Serum L C3-II levels in type 2 diabetic patients with impaired renal functions.

This study was designed to evaluate serum LC3-II, BCL-2, IL-1ß, TGF-ß1, and podocin levels in. type 2 diabetes (T2DM) patients with renal dysfunction. MATERIALS: 176 Turkish subjects were enrolled, of whom 26 were healthy, and 150 had T2DM. PATIENTS: were classified according to albumin urea ratio: 88 patients had macroalbuminuria, 20. patients had microalbuminuria, and 42 had normoalbuminuria. T2DM patients were also. classified into three groups according to proteinuria and eGFR stages. RESULTS: Increased serum LC3-II levels in patients with T2DM with increased urinary albumin. extraction and impaired renal functions. There was a strong relationship between serum. LC3-II levels and serum BCL-2, IL-1ß, TGF-ß1, and Podocin levels. The efficiency of LC3- II as a diagnostic biomarker in the differential diagnosis of DM patients with. macroproteinuria from DM patients with normoproteinuria was 75.4%. CONCLUSIONS: It was thought that increased serum LC3-II levels in T2DM patients with impaired renal. functions may cause renal podocyte damage. In these patients, serum LC3-II levels can be. evaluated as a new biomarker to follow the development of renal damage.

The Correlation of MicroRNA-21 With the Nephrin, Podocin, and Urinary Albumin-Creatinine Ratio in Patients With Type 2 Diabetes and Albuminuria: A Cross-Sectional Study.

Background: Diabetic kidney disease (DKD) is the most common and deranging microvascular complication of diabetes mellitus (DM). Podocytopathy is a key component of glomerular damage in DKD. Micro RNA-21 (miRNA-21) is an epigenetic regulator that plays a role in podocyte damage; however, the results of previous studies have not resolved the controversy about the role of miRNA-21 in the pathogenesis of DKD. Objective: The objective was to investigate the correlation between miRNA-21 levels and urinary nephrin, podocin, and urinary albumin-creatinine ratio (UACR) in patients with type 2 DM and albuminuria. Design: This is a cross-sectional study. Setting: This study was carried out in internal medicine outpatient clinic of Cipto Mangunkusumo Hospital Jakarta, Indonesia. Patients: This study consisted of 42 adults with type 2 DM and albuminuria. Measurements: The measurements include (1) Serum miRNA-21; (2) urinary podocin, nephrin, and albumin-creatinine ratio; and (3) serum miRNA-21 correlated to urinary podocin, nephrin, and albumin-creatinine ratio. Methods: The Spearman bivariate analysis to assess the correlation of miRNA-21 with nephrin, podocin, and UACR. Results: The mean relative expression of miRNA-21 was 0.069 (0.024), the median for nephrin, podocin, and UACR was 35.5 (15.75-51.25) ng/mL, 0.516 (0.442-0.545) ng/mL, and 150 (94.56-335.75) ng/mL, respectively. A correlation between miRNA-21 and nephrin was observed (r = 0.598; P < .0001). There was a correlation between miRNA-21 and UACR (r = 0.604; P < .0001). No correlation was found between miRNA-21 and podocin. Limitations: A lack of non-DM and non-albuminuric control population and small sample size. We could not exclude concurrent disease, and all other potential confounding variables, particularly those related to inflammation. Conclusions: The miRNA-21 can be considered an early biomarker for podocytopathy and albuminuria in DM, highlighting its potential for early diagnostic and therapeutic interventions. Further research is required to confirm these findings and explore their clinical applications, which could significantly alter management strategies for DKD.

Contexte: La maladie rénale diabétique (MRD) est la complication microvasculaire la plus fréquente et une des plus inquiétantes du diabète (DB). La podocytose est une composante clé des lésions glomérulaires en contexte de MRD. Le micro-ARN-21 (miARN-21) est un régulateur épigénétique impliqué dans les lésions podocytaires, mais les résultats des études précédentes n'ont pas résolu la controverse sur le rôle du miARN-21 dans la pathogenèse de la MRD. Objectif: Étudier la corrélation entre le taux de miARN-21 et la néphrine, la podocine et le rapport albumine-créatinine (RAC) urinaires chez les patients atteints de diabète de type 2 et présentant une albuminurie. Type d'étude: Étude transversale. Cadre: La clinique ambulatoire de médecine interne de l'hôpital Cipto Mangunkusumo à Jakarta (Indonésie). Sujets: 42 adultes diabétiques de type 2 présentant une albuminurie. Mesures: (1) miARN-21 sérique; (2) podocine, néphrine et rapport albumine-créatinine urinaires; (3) le miARN-21 sérique corrélé à la podocine, à la néphrine et au rapport albumine-créatinine urinaires. Méthodologie: L'analyse bivariée de Spearman a servi à évaluer la corrélation entre le taux de miARN-21 et la néphrine, la podocine et le rapport albumine-créatinine urinaires. Résultats: L'expression relative moyenne du miARN-21 était de 0,069 ng/ml (0,024). La médiane s'établissait à 35,5 (15,75­51,25) ng/ml pour la néphrine, à 0,516 (0,442­0,545) ng/ml pour la podocine et à 150 (94,56­335,75) ng/ml pour le RAC. On a observé une corrélation entre le miARN-21 et la néphrine (r = 0,598; p = < 0,0001), de même qu'entre le miARN-21 et le RAC (r = 0,604; p = <0,0001). Aucune corrélation n'a été observée entre le miARN-21 et la podocine. Limites: L'étude ne comporte pas de population témoin (non-DB et sans albuminurie) et l'échantillon est de petite taille. Il n'a pas été possible d'exclure les maladies concomitantes, de même que toutes les autres variables confondantes potentielles, en particulier celles qui sont liées à l'inflammation. Conclusion: Chez les patients diabétiques, le miARN-21 peut être considéré comme un biomarqueur précoce de la podocytose et de l'albuminurie, ce qui met en évidence son potentiel à faire partie des interventions diagnostiques et thérapeutiques précoces. D'autres recherches sont nécessaires pour confirmer ces résultats et explorer leurs applications cliniques, ce qui pourrait modifier considérablement les stratégies de prise en charge de la maladie rénale diabétique.

c-Cbl induced podocin ubiquitination contributes to the podocytes injury in diabetic nephropathy.

The ubiquitination function in diabetic nephropathy (DN) has attracted much attention, but there is a lack of information on its ubiquitylome profile. To examine the differences in protein content and ubiquitination in the kidney between db/db mice and db/m mice, we deployed liquid chromatography-mass spectrometry (LC-MS/MS) to conduct analysis. We determined 145 sites in 86 upregulated modified proteins and 66 sites in 49 downregulated modified proteins at the ubiquitinated level. Moreover, 347 sites among the 319 modified proteins were present only in the db/db mouse kidneys, while 213 sites among the 199 modified proteins were present only in the db/m mouse kidneys. The subcellular localization study indicated that the cytoplasm had the highest proportion of ubiquitinated proteins (31.87%), followed by the nucleus (30.24%) and the plasma membrane (20.33%). The enrichment analysis revealed that the ubiquitinated proteins are mostly linked to tight junctions, oxidative phosphorylation, and thermogenesis. Podocin, as a typical protein of slit diaphragm, whose loss is a crucial cause of proteinuria in DN. Consistent with the results of ubiquitination omics, the K261R mutant of podocin induced the weakest ubiquitination compared with the K301R and K370R mutants. As an E3 ligase, c-Cbl binds to podocin, and the regulation of c-Cbl can impact the ubiquitination of podocin. In conclusion, in DN, podocin ubiquitination contributes to podocyte injury, and K261R is the most significant site. c-Cbl participates in podocin ubiquitination and may be a direct target for preserving the integrity of the slit diaphragm structure, hence reducing proteinuria in DN.

A Review of Focal Segmental Glomerulosclerosis Classification With a Focus on Genetic Associations.

Focal segmental glomerulosclerosis (FSGS) defines a distinct histologic pattern observed in kidney tissue that is linked to several distinct underlying causes, all converging on the common factor of podocyte injury. It presents a considerable challenge in terms of classification because of its varied underlying causes and the limited correlation between histopathology and clinical outcomes. Critically, precise nomenclature is key to describe and delineate the pathogenesis, subsequently guiding the selection of suitable and precision therapies. A proposed pathomechanism-based approach has been suggested for FSGS classification. This approach differentiates among primary, secondary, genetic, and undetermined causes, aiming to provide clarity. Genetic FSGS from monogenic mutations can emerge during childhood or adulthood, and it is advisable to conduct genetic testing in cases in which there is a family history of chronic kidney disease, nephrotic syndrome, or resistance to treatment. Genome-wide association studies have identified several genetic risk variants, such as those in apolipoprotein L1 (APOL1), that play a role in the development of FSGS. Currently, no specific treatments have been approved to treat genetic FSGS; however, interventions targeting underlying cofactor deficiencies have shown potential in some cases. Furthermore, encouraging results have emerged from a phase 2 trial investigating inaxaplin, a novel small molecule APOL1 channel inhibitor, in APOL1-associated FSGS.

Urinary podocyte markers in diabetic kidney disease.

Podocytes are involved in maintaining kidney function and are a major focus of research on diabetic kidney disease (DKD). Urinary biomarkers derived from podocyte fragments and molecules have been proposed for the diagnosis and monitoring of DKD. Various methods have been used to detect intact podocytes and podocyte-derived microvesicles in urine, including centrifugation, visualization, and molecular quantification. Quantification of podocyte-specific protein targets and messenger RNA levels can be performed by Western blotting or enzyme-linked immunosorbent assay and quantitative polymerase chain reaction, respectively. At present, many of these techniques are expensive and labor-intensive, all limiting their widespread use in routine clinical tests. While the potential of urinary podocyte markers for monitoring and risk stratification of DKD has been explored, systematic studies and external validation are lacking in the current literature. Standardization and automation of laboratory methods should be a priority for future research, and the added value of these methods to routine clinical tests should be defined.

A small molecule chaperone rescues keratin-8 mediated trafficking of misfolded podocin to correct genetic Nephrotic Syndrome.

Podocin is a key membrane scaffolding protein of the kidney podocyte essential for intact glomerular filtration. Mutations in NPHS2, the podocin-encoding gene, represent the commonest form of inherited nephrotic syndrome (NS), with early, intractable kidney failure. The most frequent podocin gene mutation in European children is R138Q, causing retention of the misfolded protein in the endoplasmic reticulum. Here, we provide evidence that podocin R138Q (but not wild-type podocin) complexes with the intermediate filament protein keratin 8 (K8) thereby preventing its correct trafficking to the plasma membrane. We have also identified a small molecule (c407), a compound that corrects the Cystic Fibrosis Transmembrane Conductance Regulator protein defect, that interrupts this complex and rescues mutant protein mistrafficking. This results in both the correct localization of podocin at the plasma membrane and functional rescue in both human patient R138Q mutant podocyte cell lines, and in a mouse inducible knock-in model of the R138Q mutation. Importantly, complete rescue of proteinuria and histological changes was seen when c407 was administered both via osmotic minipumps or delivered orally prior to induction of disease or crucially via osmotic minipump two weeks after disease induction. Thus, our data constitute a therapeutic option for patients with NS bearing a podocin mutation, with implications for other misfolding protein disorders. Further studies are necessary to confirm our findings.

In vivo characterization of a podocyte-expressed short podocin isoform.

The most common genetic causes of steroid-resistant nephrotic syndrome (SRNS) are mutations in the NPHS2 gene, which encodes the cholesterol-binding, lipid-raft associated protein podocin. Mass spectrometry and cDNA sequencing revealed the existence of a second shorter isoform in the human kidney in addition to the well-studied canonical full-length protein. Distinct subcellular localization of the shorter isoform that lacks part of the conserved PHB domain suggested a physiological role. Here, we analyzed whether this protein can substitute for the canonical full-length protein. The short isoform of podocin is not found in other organisms except humans. We therefore analysed a mouse line expressing the equivalent podocin isoform (podocinΔexon5) by CRISPR/Cas-mediated genome editing. We characterized the phenotype of these mice expressing podocinΔexon5 and used targeted mass spectrometry and qPCR to compare protein and mRNA levels of podocinwildtype and podocinΔexon5. After immunolabeling slit diaphragm components, STED microscopy was applied to visualize alterations of the podocytes' foot process morphology.Mice homozygous for podocinΔexon5 were born heavily albuminuric and did not survive past the first 24 h after birth. Targeted mass spectrometry revealed massively decreased protein levels of podocinΔexon5, whereas mRNA abundance was not different from the canonical form of podocin. STED microscopy revealed the complete absence of podocin at the podocytes' slit diaphragm and severe morphological alterations of podocyte foot processes. Mice heterozygous for podocinΔexon5 were phenotypically and morphologically unaffected despite decreased podocin and nephrin protein levels.The murine equivalent to the human short isoform of podocin cannot stabilize the lipid-protein complex at the podocyte slit diaphragm. Reduction of podocin levels at the site of the slit diaphragm complex has a detrimental effect on podocyte function and morphology. It is associated with decreased protein abundance of nephrin, the central component of the filtration-slit forming slit diaphragm protein complex.

Podocin, mTOR, and CHOP dysregulation contributes to nephrotoxicity induced of lipopolysaccharide/diclofenac combination in rats: Curcumin and silymarin could afford protective effect.

AIM: Sepsis is a common cause of acute kidney injury (AKI). Lipopolysaccharides (LPS) are the main gram-negative bacterial cell wall component with a well-documented inflammatory impact. Diclofenac (DIC) is a non-steroidal anti-inflammatory drug with a potential nephrotoxic effect. Curcumin (CUR) and silymarin (SY) are natural products with a wide range of pharmacological activities, including antioxidant and anti-inflammatory ones. The objective of this study was to examine the protective impact of CUR and SY against kidney damage induced by LPS/DIC co-exposure. MATERIALS AND METHODS: Four groups of rats were used; control; LPS/DIC, LPS/DIC + CUR, and LPS/DIC + SY group. LPS/DIC combination induced renal injury at an LPS dose much lower than a nephrotoxic one. KEY FINDING: Nephrotoxicity was confirmed by histopathological examination and significant elevation of renal function markers. LPS/DIC induced oxidative stress in renal tissues, evidenced by decreasing reduced glutathione and superoxide dismutase, and increasing lipid peroxidation. Inflammatory response of LPS/DIC was associated with a significant increase of renal IL-1ß and TNF-α. Treatment with either CUR or SY shifted measured parameters to the opposite side. Moreover, LPS/DIC exposure was associated with upregulation of mTOR and endoplasmic reticulum stress protein (CHOP) and downregulation of podocin These effects were accompanied by reduced gene expression of cystatin C and KIM-1. CUR and SY ameliorated LPS/DIC effect on the aforementioned genes and protein significantly. SIGNIFICANCE: This study confirms the potential nephrotoxicity; mechanisms include upregulation of mTOR, CHOP, cystatin C, and KIM-1 and downregulation of podocin. Moreover, both CUR and SY are promising nephroprotective products against LPS/DIC co-exposure.

DLBS3233 enhances nephrin and podocin expression also reduces oxidative stress marker and insulin receptor serine diabetic rats' podocytes.

Numerous oxidative stresses are detected in patients with diabetic kidney disease, resulting in insulin resistance that damages the pancreas and kidney. Renal podocytes insensitive to insulin lead to decreased nephrin and podocin and increased insulin receptor serine. The authors did an experiment on diabetic rats to examine the effect of DLBS3233 on repairing insulin resistance. Materials and Methods: Thirty adult male Wistar rats were randomly divided into six groups (n=5 per group): group of nondiabetic rats as a negative control (group 1); untreated diabetic rats (group 2); diabetic rats treated with DLBS3233 4.5 mg/kg BB (group 3); 9 mg/kg BB (group 4); 18 mg/kg BB (group 5); and diabetic rats treated with pioglitazone (group 6). The authors checked Homeostatic Model Assessment for Insulin Resistance to corroborate insulin resistance prior to DLBS3233 administration in diabetic rats. Immunohistochemistry was performed to examine the expression of renal antimalondialdehyde (MDA) antibodies, nephrin, podocin, and insulin receptor serine. The data were analyzed using analysis of variance and the t-test. Result: In the DBLS3233 group, immunohistochemistry showed enhanced expression of renal nephrin and podocin, as well as diminished expression of anti-MDA antibody, along with decreased insulin receptor serine. From statistical analysis, anti-MDA antibodies and insulin receptor serine showed lower expression, whereas the expression of nephrin and podocin were enhanced compared to untreated groups (P<0.05). Conclusion: DLBS3233 reduces oxidative stress by decreasing MDA and improves insulin resistance by increasing the expression of renal nephrin and podocin as well as decreasing insulin receptor serine.

Deletion of protein tyrosine phosphatase SHP-1 restores SUMOylation of podocin and reverses the progression of diabetic kidney disease.

Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the in vivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-ß, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.

Di(2-ethylhexyl) phthalate mediates oxidative stress and activates p38MAPK/NF-kB to exacerbate diabetes-induced kidney injury in vitro and in vivo models.

Plasticizer di(2-ethylhexyl) phthalate (DEHP) is employed to make polyethylene polymers. Some studies in epidemiology and toxicology have shown that DEHP exposure over an extended period may be hazardous to the body, including nephrotoxicity, and aggravate kidney damage in the context of underlying disease. However, studies on the toxicity of DEHP in diabetes-induced kidney injury have been rarely reported. Using a high-fat diet (HFD) and streptozotocin (STZ, 35 mg/kg)-induced kidney injury in mice exposed to various daily DEHP dosages, we explored the impacts of DEHP on diabetes-induced kidney injury. We discovered that DEHP exposure significantly promoted the renal inflammatory response and oxidative stress in mice, with increased P-p38 and P-p65 protein levels and exacerbated the loss of podocin. The same findings were observed in vitro after stimulation of podocytes with high glucose (30 mmol/L) and exposure to DEHP. Our results suggest that DEHP exacerbates diabetes-induced kidney injury by mediating oxidative stress and activating p38MAPK/NF-κB.

Ameliorative effects of glycine on cobalt chloride-induced hepato-renal toxicity in rats.

BACKGROUND: The important roles of liver and kidney in the elimination of injurious chemicals make them highly susceptible to the noxious activities of various toxicants including cobalt chloride (CoCl2 ). This study was designed to investigate the role of glycine in the mitigation of hepato-renal toxicities associated with CoCl2 exposure. METHODS: Forty-two (42) male rats were grouped as Control; (CoCl2 ; 300 ppm); CoCl2 + Glycine (50 mg/kg); CoCl2 + Glycine (100 mg/kg); Glycine (50 mg/kg); and Glycine (100 mg/kg). The markers of hepatic and renal damage, oxidative stress, the antioxidant defense system, histopathology, and immunohistochemical localization of neutrophil gelatinase associated lipocalin (NGAL) and renal podocin were evaluated. RESULTS: Glycine significantly reduced the markers of oxidative stress (malondialdehyde content and H2 O2 generation), liver function tests (ALT, AST, and ALP), markers of renal function (creatinine and BUN), and decreased the expression of neutrophil gelatinase-associated lipocalin (NGAL) and podocin compared with rats exposed to CoCl2 toxicity without glycine treatment. Histopathology lesions including patchy tubular epithelial necrosis, tubular epithelial degeneration and periglomerular inflammation in renal tissues, and severe portal hepatocellular necrosis, inflammation, and duct hyperplasia were observed in hepatic tissues of rats exposed to CoCl2 toxicity, but were mild to absent in glycine-treated rats. CONCLUSION: The results of this study clearly demonstrate protective effects of glycine against CoCl2 -induced tissue injuries and derangement of physiological activities of the hepatic and renal systems in rats. The protective effects are mediated via augmentation of total antioxidant capacity and upregulation of NGAL and podocin expression.

Sitagliptin ameliorates diabetic nephropathy by upregulating renal nephrin and podocin expression through modulation of adipokines levels.

Diabetic nephropathy is the leading cause of end-stage renal failure, but the effectiveness of currently available strategies for preventing diabetic nephropathy remains unsatisfactory. This study was designed to evaluate the changes in adipokines levels caused by dipeptidyl peptidase-4 inhibitor sitagliptin therapy as one of the possible mechanisms of sitagliptin's amelioration of diabetic nephropathy. Twenty-four male Wistar rats weighing 180-200 g were taken and divided into three groups, that is, control, diseased, and treatment group. High-fat diet and streptozotocin-induced Type 2 diabetic rats were divided into diseased and treatment groups. The treatment group was given sitagliptin orally, 10 mg/kg per day for 6 weeks. Serum glucose, serum insulin, serum blood urea nitrogen, serum creatinine, and 24-h urinary protein levels were measured in serum and urine samples. mRNA expression levels of podocin, nephrin, and adipokines in renal tissues were determined. Results showed that sitagliptin treatment effectively reduced serum glucose, serum creatinine, serum blood urea nitrogen, and 24-h proteinuria, along with partial prevention of insulinopenia, in the treatment group as compared to the diseased group. The renal mRNA expression levels of podocin, nephrin, and adiponectin were significantly upregulated, while those of leptin and resistin were significantly downregulated in the diabetic rats receiving sitagliptin therapy compared to the non-treated diabetic rats. Based on these findings, it is suggested that sitagliptin, via mediating the modulation of adipokines levels, upregulates renal nephrin and podocin expression, which leads to the amelioration of diabetic nephropathy.

Endotrophin neutralization through targeted antibody treatment protects from renal fibrosis in a podocyte ablation model.

OBJECTIVE: Renal fibrosis is a hallmark for chronic kidney disease (CKD), and often leads to end stage renal disease (ESRD). However, limited interventions are available clinically to ameliorate or reverse renal fibrosis. METHODS: Herein, we evaluated whether blockade of endotrophin through neutralizing antibodies protects from renal fibrosis in the podocyte insult model (the "POD-ATTAC" mouse). We determined the therapeutic effects of endotrophin targeted antibody through assessing renal function, renal inflammation and fibrosis at histological and transcriptional levels, and podocyte regeneration. RESULTS: We demonstrated that neutralizing endotrophin antibody treatment significantly ameliorates renal fibrosis at the transcriptional, morphological, and functional levels. In the antibody treatment group, expression of pro-inflammatory and pro-fibrotic genes was significantly reduced, normal renal structures were restored, collagen deposition was decreased, and proteinuria and renal function were improved. We further performed a lineage tracing study confirming that podocytes regenerate as de novo podocytes upon injury and loss, and blockade of endotrophin efficiently enhances podocyte-specific marker expressions. CONCLUSION: Combined, we provide pre-clinical evidence supporting neutralizing endotrophin as a promising therapy for intervening with renal fibrosis in CKD, and potentially in other chronic fibro-inflammatory diseases.

ANGPTL4 promotes nephrotic syndrome by downregulating podocyte expression of ACTN4 and podocin.

BACKGROUND: lipopolysaccharide (LPS) can induce nephrotic syndrome-like features such as massive proteinuria, hyperlipidemia, and fusion of glomerular podocytes with foot processes (FPs) in mice. Angiopoietin-like protein 4 (ANGPTL4) neutralized the negative charge of glomerular basement membrane charge and aggravated renal injury. The mechanism of ANGPTL4 aggravating podocyte injury has not been well clarified. In this study, we aimed to investigate the potential role of ANGPTL4 on podocyte FPs fusion and podocyte signal molecules. METHODS: We built angptl4 gene knocked out in C57BL6 mice using CRISPR/Cas9 technique. Nephrotic model was built by LPS in wild type and angptl4-/- mice. Expression of ACTN4, podocin and TRPC6 in the glomerulus were determined by immunohistochemistry. RESULTS: In physical condition, the wild type and angptl4-/- mice showed no significant differences in biochemical indicators and kidney pathology. But in nephrotic condition, compared with wild type mice hyperlipidemia and proteinuria with the angptl4-/- mice was significantly relieved. Moreover, the degree of FPs fusion was notably improved in the nephrotic mice knocked out angptl4 gene. Expression of ACTN4 and podocin decreased drastically in the glomerulus of wild-type nephrotic mice. Different from wild-type, the ACTN4 and podocin expression showed slight weakening in angptl4-/- nephrotic mice. As transient receptor potential cation channel subfamily member, TRPC6 expression had no visible change in glomerulus of each group. CONCLUSIONS: ANGPTL4 induces hyperlipidemia and podocyte injury in nephrotic mice, thereby promoting the formation of proteinuria. Its molecular mechanism may be related to ANGPTL4 down-regulating actin cytoskeletal regulatory signals ACTN4 and podocin.

Using Electrochemical Immunoassay in a Novel Microtiter Plate to Detect Surface Markers of Preeclampsia on Urinary Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer nanovesicles secreted by cells. EVs contain biological information related to parental cells and provide biomarkers for disease diagnosis. We have previously shown that the levels of podocin and nephrin expression on urinary EVs may be used to diagnose renal injury associated with preeclampsia. This paper describes a nanoparticle-enabled immunoassay integrated with an electrochemical plate for quantifying podocin and nephrin expression in urinary EVs. The strategy entailed capturing EVs on an electrode surface and then labeling EVs with gold nanoparticles that are both functionalized with antibodies for target specificity and impregnated with redox-active metal ions for electrochemical detection. These immunoprobes produced an electrochemical redox signal proportional to the expression level of EV surface markers. Electrochemical immunoassays were carried out in a novel microtiter plate that contained 16 wells with working electrodes connected to onboard counter/reference electrodes via capillary valves. Upon validation with recombinant proteins, a microtiter plate was used for analysis of urinary EVs from healthy and preeclamptic pregnant women. This analysis revealed a higher podocin to nephrin ratio for preeclamptic women compared to healthy controls (4.31 vs 1.69) suggesting that this ratio may be used for disease diagnosis.

Biomarkers of Kidney Disease in Horses: A Review of the Current Literature.

Creatinine only allows detection of kidney disease when 60 to 75% of the glomerular function is lost and is therefore not an ideal marker of disease. Additional biomarkers could be beneficial to assess kidney function and disease. The objectives are to describe new equine kidney biomarkers. This systematic review assesses the available literature, including the validation process and reference values, following which the authors suggest recommendations for clinical use. SDMA may have some potential as equine kidney biomarker, but there is currently a lack of evidence that SDMA offers any advantage compared to creatinine in detecting Acute Kidney Injury (AKI). Cystatin C and podocin show potential as biomarkers for kidney disease (including detecting AKI earlier than creatinine) and should be studied further. NGAL has potential as a biomarker of kidney disease (including detecting AKI earlier than creatinine), and potential as an inflammatory marker. Literature on MMP-9 does not allow for conclusive statements about its potential as a biomarker for kidney disease. The future may show that NAG has potential. For all biomarkers, at this stage, available scientific information is limited or too scarce to support clinical use, and only SDMA can be measured for clinical purposes. In conclusion, there are multiple new biomarkers with the potential to diagnose kidney problems. However, there are only a few studies available and more data is needed before these biomarkers can be applied and recommended in our daily practice.

ANGPTL3 is involved in kidney injury in high-fat diet-fed mice by suppressing ACTN4 expression.

OBJECTIVE: We wanted to explore how angiopoietin-like 3 (ANGPTL3) impact hyperlipidemia-induced renal injury. METHODS: ANGPTL3 knockout mice and wild-type C57 mice were set up in four groups (N = 5) depending on a normal or 60% high-fat diet: wild-type with normal diet (WT), angptl3-/- with normal diet (KO), wild-type + high-fat diet (WT + HF) and angptl3-/- + high-fat diet (KO + HF). The detection time points were the 9th, 13th, 17th and 21st weeks after modeling. Serum lipid and urinary protein levels of mice in each group were detected, and pathological changes in the kidney were analyzed. Moreover, the expression of ANGPTL3, α-actinin-4 (ACTN4), CD2-associated protein (CD2AP) and podocin was tested in the glomerulus by immunohistochemistry (IHC). RESULTS: In the WT + HF group, hyperlipidemia and proteinuria could be observed at the 9th week and were gradually aggravated with time. Compared with WT + HF mice, the levels of serum lipids and proteinuria in KO + HF mice were significantly reduced, and the width of podocyte foot processes (FPs) fusion was also markedly improved. The IHC results suggested that in WT + HF mice, the expression of ANGPTL3 was significantly enhanced. After modeling, ACTN4 expression was markedly weakened in the glomeruli of WT + HF mice. Different to WT mice, ACTN4 expression was not observed obviously change in KO + HF mice. Compared with the normal diet group, the expression of podocin showed a decline in WT mice treated with high-fat diet and showed a significant difference from the 17th week. In addition, podocin expression in KO + HF glomeruli was also found to be weak but not significantly different from that in WT + HF glomeruli at the four time points. The expression of CD2AP showed similar results among the four groups. CONCLUSION: ANGPTL3 could play a role in the mechanism of hyperlipidemia-associated podocyte injury via ACTN4.

[Effect of PKC inhibitor on renal podocyte slit diaphragm protein expression in exhausted rats].

Objective: To investigate the expression level of podocyte slit diaphragm protein in rats after one-time exhaustive exercise, to explore the effect of PKC inhibitor on the protein expression level, and to reveal the mechanism of PKC in the formation of exercise-induced proteinuria. Methods: Thirty male SD rats were randomly divided into control group (C), exercise group (E) and exercise combining with PKC inhibitor group (EPI), with 10 rats in each group. Rats in group E and EPI performed one single bout of exhaustive exercise (25 m/min), rats in group EPI were intraperitoneally injected with a PKC inhibitor (chelerythrine, 5 mg/kg) 1 day and 1 hour before exercise respectively, while rats in group C and E were injected with the same volume of saline. Results: ①Compared with group C, the levels of urine protein, uric acid, urine sugar, blood urea, and blood uric acid of rats in group E were increased significantly (P＜0.05), the level of blood glucose was reduced significantly (P＜0.01), and renal ROS production was increased significantly (P＜0.01). The expressions of nephrin and podocin protein in renal tissue were decreased significantly (P＜ 0.05), while the expressions of PKC, Nox2, and Nox4 protein were increased significantly (P＜0.05). ②Compared with group E, the levels of urinary protein,urine glucose and blood urea in EPI group were decreased significantly (P＜0.05), the level of blood glucose was increased significantly (P＜0.01), renal ROS production was reduced significantly (P＜0.01). the expressions of nephrin and podocin protein in renal tissues of the EPI group were increased significantly (P＜0.05), while the expressions of PKC and Nox2 protein was reduced significantly (P＜0.05, P＜0.01). Conclusion: One-time exhaustive exercise can down-regulate the expressions of nephrin and podocin through PKC/Nox/ROS pathways in the kidney of rats; PKC inhibitor alleviates the decrease in the expression of podocyte slit diaphragm protein caused by exhaustive exercise, and prevents the occurrence of exercise-induced proteinuria.