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Minimal change disease (MCD) is a universal primary glomerular disease contributing to nephrotic syndrome. Lon peptidase 1 (LONP1) has been suggested to protect podocytes from damage during the progression of MCD. Accordingly, our research further explored the specific mechanisms of LONP1. Initially, the expressions of TRPC6, p-ERK1/2, and LONP1 in the kidney tissues of MCD patients were detected by immunohistochemistry and Western blot. Human podocytes AB8/13 were serially subjected to transfection with shTRPC6/shNC, and 48-h treatment with 30 µg/ml puromycin aminonucleoside (PAN). The viability, apoptosis, and migration of AB8/13 cells were assessed by cell counting kit-8, flow cytometry, and transwell assays. The mRNA and protein expressions of LONP1 were downregulated while those of TRPC6 were upregulated in the kidney tissues of MCD patients. PAN induced podocyte injury and migration and inhibited LONP1 expression, whereas TRPC6 silencing did oppositely. The phosphorylation level of ERK1/2 was reduced in MCD samples, which was negatively associated with TRPC6 expression and positively associated with LONP1 expression. Furthermore, ERK phosphorylation agonist offset the effects of TRPC6 silencing on mitigating podocyte injury and migration as well as upregulating LONP1 expression. Collectively, TRPC6 knockdown-induced ERK1/2 inactivation can ameliorate podocyte injury in MCD by increasing the expression of LONP1.

Objectives: The small GTPase Rac1 (RAC1) has been linked to podocyte disorders and steroid-sensitive nephrotic syndrome (SSNS). The aim of this study was to explore and validate the potential causal association between circulating RAC1 and SSNS., Methods: The association between circulating RAC1 and SSNS at both gene expression and proteomic levels was investigated using Mendelian randomization analysis, and further validated by single-cell RNA-sequencing, proteomic analysis, and experimental studies. The genetic instruments comprised cis-expression quantitative trait loci (cis-eQTLs) associated with RAC1 gene expression and protein QTLs correlated with plasma RAC1 protein levels. Causal associations were estimated utilizing the inverse variance weighted and MR-PRESSO methods. Validation of RAC1 expression was conducted through single-cell RNA-sequencing of peripheral blood mononuclear cells from patients with SSNS and healthy controls. Proteomic analysis was performed among patients with minimal change nephrotic syndrome. Experimental validation was conducted using a puromycin aminonucleoside (PAN)-induced nephrosis model., Results: Increased expression of RAC1 was associated with a higher risk of SSNS (gene expression level: odds ratio [OR], 1.53; 95% confidence interval [CI], 1.02-2.28; protein level: OR, 1.82; 95% CI, 1.05-3.17). The results of MR-PRESSO were consistent (gene expression level: OR, 1.49; 95% CI, 1.17-1.92; protein level: OR, 1.81; 95% CI, 1.16-2.85). Single-cell RNA sequencing and proteomic analysis confirmed elevated RAC1 expression in patients with SSNS compared to healthy controls. Experimental data further supported increased RAC1 expression in PAN-induced nephropathy., Conclusions: Increased expression of RAC1 might be causally associated with SSNS, suggesting that targeting RAC1 might represent a potential therapeutic strategy for SSNS.

Background: Recent studies have reported that helix B surface polypeptide (HBSP), an erythropoietin derivative, exhibits strong tissue protective effects, independent of erythropoietic effects, in a renal ischemia-reperfusion (IR) injury model. Meanwhile, the transforming growth factor-β (TGF-β) superfamily member glial cell line-derived neurotrophic factor (GDNF) demonstrated protective effect on podocytes in vitro . Using a rat puromycin aminonucleoside nephropathy (PAN) model, this study observed the renal protective effect of HBSP and investigated its renal protective effect on podocytes and mechanism related to GDNF., Methods: Rats nephropathy model was induced by injection of 60 mg/kg of PAN via the tail vein. Rats in the PAN + HBSP group were injected intraperitoneally with HBSP (8 nmol/kg) 4 h before the model was induced, followed by intraperitoneal injections of HBSP once every 24 h for 7 consecutive days. The 24-hour urinary protein level was measured once every other day, and blood and renal tissue samples were collected on the 7th day for the examination of renal function, complete blood count, renal pathological changes and the expression levels of GDNF., Results: Compared with the control group, the PAN nephropathy rat model showed a large amount of urinary protein. The pathological manifestations were mainly extensive fusion and disappearance of foot processes, along with vacuolar degeneration of podocytes and their separation from the glomerular basement membrane. GDNF expression was upregulated. Compared with the PAN + vehicle group, the PAN + HBSP group showed decreased urinary protein ( p  < 0.05). Pathological examination revealed ameliorated glomerular injury and vacuolar degeneration of podocytes. The expression of GDNF in the PAN nephropathy group was increased, when compared with the control group. The greatest expression of GDNF observed in the PAN + HBSP group ( p  < 0.05)., Conclusions: The expression of GDNF in the kidney of PAN rat model was increased. HBSP reduced urinary protein, ameliorated pathological changes in renal podocytes, increased the expression of GDNF in the PAN rat model. HBSP is likely to exert its protective effects on podocytes through upregulation of GDNF expression.

The endogenous opioid system regulates pain through local release of neuropeptides and modulation of their action on opioid receptors. However, the effect of opioid peptides, the enkephalins, is short-lived due to their rapid hydrolysis by enkephalin-degrading enzymes. In turn, an innovative approach to the management of pain would be to increase the local concentration and prolong the stability of enkephalins by preventing their inactivation by neural enkephalinases such as puromycin-sensitive aminopeptidase (PSA). Our previous structure-activity relationship studies offered the S-diphenylmethyl cysteinyl derivative of puromycin (20) as a nanomolar inhibitor of PSA. This chemical class, however, suffered from undesirable metabolism to nephrotoxic puromycin aminonucleoside (PAN). To prevent such toxicity, we designed and synthesized 5′-chloro substituted derivatives. The compounds retained the PSA inhibitory potency of the corresponding 5′-hydroxy analogs and had improved selectivity toward PSA. In vivo treatment with the lead compound 19 caused significantly reduced pain response in antinociception assays, alone and in combination with Met-enkephalin. The analgesic effect was reversed by the opioid antagonist naloxone, suggesting the involvement of opioid receptors. Further, PSA inhibition by compound 19 in brain slices caused local increase in endogenous enkephalin levels, corroborating our rationale. Pharmacokinetic assessment of compound 19 showed desirable plasma stability and identified the cysteinyl sulfur as the principal site of metabolic liability. We gained additional insight into inhibitor-PSA interactions by molecular modeling, which underscored the importance of bulky aromatic amino acid in puromycin scaffold. The results of this study strongly support our rationale for the development of PSA inhibitors for effective pain management. [Display omitted] • Endogenous opioid peptides, enkephalins, regulate pain though opioid receptors. • Puromycin Sensitive Aminopeptidase (PSA) modulates effects of enkephalins. • Novel PSA inhibitor offered antinociceptive effect by increasing enkephalin levels. • PSA inhibition could be an effective pain management strategy. [ABSTRACT FROM AUTHOR]

Damage to podocytes is an important determinant of renal pathology. The puromycin aminonucleoside (PAN) mice nephropathy model is commonly used in the study of renal disease with podocyte injury. Hirudin has a broad nephroprotective effect and has been shown to treat renal interstitial fibrosis in previous studies. Mice were given PAN by gavage to prepare animal models, and MPC5 cells were incubated with PAN in vitro. Twenty‐four hours urine was collected for analysis of urinary protein levels. Renal pathological changes were observed by hematoxylin and eosin staining. Immunofluorescence detection of nephrin in kidney tissues and cells. Apoptosis was analyzed with over TUNEL. Cytoskeleton, endoplasmic reticulum stress (ERS), p38 MAPK signaling, and apoptosis‐related proteins were assessed by western blot analysis. The data suggested that hirudin attenuated reduced renal injury and increased urine protein in PAN mice. Hirudin also attenuated cytoskeletal protein (synaptopodin, nephrin, and podocin) disruption, ERS activation, and apoptosis in PAN mice and PAN‐induced podocytes. In addition, hirudin inhibited the expression of p38 MAPK signaling key proteins upregulated by PAN, thereby suppressing ERS. The p38 MAPK agonist was able to partially antagonize the inhibition of p38 MAPK signaling by hirudin in PAN‐induced podocytes, thereby reactivating the ERS inhibited by hirudin, promoting cytoskeletal protein degradation and increasing the level of apoptosis. In conclusion, hirudin could decrease podocyte injury by inhibiting p38 MAPK signaling‐mediated ERS, resulting in the protection of the kidney from PAN damage. These findings may provide an experimental basis for hirudin treatment of podocyte injury diseases. [ABSTRACT FROM AUTHOR]

Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, miR-217-5p was identified as the most upregulated transcript in PAN-treated rat podocytes. MiR-217-5p overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of miR-217-5p. Of the predicted targets highly downregulated by PAN, Myosin 1d (Myo1d) is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that miR-217-5p targets Myo1d by luciferase assays, qRT–PCR, and Western blotting. Furthermore, we showed that miR-217-5p was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that miR-217-5p may serve as a therapeutic target and a biomarker for podocyte injury. [ABSTRACT FROM AUTHOR]

In the present study, we aimed to investigate the effect of puromycin aminonucleoside (PAN) on the expression and distribution of transient receptor potential canonical 6 (TRPC6) of murine podocytes and further study the protective mechanism of fluvastatin on podocyte injury by TRPC6 in vitro. Podocytes were treated by PAN at different doses and at different time points. The expressions of TRPC6 at mRNA and protein levels were assessed. An immunofluorescent assay was used to observe the distribution of TRPC6. Cultured podocytes were then divided into four groups. The expressions of TRPC6 at mRNA and protein levels were measured. The intracellular calcium concentration of podocytes was measured with a laser-scanning confocal microscope. The paracellular permeability to BSA was evaluated using Millicell-PCF Inserts. The expressions of TRPC6 at mRNA and protein levels were increased in a dose and time-dependent manner after exposure to PAN. Immunofluorescence showed that the expression intensity of TRPC6 was significantly increased, and the distribution of TRPC6 was changed after PAN was applied to podocytes. With fluvastatin intervention, PAN-induced up-regulation of TRPC6 was significantly reversed. The ratio of the peak value of intracellular calcium to the basic calcium value in the PAN group was significantly higher after TRPC6 was activated by OAG, while it is obviously reversed under the action of fluvastatin. The podocyte permeability was significantly increased after 48 h of PAN treatment, while the above situation was effectively improved after fluvastatin intervention. The changes of distribution and expression of TRPC6 were related to podocyte injury induced by PAN. Fluvastatin could exert its protective effects on podocytes by down-regulating TRPC6. [ABSTRACT FROM AUTHOR]

Nephrotic syndrome (NS) is a kidney disease characterized by hypertriglyceridemia, massive proteinuria, hypo-albuminemia and peripheral edema. Sinkihwan-gamibang (SKHGMB) was recorded in a traditional Chinese medical book named "Bangyakhappyeon (▪)" and its three prescriptions Sinkihwan, Geumgwe-sinkihwan, and Jesaeng-sinkihwan belong to Gamibang. This study confirmed the effect of SKHGMB on renal dysfunction in an NS model induced by puromycin aminonucleoside (PAN). The experimental NS model was induced in male Sprague Dawley (SD) rats through injection of PAN (50 mg·kg−1) via the femoral vein. SKHGMB not only reduced the size of the kidneys increased due to PAN-induced NS, but also decreased proteinuria and ascites. In addition, SKHGMB significantly ameliorated creatinine clearance, creatinine, and blood urea nitrogen. SKHGMB relieved glomeruli dilation and tubules fibrosis in the glomeruli of the NS model. SKHGMB inhibited the protein and mRNA levels of the NLRP3 inflammasome including NLRP3, ASC, and pro-caspase-1 in NS rats. SKHGMB reduced the protein and mRNA levels of fibrosis regulators in NS rats. The results indicated that SKHGMB exerts protective effects against renal dysfunction by inhibiting of renal inflammation and fibrosis in NS rats. [ABSTRACT FROM AUTHOR]

Glomerular filtration rate is the gold-standard method for assessment of renal function but is rarely performed in routine toxicity studies. Standard serum biomarkers of renal function are insensitive and become elevated only with significant loss of organ function. Symmetric dimethylarginine (SDMA) is a ubiquitous analyte that is freely filtered by the glomerulus and can be detected in serum. It has shown utility for the detection of renal injury in dogs and cats in clinical veterinary practice, but the potential utility of SDMA to detect renal injury in preclinical species or toxicity studies has not been thoroughly investigated. We utilized a well-characterized glomerular toxicant, puromycin aminonucleoside, to induce podocyte injury and subsequent proteinuria in young male Sprague-Dawley rats. At the end of 1 or 2 weeks, blood, urine, and kidney tissue were collected for analysis. One week following a single 50 mg/kg dose, urea nitrogen, creatinine, and albumin mean values were within historical control ranges, while SDMA was increased. Glomerular changes in these animals included periodic acid–Schiff positive globules within podocytes, podocyte hypertrophy by light microscopy, and podocyte degeneration with effacement of foot processes by electron microscopy (EM). Taken together, our data indicate that SDMA may be a useful biomarker for early detection of glomerular toxicities in rats. [ABSTRACT FROM AUTHOR]

Smad3 signaling and transgelin expression are often activated during puromycin aminonucleoside (PAN)‐induced podocyte injury. Here, we investigated whether the Smad3 inhibitor SIS3 can ameliorate damage to injured podocytes. A model of PAN‐induced podocyte injury was constructed using the MPC5 cell line. The effects of SIS3 on the expression of the podocyte cytoskeletal proteins transgelin, p15INK4B, phosphor‐smad3, phosphor‐JAK/stat3, the apoptotic marker cleaved caspase 3, and c‐myc were investigated using western blot. The distribution of F‐actin in PAN‐induced podocyte injury was observed under an immunofluorescence microscope. PAN‐induced podocyte injury altered the distribution of F‐actin and transgelin, and colocalization of these two proteins was observed. Transgelin expression and Smad3 phosphorylation were increased in the MPC5 cell line with prolonged PAN treatment. In addition, c‐myc expression, p15INK4B, and JAK phosphorylation were all increased after treatment with PAN. Treatment with the Smad3 inhibitor SIS3 reversed these phenomena and protected against PAN‐induced podocyte injury. Moreover, stimulating podocytes directly with TGFβ‐1 also led to enhanced expression of transgelin or phosphor‐JAK/stat3, and this could be inhibited by SIS3. In conclusion, transgelin expression was induced through the Smad3 signaling pathway during PAN‐induced podocyte injury, and the resulting abnormal distribution of F‐actin and the enhanced expression of transgelin could be reversed by blockade of this pathway.

Abstract Background Focal segmental glomerulosclerosis (FSGS) causes renal fibrosis and may lead to kidney failure. FSGS and its common complication, proteinuria, are challenging to treat. Corticosteroids are ineffective in many patients with FSGS, and alternative treatments often yield suboptimal responses. Repository corticotropin injection (RCI; Acthar® Gel), a naturally sourced complex mixture of purified adrenocorticotropic hormone analogs and other pituitary peptides, may have beneficial effects on idiopathic FSGS via melanocortin receptor activation. Methods Two studies in a preclinical (female Sprague-Dawley rats) puromycin aminonucleoside FSGS model assessed the effect of RCI on renal function and morphology: an 8-week comparison of a single RCI dose with methylprednisolone (N = 27), and a 12-week chronic RCI dose range study (N = 34). Primary outcomes were proteinuria and renal pathology improvements for measures of renal fibrosis, tubular damage, glomerular injury, and total kidney injury score. Impact of RCI treatment was also determined by assessing urinary biomarkers for renal injury, podocyte expression of podoplanin (a biomarker for injury), podocyte effacement by electron microscopy, and histological staining for fibrosis biomarkers. Results Compared with saline treatment, RCI 30 IU/kg significantly reduced proteinuria, with a 38% reduction in peak mean urine protein levels on day 28 in the 8-week model, and RCI 10 IU/kg, 30 IU/kg, and 60 IU/kg reduced peak mean urine protein in the 12-week model by 18, 47, and 44%, respectively. RCI also showed significant dose-dependent improvements in fibrosis, interstitial inflammation, tubular injury, and glomerular changes. Total kidney injury score (calculated from histopathological evaluations) demonstrated statistically significant improvements with RCI 30 IU/kg in the 8-week study and RCI 60 IU/kg in the 12-week study. RCI treatment improved levels of urinary biomarkers of kidney injury (KIM-1 and OPN), expression of podoplanin, and podocyte morphology. RCI also reduced levels of desmin and fibrosis-associated collagen deposition staining. Methylprednisolone did not improve renal function or pathology in this model. Conclusions These results provide evidence supporting the improvement of FSGS with RCI, which was superior to corticosteroid treatment in this experimental model. To the authors’ knowledge, this is the first evidence that a drug for the treatment of FSGS supports podocyte recovery after repeated injury.

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Podocytes, alternatively called glomerular epithelial cells, are terminally differentiated cells that wrap around glomerular capillaries and function as a part of the glomerular filtration barrier in the kidney. Therefore, podocyte injury with morphological alteration and detachment from glomerular capillaries leads to severe proteinuria and subsequent renal failure through glomerulosclerosis. Previous RNA sequencing analysis of primary rat podocytes exposed to puromycin aminonucleoside (PAN), a well-known experimental model of injured podocytes, identified several transcripts as being aberrantly expressed. However, how the expression of these transcripts is regulated remains unclear. MicroRNAs (miRNAs) are small noncoding RNAs that posttranscriptionally inhibit the expression of their target transcripts. In this study, using small RNA sequencing analysis, miR-217-5p was identified as the most upregulated transcript in PAN-treated rat podocytes. MiR-217-5p overexpression in E11 podocyte cells led to shrunken cells with abnormal actin cytoskeletons. Consistent with these changes in cell morphology, gene ontology (GO) enrichment analysis showed that interactive GO terms related to cell morphogenesis were enriched with the predicted targets of miR-217-5p. Of the predicted targets highly downregulated by PAN, Myosin 1d (Myo1d) is a nonmuscle myosin predicted to be involved in actin filament organization and thought to play a role in podocyte morphogenesis and injury. We demonstrated that miR-217-5p targets Myo1d by luciferase assays, qRT–PCR, and Western blotting. Furthermore, we showed that miR-217-5p was present in urine from PAN- but not saline-administrated rats. Taken together, our data suggest that miR-217-5p may serve as a therapeutic target and a biomarker for podocyte injury.

Background: Glucocorticoids are the treatment of choice for proteinuric patients with minimal change disease (MCD) and primary focal segmental glomerulosclerosis (FSGS). Immunosuppressive as well as direct effects on podocytes are believed to mediate their actions. In this study, we analyzed the anti-proteinuric effects of inhibition of the glucocorticoid receptor (GR) in glomerular epithelial cells, including podocytes., Methods: We employed genetic and pharmacological approaches to inhibit the GR. Genetically, we used Pax8-Cre/GRfl/fl mice to specifically inactivate the GR in kidney epithelial cells. Pharmacologically, we utilized a glucocorticoid antagonist called mifepristone., Results: Genetic inactivation of GR, specifically in kidney epithelial cells, using Pax8-Cre/GRfl/fl mice, ameliorated proteinuria following protein overload. We further tested the effects of pharmacological GR inhibition in three models and species: the puromycin aminonucleoside-induced nephrosis model in rats, the protein overload model in mice and the inducible transgenic NTR/MTZ zebrafish larvae with specific and reversible podocyte injury. In all three models, both pharmacological GR activation and inhibition consistently and significantly ameliorated proteinuria. Additionally, we translated our findings to humans, where three nephrotic adult patients with MCD or primary FSGS with contraindications or insufficient responses to corticosteroids were treated with mifepristone. This treatment resulted in a clinically relevant reduction of proteinuria., Conclusions: Thus, across multiple species and proteinuria models, both genetic and pharmacological GR inhibition was at least as effective as pronounced GR activation. While the mechanism remains perplexing, GR inhibition may be a novel and targeted therapeutic approach to treat glomerular proteinuria potentially bypassing adverse actions of steroids., (© The Author(s) 2023. Published by Oxford University Press on behalf of the ERA.)

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.

Background: TLR4 signaling is known to be involved in podocyte injury. We have previously shown that Salvia przewalskii extract of total phenolic acids (SPE) and its active monomer salvianolic acid B (SalB) and rosmarinic acid (RA) protect podocytes from injury induced by PAN. In the present study, we test whether SPE inhibits TLR4 signaling. Methods: The conditionally immortalized mouse podocytes were treated with SPE, SalB, RA, SalB + RA or tacrolimus for 30 min, followed by PAN (100 μg/mL) for 24 h. The F-actin staining with phalloidin was used to assess cytoskeletal injury in the podocytes. Western blotting and semi-quantitatives RT-PCR were used to assess the changes of the components in the TLR4 signaling pathway. Results: (1) The F-actin stress fibers of podocytes were almost completely disrupted after PAN treatment for 24 h, and the disruption was significantly alleviated by SPE; (2) the PAN-induced elevation of mRNA levels of TLR4, MyD88 and p65 were inhibited except p65 with high-dose SalB; (3) consistently, the protein levels of TLR4, MyD88 and pp65 were significantly elevated by PAN, and SPE, SalB, RA and admixture, respectively, attenuated the elevations of TLR4 and pp65 proteins; (4) SPE and tacrolimus have a similarly strong effect on inhibition of the expression of TLR4 signaling components. Conclusions: SPE protects podocytes from PAN-induced injury at least partly through inhibiting TLR4 signaling. SPE is as strong as tacrolimus in inhibiting TLR4 signaling in podocytes.

Background: Puromycin aminonucleoside (PAN) is a known podocytotoxin. PAN-induced nephrosis is a widely used animal model for studying human idiopathic nephrotic syndrome. Abnormal protein accumulation associated with podocyte-specific endoplasmic reticulum (ER) stress damages cells structurally and functionally, which in turn induces apoptosis and severe proteinuria. In the present study, we investigated the effect of PAN on ER stress and apoptosis in podocytes in vitro. Methods: Mouse podocytes were cultured and treated with various concentrations of PAN. ER stress markers were then evaluated by western blotting, and apoptosis was evaluated by fluorescence-activated cell sorting (FACS) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays. Results: PAN treatment increased ER stress markers such as activating transcription factor (ATF) 6α and caspase-12 in a dose-dependent manner at 12 and 24 hours, respectively. These markers were reduced by chemical chaperones, such as sodium 4-phenylbutyric acid and tauroursodeoxycholic acid. PAN treatment also increased 78 kD glucose-regulated protein (GRP78)/binding immunoglobulin protein (BiP) at the earlier stage of 12 hours. PAN significantly induced podocyte apoptosis in concentration- and time-dependent manners, as seen using FACS and TUNEL assays. This result was improved by Nox4 siRNA, ATF6 siRNA, and chemical chaperones. LY294002, a PI3-kinase inhibitor, significantly boosted ER stress and apoptosis. PAN-induced ER stress increased oxidative stress and subsequently induced apoptosis, and could be mitigated by inhibition of PI3-kinase signaling. Conclusion: Our findings suggest that PAN induces ER stress in podocytes mainly through the GRP78/BiP, ATF6α, and caspase-12 pathways, which trigger apoptosis via induction of oxidative stress. This stress is mitigated by inhibiting PI3-kinase signaling.

Smad3 signaling and transgelin expression are often activated during puromycin aminonucleoside (PAN)‐induced podocyte injury. Here, we investigated whether the Smad3 inhibitor SIS3 can ameliorate damage to injured podocytes. A model of PAN‐induced podocyte injury was constructed using the MPC5 cell line. The effects of SIS3 on the expression of the podocyte cytoskeletal proteins transgelin, p15INK4B, phosphor‐smad3, phosphor‐JAK/stat3, the apoptotic marker cleaved caspase 3, and c‐myc were investigated using western blot. The distribution of F‐actin in PAN‐induced podocyte injury was observed under an immunofluorescence microscope. PAN‐induced podocyte injury altered the distribution of F‐actin and transgelin, and colocalization of these two proteins was observed. Transgelin expression and Smad3 phosphorylation were increased in the MPC5 cell line with prolonged PAN treatment. In addition, c‐myc expression, p15INK4B, and JAK phosphorylation were all increased after treatment with PAN. Treatment with the Smad3 inhibitor SIS3 reversed these phenomena and protected against PAN‐induced podocyte injury. Moreover, stimulating podocytes directly with TGFβ‐1 also led to enhanced expression of transgelin or phosphor‐JAK/stat3, and this could be inhibited by SIS3. In conclusion, transgelin expression was induced through the Smad3 signaling pathway during PAN‐induced podocyte injury, and the resulting abnormal distribution of F‐actin and the enhanced expression of transgelin could be reversed by blockade of this pathway. [ABSTRACT FROM AUTHOR]

Background: Focal segmental glomerulosclerosis (FSGS) causes renal fibrosis and may lead to kidney failure. FSGS and its common complication, proteinuria, are challenging to treat. Corticosteroids are ineffective in many patients with FSGS, and alternative treatments often yield suboptimal responses. Repository corticotropin injection (RCI; Acthar® Gel), a naturally sourced complex mixture of purified adrenocorticotropic hormone analogs and other pituitary peptides, may have beneficial effects on idiopathic FSGS via melanocortin receptor activation. Methods: Two studies in a preclinical (female Sprague-Dawley rats) puromycin aminonucleoside FSGS model assessed the effect of RCI on renal function and morphology: an 8-week comparison of a single RCI dose with methylprednisolone (N = 27), and a 12-week chronic RCI dose range study (N = 34). Primary outcomes were proteinuria and renal pathology improvements for measures of renal fibrosis, tubular damage, glomerular injury, and total kidney injury score. Impact of RCI treatment was also determined by assessing urinary biomarkers for renal injury, podocyte expression of podoplanin (a biomarker for injury), podocyte effacement by electron microscopy, and histological staining for fibrosis biomarkers. Results: Compared with saline treatment, RCI 30 IU/kg significantly reduced proteinuria, with a 38% reduction in peak mean urine protein levels on day 28 in the 8-week model, and RCI 10 IU/kg, 30 IU/kg, and 60 IU/kg reduced peak mean urine protein in the 12-week model by 18, 47, and 44%, respectively. RCI also showed significant dose-dependent improvements in fibrosis, interstitial inflammation, tubular injury, and glomerular changes. Total kidney injury score (calculated from histopathological evaluations) demonstrated statistically significant improvements with RCI 30 IU/kg in the 8-week study and RCI 60 IU/kg in the 12-week study. RCI treatment improved levels of urinary biomarkers of kidney injury (KIM-1 and OPN), expression of podoplanin, and podocyte morphology. RCI also reduced levels of desmin and fibrosis-associated collagen deposition staining. Methylprednisolone did not improve renal function or pathology in this model. Conclusions: These results provide evidence supporting the improvement of FSGS with RCI, which was superior to corticosteroid treatment in this experimental model. To the authors' knowledge, this is the first evidence that a drug for the treatment of FSGS supports podocyte recovery after repeated injury. [ABSTRACT FROM AUTHOR]

Objective: The aim of our study is to investigate the relationship between podocyte autophagy and apoptosis induced by Puromycin Aminonucleoside (PAN) and to clarify its mechanism. Methods: Podocytes were cultured in vitro. The apoptosis rates of each group were detected using flow cytometry. The expression of LC3-II protein and changes in distribution were detected through laser scanning confocal microscope, and the western blot protocol was employed for detection of protein expression of LC3-II. The autophagosomes were detected by transmission electron microscopy. Results: In this study, We found that autophagosome increased followed by apoptosis after podocyte injury. Furthermore, we conformed that the activation of autophagy could inhibit the apoptosis to alleviate the injury of podocyte at an early stage. Conclusions: Autophagy occurred earlier before apoptosis and autophagy mediated podocyte apoptosis induced by PAN. These findings indicate that autophagy may become a novel therapeutic target for the treatment of podocyte injury and proteinuria in the future. [ABSTRACT FROM AUTHOR]

Experimental glomerulonephritis results in hypertension that is sensitive to salt. Nevertheless, salt retention alone cannot explain the increase in blood pressure. Angiotensin antagonistic therapy reduces hypertension caused by puromycin amino nucleosides (PAN). We investigated the hypothesis that PAN modifies renal vascular reactivity through processes dependent on angiotensin. Long-Evans rats were given an intraperitoneal injection of either puromycin (150 mg/kg) or saline (controls). Group 1 was fed a normal sodium diet (NSD, n = 9). Group 2 was given 30 mg/L of quinapril (Q) in addition to NSD (NSD + Q; n = 6). Group 3 received a high sodium diet (HSD, n = 7), and Group 4 received HSD + Q ( n = 7). Systolic blood pressure (SBP), plasma creatinine, proteinuria, and sodium balance were monitored for 12 days. On day 15, renal vascular reactivity was assessed by administering increasing doses of angiotensin II, acetylcholine (ACh), and sodium nitroprusside (SNP) directly into the renal artery. SBP progressively increased in all PAN groups. This increase in SBP was greater in the HSD groups and was not significantly altered by Q treatment. SBP increased by 22 ± 4% (NSD), 51 ± 5% (NSD + Q), 81 ± 10% (HSD), and 65 ± 8% (HSD + Q). The renal blood flow of PAN rats did not return to baseline despite their normal renal vasoconstrictor responses to angiotensin II. Additionally, they showed reduced renal vasodilator responses to SNP and Ach. The vasodilator responses to both vasodilators were surprisingly unaffected by the inhibition of the angiotensin-converting enzyme (ACE). Renal vasodilator responses to both endothelium-dependent and independent variables were reduced in early PAN-induced hypertension. We found that the angiotensin-mediated mechanism is not responsible for this altered renal vasoreactivity., Competing Interests: The authors declare no conflict of interest., (© 2024 by the authors.)

Podocytes are terminally differentiated cells that function as the glomerular filtration barrier in the kidney, and podocyte injury leads to serious proteinuria and podocyte leakage into urine. Recent studies have demonstrated that the number of urinary podocytes is correlated with the progression of glomerular diseases. Therefore, urinary podocytes may serve as an indicator of podocyte injury. In this study, to explore podocyte injury-related genes, we performed comprehensive transcriptome analysis of primary rat podocytes cultured in the presence or absence of puromycin aminonucleoside (PAN), an agent commonly used to induce podocyte injury. RNA-seq revealed that a transcript containing the intronic sequence of small nucleolar RNA host gene 4 (Snhg4) was expressed in podocytes and upregulated by PAN. RT-qPCR analysis demonstrated that this transcript, but not Snhg4, was selectively expressed in podocytes. Therefore, we designated the novel transcript Snhg4-pod. 5′- and 3′-RACE experiments revealed that Snhg4-pod is a novel splice variant of Snhg4 lacking a poly(A) tail. PAN induced Snhg4-pod expression in podocytes in a dose-dependent manner along with their mitochondria-mediated apoptotic cell death. Further, Snhg4-pod was detected in urinary sediments from PAN-induced nephrotic rats. Our findings suggest that Snhg4-pod may serve as a novel marker for the diagnosis of glomerular injury. [ABSTRACT FROM AUTHOR]

This study aims to reveal the endogenous metabolic characteristics of acteoside in the young rat model of purinomycin aminonucleoside nephropathy(PAN) by non-targeted urine metabolomics and decipher the potential mechanism of action. Biochemical indicators in the urine of rats from each group were determined by an automatic biochemical analyzer. The potential biomarkers and related core metabolic pathways were identified by ultra-high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(UHPLC-LTQ-Orbitrap MS) combined with principal component analysis(PCA) and orthogonal partial least squares-discriminant analysis(OPLS-DA). MetaboAnalyst 5.0 was used to establish the receiver operating characteristic(ROC) curve for evaluating the clinical diagnostic performance of core metabolites. The results showed that acteoside significantly decreased urinary protein-to-creatinine ratio in PAN young rats. A total of 17 differential metabolites were screened out by non-targeted urine metabolomics in PAN young rats and they were involved in phenylalanine metabolism and phenylalanine, tyrosine and tryptophan biosynthesis. Thirtten differential metabolites were screened by acteoside intervention in PAN young rats, and they were involved in phenylalanine metabolism and arginine and proline metabolism. Among them, leucylproline and acetophenone were the differential metabolites that were significantly recovered after acteoside treatment. These pathways suggest that acteoside treats PAN in young rats by regulating amino acid metabolism. The area under the curve of two core biomarkers, leucylproline and acetophenone, were both greater than 0.9. In summary, acteoside may restore amino acid metabolism by regulating endogenous differential metabolites in PAN young rats, which will help to clarify the mechanism of acteoside in treating chronic glomerulonephritis in children. The characteristic biomarkers screened out have a high diagnostic value for evaluating the treatment of chronic glomerulonephritis in children with acteoside.

Background: TLR4 signaling is known to be involved in podocyte injury. We have previously shown that Salvia przewalskii extract of total phenolic acids (SPE) and its active monomer salvianolic acid B (SalB) and rosmarinic acid (RA) protect podocytes from injury induced by PAN. In the present study, we test whether SPE inhibits TLR4 signaling. Methods: The conditionally immortalized mouse podocytes were treated with SPE, SalB, RA, SalB + RA or tacrolimus for 30 min, followed by PAN (100 μg/mL) for 24 h. The F-actin staining with phalloidin was used to assess cytoskeletal injury in the podocytes. Western blotting and semi-quantitatives RT-PCR were used to assess the changes of the components in the TLR4 signaling pathway. Results: (1) The F-actin stress fibers of podocytes were almost completely disrupted after PAN treatment for 24 h, and the disruption was significantly alleviated by SPE; (2) the PAN-induced elevation of mRNA levels of TLR4, MyD88 and p65 were inhibited except p65 with high-dose SalB; (3) consistently, the protein levels of TLR4, MyD88 and pp65 were significantly elevated by PAN, and SPE, SalB, RA and admixture, respectively, attenuated the elevations of TLR4 and pp65 proteins; (4) SPE and tacrolimus have a similarly strong effect on inhibition of the expression of TLR4 signaling components. Conclusions: SPE protects podocytes from PAN-induced injury at least partly through inhibiting TLR4 signaling. SPE is as strong as tacrolimus in inhibiting TLR4 signaling in podocytes. [ABSTRACT FROM AUTHOR]

Diabetic nephropathy (DN) is one of the most common microvascular complications associated with diabetes mellitus (DM); the incidence has been predicted to reach 7.7% by 2030 on a global scale. Krüppel-like factor 5 (KLF5) is involved in numerous important biological processes; however, the potential effects of KLF5 on podocytes in patients with diabetic nephrotic (DN) have not yet been investigated. In the present study, synaptopodin expression in podocytes was investigated using an immunofluorescence assay. Following this, the proliferation of podocytes was investigated using an MTT assay. In addition, KLF5 was overexpressed in podocytes, and cell cycle arrest and apoptosis was subsequently investigated using flow cytometry. Western blotting and reverse transcription-quantitative polymerase chain reaction assays were performed to detect the expression levels of genes involved in the cell cycle and apoptosis, and the extracellular signal-regulated protein kinase (ERK)/p38 mitogen-activated protein (MAP) kinase pathway. The results demonstrated that treatment with puromycin aminonucleoside (PAN) suppressed the proliferation of podocytes in a dose- and time-dependent manner, and overexpression of KLF5 induced cell cycle arrest of podocytes regulated by PAN. Furthermore, overexpression of KLF5 was revealed to have inhibited PAN-induced apoptosis of podocytes, and that overexpression of KLF5 suppressed the ERK/p38 MAP kinase pathway in podocytes induced by PAN. Therefore, the results of the present study suggested that KLF5 may represent a potential therapeutic target for treatment of patients with DN. [ABSTRACT FROM AUTHOR]

The glomerular visceral epithelial cells, also termed podocytes, are key in maintaining the normal renal filtration barrier. Although it has been demonstrated that stimulation of c‑Maf inducing protein (CMIP) expression is involved in podocyte damage, the molecular events during this process remain unclear. In the current study, CMIP‑induced proximal signaling was investigated by focusing on its effect on cofilin‑1 activity in puromycin aminonucleoside (PA)‑damaged podocytes. An obvious elevation of CMIP expression and phosphorylated (p) cofilin‑1 levels was detected in cultured podocytes treated with PA and in glomeruli isolated from PA‑induced nephropathy rats. Stable knockdown of CMIP prevented upregulation of p‑cofilin‑1 and reorganization of actin cytoskeleton in PA‑treated podocytes. The activity of the Src family kinase Fyn was reduced, whereas small GTPase Ras homolog gene family, member A (RhoA) activity was increased in PA‑treated podocytes. Stimulation of CMIP expression inhibited Fyn activation and decreased the expression level of p‑p190RhoGAP, a negative regulator of RhoA activity. The level of p‑LIM domain kinase 1 (LIMK1), a downstream effector of RhoA, increased significantly in PA‑treated podocytes. Notably, the applications of RhoA inhibitor or knockdown of LIMK prevented increase of the p‑cofilin‑1 level in PA‑treated podocytes. Thus, the current data provided evidence that the CMIP/Fyn/RhoA/cofilin‑1 signaling pathway may be associated with actin disorganization and podocyte foot process spreading following podocyte injury. [ABSTRACT FROM AUTHOR]

PurposePuromycin aminonucleoside (PAN) specifically injures podocytes, leading to foot process effacement, actin cytoskeleton disorganization, and abnormal distribution of slit diaphragm proteins. p130Cas is a docking protein connecting F-actin fibers to the glomerular basement membrane (GBM) and adapter proteins in glomerular epithelial cells (GEpCs; podocytes). We investigated the changes in the p130Cas expression level in the PAN-induced pathological changes of podocytes in vitro.MethodsWe observed changes in the p130Cas expression in cultured rat GEpCs and mouse podocytes treated with various concentrations of PAN and antioxidants, including probucol, epigallocatechin gallate (EGCG), and vitamin C. The changes in the p130Cas expression level were analyzed using confocal immunofluorescence imaging, Western blotting, and polymerase chain reaction.ResultsIn the immunofluorescence study, p130Cas showed a diffuse cytoplasmic distribution with accumulation at distinct sites visible as short stripes and colocalized with P-cadherin. The fluorescences of the p130Cas protein were internalized and became granular by PAN administration in a dose-dependent manner, which had been restored by antioxidants, EGCG and vitamin C. PAN also decreased the protein and mRNA expression levels of p130Cas at high doses and in a longer exposed duration, which had been also reversed by antioxidants.ConclusionThese findings suggest that PAN modulates the quantitative and distributional changes of podocyte p130Cas through oxidative stress resulting in podocyte dysfunction.

Focal and segmental glomerulosclerosis (FSGS) is a chronic renal disorder characterized by segmental glomerular lesions and widespread podocyte foot process effacement. We have previously shown that glomerular enlargement (hypertrophy) precedes the development of FSGS in an animal model not previously thought to involve glomerular hypertrophy. This hypertrophy involved growth of glomerular capillaries. The aim of the present study was to determine whether the capillary growth involved an increase in the number of capillaries per glomerulus, or lengthening of existing capillaries. In addition, we examined the contribution of glomerular cell hyperplasia to the hypertrophy. We found that glomerular capillary growth in this model appears to primarily involve lengthening of existing capillaries rather that sprouting of new capillaries, and that glomerular cell proliferation contributes to the glomerular hypertrophy.

Podocyte injury is an early pathological change of many kidney diseases. In particular, the actin cytoskeleton plays an important role in maintaining the normal function of podocytes. Disruption of the actin cytoskeleton is a feature of podocyte injury in proteinuric nephropathies. Recent studies showed that myosin-9 was localized in the podocyte foot processes and was necessary in maintaining podocyte structural homeostasis. However, it is unclear whether myosin-9 maintains podocyte structure by affecting actin cytoskleton. Here, the role of myosin-9 in puromycin aminonucleoside (PAN)-induced podocyte injury was explored bothin vitroandin vivo. In cultured mouse podocytes (MPC5), it was determined that PAN downregulated myosin-9 expression, disrupted the actin cytoskeleton and reduced the adhesion ability. Reduced myosin-9 expression by siRNA precipitated podocyte cytoskeletal damage and accelerated PAN-induced podocyte detachment. Overexpression of myosin-9 protected against PAN-induced podocyte detachment. Furthermore, administration of an antioxidant Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP) inhibited PAN-induced podocyte cytoskeletal damage and podocyte detachment by restoring the expression of myosin-9. In the rat PAN nephropathy model, MnTBAP could also attenuate PAN-induced reduction of myosin-9 and podocyte loss. Taken together, these findings pinpointed that oxidative stress contributed to PAN-induced podocyte injury through the repression of a cytoskeletal protein myosin-9, which provided novel insights into a potential target for the treatment of podocyte injury-associated glomerulopathies. [ABSTRACT FROM PUBLISHER]