rats, treatment with PAN substantially downregulated podocyte miR-30s and induced proteinuria and podocyte injury; however, transfer of exogenous miR-30a to podocytes of PAN-treated rats ameliorated proteinuria and podocyte injury and reduced Notch1 activation. Finally, we demonstrated that glucocorticoid treatment maintains miR-30 expression in cultured podocytes treated with TGF-ß, LPS, or PAN and in the podocytes of PAN-treated rats. Glucocorticoid-sustained miR-30 expression associated with reduced Notch1 activation and alleviated podocyte damage. Taken together, these findings demonstrate that miR-30s protect podocytes by targeting Notch1 and p53 and that the loss of miR-30s facilitates podocyte injury. In addition, sustained miR-30 expression may be a novel mechanism underlying the therapeutic effectiveness of glucocorticoids in treating podocytopathy.

A novel source of cultured podocytes.

Amniotic fluid is in continuity with multiple developing organ systems, including the kidney. Committed, but still stem-like cells from these organs may thus appear in amniotic fluid. We report having established for the first time a stem-like cell population derived from human amniotic fluid and possessing characteristics of podocyte precursors. Using a method of triple positive selection we obtained a population of cells (hAKPC-P) that can be propagated in vitro for many passages without immortalization or genetic manipulation. Under specific culture conditions, these cells can be differentiated to mature podocytes. In this work we compared these cells with conditionally immortalized podocytes, the current gold standard for in vitro studies. After in vitro differentiation, both cell lines have similar expression of the major podocyte proteins, such as nephrin and type IV collagen, that are characteristic of mature functional podocytes. In addition, differentiated hAKPC-P respond to angiotensin II and the podocyte toxin, puromycin aminonucleoside, in a way typical of podocytes. In contrast to immortalized cells, hAKPC-P have a more nearly normal cell cycle regulation and a pronounced developmental pattern of specific protein expression, suggesting their suitability for studies of podocyte development for the first time in vitro. These novel progenitor cells appear to have several distinct advantages for studies of podocyte cell biology and potentially for translational therapies.

Nephrin phosphorylation regulates podocyte adhesion through the PINCH-1-ILK-α-parvin complex.

Nephrin, a structural molecule, is also a signaling molecule after phosphorylation. Inhibition of nephrin phosphorylation is correlated with podocyte injury. The PINCH-1-ILK-α-parvin (PIP) complex plays a crucial role in cell adhesion and cytoskeleton formation. We hypothesized that nephrin phosphorylation influenced cytoskeleton and cell adhesion in podocytes by regulating the PIP complex. The nephrin phosphorylation, PIP complex formation, and F-actin in Wistar rats intraperitoneally injected with puromycin aminonucleoside were gradually decreased but increased with time, coinciding with the recovery from glomerular/podocyte injury and proteinuria. In cultured podocytes, PIP complex knockdown resulted in cytoskeleton reorganization and decreased cell adhesion and spreading. Nephrin and its phosphorylation were unaffected after PIP complex knockdown. Furthermore, inhibition of nephrin phosphorylation suppressed PIP complex expression, disorganized podocyte cytoskeleton, and decreased cell adhesion and spreading. These findings indicate that alterations in nephrin phosphorylation disorganize podocyte cytoskeleton and decrease cell adhesion through a PIP complex-dependent mechanism.

Functional metabotropic glutamate receptors 1 and 5 are expressed in murine podocytes.

In non-neuronal cells, glutamate is an extracellular signaling mediator. Since podocytes have glutamate-containing vesicles, we sought to determine glutamate receptor presence and action in glomerular cells. The metabotropic glutamate receptors (mGluR) 1, 5, 6, and 8 were found to be expressed in mouse brain and glomeruli; predominantly in podocytes. In two models of proteinuria (BalB/C mice with puromycin aminonucleoside- and doxorubicin-induced podocyte injury) we found that the selective mGluR1/5 agonist (S)-3,5-dihydroxyphenylglycine (DHPG) attenuated albuminuria and improved the expression of the podocyte marker WT-1. TUNEL staining showed that the number of podocytes undergoing apoptosis was inversely correlated with the number of WT-1-positive cells in glomeruli. When podocytes were treated with DHPG in vitro, they generated cyclic AMP and activated CREB (cyclic AMP response element binding protein). The selective mGluR1/5 antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid, the adenylate cyclase inhibitor SQ22536, and RNA interference knockdown of mGluR1 or mGluR5 all prevented DHPG-induced cAMP generation and CREB activation. DHPG inhibited apoptosis and the decrease of aminonucleoside-induced mitochondrial membrane potential in podocytes but had no effect in the presence of SQ22536 with knockdown mGluR1 or mGluR5. Thus, functional mGluR1 and mGluR5 are expressed in podocytes and their activation protects against albuminuria and podocyte apoptosis, processes that are, at least in part, dependent on cAMP.