Nephrin Levels Research Articles

OTUB1 regulation of ferroptosis and the protective role of ferrostatin-1 in lupus nephritis

Lupus nephritis (LN) is a prevalent and severe manifestation of systemic lupus erythematosus (SLE), leading to significant morbidity and mortality. OTUB1, a deubiquitinating enzyme, has emerged as a potential therapeutic target due to its role in cellular protection and regulation of ferroptosis, a form of cell death linked to LN. Our study revealed significantly reduced OTUB1 expression in the glomeruli of LN patients and podocytes, correlated with disease severity. CRISPR/Cas9-mediated OTUB1 knockout in podocytes resulted in pronounced injury, indicated by decreased levels of nephrin and podocin. Ferrostatin-1 treatment effectively mitigated this injury, restoring SLC7A11 expression and significantly reducing MDA levels, Fe2+ levels, BODIPY C11 expression, and normalized cysteine and glutathione expression. In the MRL/lpr mouse model, Ferrostatin-1 significantly improved renal function decreased proteinuria, and ameliorated renal histopathological changes, including reduced glomerular endothelial swelling, mesangial cell proliferation, and leukocyte infiltration. These results underscore the protective role of Ferrostatin-1 in modulating the pathogenesis of LN. OTUB1 plays a crucial protective role against podocyte injury in LN by regulating ferroptosis. Ferrostatin-1 effectively mitigates podocyte damage induced by OTUB1 deficiency, suggesting that targeting ferroptosis could be a promising therapeutic strategy for LN.

Magnolol Inhibits High Fructose-Induced Podocyte Inflammation via Downregulation of TKFC/Sp1/HDAC4/Notch1 Activation

Background/Objectives: High fructose has been implicated as an important trigger of kidney inflammation in patients and experimental models. Magnolol, isolated from Magnolia officinalis, has an anti-inflammatory effect, but its protective role in podocytes remains underexplored. This study explored the protective effects and underlying mechanism of magnolol against high fructose-induced podocyte inflammation. Methods: The effects of magnolol on high fructose-induced podocyte inflammation were assessed in male Sprague Dawley rats administered 10% (w/v) fructose water for 12 weeks and heat-sensitive human podocyte cell lines (HPCs) exposed to 5 mM fructose. Podocyte foot processes were examined using transmission electron microscopy. The expression levels of nephrin, podocin, tumor necrosis factor-α (TNF-α), Notch1 intracellular domain (NICD1), triokinase/FMN cyclase (TKFC), specificity protein 1 (Sp1) and histone deacetylase 4 (HDAC4) were determined by Western blot, immunofluorescence and real-time quantitative polymerase chain reaction (qRT-PCR). The chromatin immunoprecipitation (ChIP) assay was performed to evaluate the interaction between Sp1 and the promoter region of HDAC4. Results: Magnolol mitigated the impairment of glomerular filtration function in high fructose-fed rats. Besides, it significantly alleviated the inflammatory responses in glomeruli and HPCs, evidenced by decreased protein levels of TNF-α and NICD1. Increased protein levels of TKFC, Sp1 and HDAC4 were observed in high fructose-stimulated HPCs and rat glomeruli. TMP195, an HDAC4 inhibitor, reduced TNF-α and NICD1 protein levels in high fructose-exposed HPCs. The increased Sp1 was shown to associate with the promoter region of HDAC4, promoting HDAC4 protein expression in high fructose-exposed HPCs. The knockdown of TKFC in HPCs by TKFC siRNA decreased Sp1, HDAC4 and NICD1 protein levels, alleviating podocyte inflammatory response. Furthermore, magnolol inhibited TKFC/Sp1/HDAC4/Notch1 activation in vivo and in vitro. Conclusions: Magnolol attenuated high fructose-induced podocyte inflammation possibly through the suppression of TKFC/Sp1/HDAC4/Notch1 activation, providing new evidence for its potential role in podocyte protection.

The Use of Kidney Biomarkers, Nephrin and KIM-1, for the Detection of Early Glomerular and Tubular Damage in Patients with Acromegaly: A Case-Control Pilot Study.

Acromegaly is a rare disorder caused by excessive growth hormone (GH) secreted from a pituitary tumor. High levels of GH and insulin growth factor-1 can lead to renal hypertrophy, as well as to diabetes mellitus and hypertension, which negatively impact kidney function. It is believed that high GH may also be involved in the onset of diabetic nephropathy, the main cause of end-stage kidney disease in developed countries. This case-control study was conducted on 23 acromegalic patients and on a control group represented by 21 healthy subjects. The following parameters were determined for all the subjects: serum creatinine, serum urea, estimated glomerular filtration rate (eGFR), urinary albumin/creatinine ratio (UACR), nephrin and kidney injury molecule 1 (KIM-1). Patients with acromegaly showed higher levels of UACR and lower levels of eGFR as compared to healthy subjects. No significant correlations were found between clinical or biochemical parameters associated with acromegaly and nephrin or KIM-1. There was no glomerular or proximal tubular damage at the time of the study, as proven by the normal levels of the biomarkers nephrin and KIM-1. Studies including more patients with uncontrolled disease are needed to clarify the utility of nephrin and KIM-1 for the detection of early kidney involvement in acromegalic patients.

Loss of 3-O-sulfotransferase enzymes, Hs3st3a1 and Hs3st3b1, reduces kidney and glomerular size and disrupts glomerular architecture

Heparan sulfate (HS) is an important component of the kidney anionic filtration barrier, the glomerular basement membrane (GBM). HS chains attached to proteoglycan protein cores are modified by sulfotransferases in a highly ordered series of biosynthetic steps resulting in immense structural diversity due to negatively charged sulfate modifications. 3-O-sulfation is the least abundant modification generated by a family of seven isoforms but creates the most highly sulfated HS domains. We analyzed the kidney phenotypes in the Hs3st3a1, Hs3st3b1 and Hs3st6 -knockout (KO) mice, the isoforms enriched in kidney podocytes. Individual KO mice show no overt kidney phenotype, although Hs3st3b1 kidneys were smaller than wildtype (WT). Furthermore, Hs3st3a1-/-; Hs3st3b1-/- double knockout (DKO) kidneys were smaller but also had a reduction in glomerular size relative to wildtype (WT). Mass spectrometry analysis of kidney HS showed reduced 3-O-sulfation in Hs3st3a1-/- and Hs3st3b1-/-, but not in Hs3st6-/- kidneys. Glomerular HS showed reduced HS staining and reduced ligand-and-carbohydrate engagement (LACE) assay, a tool that detects changes in binding of growth factor receptor-ligand complexes to HS. Interestingly, DKO mice have increased levels of blood urea nitrogen, although no differences were detected in urinary levels of albumin, creatinine and nephrin. Finally, transmission electron microscopy showed irregular and thickened GBM and podocyte foot process effacement in the DKO compared to WT. Together, our data suggest that loss of 3-O-HS domains disrupts the kidney glomerular architecture without affecting the glomerular filtration barrier and overall kidney function.

Anatomical and molecular insights into the antennal gland of the giant freshwater prawn Macrobrachium rosenbergii

In this study, the complex organization of the AnG in the giant freshwater prawn Macrobrachium rosenbergii was revealed using various techniques, including conventional histology, histochemistry, scanning electron microscopy, and X-ray tomography. The results showed the diversity of cells in the AnG and the detailed organization of the labyrinth’s tubule into four radiated areas from the central to peripheral zones. The study also demonstrated the expression of some vertebrate kidney-associated homolog genes, aquaporin (AQP), solute carrier family 22 (SLC-22), nephrin, and uromodulin, in the AnG by qPCR. The result of in situ hybridization further showed the localization of SLC-22 and AQP transcript in the bladder and labyrinth’s epithelium, specifically in regions 2, 3, and 4. Additionally, the study revealed neuropeptide expressions in the AnG by qPCR and in situ hybridization, i.e., crustacean hyperglycemic hormone (CHH) and molt inhibiting hormone (MIH), implying that the AnG may have a role in hormone production. Moreover, male and female prawns exhibited different levels of AQP, SLC-22, nephrin, and CHH expressions during the premolt and intermolt stages, suggesting a crucial role relevant to the molting stages. In conclusion, this study clarified the complex structure of the AnG in M. rosenbergii and demonstrated for the first time the expression of vertebrate kidney-associated genes and the possible endocrine role of the AnG. Further investigation is needed to clarify the role of these genes, particularly during ecdysis. The implications of these findings could significantly advance our understanding of the AnG in decapod crustaceans.

Neutrophil-to-lymphocyte ratio is elevated in acute hantavirus infection and correlates with markers of disease severity.

Pathogenic Eurasian hantaviruses cause hemorrhagic fever with renal syndrome (HFRS), which is characterized by acute kidney injury. The clinical course shows a broad range of severity and is influenced by direct and immune-mediated effects. The neutrophil-to-lymphocyte ratio (NLR) is a marker of systemic inflammation and predicts severity and outcome in various diseases. Therefore, we examined the role of NLR in HFRS caused by hantavirus Puumala (PUUV) and its association with disease severity and kidney injury. We detected elevated NLR levels on admission (NLRadm: median 3.82, range 1.75-7.59), which increased during acute HFRS. Maximum NLR levels (NLRmax: median 4.19, range 1.75-13.16) were 2.38-fold higher compared to the reference NLR level of 1.76 in the general population. NLR levels on admission correlate with markers of severity (length of hospital stay, serum creatinine) but not with other markers of severity (leukocytes, platelets, C-reactive protein, lactate dehydrogenase, serum albumin, proteinuria). Interestingly, levels of nephrin, which is a specific marker of podocyte damage in kidney injury, are highest on admission and correlate with NLRmax, but not with NLRadm. Together, we observed a correlation between systemic inflammation and the severity of HFRS, but our results also revealed that podocyte damage precedes these inflammatory processes.

Aminophylline suppresses chronic renal failure progression by activating SIRT1/AMPK/mTOR-dependent autophagy.

Chronic renal failure (CRF) is a severe syndrome affecting the urinary system for which there are no effective therapeutics. In this study, we investigate the effects and mechanisms of aminophylline in preventing CRF development. A rat model of chronic renal failure is established by 5/6 nephrectomy. The levels of serum creatinine (SCR), urinary protein (UPR), and blood urea nitrogen (BUN) are detected by ELISA. Histological evaluations of renal tissues are performed by H&E, Masson staining, and PAS staining. Functional protein expression is detected by western blot analysis or immunofluorescence microscopy. Glomerular cell apoptosis is determined using the TUNEL method. Results show that Aminophylline significantly reduces the levels of SCR, UPR, and BUN in the CRF model rats. Histological analyses show that aminophylline effectively alleviates renal tissue injuries in CRF rats. The protein expression levels of nephrin, podocin, SIRT1, p-AMPK, and p-ULK1 are greatly increased, while p-mTOR protein expression is markedly decreased by aminophylline treatment. Additionally, the protein level of LC3B in CRF rats is significantly increased by aminophylline. Moreover, aminophylline alleviates apoptosis in the glomerular tissues of CRF rats. Furthermore, resveratrol promotes SIRT1, p-AMPK, and p-ULK1 protein expressions and reduces p-mTOR and LC3B protein expressions in CRF rats. Selisistat (a SIRT1 inhibitor) mitigates the changes in SIRT1, p-AMPK, p-ULK1, p-mTOR, and LC3B expressions induced by aminophylline. Finally, RAPA alleviates renal injury and apoptosis in CRF rats, and 3-MA eliminates the aminophylline-induced inhibition of renal injury and apoptosis in CRF rats. Aminophylline suppresses chronic renal failure progression by modulating the SIRT1/AMPK/mTOR-mediated autophagy process.

Hypoxia-inducible factor-1α attenuates renal podocyte injury in male rats in a simulated high-altitude environment by upregulating Krüppel-like factor 4 expression.

Previous studies have shown that podocyte injury is involved in the development of proteinuria in rats under hypobaric hypoxia conditions. Prolyl hydroxylase inhibitors (PHIs) may reduce proteinuria. This study aimed to further investigate whether the protective effects of hypoxia-inducible factor 1α (HIF1α) on podocyte injury induced by hypobaric hypoxia are related to Krüppel-like factor 4 (KLF4). Rats were housed in a low-pressure oxygen chamber to simulate a high-altitude environment (5000m), and a PHI was intraperitoneally injected. Urinary protein electrophoresis was performed and the morphology of the podocytes was observed by electron microscopy. Rat podocytes were cultured under 1% O2, and siRNA was used to interfere with KLF4 expression. The protein expression levels of HIF1α, KLF4, CD2-associated protein (CD2AP) and nephrin were determined by western blotting. Compared with those in the experimental group, the rats in the intervention group on day 14 had lower urinary protein levels, increased protein expression levels of CD2AP and nephrin, and reduced podocyte injury. The results of in vitro experiments showed that the protein expression levels of KLF4, CD2AP and nephrin were greater in the PHI intervention group and lower in the HIF1α inhibitors group than in the low-oxygen group. The protein expression of CD2AP and nephrin in the siKLF4-transfected podocytes treated with PHI and HIF1α inhibitors did not differ significantly from that in the low-oxygen group. HIF1α may be involved in reducing progressive high-altitude proteinuria by regulating KLF4 expression and contributing to the repair of podocyte injury induced by hypobaric hypoxia.

Indoxyl Sulfate Aggravates Podocyte Damage through the TGF-β1/Smad/ROS Signalling Pathway.

Hyperglycaemia induces the production of a large quantity of reactive oxygen species (ROS) and activates the transforming growth factor β1 (TGF-β1)/Smad signalling pathway, which is the main initiating factor in the formation of diabetic nephropathy. Indoxyl sulphate (IS) is a protein-binding gut-derived uraemic toxin that localizes to podocytes, induces oxidative stress, and inflames podocytes. The involvement of podocyte damage in diabetic nephropathy through the TGF-β1 signalling pathway is still unclear. In this study, we cultured differentiated rat podocytes in vitro and measured the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, and α-SMA by quantitative real-time PCR (qRT-PCR) and Western blotting after siRNA-mediated TGF-β1 silencing, TGF-β1 overexpression, and the presence of the ROS inhibitor acetylcysteine. We detected the expression levels of nephrin, synaptopodin, CD2AP, SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, NADPH oxidase 4 (NOX4), and ROS levels under high glucose (HG) and IS conditions. The results indicated that nephrin, synaptopodin, CD2AP, and SRGAP2a expressions were significantly upregulated, and α-SMA expression was significantly downregulated in the presence of HG under siRNA-mediated TGF-β1 silencing or after the addition of acetylcysteine. However, in the presence of HG, the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a were significantly downregulated, and the expression of α-SMA was significantly upregulated with the overexpression of TGF-β1. IS supplementation under HG conditions further significantly reduced the expressions of nephrin, synaptopodin, CD2AP, and SRGAP2a; altered the expressions of Smad2/3, p-Smad2/3, Smad7, and NOX4; and increased ROS production in podocytes. This study suggests that IS may modulate the expression of nephrin, synaptopodin, CD2AP, and SRGAP2a by regulating the ROS and TGF-β1/Smad signalling pathways, providing new theoretical support for the treatment of diabetic nephropathy.

Xuebijing injection and its bioactive components alleviate nephrotic syndrome by inhibiting podocyte inflammatory injury

Xuebijing injection (XBJ) is widely used to treat nephrotic syndrome (NS) in clinic, but its bioactive components and therapeutic mechanism are still unclear. In this study, the bioactive components of XBJ were determined by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometry (UPLC-Q-TOF/MS). The therapeutic effect of XBJ on NS was evaluated in BALB/c mice induced by adriamycin (ADR, 10 mg/kg) via a single tail vein. The protective effect of XBJ and its bioactive components on podocytes was demonstrated using mouse podocytes (MPC-5) induced by lipopolysaccharide (LPS, 4 μg/mL). The results show that 33 components of XBJ were identified. Furthermore, 12 bioactive components were detected in blood, including protocatechuic acid, salvianolic acid C, benzoyloxypaeoniflorin, danshensu, salvianolic acid A, salvianolic acid B, catechin, caffeic acid, galloylpaeoniflorin, oxypaeoniflorin, hydroxysafflor yellow A, rosmarinic acid. The relative content (%) of the bioactive components were 59.32, 16.01, 9.97, 9.73, 8.72, 8.31, 7.92, 6.54, 1.54, 1.30, 0.68 and 0.59 in this order. After XBJ treatment, the renal function, hyperlipidemia and renal pathological damage were improved in NS model mice. Moreover, the levels of nephrin and desmin which are functional proteins in podocytes were reversed, and the levels of pro-inflammatory factors were reduced by XBJ. Interestingly, protocatechuic acid and salvianolic acid C also showed good protective effects on podocyte function and reduced the level of inflammation in LPS-induced MPC-5. The study is the first time to elucidate the bioactive components of XBJ and its potential therapeutic mechanism for treating NS by protecting podocyte function.

The potential role of differentially expressed tRNA-derived fragments in high glucose-induced podocytes

The prevalence of diabetic kidney disease (DKD) is increasing annually. Damage to and loss of podocytes occur early in DKD. tRNA-derived fragments (tRFs), originating from tRNA precursors or mature tRNAs, are associated with various illnesses. In this study, tRFs were identified, and their roles in podocyte injury induced by high-glucose (HG) treatment were explored. High-throughput sequencing of podocytes treated with HG was performed to identify differentially expressed tRFs. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed. The expression levels of nephrin, podocin, and desmin were measured in podocytes after overexpression of tRF-1:24-Glu-CTC-1-M2 (tRF-1:24) and concomitant HG treatment. A total of 647 tRFs were identified, and 89 differentially expressed tRFs (|log2FC| ≥ 0.585; p ≤ .05) were identified in the HG group, of which 53 tRFs were downregulated and 36 tRFs were upregulated. The 10 tRFs with the highest differential expression were detected by real-time quantitative polymerase chain reaction (RT-qPCR), and these results were consistent with the sequencing results. GO analysis revealed that the biological process, cellular component, and molecular function terms in which the tRFs were the most enriched were cellular processes, cellular anatomical entities, and binding. KEGG pathway analysis revealed that tRFs may be involved in signaling pathways related to growth hormones, phospholipase D, the regulation of stem cell pluripotency, and T-/B-cell receptors. Overexpression of tRF-1:24, one of the most differentially expressed tRFs, attenuated podocyte injury induced by HG. Thus, tRFs might be potential biomarkers for podocyte injury in DKD.

Urinary podocyte stress marker as a prognostic indicator for diabetic kidney disease

BackgroundDiabetic kidney diseases (DKD) is a the most common cause of end-stage kidney disease (ESKD) around the world. Previous studies suggest that urinary podocyte stress biomarker, e.g. podocin:nephrin mRNA ratio, is a surrogate marker of podocyte injury in non-diabetic kidney diseases.MethodWe studied 118 patients with biopsy-proved DKD and 13 non-diabetic controls. Their urinary mRNA levels of nephrin, podocin, and aquaporin-2 (AQP2) were quantified. Renal events, defined as death, dialysis, or 40% reduction in glomerular filtration rate, were determined at 12 months.ResultsUrinary podocin:nephrin mRNA ratio of DKD was significantly higher than the control group (p = 0.0019), while urinary nephrin:AQP2 or podocin:AQP2 ratios were not different between groups. In DKD, urinary podocin:nephrin mRNA ratio correlated with the severity of tubulointerstitial fibrosis (r = 0.254, p = 0.006). and was associated with the renal event-free survival in 12 months (unadjusted hazard ratio [HR], 1.523; 95% confidence interval [CI] 1.157–2.006; p = 0.003). After adjusting for clinical and pathological factors, urinary podocin:nephrin mRNA ratio have a trend to predict renal event-free survival (adjusted HR, 1.327; 95%CI 0.980–1.797; p = 0.067), but the result did not reach statistical significance.ConclusionUrinary podocin:nephrin mRNA ratio has a marginal prognostic value in biopsy-proven DKD. Further validation is required for DKD patients without kidney biopsy.

QiDiTangShen granules alleviates diabetic nephropathy podocyte injury: A network pharmacology study and experimental validation in vivo and vitro

BackgroundQiDiTangShen granules (QDTS), a traditional Chinese medicine (TCM) compound prescription, have remarkable efficacy in diabetic nephropathy (DN) patients, and their pharmacological mechanism needs further exploration. MethodsAccording to the active ingredients and targets of the QDTS in the TCMSP database, the network pharmacology of QDTS was investigated. The potential active ingredients were chosen based on the oral bioavailability and the drug similarity index. At the same time, targets for DN-related disease were obtained from GeneCards, OMIM, PharmGKB, TTD, and DrugBank. The TCM-component-target network and the protein-protein interaction (PPI) network were constructed with the Cytoscape and STRING platforms, respectively, and then the core targets of DN were selected with CytoNCA. GO and KEGG enrichment analysis using R software. Molecular docking to identify the core targets of QDTS for DN. In vivo, db/db mice were treated as DN models, and the urine microalbuminuria, the pathological changes in the kidney and the protein expression levels of p-PI3K, p-Akt, JUN, nephrin and synaptopodin were detected by immunohistochemistry, immunofluorescence method and Western blotting. After QDTS was used in vitro, the protein expression of mouse podocyte clone-5 (MPC5) cells was detected by immunohistochemistry, immunofluorescence and Western blot. ResultsThrough network pharmacology analysis, 153 potential targets for DN in QDTS were identified, 19 of which were significant. The KEGG enrichment analysis indicated that QDTS might have therapeutic effects on IL-17, TNF, AGE-RAGE, PI3K-Akt, HIF-1, and EGFR through interfering with Akt1 and JUN. The main active ingredients in QDTS are quercetin, β-sitosterol, stigmasterol and kaempferol. Both in vivo and in vitro studies showed that QDTS could decrease the urine microalbuminuria and renal pathology of db/db mice, and alleviate podocyte injuries through the PI3K/Akt signaling pathway. ConclusionThrough network pharmacology, in vivo and in vitro experiments, QDTS has been shown to improve the urine microalbuminuria and renal pathology in DN, and to reduce podocyte damage via the PI3K/Akt pathway.

Urinary Nephrin Levels Among Pregnant Women With Preeclampsia in Lagos, Southwest Nigeria: An Analytical Cross-Sectional Study.

Hypertensive disorders in pregnancy are one of the leading causes of maternal and perinatal morbidity and mortality worldwide. The clinical utility of urinary nephrin as a diagnostic biomarker of preeclampsia is currently of research interest. However, this is yet to gain significant traction within clinical settings. We evaluated the association between maternal urinary nephrin levels and the occurrence and severity of preeclampsia among pregnant women in Lagos, Nigeria. We conducted an analytical cross-sectional study involving pregnant women diagnosed with preeclampsia as well as their age- and gestational-age-matched normotensive counterparts. We tested the association between high maternal urinary nephrin levels and the occurrence of preeclampsia without and with severe features. P < 0.05 was reported as statistically significant. The study showed that for every unit increase in urinary nephrin levels, the odds of preeclampsia increased by about ninefold (adjusted Odds ratio = 8.9, 95% confidence interval: 2.8-29.2, P < 0.001). The levels of urinary nephrin increased steadily with increasing severity of the disease: 1.9 ± 0.8 ng/mL in preeclampsia without severe features, 2.7 ± 0.7 ng/mL in preeclampsia with at least one severe feature, and 3.3 ±1.1 ng/mL in eclampsia. There was an association between elevated levels of urinary nephrin and preeclampsia and its severe variant. However, there is a need for more robust studies with a longitudinal characterization of urinary nephrin levels to establish causal relationships with preeclampsia, explore other potential risk factors of preeclampsia, and define the clinical usefulness of urinary nephrin as a potentially reliable and accurate predictive marker of preeclampsia among women in low- and middle-income countries (LMIC) settings.

Qufeng tongluo decoction decreased proteinuria in diabetic mice by protecting podocytes via promoting autophagy

BackgroundDiabetic kidney disease (DKD) is one of diabetic complications, which has become the leading cause of end-stage kidney disease. In addition to angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker(ACEI/ARB) and sodium-glucose cotransporter-2 inhibitor (SGLT2i), traditional Chinese medicine (TCM) is an effective alternative treatment for DKD. In this study, the effect of Qufeng Tongluo (QFTL) decoction in decreasing proteinuria has been observed and its mechanism has been explored based on autophagy regulation in podocyte. MethodsIn vivo study, db/db mice were used as diabetes model and db/m mice as blank control. Db/db mice were treated with QFTL decoction, rapamycin, QFTL + 3-Methyladenine (3-MA), trehalose, chloroquine (CQ) and QFTL + CQ. Mice urinary albumin/creatinine (UACR), nephrin and autophagy related proteins (LC3 and p62) in kidney tissue were detected after intervention of 9 weeks. Transcriptomics was operated with the kidney tissue from model group and QFTL group. In vitro study, mouse podocyte clone-5 (MPC-5) cells were stimulated with hyperglycemic media (30 mmol/L glucose) or cultured with normal media. High-glucose-stimulated MPC-5 cells were treated with QFTL freeze-drying powder, rapamycin, CQ, trehalose, QFTL+3-MA and QFTL + CQ. Cytoskeletal actin, nephrin, ATG-5, ATG-7, Beclin-1, cathepsin L and cathepsin B were assessed. mRFP-GFP-LC3 was established by stubRFP-sensGFP-LC3 lentivirus transfection. ResultsQFTL decoction decreased the UACR and increased the nephrin level in kidney tissue and high-glucose-stimulated podocytes. Autophagy inhibitors, including 3-MA and chloroquine blocked the effects of QFTL decoction. Further study showed that QFTL decoction increased the LC3 expression and relieved p62 accumulation in podocytes of db/db mice. In high-glucose-stimulated MPC-5 cells, QFTL decoction rescued the inhibited LC3 and promoted the expression of ATG-5, ATG-7, and Beclin-1, while had no effect on the activity of cathepsin L and cathepsin B. Results of transcriptomics also showed that 51 autophagy related genes were regulated by QFTL decoction, including the genes of ATG10, SCOC, ATG4C, AMPK catalytic subunit, PI3K catalytic subunit, ATG3 and DRAM2. ConclusionQFTL decoction decreased proteinuria and protected podocytes in db/db mice by regulating autophagy.

TRF-003634 alleviates adriamycin-induced podocyte injury by reducing the stability of TLR4 mRNA.

Podocyte injury plays a key role in the production of proteinuria and is closely related to the progression of chronic kidney disease (CKD). Alleviating podocyte injury is beneficial to prevent the occurrence and development of CKD. tRNA-derived RNA fragments (tRFs) are associated with podocytes injury processes such as protein binding, cell adhesion, synapses, the actin cytoskeleton. Our previous data showed that tRF-003634 tightly correlated with podocyte injury, while its effect remains unclear. This study aimed to investigate the role of tRF-003634 in podocyte injury and the potential mechanisms. The expression level of tRF-003634, nephrin, podocin and tRF-003634 targeted toll-like receptor 4 (TLR4) in podocytes and kidney tissues were examined by quantitative real-time PCR (qRT-PCR), western blot and immunohistochemistry. The biochemical indices were monitored and renal pathological changes were assessed by hematoxylin and eosin PAS staining. Furthermore, potential target genes of tRF-003634 were screened using high-throughput mRNA sequencing, and then confirmed by RNA pulse-chase analysis. The results showed that tRF-003634 was downregulated in adriamycin (Adr)-induced podocyte injury. Overexpression of tRF-003634 increased the expression of nephrin and podocin in vivo and in vitro and alleviated podocyte injury. Meanwhile, overexpression of tRF-003634 alleviated proteinuria and renal pathological damage. In addition, high-throughput sequencing after overexpression of tRF-003634 showed that TLR4 might be a downstream target gene. tRF-003634 can alleviate podocyte injury by reducing the stability of TLR4 mRNA, possibly by competing with TLR4 mRNA to bind to YTH domain-containing protein 1 (YTHDC1). In conclusion, tRF-003634 was underexpressed in Adr-induced podocyte injury, and its overexpression alleviated podocyte injury in vitro and in vivo by reducing the stability of TLR4 mRNA.

Abstract P3164: Podocalyxin, A Renal Podocyte Specific Protein Expression In Urine Exosome Acts As A Marker For Podocyte Injury During Sars-cov-2 Infection

COVID-19 attributed to SARS-CoV2 infection has been associated with cardiovascular disease and diabetes. COVID-19 has been associated with endothelial cell dysfunction (ECD), which is manifested by entities such as microvascular thrombosis. ECD is quite common in type 2 diabetes mellitus (T2DM) where the renal podocyte dysfunction is often an early manifestation of microvascular complication. We explored whether presence of hyperglycemia predisposes to increased SARS-CoV2 infection and whether SARS-CoV2 infection puts an individual at a higher risk of developing cardio-metabolic complications such as kidney disease (DKD), with podocyte damage. We evaluated albuminuria and podocyte protein in urine derived exosomes in urine exosomes from SARS-CoV2 patients at 10 days, 6 months and 12 months post SARS-CoV2 infection. Methods: Blood and Urine samples from SARS-CoV2 patients post-acute phase of infection were procured. Peripheral blood mononuclear cells (PBMNCs) and urine exosomes were isolated and podocyte protein marker Podocalyxin (PODXL) was identified by western blot analysis. Results: Podocyte specific proteins were examined in urine exosomal fraction such as Podocalyxin and Nephrin (normalized to CD9 exosomal protein band intensities) levels increased significantly compared to urine samples from type-2 diabetes subjects with documented evidence of nephropathy. Podocalyxin levels were significantly high at 12month time point (p=0.001; n=9) and Nephrin levels were noted to be high at 10week (p=0.01; n=8), 6months (p=0.04; n=14 ) and 12Months (p=0.001; n=6) time points. Podocalyxin and Nephrin obtained from urine exosome samples collected from non diabetic subjects who never had COVID-19, showed little or barely detectable corresponding western blot bands. Interestingly, there was no significant differences noted on urine albumin: creatinine (UAR) ratios between the two groups, indicating that urine exosomal protein estimation may be an ideal biomarker to monitor podocyte damage in early phases of CKD. Conclusion: A persistent high levels of podocyte specific proteins was noted in urinary exosomes even at 12 months post acute Covid which may be secondary to long-standing podocyte inflammation leading to long term renal damage.

Correlation of Kidney Injury Molecule-1 and Nephrin Levels in Iraqi Patients with Diabetic Nephropathy

Background: Diabetic nephropathy is characterized by persistent microalbuminuria and metabolic changes that decline renal functions. Researchers have been prompted to explore new biomarkers such as KIM-1 and nephrin that may enhance the identification of disease. Objective: To Evaluate biomarker levels of kidney injury molculre-1 (KIM-1) concentration and nephrin as early and sensitive markers of nephropathy in type 2 diabetic patients. Method: One hundred T2DM patients were included in a cross-sectional study at the specialized center for endocrinology and diabetes, Baghdad. The first group includes 50 diabetic nephropathy (DN) patients, and the second group includes 50 T2DM patients without DN. Biochemical and clinical parameters were reported for participants, and serum and urine levels of KIM-1 and nephrin were analyzed by Enzyme-linked immunosorbent assay. Results: The study showed a significant increase in serum and urinary levels of KIM-1 and nephrin in DN patients compared to the control group. Serum nephrin is positively correlated with urinary nephrin, serum creatinine, ACR ratio, serum and urine KIM-1, and negatively correlated with the estimated glomerular filtration rate. Urinary nephrin was positively correlated with urinary albumin/creatinine ratio, KIM-1 level in both serum and urine, and negatively correlated with estimated glomerular filtration rate. Conclusion: KIM-1 and nephrin are specific and sensitive indicators of early-stage diabetic nephropathy-associated renal damage.

Risk factors and early biomarkers of nephrotoxicity associated with the use of anti-VEGF drugs.

e24079 Background: Anti-angiogenic anticancer drugs that block VEGF can cause kidney damage (Fujii et al., 2019). Among the mechanisms of nephrotoxicity thrombotic microangiopathy (TMA) of renal vessels can be distinguished (Estrada et al., 2019). TMA can be accompanied the development of anemia, excessive schistocytes, as well as increased LDH levels and a low platelet count, which indicated the occurrence of endothelial dysfunction and microangiopathic processes (Hauser et al, 2009). The goal of this study was to assess the risk factors of early nephrotoxicity and the utility of NGAL, KIM-1, HIF-1α and nephrin levels in urine as early biomarkers of nephrotoxicity. Methods: The study included 50 patients who received anti-VEGF drugs (aflibercept, bevacizumab or ramucirumab). Nephrotoxicity was determined as a decrease in glomerular filtration rate (GFR) to &lt; 60 ml/min/1.73m2 at 8 weeks from the initiation of therapy. The laboratory parameters and the levels of KIM-1, NGAL, HIF-1α and nephrin in urine samples were determined by ELISA before treatment and after 1, 2, 4 and 8 weeks of treatment. To assess risk factors for nephrotoxicity we analyzed primary clinical and laboratory parameters. Results: Nephrotoxicity at 8 weeks was achieved in 21 (42%) of patients. A statistically significant decrease in hemoglobin and platelet count by 20 and 26%, respectively, from the initial level was observed. An increase in the number of schistocytes and a slight increase in LDH levels were also observed. The increase in NGAL, KIM-1 and nephrin levels was statistically significant in 1 weeks and the increase of HIF-1α in 2 weeks after the start of therapy (p = 0.001). The unfavorable predictors of nephrotoxicity as well as the favorable predictors in terms of decreasing renal function are presented in Table. ROC analysis demonstrated high sensitivity and specificity: NGAL – AUC 0.7; KIM-1 – AUC 0.69; HIF-1α – 0.7; nephrin – AUC 0.7. Conclusions: We report a significant increase in the parameters of microangiopathic hemolysis and in urine biomarkers of tubular and podocytic damage as well as hypoxia by the 8th week of treatment. We suggest that the TMA process can take part in renal damage. Increased levels of NGAL, KIM-1, HIF-1α and nephrin in the urine reflect the renal damage processes and have higher sensitivity and specificity then serum creatitine. The independent risk factors for nephrotoxicity are determined. [Table: see text]

MTOR/ULK1 signaling axis mediated ultrashort wave regulation of autophagy to alleviate diabetic kidney disease.

Diabetic kidney disease (DKD) is closely related to autophagy and inflammation. The mTOR/unc-51 like autophagy activating kinase 1 (ULK1) signaling axis is involved in the regulation of autophagy. Ultrashort wave (USW) therapy has been extensively studied in inflammatory diseases. However, the therapeutic effect of USW on DKD and the role of the mTOR/ULK1 signaling axis in USW interventions remain uncertain. This study aimed to explore the therapeutic effects of USW on DKD rats and the role of the mTOR/ULK1 signaling axis in USW interventions. A DKD rat model was established using a high-fat diet (HFD)/sugar diet and streptozocin (STZ) induction. The optimal duration of USW intervention was determined using different USW treatments. The levels of metabolism, inflammation and fibrosis associated with kidney injury in rats were measured. Western blot analysis was performed on the related indexes of autophagy and the mTOR/ULK1 signaling axis. In DKD rats, microalbuminuria (MAU), glucose (GLU), creatinine (CRE), and blood urea nitrogen (BUN) levels decreased after the USW intervention. Levels of interleukin (IL)-1β, inducible nitric oxide synthase (iNOS), immunoglobulin M (IgM), immunoglobulin G (IgG), IL-18, tumor necrosis factor alpha (TNF-α), and IL-6 decreased in the USW group compared to the model group. The IL-10 and arginase (Arg-1) levels were increased in the USW group. The content of fibrosis-related indexes (vascular endothelial growth factor (VEGF), fibronectin (FN), type IV collagen, and type I collagen) decreased in the urine of the DKD rats. After USW treatment, LC3B and Beclin1 levels increased, while the level of p62 decreased. The levels of nephrin, podocin and synaptopodin increased. Ultrashort wave could reduce p-mTOR/mTOR ratios and increase ULK1 expression. After the overexpression of ULK1, the levels of LC3B and Beclin1 were higher in the overexpression (oe)-ULK1 group than in the oe-negative control (NC) group, while the level of p62 decreased. After mTOR activation, LC3B and ULK1 expression decreased, while CRE, BUN, MAU, and GLU levels increased. Ultrashort wave alleviated kidney injury induced by the HFD/sugar diet and STZ. The USW intervention reversed the decreased autophagy levels in the DKD rats. The mTOR/ULK1 signaling axis mediated USW to promote autophagy.