Linggui Zhugan Decoction Improves High Glucose-Induced Autophagy in Podocytes

Objective To explore the influence of Linggui Zhugan Decoction(LGZGD) on high glucose induced podocyte autophagy.Methods LGZGD containing serum was prepared by intragastric administation of 4.2 g/kg(low dose), 8.4 g/kg(medium dose), and 12.6 g/kg(high dose) LGZGD into SD rats respectively. MPC5 and AB8/13 podocyte cells were treated with 60 mmol/L glucose to establish diabetic nephropathy podocyte model in vitro. Both podocytes were divided into control group, high glucose group, low dose LGZGD group, medium dose LGZGD group, and high dose LGZGD group, respectively. For the three LGZGD groups, before LGZGD intervention, podocytes were treated with 60 mmol/L glucose for 3 days. After treated with LGZGD containing serum, cells were collected to analyze cell migration using Transwell assay, proliferation using CCK8, apoptosis and cell cycle using flow cytometry, autophagosome formation using transmission electron microscopy, and expression levels of Beclin-1, Atg5, LC3II/I, and P62 proteins using Western blot.Results Compared with the control group, the proliferation and migration of MPC5 and AB8/13 cells in the high glucose group slightly decreased, whereas these parameters restored after intervention with low and medium concentrations of LGZGD, with the medium dose LGZGD having the better effect(P < 0.05). Flow cytometry showed that the medium dose LGZGD group had a significantly lower apoptosis rate(P < 0.05) and higher survival rate(P > 0.05) compared to the high dose LGZGD group. High glucose arrested podocytes in G1 phase, whereas LGZGD shifted podocytes from being predominant in G1 phase to G2 phase. High dose LGZGD significanly reduced high glucose-increased autophagosome formation in both podocytes(P < 0.05). Western blot analysis showed that Beclin-1, Atg5, LC3II/I, and P62 expressions were increased in MPC5 cells treated with high glucose and reversed after adminstration of low and medium doses of LGZGD(P < 0.05).Conclusion LGZGD reduced apoptosis and enhanced autophagy in high glucose treated podocytes via regulating Beclin-1/LC3II/I/Atg5 expression.

Foodborne toxin Aflatoxin B_(1)induced glomerular podocyte inflammation through proteolysis of RelA,downregulation of miR-9 and CXCR4/TXNIP/NLRP3 pathway

Aflatoxin B_(1)(AFB_(1))is a naturally-occurring mycotoxin and recognized as the most toxic foodborne toxin,particularly causing damages to kidney.Glomerular podocytes are terminally differentiated epithelial cells.AFB_(1)induces podocyte inflammation,proteinuria and renal dysfunction.Studying the mechanism of AFB_(1)-induced podocyte inflammation and murine kidney dysfunction,we detected that AFB_(1)increased ubiquitindependent degradation of the transcription factor RelA through enhanced interaction of RelA with E3 ubiquitin ligase tripartite motif containing 7(TRIM7)in mouse podocyte clone-5(MPC-5)and mouse glomeruli.Reduction of RelA resulted in decreasing microRNA-9(miR-9)and activating the chemokine receptor 4(CXCR4),thioredoxin interacting protein(TXNIP),and NOD-like receptor pyrin domain-containing 3(NLRP3)signaling axis(CXCR4/TXNIP/NLRP3 pathway),leading to podocyte inflammation.We also determined that downregulation of miR-9 led to CXCR4 expression and the downstream TXNIP/NLRP3 pathway activation.Overexpression of miR-9 or deletion of CXCR4 suppressed AFB_(1)-induced CXCR4/TXNIP/NLRP3 pathway,resulting in alleviating podocyte inflammation and kidney dysfunction.Our findings indicated that ubiquitin-dependent proteolysis of RelA,downregulation of miR-9,and activation of CXCR4/TXNIP/NLRP3 pathway played an essential role in AFB_(1)-induced glomerular podocyte inflammation.Our study revealed a novel mechanism,via RelA,for the control of AFB_(1)’s nephrotoxicity,leading to an effective protection of food safety and public health.

Changes in macrophage infiltration and podocyte injury in lupus nephritis patients with repeated renal biopsy: Report of three cases

BACKGROUND In this study,we retrospectively analysed macrophage infiltration and podocyte injury in three patients with diffuse proliferative lupus nephritis(LN)who un-derwent repeated renal biopsy.CASE SUMMARY Clinical data of three diffuse proliferative LN patients with different pathological characteristics(case 1 was LN IV-G(A),case 2 was LN IV-G(A)+V,and case 3 was LN IV-G(A)+thrombotic microangiopathy)were reviewed.All patients underwent repeated renal biopsies 6 mo later,and renal biopsy specimens were studied.Macrophage infiltration was assessed by CD68 expression detected by immunohistochemical staining,and an immunofluorescence assay was used to detect podocin expression to assess podocyte damage.After treatment,Case 1 changed to LN III-(A),Case 2 remained as type V LN lesions,and Case 3,which changed to LN IV-S(A),had the worst prognosis.We observed reduced macro-phage infiltration after therapy.However,two of the patients with active lesions after treatment still showed macrophage infiltration in the renal interstitium.Before treatment,the three patients showed discontinuous expression of podocin.Notably,the integrity of podocin was restored after treatment in Case 1.CONCLUSION It may be possible to reverse podocyte damage and decrease the infiltrating ma-crophages in LN patients through effective treatment.

Corilagin alleviates podocyte injury in diabetic nephropathy by regulating autophagy via the SIRT1-AMPK pathway

BACKGROUND Diabetic nephropathy(DN)is the most frequent chronic microvascular consequence of diabetes,and podocyte injury and malfunction are closely related to the development of DN.Studies have shown that corilagin(Cor)has hepatoprotective,anti-inflammatory,antibacterial,antioxidant,anti-hypertensive,antidiabetic,and anti-tumor activities.AIM To explore the protective effect of Cor against podocyte injury in DN mice and the underlying mechanisms.METHODS Streptozotocin and a high-fat diet were combined to generate DN mice models,which were then divided into either a Cor group or a DN group(n=8 in each group).Mice in the Cor group were intraperitoneally injected with Cor(30 mg/kg/d)for 12 wk,and mice in the DN group were treated with saline.Biochemical analysis was used to measure the blood lipid profiles.Hematoxylin and eosin staining was used to detect pathological changes in kidney tissue.Immunohistochemistry and Western blotting were used to assess the protein expression of nephrin and podocin.Mouse podocyte cells(MPC5)were cultured and treated with glucose(5 mmol/L),Cor(50μM),high glucose(HG)(30 mmol/L),and HG(30 mmol/L)plus Cor(50μM).Real-time quantitative PCR and Western blotting RESULTS Compared with the control group,the DN mice models had increased fasting blood glucose,glycosylated hemoglobin,triglycerides,and total cholesterol,decreased nephrin and podocin expression,increased apoptosis rate,elevated inflammatory cytokines,and enhanced oxidative stress.All of the conditions mentioned above were alleviated after intervention with Cor.In addition,Cor therapy improved SIRT1 and AMPK expression(P<0.001),inhibited reactive oxygen species and oxidative stress,and elevated autophagy in HG-induced podocytes(P<0.01).CONCLUSION Cor alleviates podocyte injury by regulating autophagy via the SIRT1-AMPK pathway,thereby exerting its protective impact on renal function in DN mice.

Long noncoding RNA protein-disulfide isomerase-associated 3 regulated high glucose-induced podocyte apoptosis in diabetic nephropathy through targeting miR-139-3p

BACKGROUND Podocyte apoptosis plays a vital role in proteinuria pathogenesis in diabetic nephropathy(DN).The regulatory relationship between long noncoding RNAs(lncRNAs)and podocyte apoptosis has recently become another research hot spot in the DN field.AIM To investigate whether lncRNA protein-disulfide isomerase-associated 3(Pdia3)could regulate podocyte apoptosis through miR-139-3p and revealed the underlying mechanism.METHODS Using normal glucose or high glucose(HG)-cultured podocytes,the cellular functions and exact mechanisms underlying the regulatory effects of lncRNA Pdia3 on podocyte apoptosis and endoplasmic reticulum stress(ERS)were explored.LncRNA Pdia3 and miR-139-3p expression were measured through quantitative real-time polymerase chain reaction.Relative cell viability was detected through the cell counting kit-8 colorimetric assay.The podocyte apoptosis rate in each group was measured through flow cytometry.The interaction between lncRNA Pdia3 and miR-139-3p was examined through the dual luciferase reporter assay.Finally,western blotting was performed to detect the effect of lncRNA Pdia3 on podocyte apoptosis and ERS via miR-139-3p.RESULTS The expression of lncRNA Pdia3 was significantly downregulated in HG-cultured podocytes.Next,lncRNA Pdia3 was involved in HG-induced podocyte apoptosis.Furthermore,the dual luciferase reporter assay confirmed the direct interaction between lncRNA Pdia3 and miR-139-3p.LncRNA Pdia3 overexpression attenuated podocyte apoptosis and ERS through miR-139-3p in HG-cultured podocytes.CONCLUSION Taken together,this study demonstrated that lncRNA Pdia3 overexpression could attenuate HG-induced podocyte apoptosis and ERS by acting as a competing endogenous RNA of miR-139-3p,which might provide a potential therapeutic target for DN.

Glycyrrhizic Acid Protects Glomerular Podocytes Induced by High Glucose by Modulating SNARK/AMPK Signaling Pathway

Objective:Diabetic nephropathy is one of the most important microvascular complications of diabetes,which mainly refers to glomerular capillary sclerosis.Podocytes are an important part of glomerular capillaries.Previous clinical and basic studies have shown that fibrosis is the main factor of diabetic nephropathy.This study aimed to assess the protective mechanism of glycyrrhizic acid(GA)on glomerular podocytes induced by high glucose as we hypothesized that GA may have antifibrotic and anti-inflammatory effects on podocytes through regulation of the adenosine 5'-monophosphate-activated protein kinase(AMPK)/sucrose nonfermenting AMPK-related kinase(SNARK)signaling pathway.Methods:SNARK siRNA was used to transfect podocytes.Real-time quantitative polymerase chain reaction and immunofluorescence staining assays were used for molecular and pathological analysis.The expression levels of key pathway proteins(including TGF-β1,α-SMA,SITR1,AMPKα,LKB1,PGC-1α,NF-κB,IL-6,and TNF-α)were verified by Western blotting.The expression of inflammatory factors in podocytes was detected by ELISA.Results:We demonstrated that GA decreased the expression of podocyte fibrosis signaling pathway-related factors by upregulating the AMPK pathway and its related factors.However,after transfection of podocytes with SNARK siRNA,there was an increased expression of fibrosis-related factors and inflammation-related factors.Conclusion:GA can protect podocytes and alleviate fibrosis and inflammation induced by high glucose,which is related to the AMPK signaling pathway.Meanwhile,knockdown of SNARK protein can inhibit the AMPK signaling pathway,aggravate fibrosis,and increase inflammation.

Exosomal miR-30a-5p targets NLRP3 to suppress podocyte pyroptosis in diabetic nephropathy

Background:Mesenchymal stem cell(MSC)-derived exosomes are closely related to pyroptosis in diabetic nephropathy(DN).This study aimed to explore the protective effect of exosomal miR-30a-5p on podocyte pyroptosis in DN.Methods:Streptozotocin was used to establish the mouse model of DN.Human bone marrow MSC-derived exosomes were extracted and identified via transmission electron microscopy,nanoparticle tracking analysis,and western blotting.MiR-30a-5p mimics and non-control(NC)mimics were transfected into MSCs and podocytes,and exosomes were isolated from the MSCs.High glucose(HG)-induced podocyte model was established to determine the effect of exosomal miR-30a-5p on pyroptosis and inflammation in vitro.Results:MiR-30a-5p was expressed at low levels in DN models,while NLR family pyrin domain containing 3(NLRP3),caspase-1,gasdermin-N(GSDMD-N),and pro-inflammatory factors(tumor necrosis factor-alpha,interleukin(IL)-1beta,and IL-18)were augmented.In vitro,miR-30a-5p expression in the HG-damaged podocytes was down-regulated,while NLRP3 was up-regulated.Interestingly,miR-30a-5p overexpression diminished HG-induced podocyte injury,as proven by increased activity and decreased pyroptosis of podocytes.Concurrently,the up-regulation of miR-30a-5p could inhibit the expression of pro-inflammatory factors,caspase-1,GSDMD-N,and NLRP3 in HG-induced podocytes.MSC-derived exosomal miR-30a-5p treatment of HG-damaged cells has similar effects to miR-30a-5p mimics treatment.Overexpression of NLRP3 reversed the effect of miR-30a-5p mimics on HG-induced podocytes.Conclusion:This research confirmed that exosomal miR-30a-5p regulates pyroptosis via mediating NLRP3 in DN.

Total flavonoids of Scutellaria barbata improving podocyte injury induced by high glucose through Smad4/PKM2/HIF‑1α pathway

Objective: To observe the effect of total flavonoids of Scutellaria barbata (TF‑SB) on the injury of high glucose induced podocytes (MPC‑5) and the influence of Smad4/PKM2/HIF‑1α pathway. Methods: Firstly, CCK8 was used to analyze the safety and efficacy concentration of TF‑SB on MPC‑5 cells. Then, MPC‑5 was then divided into the control group, model group and TF‑SB group. In addition to the control group, model group and TF‑SB group were induced by high glucose to establish MPC‑5 cell injury model. The effects of TF‑SB on ATP, apoptosis and ROS levels of MPC‑5 cells were detected respectively. The contents of IL‑1β, TNF‑α, and MCP‑1 were determined by ELISA, the expression abundance of glycolytic genes (GLU1, PFK1 and HK1) were detected by RT‑PCR. Western blot method was used to detect the expression level of related proteins in Smad4/PKM2/HIF‑1α pathway. Results: Compared with the blank group, ATP content, GLU1, PKF1 and HK1 expression abundance of MPC‑5 cells in the model group decreased significantly, apoptosis, ROS level and IL‑1 β、 TNF‑ α And MCP‑1 significantly increased (P<0.01);Compared with model group, ATP content, GLU1, PKF1 and HK1 expression abundance, apoptosis, ROS level and IL‑1β in TF‑SB group were significantly increased , TNF‑ α The contents of MCP‑1 and MCP‑1 decreased significantly (P<0.01). In addition, compared with the blank group, the model group Smad4 and HIF‑1 α The protein expression and PKM2 expression in nucleus were significantly increased, while PKM2 expression in cytoplasm was significantly decreased (P<0.01);Compared with model group, TF‑SB group Smad4, HIF‑1 α The expression of PKM2 in the nucleus and expression of PKM2 were significantly decreased, while the expression of PKM2 in the cytoplasm was significantly increased (P<0.01). Conclusion: TF‑SB promotes the mitochondrial activity of MPC‑5 cells to induce glycolysis, and then inhibits the secretion of inflammation, which may play a role in treating diabetes nephropathy by inhibiting Smad4/PKM2/HIF‑1α signaling pathway.

Effect of Down-regulation of TRPC6 on the Puromycin Aminonucleoside-induced Apoptosis of Mouse Podocytes

Eukaryotic expression vectors carrying the small hairpin RNA （shRNA） for TRPC6 mRNA were constructed, and the effects of knocking-down TRPC6 on puromycin aminonucleoside （PAN）-induced apoptosis of mouse podocytes were observed. Two eukaryotic expression vectors containing small hairpin structure targeting TRPC6 named pGCsi-TRPC6A and pGCsi-TRPC6B were designed and synthesized. The plasmids were transfected into conditionally immortalized murine podocyte cell line by liposome. The changes in the TRPC6 mRNA and protein expression were observed by RT-PCR and Western blot after 48 h. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN treatment＋shRNA transfection group, and PAN treatment＋negative control group. The expression of Bax and Bcl-2 mRNA and proteins was detected by RT-PCR and Western-blot respectively. The apoptotic rate of podocytes was measured by flow cytometry. The results showed that the expression of TRPC6 mRNA and protein was decreased in the podocytes when transfected with pGCsi-TRPC6A, and pGCsi-TRPC6B. The expression of Bax was increased, and that of Bcl-2 was decreased at protein and mRNA levels in the podocytes after treated with PAN for 48 h. These changes was attenuated by knocking-down TRPC6. Knocking-down TRPC6 could effectively decrease the PAN-induced apoptosis of podocytes. It was concluded that TRPC6 may play an important role in the PAN-induced apoptosis of podocytes. Knocking-down TRPC6 gene could effectively prevent the podocytes from apoptosis induced by PAN.

Effect of TRPC6 Knockdown on Puromycin Aminonucleoside-induced Podocyte Injury

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA （shRNA） targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside （PAN）-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein （GFP）-contained plasmids （pGC） to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein ex-pression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups： control group, PAN treatment group, PAN＋TRPC6 shRNA transfected group and PAN＋scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

Protective Effect of Sulodexide on Podocyte Injury in Adriamycin Nephropathy Rats

This study examined the effect of sulodexide on podocyte injury in rats with adriamycin nephropathy （AN）. A total of 36 healthy male SD rats were randomly assigned to three groups： control group, AN group and sulodexide treatment group. Rat models of AN were established by a single tail intravenous injection of adriamycin （6.5 mg/kg） in both AN group and sulodexide treatment group. Sulodexide （10 mg/kg） was administered the rats in the treatment group once daily by garage from the first day of model establishment until the 14th day or the 28th day. Samples of 24-h urine and renal cortex tissues were harvested at day 14, 28 after the model establishment. Excretion of 24-h urinary protein was measured by Coomassie brilliant blue method. The pathological changes in renal tissues were observed by light microscopy and electron microscopy respectively. Heparanase mRNA was detected by RT-PCR. Expressions of desmin, CD2AP and heparanase were determined by immunohistological staining. The results showed that the expressions of heparanase mRNA and protein were increased in the glomeruli of AN rats at day 14 and 28 after the model establishment, which was accompanied by the increased expression of desmin and CD2AP. The mRNA and protein expression of heparanase was decreased in the sulodexide-treated rats as compared with AN rats at day 14 and 28. And, the protein expression of desmin and CD2AP was reduced as with heparanase in the sulodexide-treated rats. Proteinuria and podocyte foot process effacement were alleviated in the AN rats after sulodexide treatment. There was a positive correlation between the expression of heparanase and the expression of desmin and CD2AP （as well as 24-h urinary protein excretion）. It was concluded that increased heparanase is involved in podocyte injury. Sulodexide can maintain and restore podocyte morphology by inhibiting the expression of heparanase in AN.

Human podocyte injury in the early course of hypertensive renal injury

BACKGROUND Hypertension is prevalent in the general population and is regarded as the second leading cause of renal damage and dysfunction,outnumbered only by diabetes.However,the mechanisms remain unclear.AIM To investigate podocyte injury induced by hypertension in the early course without massive proteinuria or renal dysfunction.METHODS The hypertension group comprised 18 patients with hypertension accompanied by microalbuminuria,diagnosed with hypertensive renal injury according to biopsy results.For a comparison of pathological changes in renal tissue,control group 1 comprised 10 healthy volunteers,and control group 2 comprised 16 patients who underwent surgery for renal trauma.RESULTS The hypertension group had significantly higher blood pressure(P=0.000)and microalbuminuria(P=0.000)compared with control group 1.In the hypertension group,urinary podocytes were detected following positive staining of podocytespecific nephrin and/or CD2-associated protein(CD2AP)in urine sediment.Podocyte foot process fusion and a significant decrease in nephrin and/or CD2AP expression in glomeruli were observed in the hypertension group compared with control group 2.This indicated that hypertension caused podocyte injury and detachment from the glomerular basement membrane,which was consistent with urinary detection of podocytes.CONCLUSION Our results suggest that podocyturia appears early in the course of hypertensive renal injury,and may be a sensitive marker for early prediction of hypertensive renal injury.

Correlation between podocyte excretion and proteinuria in diabetic nephropathy patients

Objective： To observe the podocyte injury in diabetic nephropathy （DN） patients by identifying the urinary podocytes and the situation of detached podocytes in glomeruli and to demonstrate the correlation between podocyte excretion and proteinuria, blood glucose, serum creatinine in different phases in DN patients. Methods： Urinary podocytes and the podocalyxin （PCX） expression state of podocytes in glomeruli were identified and observed by indirect immunofluorescent method. The DN patients were divided into three groups according to the volume of proteinuria, namely small, medium and large volume proteinuria groups. The podocytes in the urine of every group were calculated. The DN patients were divided into five groups according to the chronic kidney disease （CKD） phases, then the positive podocytes in urine were calculated. Meanwhile, the 24-hour protein in urine, fasting blood glucose （FBG） and the serum creatinine of DN patients were tested. The correlations among the proteinuria, serum creatinine, FBG and the number of positive podocytes in the urine of DN patients were statistically analyzed. Results： Urinary positive podocytes were found in 88% of the patients with DN, whereas podocytes were found in 0% of patients with minimal changed disease （MCD） and healthy cases. The expression of PCX was absent in DN patients. In contrast, PCX was expressed integrally in MCD patients. The positive podocytes was 1.49±0.95/ml in small-volume proteinuria group, 2.15±0.70/ml in the medium-volume proteinuria group, and 3.48±1.27/ml in the large-volume proteinuria group. There was no significant difference between the small- and medium- volume proteinuria groups, and there were significant differences between other groups （P〈0.05）. The positive podocyte number tended to increase as proteinuria was increased. By Pearson analysis, the correlation between podocyte number and proteinuria was podocytes in urine from different groups of DN patients, CKD pc I sitive statistically. The difference of the number of positive -V group was significant statistically. The correlation between serum creatinine of CKD Ⅰ -Ⅲ group and positive podocytes in urine was positive statistically. The correlation between serumcreatinine of CKD Ⅳ- Ⅴ group and positive podocytes in urine was not significant statistically. The correlation between FBG and positive podocytes in urine was not significant either. Conclusion： The mechanism of the podocyte injury in DN patients is present. The podocyte injury in DN may positively correlate to proteinuria and serum creatinine of CKD Ⅰ -ⅢDN patients, but not to the FBG and serum creatinine of CKD Ⅳ-Ⅴ patients.

Protective Effects of Eplerenone on Podocyte Injury in Adriamycin Nephropathy Rats

To investigate the protective effects of eplerenone on adriamycin-induced renal injury and the possible mechanisms involved,36 male Sprague-Dawley rats were randomly divided into control group,adriamycin nephropathy（AN） group and eplerenone-treated group（100 mg.kg-1.d-1 eplerenone）.Blood pressure,24-h urinary protein,serum potassium,sodium and creatinine were measured 28 days after adriamycin injection（a single tail intravenous injection of 6.5 mg/kg adriamycin）.The morphological changes of renal tissues were observed by light and electron microscopy.Immunohistochemistry and Western blotting were performed to examine the expression of TGF-β1 and desmin in renal cortex.The results showed that 28 days after adriamycin injection,there were no significant changes in the level of serum potassium,sodium,creatinine concentrations and blood pressure values in the rats of the three groups.Meanwhile,the 24-h proteinuria excretion in the AN group was significantly higher than that in the control group（P0.01）,but that in the eplerenone-treated group was substantially reduced when compared with that in the AN group（P0.05）.Mild mesangial cell proliferation and matrix expansion,diffuse deformation and confluence of foot processes in podocytes were found in the AN group.By contrast,rats in the eplerenone-treated group exhibited obvious attenuation of these morphological lesions.The protein expression of TGF-β1 and desmin in the AN group was markedly up-regulated in contrast to that in the control group（P0.01）,whereas that in the eplerenone-treated group was much lower than in the AN group（P0.05）.It was concluded that eplerenone may ameliorate the proteinuria and the development of pathological alteration in adriamycin-induced nephropathy presumably via the inhibition of cytokine release,and restore the morphology of podocytes independent of its blood pressure-lowing effects.

Podocyte loss and albuminuria of KK-Ay mouse: A spontaneous animal model for human type 2 diabetic nephropathy

Podocyte loss was well known in type 2 diabetic nephropathy patients. The objective of the present study was to determine the number of podocytes and the degree of albuminuria in diabetic KK-Ay/Ta (KK-Ay) mice which had been reported as diabetic nephropathy model. Diabetic KK-Ay mice, diabetic KK/Ta mice and non-diabetic BALB/cA Jcl (BALB/cA) mice were studied. We analyzed glomerular lesions in all mice by morphometric analysis and immunofluorescence to determine the number of podocytes. Level of urinary albumin was also measured. Glomerular enlargement and mesangial expansion were observed in KK-Ay mice. Mean number of podocytes per glomerulus (NG pod) in diabetic KK-Ay mice was significantly lower than that in non-diabetic BALB/cA mice. Mean NG pod/glomerular area (GA) per glomerulus was also significantly decreased in diabetic KK-Ay mice. The level of urinary albumin/creatinine ratio (ACR) in diabetic KK-Ay mice was significantly higher than that in non-diabetic BALB/cA mice. These data suggest that podocyte loss might induce albuminuria in KK-Ay mice. This finding confirmed our previous report that KK-Ay mice, especially in terms of histological findings, are a suitable animal model for glomerular injury in type 2 diabetic nephropathy.

miRNAs Expression and Role of Dicer on Podocyte Injury in PAN Nephrosis Rats

Objective: microRNAs (miRNAs) are regulatory RNAs that act as important players in diverse biologic and pathologic processes. Under circumstance as podocye-injury triggering proteinuria, which miRNAs are up-regulated or down-regulated? This experiment aims at detecting miRNAs changes in PAN nephrosis rats based on miRNA arrays and exploring the therapeutic targets of Leizhi capsule. Methods: Fifty male wistar rats were randomly divided into five groups, including control group, model group, leizhi capsule group, Tripterygium glucosides group, and valsartan group. PAN nephrosis models were made by jugular vein injection of PAN (100 mg/kg body weight, dissolve in physiological saline), while control group rats were made by jugular vein injection of physiological saline with equal volume. Other groups rats had been given medicines by irrigating stomach once a day for ten days. Blood and urine samples were collected, and renal tissues were processed after rats being euthanasised. The 24 h urinary protein excretion and blood biochemistry parameters were measured by routine methods. The glomerular morphology and podocyte ultrastructure were observed with light microscopy and transmission electron microscopy respectively. miRNA expression profile was detected by Exiqon miRNA Array. Real time RT-PCR analysis for mature miRNAs was used to validate differentially expressed miRNAs. Results: 1) In day 3 - 5, model rats had decreased urine volume, ascites, malnutrition and wight loss. From day 7 to day 10, the nephrotic syndromes were worst in model rats, but which had no skin edema. Some rats died in serious ascites, the mortality is 3/10. 2) miRNA array detection shows 106 miRNAs up regulated and 62 miRNAs down regulated in PAN nephrosis rats. Fold change (model vs. control group) varies from 1.8 to 7.0. For leizhi capsule group and model sample, there are 90 miRNAs differentially expressed, with 65 miRNAs up and 25 miRNAs down. The most important finding in our research is the discovery of the specific miRNAs related to PAN nephrosis (rno-miR23a, rno-miR-24, rno-miR-30c and rno-miR-300-3p), which have been validated by Real time RT-PCR analysis. 3) Compared with control sample, immune fluorescence intensity of dicer, expression profile of nephrin, podocin and synaptopodin mRNA and protein decrease in PAN nephrosis rats. After treated with Leizhi Capsule, immune fluorescence intensity of the above molecules improved. Conclusion: 1) Characteristic miRNAs of PAN nephrosis were screening. Up-regulated miRNAs (rno-miR-23a, rno-miR-300-3p) may trigger podocyte injury and proteinuria, while down-regulated miRNAs (rno-miR-24, rno-miR-30c) may be protective factors by anti-apoptosis. 2) Dicer and these miRNAs (rno-miR-24, rno-miR-30c, rno-miR-23a) may be are probably key molecules therapeutic targets of Leizhi capsule.

A Potential Pathological Role of Angiopoietins Expression in Glomeruli during Progressive Glomerulisclerosis Related to Podocyte Injury

A potential pathological role of angiopoietins （Ang） in glomeruli following podocyte injury-induced progressive glomerulosclerosis was explored. Eighty male Wistar rats were randomly allocated into sham operation group （Sham, n= 25）, Uninephrectomy group （UPHT, n= 25） and Uninephrectomy＋Daunorubicin group （DRB, n= 30）. In DRB group, daunorubicin （5 mg/kg） was injected via tail vein on the 7th and 14th day after uninephrectomy. At week 1, 2, 4, 6 and 8 respectively following establishment of the animal model, 5 rats in Sham group and UPHT group, and 6 in DRB group were taken respectively for determining 24-h urinary protein excretion rate （24hUPER）, blood urea nitrogen （BUN） and serum creatinine （Scr）. The sections of kidneys were examined by an electric microscope, PAS staining, immunohistochemical staining and in situ hy-bridization histochemistry. The results showed that 24hUPER, BUN and Scr in DRB group were more than those in Sham group and UPHT group at the same time points, and there was a trend towards an increase on level of GSI in DRB group from week 2 to week 8. Electric microscopy revealed that podocyte injury presented in DRB group. The expression of Angl mRNA and protein in glomeruli of DRB group was decreased, while the expression of Ang2 protein in glomeruli of DRB group increased. Meanwhile, the expression of Angl mRNA had a negative correlation with the expression of Ang2 mRNA, and the expression of Angl protein had a positive correlation with the expression of Angl mRNA, and had a negative correlation with 24hUPER, BUN, Scr, glomerular sclerotic index （GSI）, the expression of Ang2 protein and CoIV protein. The expression of Ang2 protein had a positive correlation with the expression of Ang2 mRNA, and had a positive correlation with 24hUPER, BUN, Scr, GSI, the expression of CoIV protein. It was concluded that podocyte injury might lead to an alteration in the expression of Angl and Ang2 within glomeruli. Ang2 may get rid of inhibition from Angl for downregulation of the Angl expression, which facilitate upregulation of the Ang2 expression in glomeruli to promote progressive glomerulosclerosis in the rats.

Losartan Protects Podocytes against High Glucose-induced Injury by Inhibiting B7-1 Expression

The role of B7-1 in podocyte injury has received increasing attention.The aim of this study was to investigate whether losartan protects podocytes of patients with diabetic kidney disease(DKD)by regulating B7-1 and the underlying mechanisms.Rats with streptozotocin-induced DKD were treated with losartan for 8 weeks.Biochemical changes in blood and urine were analyzed.Kidneys were isolated for electron microscopy,immunofluorescence,real-time quantitative PCR(RT-PCR),and Western blot analysis.Immortalized mouse podocyte cells were cultured in normal or high glucose medium in the presence or absence of losartan for 48 h,and then the cells were collected for immunofluorescence,PCR,Western blotting and monolayer permeability detection.The phosphatidylinositol 3-kinase(PI3K)110a subunit and angiotensin II type 1 receptor(AT1R)plasmids were transfected into podocytes,respectively,and then Western blotting was performed to assess the expression of B7-1 protein.The results showed that losartan ameliorated podocyte structure and function in the rat model of DKD,and reduced the expression of B7-1 protein.Overexpression of PI3K 110a subunit in podocytes attenuated the inhibitory effect of losartan on B7-1 expression in high glucose-stimulated podocytes.The expression of B7-1 was significantly increased by overexpression of ATI R and significantly reduced by blocking PI3K 110a subunit.We conclude that losartan protects podocytes against high glucose-induced injury by inhibiting AT1R-mediated B7-1 expression.This effect is dependent on the AT1R-PI3K 110a subunit pathway.

Albumin Modulates the Production of Matrix Metalloproteinases-2 and -9 in Podocytes

To investigate the effects of albumin on the production of matrix metalloproteinases-2 （MMP-2） and matrix metalloproteinases-9 （MMP-9）in podocytes. Podocytes were treated with bovine serum albumin （BSA） at the concentration of 0.1, 0.5, 1, 2 g/L, respectively. Conditioned media were harvested 12, 24, 48 and 72 h after the treatment. The expression of MMP-2 and MMP-9 was assayed by gelatin zymography, RT-PCR and Western blotting analysis. Our results showed that in comparison with the control group, BSA increased the expression of MMP-2 and MMP-9 mRNA and protein in a doseand time-dependent manner （P〈0.05）. Meanwhile, the enzymatic activities of MMP-2 and MMP-9 in the culture supernatants of podocytes were also increased （P〈0.05）. It is concluded that albumin up-regulated the expression of MMP-2 and MMP-9 at gene and protein levels in a time-and dose-dependent manner.

Overview of effect of traditional Chinese medicine on diabetic kidney disease by regulating podocyte

Diabetic kidney disease has now become the leading cause of end-stage renal disease.Podocytes are an important filtration barrier of the glomerulus,and their damage plays an important role in the occurrence and progression of glomerular sclerosis and DKD.This article discusses the molecular mechanism of traditional Chinese medicine on the protection of podocyte damage in diabetic kidney disease from the aspects of anti-oxidative stress,activating autophagy,and regulating signal pathways,in order to further deepen the modern material basis theory of traditional Chinese medicine treatment and provide reference for the treatment of DKD.