Pathological Changes In Renal Tissues Research Articles

Ameliorating role of microRNA-378 carried by umbilical cord mesenchymal stem cells-released extracellular vesicles in mesangial proliferative glomerulonephritis

BackgroundMesenchymal stem cells (MSCs) and their released extracellular vesicles (Evs) have shown protective effects against kidney diseases. This study aims to study the functions of umbilical cord MSCs-released Evs (ucMSC-Evs) and their implicated molecules in mesangial proliferative glomerulonephritis (MsPGN).MethodsA rat model of MsPGN was induced by anti-Thy-1.1, and rat mesangial cells (rMCs) HBZY-1 were treated with PDGF-BB/DD to mimic MsPGN condition in vitro. Rats and cells were treated with different doses of ucMSC-Evs, and then the pathological changes in renal tissues and proliferation of rMCs were determined. Differentially expressed microRNAs (miRNAs) after Evs treatment were screened by microarray analysis. The interactions among miR-378, PSMD14, and TGFBR1 were analyzed. Gain- and loss-of function studies of miR-378 and PSMD14 were performed to explore their effects on tissue hyperplasia and rMC proliferation and their interactions with the TGF-β1/Smad2/3 signaling pathway.ResultsThe ucMSC-Evs treatment ameliorated mesangial hyperplasia and fibrosis in rat renal tissues and suppressed the aberrant proliferation of rMCs in a dose-dependent manner. miR-378 was the most upregulated miRNA in tissues and cells after ucMSC-Evs treatment. miR-378 directly targeted PSMD14, and PSMD14 maintained the stability of TGFBR1 through deubiquitination modification, which led to TGF-β1/Smad2/3 activation. Either miR-378 knockdown or PSMD14 overexpression diminished the protective functions of ucMSC-Evs by activating the TGF-β1/Smad2/3 signaling pathway.ConclusionUcMSC-Evs ameliorate pathological process in MsPGN through the delivery of miR-378, which suppresses PSMD14-mediated TGFBR1 stability and inactivates the TGF-β1/Smad2/3 signaling pathway to reduce tissue hyperplasia and rMC proliferation.Graphical 6tcW_AV6VyAYJya28kLbKyVideo abstract

Knockout of farnesoid X receptor gene aggravates cisplatin-induced kidney injury.

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily of ligand activated transcription factors and belongs to bile acid receptor. Studies have shown that the expression of FXR in renal tissue can reduce renal injury via regulation of glucose and lipid metabolism, inhibition of inflammatory response, reduction of oxidative stress and renal fibrosis. However, it is unclear whether FXR is involved in autophagy in renal diseases. This study aims to investigate the role of FXR in cisplatin-induced acute renal injury and whether its mechanism is related to autophagy regulation. Twelve male WT or FXR-KO mice at 12 weeks were randomly divided into a WT group, a WT+cisplatin group, a FXR-KO group, and a FXR-KO+cisplatin group, with 6 mice in each group. The WT+cisplatin group and the FXR-KO+cisplatin group were intraperitoneally injected with cisplatin (20 mg/kg), and the WT group and the FXR-KO group were intraperitoneally injected with equal volume of cisplatin solvent. Seventy-two hours later, the mice were killed and blood and renal tissue samples were collected. The levels of SCr and BUN were detected by immunoturbidimetry. After the staining, the pathological changes of renal tissue were observed under optical microscope. The protein levels of LC3 and p62 were detected by Western blotting and immunohistochemistry. The clearance of damaged mitochondria and the accumulation of lysosomal substrate were observed under electron microscope. The apoptosis of renal tubular epithelial cells was detected by TUNEL. Compared with the WT group or the FXR-KO group, both SCr and BUN levels in the WT+cisplatin group or the FXR-KO+cisplatin group were significantly increased (P<0.01 or P<0.001), and SCr and BUN levels in the FXR-KO+cisplatin group were significantly higher than those in the WT+cisplatin group (both P<0.05). Under the light microscope, there were no obvious pathological changes in the renal tissue of mice in the WT group and the FXR-KO group. Both the WT+cisplatin group and the FXR-KO+cisplatin group had vacuolar or granular degeneration of renal tubular epithelial cells, flat cells, lumen expansion, brush edge falling off, and even exposed basement membrane and tubular formation. The scores of renal tubular injury in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the score in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Under the transmission electron microscope, the mitochondria of mouse tubular epithelial cell in the WT+cisplatin group and the FXR-KO+cisplatin group was swollen, round, vacuolated, cristae broken or disappeared; the lysosome was uneven and high-density clumps, and the change was more obvious in the FXR-KO+cisplatin group. Western blotting showed that the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased in the WT+cisplatin group compared with the WT group and the FXR-KO+cisplatin group compared with FXR-KO group (P<0.05 or P<0.01); compared with the FXR-KO group, the ratio of LC3-II to LC3-I was decreased and the expression of p62 was increased significantly in the FXR-KO+cisplatin group (both P<0.05). Immunohistochemistry results showed that the expression of total LC3 and p62 in renal cortex of the WT+cisplatin group and the FXR-KO+cisplatin group was increased significantly, especially in the FXR-KO+cisplatin group. TUNEL results showed that the mice in the WT group and the FXR-KO group had negative staining or only a few apoptotic tubular epithelial cells, and the number of apoptotic cells in the WT+cisplatin group and the FXR-KO+cisplatin group were increased. The apoptosis rates of renal tubular epithelial cells in the WT+cisplatin group and the FXR-KO+cisplatin group were significantly higher than those in the WT group and the FXR-KO group, respectively (both P<0.001), and the apoptosis rate in the FXR-KO+cisplatin group was significantly higher than that in the WT+cisplatin group (P<0.05). Knockout of FXR gene aggravates cisplatin induced acute renal injury, and its mechanism may be related to inhibiting autophagy and promoting apoptosis.

ADAMTS-13-regulated nuclear factor E2-related factor 2 signaling inhibits ferroptosis to ameliorate cisplatin-induced acute kidney injuy

ABSTRACT ADAMTS-13 plays an important role in acute kidney injury (AKI), but the mechanism of cisplatin (CP) induced AKI remains unclear. Ferroptosis is increased in CP-induced AKI, and ADAMTS13 levels are associated with ferritin expression. In this article, we will explore the relationship between the three. After CP induction, mice were given 0.1 and 0.3 nmol/kg ADAMTS-13, and then serum creatinine (Scr) and blood urea nitrogen (BUN) were detected by the kits. The pathological changes of renal tissue were observed by staining with HE and PAS staining, and Western blot detected the expressions of KIM1 and NGAL in renal tissu. Perl’s staining detected iron deposition in renal tissues, the kits detected iron levels, and western blot detected the expression of ferroptosis related proteins. Then the mechanism was further explored by adding ferroptosis inhibitors Ferrostatin 1 (Fer-1) and iron supplements Fe. The expression of Nrf2 pathway related proteins were detected by Western blot. We found that ADAMTS13 alleviated CP-induced ferroptosis in AKI mice with renal function impairment and tubular damage. Fer-1partially reversed CP-induced AKI, and Fe exacerbated this effect. ADAMTS13 alleviated CP-induced inflammatory response and oxidative stress in AKI mice, during which the Nrf2 signaling pathway was abnormal. Overall, ADAMTS-13-regulated Nrf2 signaling inhibits ferroptosis to ameliorate CP-induced AKI.

Influence of long non-coding RNA Gm15645 on the podocyte injury in diabetic nephropathy

To explore the influence of long non-coding (lnc) RNA Gm15645 on the podocyte injury in mice with diabetic nephropathy. Male db/db mice (with Type 2 diabetes) with a genetic background of C57BLKs/J and db/m mice (healthy) born in littermates were randomly divided into three groups. db/db group was injected with lncRNAGm15645 shRNA lentivirus with a podocyte-specific marker NPHS2; db/db blank group was injected with saline, and db/db control group was injected withnon-sense lentivirus. The results of PAS staining, pathological changes of renal tissue, relative expression of GSK-3beta, and podocin expression were compared. lncRNAGm15 645 was overexpressed and podocin was down-regulated in the lentivirus overexpressed group. Mesangial cell proliferation, mesangial matrix hyperplasia, thickened basement membrane, widely fused foot process, and podocyte injury were observed by PAS staining. The expression of Gm15645 in the db/db group was significantly lower than that of the db/db blank group and db/db control group (P< 0.05), while the expression of podocin was higher (P< 0.05). Gm15645 was co-stained with podocin in renal tissue, and the target gene was GSK-3beta. lncRNAGm15645 may provide an early biomarker for the occurrence of podocyte injury in diabetic nephropathy. The mechanism may be related to the feedback regulation of GSK-3beta gene.

MicroRNA-199a downregulation alleviates hyperuricemic nephropathy via the PPARγ/β-catenin axis

Hyperuricemia always develops into hyperuricemic nephropathy (HN). The role of microRNA (miR) in HN is less studied. We aimed to discuss the role of miR-199a in HN. The expression of miR-199a and PPARγ in renal tissues of HN rats was detected. The targeting relation between miR-199a and PPARγ was verified. The contents of SCr, UA, BUN, and mALB, renal injury-relevant biomarkers were detected, and the pathological changes of renal tissue and renal interstitial fibrosis were observed by histological staining. After miR-199a and PPARγ knockdown, the contents of SCr, BUN, and mALB and renal interstitial fibrosis were estimated. Collectively, overexpression of miR-199a aggravated the renal injury in HN rats. By contrast, inhibition of miR-199a weakened renal injury, as evidenced by decreased contents of SCr, UA, BUN, and mALB, and mitigated renal interstitial fibrosis. miR-199a targeted PPARγ. Silencing of PPARγ upregulated the levels of downstream genes of β-catenin and the contents of SCr, UA, BUN, and mALB and deteriorated renal interstitial fibrosis. Moreover, the silencing of PPARγ blocked the alleviative effects of miR-199a inhibitor on the renal injury. Overall, miR-199a targets PPARγ and activates the β-catenin pathway, thus aggravating HN, which might provide a future target for the treatment of HN.

Ginkgo Biloba Extract EGB761 Ameliorates the Extracellular Matrix Accumulation and Mesenchymal Transformation of Renal Tubules in Diabetic Kidney Disease by Inhibiting Endoplasmic Reticulum Stress.

The study is aimed at investigating the effects of Ginkgo biloba extract EGB761 on renal tubular damage and endoplasmic reticulum stress (ERS) in diabetic kidney disease (DKD). A total of 50 C57BL/6 N mice were randomly divided into the normal group, DKD group, DKD+EGB761 group (36 mg/kg), and DKD+4-phenylbutyrate (4-PBA) group (1 g/kg). The DKD model was replicated by high-fat diet combined with intraperitoneal injection of streptozotocin (STZ). Renal tubular epithelial cells (HK-2) were divided into the control group, high-glucose group (30 mmol/L), EGB761 group (40 mg/L, 20 mg/L, 10 mg/L), TM group, and TM+4-PBA group. After 8 weeks of administration, expressions of serum creatinine (Scr), blood urea nitrogen (BUN), 24 h urinary protein (24 h Pro), fasting blood glucose (FBG), β2-microglobulin (β2-MG), and retinol binding protein 4 (RBP4) of mice were tested. The pathological changes of renal tissue were observed. The expressions of extracellular matrix (ECM) accumulation and epithelial-mesenchymal transition (EMT) markers α-smooth muscle actin (α-SMA), E-cadherin, fibronectin, and collagen IV, as well as the ERS markers GRP78 and ATF6, were tested by Western blot, qPCR, immunohistochemistry, or immunofluorescence. EGB761 could decrease the Scr, BUN, 24 h Pro, and FBG levels in the DKD group, alleviate renal pathological injury, decrease urine β2-MG, RBP4 levels, and decrease the expression of α-SMA, collagen IV, fibronectin, and GRP78, as well as ATF6, while increase the expression of E-cadherin. These findings demonstrate that EGB761 can improve renal function, reduce tubular injury, and ameliorate ECM accumulation and EMT in DKD kidney tubules, and the mechanism may be related to the inhibition of ERS.

Silence of Long Noncoding RNA SNHG14 Alleviates Ischemia/Reperfusion-Induced Acute Kidney Injury by Regulating miR-124-3p/MMP2 Axis.

Purpose Ample evidence has proved that lncRNAs are pivotal regulators in acute kidney injury (AKI). Here, we focus on the role and mechanism of lncRNA SNHG14 in ischemia/reperfusion- (I/R-) caused AKI. Methods I/R and hypoxia/reoxygenation (H/R) were applied to induce rats and HK-2 cells to establish AKI models in vivo and in vitro. Relative expression of SNHG14, miR-124-3p, and MMP2 was determined by qRT-PCR. HE staining was used to evaluate pathological changes in renal tissues, and acute tubular necrosis (ATN) score was calculated. Renal function was evaluated by measuring serum creatinine content and blood urea nitrogen content. Levels of IL-1β, IL-6, and TNF-α were measured by ELISA. Cell viability was examined by MTT assay. Oxidative stress was assessed by measuring SOD, MDA, and ROS levels. The target of SNHG14 or miR-124-3p was verified by DLR assay. Protein expression of MMP2 was examined by western blot. Results SNHG14 was boosted in renal tissues of I/R-stimulated rats and H/R-induced HK-2 cells, while miR-124-3p was diminished in H/R-stimulated HK-2 cells. Si-SNHG14 or miR-124-3p mimics repressed inflammation and oxidative stress and enhanced cell viability in H/R-stimulated HK-2 cells. Sh-SNHG14 mitigated I/R-induced AKI in rats. MiR-124-3p was targeted by SNHG14, and MMP2 was targeted by miR-124-3p. Inhibition of miR-124-3p or upregulation of MMP2 reversed inhibitory effects of SNHG14 silence on inflammation and oxidative stress as well as the promoting effect of SNHG14 silence on cell viability in H/R-induced HK-2 cells. Conclusion Knockdown of SNHG14 alleviated I/R-induced AKI by miR-124-3p-mediated downregulation of MMP2.

Withaferin A protects against hyperuricemia induced kidney injury and its possible mechanisms

ABSTRACT The study was designed to explore the effects of Withaferin A (WFA) on hyperuricemia-induced kidney injury and its action mechanism. Potassium oxonate (PO) was employed to establish the hyperuricemic mouse model. The pathological changes of renal tissue were evaluated by hematoxylin-eosin and masson trichrome staining. The levels of creatinine, blood urea nitrogen (BUN), uric acid (UA) and xanthine oxidase (XOD) were detected using corresponding commercial kits. Expressions of collagen-related and apoptosis-associated proteins in renal tissues were, respectively, evaluated by immunofluorescence and western blotting. Cell apoptosis was detected by TUNEL assay, and transporter expressions using western blotting. Followed by WFA, NRK-52E cells were treated with UA before evaluation of apoptosis and fibrosis. Results indicated that WFA ameliorated renal damage, improved kidney function, and decreased levels of creatinine, BUN, UA, and XOD in PO-induced hyperuricemic mice. Furthermore, WFA significantly prevented renal fibrosis and increased the expression of collagen-related proteins. Similarly, WFA markedly inhibited renal apoptosis, accompanied by changes of apoptosis-related proteins. Importantly, expression of transporters responsible for the secretion of organic anion transporter 1 (OAT1), OAT3, ATP-binding cassette subfamily G member 2 (ABCG2) was remarkably enhanced whereas that of urate transporter 1 (URAT1) and glucose transporter 9 (GLUT9) was reduced in renal tissues of mice with hyperuricemia. In vitro study revealed that WFA notably ameliorated UA-induced cell fibrosis and apoptosis. Taken together, WFA improves kidney function by decreasing UA via regulation of XOD and transporter genes in renal tubular cells.

Effect of liraglutide on endoplasmic reticulum stress in the renal tissue of type 2 diabetic rats.

BACKGROUNDLiraglutide is a glucagon-like peptide 1 receptor agonist analog that has been found to have a therapeutic effect in diabetes. In addition to its ability to treat diabetes, liraglutide has beneficial effects on the cardiovascular system and kidney as well as other beneficial effects, but its specific mechanism is not clear. In this study, a rat model of type 2 diabetes was established by administration of a high-sugar, high-fat diet combined with low-dose streptozotocin (STZ) to observe the effect of liraglutide on the kidneys of type 2 diabetes rats and the possible underlying mechanisms.AIMTo explore whether liraglutide has a protective effect on type 2 diabetic rat kidneys and the underlying mechanisms.METHODSEight-week-old male Sprague-Dawley rats were randomly divided into a control group, model group, low-dose liraglutide group, and high-dose liraglutide group. Control rats were fed a standard diet, while model group and intervention group rats were fed high-sugar, high-fat feed for 1 mo and then intraperitoneally injected with 40 mg/kg STZ to induce type 2 diabetes. The low-dose and high-dose intervention groups received 100 µg/kg and 200 µg/kg liraglutide, respectively, once daily by subcutaneous injection. The control and model groups were given an equivalent volume of physiological saline for 8 wk. Pathological changes in renal tissues were observed by hematoxylin and eosin staining and periodic acid-Schiff staining, and GRP78 and caspase-12 expression was detected by Western blot and reverse transcription-polymerase chain reaction (RT-PCR).RESULTSWestern blot analysis showed that GRP78 and caspase-12 protein expression in kidney tissue was significantly higher in model rats than in normal rats and lower in the liraglutide-treated groups than in the model group, with a more significant decrease being observed in the high-dose group than in the low-dose group. RT-PCR showed that the mRNA expression of GRP78 and caspase-12 was higher in model rats than in control rats and lower in the liraglutide-treated groups than in the model group, with the high-dose group exhibiting a more significant decrease than the low-dose group.CONCLUSIONLiraglutide may delay the progression of diabetic nephropathy by reducing endoplasmic reticulum stress and protect the kidneys in a dose-dependent manner.

Safety evaluation of Eucommia ulmoides extract

Eucommia ulmoides Oliver is native to China and frequently used in traditional Chinese medicine formulations. However, studies show that Eucommia ulmoides extract (EUE) are potentially genotoxic and nephrotoxic. To evaluate its safety, the Ames test, bone marrow micronucleus assay and chromosomal aberration assay, along with acute (24 h) and sub-chronic (13 weeks) toxicity were conducted. EUE was non-genotoxic within the dose ranges of 0.0352–22 mg/plate (raw plant equivalent as below), 22–88 g/kg body weight and 2–20 mg/mL. The maximum tolerated dose of EUE was not less than 168 g/kg, which is 1260 times that of clinical doses in human adults. Long-term (13 weeks) administration led to dose-dependent increase in nephrotoxicity-related indices, and pathological changes in renal tissues. These changes were alleviated 5 weeks after ceasing the low dosage of 11.2 g/kg but persisted at the high dosage of 56 g/kg. Conclusively, EUE is non-genotoxic, and do not result in acute toxicity. However, long-term and high-dose administration can lead to partly reversible nephrotoxicity.

The role of lipoprotein-associated phospholipase A2 in acute kidney injury of septic mice.

BackgroundThis experiment aimed to investigate the role and mechanism of lipoprotein-associated phospholipase A2 (Lp-PLA2) in kidney injury in septic mice induced by cecal ligation and perforation (CLP).MethodsMale BALB/c mice were randomly divided into two groups: sham-operation group (Sham group) and septic group (CLP group). The septic model was simulated by cecal ligation and puncture method, but only cecal ligation was used for the sham operation group. The whole serum and renal tissue samples of the mice were collected 24 hours after modeling in both groups. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of renal tissue, the renal injury score was recorded, and the creatinine (Cr) and blood urea nitrogen (BUN) levels were detected by automatic biochemical analyzer, while the serum Lp-PLA2 level was detected by enzyme-linked immunosorbent assay (ELISA). The 7-day survival rate and the survival curve of the two groups were statistically analyzed.ResultsCompared with the Sham group, the pathological score of renal injury in the CLP Group was higher, the level of Lp-PLA2 in serum was significantly increased (all P<0.01), and the expression of Lp-PLA2 in renal tissue was significantly elevated (all P<0.01). Furthermore, the 7-day survival rate of the Sham group was 90%, while that of CLP group was 25%.ConclusionsThe expression level of Lp-PLA2 in blood and kidney tissue of septic mice was increased and correlated with prognosis. However, the predictive value of Lp-PLA2 for prognosis in septic mice needs further study.

CP-25 improves nephropathy in collagen-induced arthritis rats by inhibiting the renal inflammatory response

Paeoniflorin-6′-O-benzene sulfonate (CP-25) is a derivative of paeoniflorin. We previously confirmed that CP-25 inhibits inflammatory responses in several arthritis animal models. The aim of the present study was to investigate the beneficial effects of CP-25 on renal damage in rats with collagen-induced arthritis (CIA). CIA was induced in rats, which were orally administered CP-25 (25, 50 and 100 mg/kg/day) for 24 days. The levels of plasma blood urea nitrogen (BUN) and urine protein in CIA rats were measured. Pathological changes in renal tissues and joints were observed, and inflammatory cell infiltration was evaluated by immunohistochemistry. Moreover, renal inflammatory mediators and transporters were measured by western blotting. We found that CP-25 not only inhibited arthritis manifestations but also improved renal pathological manifestations and kidney injury by decreasing serum BUN and urine protein levels. Further study revealed that CP-25 treatment reduced the number of renal CD68+ cells and downregulated the levels of MCP-1, TNF-α and IL-6 in CIA rats. On the other hand, we noted that CP-25 decreased the ratios of phosphorylated NF-κB p65 (p-p65) to total p65 and p-IκBα to total IκBα in CIA rats, suggesting that CP-25 blocked NF-κB activation. Finally, we observed that CP-25 restored the abnormal expression of OAT1 and OCT1 in the renal tissues of CIA rat. Our data indicate that CP-25 ameliorates kidney damage in CIA rats, and this beneficial effect is closely related to inhibiting renal inflammation and the abnormal expression of transporters.

Protective Effects of Astaxanthin on Nephrotoxicity in Rats with Induced Renovascular Occlusion.

Various effects of Astaxanthin were shown in the studies, including its antioxidant, anti-inflammatory, anti-tumor and immunoregulatory effects. The aim of this study was to evaluate the beneficial effects of Astaxanthin on renovascular occlusion induced renal injury and to investigate the possible mechanisms. The rats were randomly assigned into three groups as follows: Group 1: control group (n=12), Group 2: renal ischemia-reperfusion injury group (n=12), Group 3: renal ischemia-reperfusion + asthaxantine treated group (n=12). The control group and the renal ischemia-reperfusion group were given 2cc/kg/g olive oil for 7 days before establishing ischemia to renal tissue. Astaxanthin dissolved in olive oil was given orally to the renal ischemia+astaxanthin group for 7 days before inducing renal ischemia. Caspase-(3, 8, 9), GSH, SOD, Total Thiol, TNF-α, IL-6, 8-OHdG were evaluated in each group. Renal IRI was verified by analysing the pathological changes of renal tissues and the renal functions after renal reperfusion. Much less renal tubular damage was determined in the IRI+ASX group in comparison to the IRI group. Caspase-8, -9 and -3 immunoreactivity was observed to be minimal in the control group. Apoptosis was observed to be significantly reduced in the IRI + ASX group with respect to the IRI group and close to the level of the control group (p <0.05). Caspase-3 levels of tissue samples were significantly increased in the IRI group compared to the other groups, but significantly lower in the IRI+ASX group with respect to the IRI group (p<0.05). The TOS and OSI levels, indicating increased oxidative stress, were significantly lower in the IRI+ASX group with respect to the IRI group (p <0.001), but still higher than the control group (p <0.001). In addition to GSH, SOD and Total Thiol levels, TAS levels were also significantly higher in the IRI + ASX group in comparison to the IRI group (p <0.05). TNF-α, IL-6, lipid hydroperoxide, AOPP and 8-OHdG levels were lower in the IRI+ASX group than the IRI group (p <0.001). MPO, IL-6, TNF-α levels, representing the parameters indicating neutrophil infiltration and inflammation of the renal tissues, significantly increased in the IRI group with respect to the other groups (p <0.005). When all the data obtained in our study were evaluated, ASX was determined to prevent renal damage due to renovascular occlusion to a great extent, through complex mechanisms involving antioxidant, anti-inflammatory and antiapoptotic effects. Biochemical, histological and oxidative stress parameters were improved due to ASX.

Berberine alleviates cisplatin-induced acute kidney injury by regulating mitophagy via PINK 1/Parkin pathway.

BackgroundTo study the protective effect of berberine (BBR) on cisplatin-induced acute kidney injury (AKI) and its effect on mitophagy.Methods(I) Male C57BL/6 mice aged 6–8 weeks were randomly divided into control group (saline), cisplatin group (cisplatin), and cisplatin + BBR (5, 10 mg/kg) groups. In the cisplatin group and BBR groups, mice were injected intraperitoneally with 15 mg/kg of cisplatin. Mice in BBR groups were given BBR at 72, 48, 24, 0.5 h before and 24, 48 h after cisplatin injection. Mice were sacrificed 72 h after cisplatin injection, and blood were collected for detecting serum creatinine (SCr) and blood urea nitrogen (BUN) levels. Kidneys were collected for detecting protein expression levels of Kidney injury molecule 1 (KIM-1), LC3 II/LC3 I, p62, PINK 1, Parkin in the renal tissue by Western blotting. The pathological changes in renal tissues were observed using periodic acid-Schiff (PAS) staining. (II) Renal tubular epithelial cells (RTECs) were pretreated with different concentrations (1, 2, and 4 µM) of BBR, and then incubated with cisplatin. Changes in autophagy proteins LC3 II/LC3 I, p62, PINK 1, and Parkin were detected by Western blotting, and changes in cellular reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry.Results(I) Mice treated with BBR at dosage of 5 and 10 mg/kg for 6 days showed significant reduction in SCr and BUN compared to that in mice treated with cisplatin. PAS staining and immunohistochemistry showed that BBR ameliorated cisplatin-induced nephrotoxicity and reduced cisplatin-induced increase in protein expression levels of KIM-1. Compared to cisplatin-treated mice, the mice treated with BBR showed increased LC3 II/LC3 I, PINK 1, and Parkin, and decreased p62 protein expression. (II) Compared to cisplatin-incubated RTECs, cells pretreated with BBR for 24 h exhibited increased protein expressions of LC3 II/LC3 I, PINK1, and Parkin and decreased protein expression of p62. BBR reversed cellular ROS and cell MMP level induced by cisplatin.ConclusionsBBR plays a protective role in cisplatin-induced AKI by up-regulating mitophagy in RTECs.

Resveratrol Plays Protective Roles on Kidney of Uremic Rats via Activating HSP70 Expression.

Objective To investigate the protective effects of resveratrol on kidney of uremic rats and to explore whether the mechanism is associated with heat shock protein 70 (HSP70) expression. Methods Sixty male Sprague Dawley rats were randomly separated into 5 groups, including sham group, uremic model group, and different doses of resveratrol group (5 mg/kg, 10 mg/kg, and 20 mg/kg). The serum creatinine (Cr) and urea nitrogen (BUN) levels were detected by Automatic Biochemical Analyzer (ABA). The pathological changes of renal tissues and the renal interstitial fibrosis were analyzed by hematoxylin-eosin (HE) and Masson, respectively. The expression of HSP70 protein in renal tissues was detected by immunohistochemistry. The expression of HSP70 and NF-κB pathway-related proteins were detected by Western blot. To further validate the protective role of resveratrol through activating HSP70 in uremic rats, HSP70 activator (17-AAG) and HSP70 inhibitor group (MKT-077) were used. Results In the model group, the levels of Cr and BUN in serum were significantly increased, and the renal interstitial collagen deposition was also obviously increased (p < 0.05). Compared with the model group, the levels of Cr and BUN in different doses of resveratrol groups were remarkably declined, and the renal interstitial collagen deposition was declined (p < 0.05). Resveratrol also significantly improved the renal tissue lesions when compared with the model group. In renal tissues, different doses of resveratrol treatment remarkably raised HSP70 and p-IκBα expression and also remarkably declined the level of p-P65 protein (p < 0.05). Meanwhile, the effect of 17-AAG was similar to 20 mg/kg resveratrol on NF-κB pathway-related proteins expression. After the added MKT-077 in the resveratrol treatment group, the levels of HSP70 and p-IκBα in the renal tissue were remarkably declined; however, the levels of p-P65 protein was remarkably raised (p < 0.05). Conclusion Resveratrol played a protective role on the kidney of uremic rats through activating HSP70 expression.

Downregulation of lncRNA TUG1 attenuates inflammation and apoptosis of renal tubular epithelial cell induced by ischemia-reperfusion by sponging miR-449b-5p via targeting HMGB1 and MMP2.

We aimed to evaluate the functions of long non-coding RNA taurine upregulated gene 1 (lncRNA TUG1) in renal ischemia-reperfusion (I/R) injury and identify the potential mechanisms. Pathological changes of renal tissues were examined using H&E staining after mimic renal I/R injury in vivo. The contents of serum renal functional parameters and inflammatory factors were measured. The expression of TUG1 and miR-449b-5p in renal tissues and HK-2 cells stimulated by I/R were detected. Then, the effects of TUG1 silencing on inflammation and apoptosis of cells were evaluated. Dual luciferase reporter assays were executed for determining the correlation between miR-449b-5p and TUG1, high mobility group box 1 (HMGB1), or matrix metalloproteinase 2 (MMP2). Subsequently, cells were co-transfected with miR-449b-5p mimic and pcDNA3.1 TUG1. The levels of inflammation, apoptosis, and the expression of HMGB1 and MMP2 were detected. The results revealed that renal tissues were obviously damaged after I/R accompanied by changes in renal functional markers and inflammatory factors. TUG1 was highly expressed whereas miR-449b-5p was lowly expressed. TUG1 silencing reduced the inflammation and apoptosis. Dual luciferase reporter assays confirmed that miR-449b-5p was a target of TUG1 as well as HMGB1 and MMP2 were direct targets of miR-449b-5p. Meanwhile, miR-449b-5p mimic presented the same results with TUG1 silencing, which were reversed after TUG1 overexpression. Moreover, MMP2 and HMGB1 expression was decreased after miR-449b-5p overexpression while that of was increased after TUG1 overexpression. These findings demonstrated that TUG1 silencing attenuates I/R-induced inflammation and apoptosis via targeting miR-449b-5p and regulating HMGB1 and MMP2 expression.

ErHuang Formula Improves Renal Fibrosis in Diabetic Nephropathy Rats by Inhibiting CXCL6/JAK/STAT3 Signaling Pathway.

Diabetic nephropathy (DN) is one of the main causes of renal fibrosis and is associated with high morbidity and mortality. Traditional Chinese Medicine (TCM) therapy has a long history of usage in a clinical setting and its usage is increasing. ErHuang Formula (EHF), a Chinese herbal compound, has been clinically used in treating DN for more than 30 years. However, its mechanism of action is still unknown. This study was conducted to evaluate the effect of EHF on renal fibrosis in a DN rat model and explore its underlying mechanism. The DN rat model was established by high-sugar-fat diet combined with a single intraperitoneal injection of streptozotocin (STZ), and EFH extract (4, 2, 1 g/kg d−1) was administered orally for 8 weeks. The biochemical parameters (blood glucose, weight, Scr, BUN, UA, U-Alb and UAE) were analyzed. The pathological changes in renal tissue were observed by histological staining with H&E and Masson. The effect of EHF on the proliferation of NRK-49F cells was examined by CCK-8 assay and the levels of several inflammation and fibrosis related cytokines (IL-6, TNF-α, TGF-β1, Collagen I/III, MMP2/9) in serum and NRK-49F cell culture supernatants were detected by enzyme-linked immunoassay (ELISA). The mRNA levels of CXCL6, CXCR1, Collagen I/III, MMP2/9 in renal tissue were also measured by quantitative RT-PCR. Furthermore, the protein expression of PCNA, Collagen I/III, MMP2/9, CXCL6, CXCR1, p-STAT3, STAT3 in renal tissue and NRK-49F cells were determined by western blot. EHF improved the abnormal biochemical parameters and ameliorated the abnormal histology and fibrosis of renal tissue in a dose-dependent manner. EHF inhibited NRK-49F proliferation and decreased the expressions of inflammation and fibrosis related factors both in vitro and in vivo. Interestingly, the levels of Collagen I/III, PCNA, MMP2/9 and p-STAT3 were positively correlated with CXCL6. The amelioration of renal fibrosis in DN by EHF is related to CXCL6/JAK/STAT3 signal pathway, which is associated with inflammation and fibrosis of the tissue. These findings may have clinical implications for the treatment of DN.

Expression characteristics of inflammatory and apoptosis factors and regulatory effect of anemoside B4 in mice with acute kidney injury

This paper was aimed to observe the effect of anemoside B4(hereinafter referred to as B4) on cisplatin-induced acute kidney injury in mice, and to investigate its possible mechanism in renal protection from inflammation and apoptosis aspects. Mice were divided into normal group, model group, dexamethasone positive group and B4 high, middle and low dose groups(5, 2.5, and 1.25 mg·kg~(-1 )doses). All the other mice groups except normal group were given with tail vein injection of cisplatin(15 mg·kg~(-1)) to induce acute kidney injury models. The drug administration was started on the day of modeling, and lasted for 4 days. After 1 hour of the last injection, orbital blood was collected. After the serum was separated, serum urea nitrogen(BUN), creatinine(Cre), total protein(TP), and albumin(ALB) were tested by using an automatic biochemical analyzer; the changes of kidney pathological morphology were observed by PAS staining; the protein expression levels of inflammatory factors including nucleotide binding oligomerization domain-like receptor(NLRP3), cysteinyl aspartate specific proteinase 1(caspase-1), interleukin-18(IL-18), interleukin-1β(IL-1β), tumor necrosis factor(TNF-α), and interleukin-6(IL-6) and apoptosis factors including p53, caspase-3, cleaved-caspase-3, Bcl-2 associated X protein(Bax), and B-cell lymphoma-2(Bcl-2) were analyzed by Western blot. The results showed that B4 significantly reduced the serum BUN and Cre contents, and alleviated pathological changes in renal tissues, such as the shedding and degeneration of renal tubular epithelial cells, tubulin tubule type. B4 significantly down-regulated the protein expressions of p53, Bax, cleaved-caspase-3 in the kidney and up-regulated the expression of Bcl-2/Bax. In model group, however, no significant up-regulation was observed in the protein expression levels of inflammatory cytokines(NLRP3, pro-caspase-1, IL-18, IL-1β, TNF-α, IL-6). The results suggested that B4 had a certain protective effect on cisplatin-induced acute kidney injury, and could activate p53 signaling pathway related apoptotic factors. B4 renal protective effect was mainly related to the regulation of p53 signaling pathway, while NLRP3 inflammasome and related inflammatory factors had no obvious response in this model.

Ursolic Acid Treatment Alleviates Diabetic Kidney Injury By Regulating The ARAP1/AT1R Signaling Pathway.

PurposeThis study aimed to investigate whether ursolic acid (UA) mitigates renal inflammation, oxidative stress and fibrosis by regulating the angiotensin II type 1 receptor-associated protein (ARAP1)/angiotensin II type 1 receptor (AT1R) signaling pathway and subsequently alleviating renal damage.Methodsdb/db mice were divided randomly into a diabetic nephropathy (DN) group and a UA treatment group. Light microscopy and electron microscopy were used to observe pathological changes in renal tissues. Immunohistochemistry (IHC) was employed to examine changes in the expression of ARAP1, AT1R, 8-hydroxydeoxyguanosine (8-OHdG), NADPH oxidase 2 (NOX2), the extracellular matrix protein fibronectin (FN), IL-1β and IL-18 in renal tissues. Western blotting and RT-qPCR were used to detect the respective changes in the protein and mRNA levels of ARAP1, AT1R, NOX4, NOX2, transforming growth factor-β1 (TGF-β1), FN, collagen IV, IL-1β and IL-18 in renal tissues and mesangial cells. In addition, immunofluorescence staining was employed to examine changes in FN and NOX2 expression in mesangial cells.ResultsUA treatment effectively reduced the body weights and blood glucose levels of db/db mice (p<0.05) as well as the urinary albumin/creatinine ratio (p<0.05). In addition, the renal tissue lesions and glomerulosclerosis index of the db/db mice were significantly improved after treatment (p<0.01). Histochemical analysis results showed significantly lower expression levels of ARAP1, AT1R, FN, NOX2, 8-OHdG, IL-1β and IL-18 in renal tissues in the UA treatment group than in the DN group. Western blotting and RT-qPCR data also revealed UA-induced decreases in the renal levels of the ARAP1, AT1, NOX4, NOX2, TGF-β1, FN, collagen IV, IL-1β and IL-18 proteins in vivo and/or in vitro (p<0.01). ARAP1 knockdown effectively reduced the expression of NOX2 and FN in vitro.ConclusionUA alleviated renal damage in type 2 diabetic db/db mice by downregulating proteins in the ARAP1/AT1R signaling pathway to inhibit extracellular matrix accumulation, renal inflammation, fibrosis and oxidative stress.

Urinary proteome of dogs with kidney injury during babesiosis

BackgroundAcute kidney injury is the most frequent complication of babesiosis in dogs and may provide a natural model for identifying early and specific markers of kidney injury in this species. There are limited data on urine proteomics in dogs, and none of the effect of babesiosis on the urine proteome. This study aimed to identify urinary proteins of dogs with kidney injury during the natural course of babesiosis caused by Babesia canis, and to compare them with proteins in a control group to reveal any potential biomarkers predicting renal injury before the presence of azotemia.Urine samples were collected from 10 dogs of various breeds and sex with naturally occurring babesiosis, and 10 healthy dogs. Pooled urine samples from both groups were separated by 2D (two-dimensional) electrophoresis, followed by protein identification using MALDI-TOF (matrix-assisted laser desorption ionization time of flight) mass spectrometry.ResultsIn total, 176 proteins were identified in the urine samples from healthy dogs, and 403 proteins were identified in the urine samples from dogs with babesiosis. Of the 176 proteins, 146 were assigned exclusively to healthy dogs, and 373 of the 403 proteins were assigned exclusively to dogs with babesiosis; 30 proteins were common for both groups. Characteristic analysis of 373 proteins found in dogs with babesiosis led to the isolation of 8 proteins associated with 10 metabolic pathways involved in immune and inflammatory responses.ConclusionsIt was hypothesized that epithelial-mesenchymal transition might play an important role in the mechanisms underlying pathological changes in renal tissue during babesiosis, as indicated by a causal relationship network built by combining 5 of the 10 selected metabolic pathways, and 4 of the 8 proteins associated with these pathways; this network included cadherins, gonadotropin releasing hormone receptors, inflammatory responses mediated by chemokine and cytokine signalling pathways, integrins, interleukins, and TGF-β (transforming growth factor β) pathways. Those pathways were linked by interleukin-13, bone morphogenetic protein 7, α2(1) collagen, and tyrosine protein kinase Fer, which are potential biomarkers of damage during babesiosis in dogs, that might indicate early renal injury.