Human Adipose Tissue-Derived Stromal Cells Ameliorate Adriamycin-Induced Nephropathy by Promoting Angiogenesis.

This study is to investigate the therapeutical effect and mechanisms of human-derived adipose mesenchymal stem cells (ADSC) in relieving adriamycin (ADR)-induced nephropathy (AN). SD rats were separated into normal group, ADR group, ADR+Losartan group (20 mg/kg), and ADR + ADSC group. AN rats were induced by intravenous injection with adriamycin (8 mg/kg), and 4 d later, ADSC (2 × 105 cells/mouse) were administrated twice with 2 weeks interval time (i.v.). The rats were euthanized after the 6 weeks' treatment. Biochemical indicators reflecting renal injury, such as blood urea nitrogen (BUN), neutrophil gelatinase alpha (NGAL), serum creatinine (Scr), inflammation, oxidative stress, and pro-fibrosis molecules, were evaluated. Results demonstrated that we obtained high qualified ADSCs for treatment determined by flow cytometry, and ADSCs treatment significantly ameliorated renal injuries in DN rats by decreasing BUN, Scr and NGAL in peripheral blood, as well as renal histopathological injuries, especially protecting the integrity of podocytes by immunofluorescence. Furthermore, ADSCs treatment also remarkably reduced the renal inflammation, oxidative stress, and fibrosis in DN rats. Preliminary mechanism study suggested that the ADSCs treatment significantly increased renal neovascularization via enhancing proangiogenic VEGF production. Pharmacodynamics study using in vivo imaging confirmed that ADSCs via intravenous injection could accumulate into the kidneys and be alive at least 2 weeks. In a conclusion, ADSC can significantly alleviate ADR-induced nephropathy, and mainly through reducing oxidative stress, inflammation and fibrosis, as well as enhancing VEGF production.

Hydrangea paniculata coumarins alleviate adriamycin-induced renal lipotoxicity through activating AMPK and inhibiting C/EBPß.

ETHNOPHARMACOLOGICAL RELEVANCE: Throughout Chinese history, Hydrangea paniculata Siebold has been utilized as a traditional medicinal herb to treat a variety of ailments associated to inflammation. In a number of immune-mediated kidney disorders, total coumarins extracted from Hydrangea paniculata (HP) have demonstrated a renal protective effect. AIM OF THE STUDY: To investigate renal beneficial effect of HP on experimental Adriamycin nephropathy (AN), and further clarify whether reversing lipid metabolism abnormalities by HP contributes to its renoprotective effect and find out the underlying critical pathways. MATERIALS AND METHODS: After establishment of rat AN model, HP was orally administrated for 6 weeks. Biochemical indicators related to kidney injury were determined. mRNAs sequencing using kidney tissues were performed to clarify the underlying mechanism. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, western blot, molecular docking, and drug affinity responsive target stability (DARTS) assay was carried out to further explore and confirm pivotal molecular pathways and possible target by which HP and 7-hydroxylcoumarin (7-HC) played their renal protection effect via modulating lipid metabolism. RESULTS: HP could significantly improve renal function, and restore renal tubular abnormal lipid metabolism and interstitial fibrosis in AN. In vitro study demonstrated that HP and its main metabolite 7-HC could reduce ADR-induced intracellular lipid deposition and fibrosis characteristics in renal tubular cells. Mechanically, HP and 7-HC can activate AMP-activated protein kinase (AMPK) via direct interaction, which contributes to its lipid metabolism modulation effect. Moreover, HP and 7-HC can inhibit fibrosis by inhibiting CCAAT/enhancer binding protein beta (C/EBPß) expression in renal tubular cells. Normalization of lipid metabolism by HP and 7-HC further provided protection of mitochondrial structure integrity and inhibited the nuclear factor kappa-B (NF-κB) pathway. Long-term toxicity using beagle dogs proved the safety of HP after one-month administration. CONCLUSION: Coumarin derivates from HP alleviate adriamycin-induced lipotoxicity and fibrosis in kidney through activating AMPK and inhibiting C/EBPß.

Minnelide Markedly Reduces Proteinuria in Mice with Adriamycin Nephropathy by Protecting Against Podocyte Injury.

Minimal change disease (MCD) is the most common cause of idiopathic nephrotic syndrome in children. The current major therapy is hormones for most steroid-sensitive patients. However, many patients have recurrent relapses of the disease and require long-term immunosuppression, leading to significant morbidity due to the side effects of the drugs. Therefore, better drugs need to be urgently explored to treat nephrotic syndrome while avoiding the side effects of drugs. Minnelide, a water-soluble prodrug of triptolide, has been proved to be effective in treating cancers in many clinical trials. This study aimed to investigate the therapeutic effect of minnelide in mice with adriamycin (ADR) nephropathy, its underlying protection mechanisms, and its reproductive toxicity. Minnelide was administered intraperitoneally to 6-8-week female mice with adriamycin nephropathy for 2 weeks, and the urine, blood, and kidney tissues were taken to analyze the therapeutic effect. In addition, we evaluated reproductive toxicity by measuring the levels of gonadal hormones and observing the histological changes in ovaries and testes. Primary mouse podocytes were exposed to puromycin (PAN) to damage the cytoskeleton and induce apoptosis, and then, triptolide was used to evaluate the therapeutic effect and underlying protection mechanisms in vitro. It was observed that minnelide dramatically alleviated proteinuria and apoptosis in mice with adriamycin nephropathy. In vitro, triptolide ameliorated puromycin-induced cytoskeletal rearrangement and apoptosis via reactive oxygen species-mediated mitochondrial pathway. In addition, minnelide caused no reproductive toxicity to male and female mice. The results suggested that minnelide might be a promising drug for nephrotic syndrome.

Relative comparison of chronic kidney disease-mineral and bone disorder rat models.

Objective: The aim of this study is to establish a suitable animal model of chronic kidney disease-mineral and bone disorder (CKD-MBD) by comparing CKD-MBD rat models induced by 5/6 Nx, AN, and UUO, accompanied by a low-calcium and high-phosphorus diet. Methods: Sprague‒Dawley rats were randomly divided into four groups: control group, 5/6 nephrectomy (5/6 Nx) group, Adriamycin nephropathy (AN) group, and unilateral ureteral obstruction (UUO) group. Serum biochemical indices were measured to evaluate renal function, mineral and bone metabolism, the severity of CKD-MBD, and the status of bone transformation. Hematoxylin-eosin staining (HE) and Masson's trichrome (Masson) staining were used for histopathological analysis of the kidney. Goldner's trichrome (Goldner) and tartrate-resistant acid phosphatase (TRAP) staining were utilized to observe bone mineralization and osteoclasts in the femur, respectively. Micro-CT images were applied to study the structure of the femur. The expression levels of osterix and cathepsin K in the femur were measured by immunohistochemistry (IHC) to confirm the status of bone transformation. Results: The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) in the 5/6 Nx and AN group rats were significantly higher than those in the control rats, and this change was accompanied by marked changes in the levels of calcium (Ca), phosphate (Pi), intact parathyroid hormone (i-PTH), fibroblast growth factor 23 (FGF23), osteocalcin (OC), and cross-linked C-telopeptide of type 1 collagen (CTX-1); UUO group rats exhibited slight and inconsistent variations in the levels of Scr, BUN, Ca, Pi, i-PTH, FGF23, OC, and CTX-1 in serum. Histopathological analysis of the kidney showed that the UUO group rats suffered serious fibrosis and 5/6 Nx group rats exhibited severe focal calcification. Histopathological analysis of the femur showed that the AN group rats had minimal bone mineralization and that the 5/6 Nx group rats had overactive osteoclasts. Micro-CT revealed that the AN model had the most severe bone destruction and that the 5/6 Nx model had the least severe bone loss among the three models. The expression of cathepsin K in the femur was significantly increased in all models, while the expression of osterix in the femur was only significantly increased in the 5/6 Nx model. Conclusion: 5/6 Nx, AN, and UUO accompanied by a low-calcium and high-phosphorus diet successfully induced CKD-MBD in rats. The 5/6 NX model presented the progression of high-turnover bone disease, with consistency between biochemical indices in serum and histomorphometric analysis of the femur, and the AN and UUO models developed a severe deterioration in bone quantity and severe bone resorption; however, the changes in biochemical indices were subtle in the UUO model, and liver injury was obvious in the AN model.

Minnelide combined with Angptl3 knockout completely protects mice with adriamycin nephropathy via suppression of TGF-ß1-Smad2 and p53 pathways.

Minimal change disease (MCD) is the common type of nephrotic syndrome in children. There is an urgent need to explore new treatment methods as current treatments have many drawbacks and cause significant side effects. Our group found that Angiopoietin-like protein 3 (Angptl3) is closely related to renal disease and Angptl3 knockout significantly alleviated proteinuria in mice with adriamycin nephropathy (AN), however, some proteinuria was still present. Minnelide is a water-soluble prodrug of triptolide which has been used for the treatment of glomerular diseases. Therefore, this study aimed to investigate whether minnelide, combined with Angptl3 knockout, could completely protect mice with AN and its mechanism. AN was induced in B6;129S5 female mice by tail vein injection of 25 mg/kg of Adriamycin (ADR), and treatment with 200 ug/kg/d of minnelide. The results showed that minnelide combined with Angptl3 knockout completely reduced proteinuria and restored the foot processes in mice with AN. Moreover, in Angptl3 knockout mice with AN, minnelide restored the distribution of nephrin, podocin and cd2ap and reduced inflammatory factors (Tumor necrosis factor alpha (TNF-α), Interleukin-6 (IL-6) and Interleukin-1ß (IL-1ß)). Through RNA sequencing and related experiments, we found minnelide could ameliorate fibrosis and apoptosis by inhibiting TGF-ß1-Smad2 and p53 pathways in Angptl3 knockout mice with AN, respectively. In Angptl3 knockout primary podocytes, triptolide alleviates ADR-induced decreases in nephrin, podocin and cd2ap, upregulation of Bax and downregulation of Bcl-2. Overall, our study shows that minnelide combined with Angptl3 knockout completely protects mice with AN by inhibiting the TGF-ß1-smad2 and p53 pathways.

Heparanase Increases Podocyte Survival and Autophagic Flux after Adriamycin-Induced Injury.

The kidney glomerular filtration barrier (GFB) is enriched with heparan sulfate (HS) proteoglycans, which contribute to its permselectivity. The endoglycosidase heparanase cleaves HS and hence appears to be involved in the pathogenesis of kidney injury and glomerulonephritis. We have recently reported, nonetheless, that heparanase overexpression preserved glomerular structure and kidney function in an experimental model of Adriamycin-induced nephropathy. To elucidate mechanisms underlying heparanase function in podocytes-key GFB cells, we utilized a human podocyte cell line and transgenic mice overexpressing heparanase. Notably, podocytes overexpressing heparanase (H) demonstrated significantly higher survival rates and viability after exposure to Adriamycin or hydrogen peroxide, compared with mock-infected (V) podocytes. Immunofluorescence staining of kidney cryo-sections and cultured H and V podocytes as well as immunoblotting of proteins extracted from cultured cells, revealed that exposure to toxic injury resulted in a significant increase in autophagic flux in H podocytes, which was reversed by the heparanase inhibitor, Roneparstat (SST0001). Heparanase overexpression was also associated with substantial transcriptional upregulation of autophagy genes BCN1, ATG5, and ATG12, following Adriamycin treatment. Moreover, cleaved caspase-3 was attenuated in H podocytes exposed to Adriamycin, indicating lower apoptotic cell death in H vs. V podocytes. Collectively, these findings suggest that in podocytes, elevated levels of heparanase promote cytoprotection.

Astragalus polysaccharides affects multidrug resistance gene 1 and P-glycoprotein 170 in adriamycin nephropathy rats via regulating microRNA-16/NF-κB axis. / 黄芪多糖调控微RNA-16/NF-κBè½´å¯¹é˜¿éœ‰ç´ è‚¾ç— å¤§é¼ å¤šè¯è€è¯åŸºå› 1和P-糖蛋白170的影响.

OBJECTIVES: Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms. METHODS: A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1ß, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB. RESULTS: The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1ß, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (all P<0.05); the levels of ALB and miR-16 were significantly decreased (both P<0.05). Compared with the model group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1ß, TNF-α, and MDR1 mRNA, and the protein levels of pNF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased (all P<0.05); and the levels of ALB and miR-16 were significantly increased (both P<0.05). Compared with APS+miR-16 antagomir control group, the UP, CHOL, BUN, SCr, IL-6, IL-1ß, and TNF-α levels, MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased (all P<0.05). The levels of ALB and miR-16 were significantly decreased in the APS+miR-16 antagomir group compared with the APS+miR-16 antagomir control group (both P<0.05). CONCLUSIONS: APS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.

Artemether attenuates renal tubular injury by targeting mitochondria in adriamycin nephropathy mice.

Chronic kidney disease (CKD) is complex and current treatment remains limited. As we know, glomerular injury plays a dominant role in kidney disease progression. However, accumulating evidence demonstrated that renal tubules, rather than being victims or bystanders, are major initiators in renal fibrosis progression. Renal tubules are rich in mitochondria and mitochondrial dysfunction may participate in renal tubular phenotypic changes and ultimately promote renal fibrosis. Previous studies have proved that artemether displayed renal protective effects, but the mechanisms remain unclear. In this experiment, we showed that artemether reduced urinary protein/creatinine ratio and attenuated renal tubular injury. Both in vivo and in vitro results indicated that artemether could restore renal tubular phenotypic alterations. Meanwhile, the unbalanced expressions of Bax and Bcl-xL in renal tubules were restored by artemether. In addition, artemether also regulated mitochondrial pyruvate metabolism, increased mitochondrial biogenesis, and improved mitochondrial function. Taken together, this study suggested that artemether could attenuate renal tubular injury by regulating mitochondrial biogenesis and function. It has great potential to be translated to the clinic as a therapeutic agent for treating kidney diseases, especially those associated with renal tubular injury.

Effects of a Chinese herbal extract on the intestinal tract and aquaporin in Adriamycin-induced nephropathy.

Wuling Decoction is a traditional Chinese medicine that has been used to open knots, benefit water, transform Qi, return fluid, and has a significant effect on strengthening the spleen and removing dampness. To explore the effects of Wuling Decoction on the intestinal tract and aquaporin in Adriamycin-induced nephropathy, 45 specific pathogen free (SPF) Wistar rats were randomly divided into a blank control group (5 rats), Dosing control group (10 rats), Adriamycin nephropathy model group (10 rats), diarrhea group (10 rats), and an Adriamycin nephropathy diarrhea model group (10 rats). The tissue localization of aquaporin (AQP) was determined by immunohistochemistry. The expression of AQP mRNA and protein was measured by RT-PCR and western blot analysis, respectively. The results indicated that Wuling Decoction causes excretion of AQP2 through the urine, regulates AQP2 levels, and exerts diuretic and anti-diarrheal effects. It also regulates the levels of antidiuretic hormone (ADH) and arginine vasopressin (AVP), affects water absorption rate, and reduces the level of cyclic adenosine monophosphate (cAMP) in each tissue, thus reducing the absorption of AQP2 to water. Wuling Decoction promoted AQP2 expression in the nephropathy model group and inhibited AQP2 expression in the diarrhea group. Wuling Decoction increased the expression of aquaporin in the intestinal tract, reduced the water content of stool by promoting the absorption of water in the intestinal tract, inhibited the expression of aquaporin and its regulatory factors in nephridia tissue, and reduced the reabsorption of water to increase urine volume, to decrease the occurrence of diarrhea.

Astragalus polysaccharides affects multidrug resistance gene 1 and P-glycoprotein 170 in adriamycin nephropathy rats via regulating microRNA-16/NF-κB axis / 中南大学学报(医学版)

OBJECTIVES@#Nephrotic syndrome is a common disease of the urinary system. The aim of this study is to explore the effect of astragalus polysaccharides (APS) on multidrug resistance gene 1 (MDR1) and P-glycoprotein 170 (P-gp170) in adriamycin nephropathy rats and the underlying mechanisms.@*METHODS@#A total of 72 male Wistar rats were divided into a control group, a model group, an APS low-dose group, an APS high-dose group, an APS+micro RNA (miR)-16 antagomir group and an APS+miR-16 antagomir control group, with 12 rats in each group. Urine protein (UP) was detected by urine analyzer, and serum cholesterol (CHOL), albumin (ALB), blood urea nitrogen (BUN), and creatinine (SCr) were detected by automatic biochemical analyzer; serum interleukin-6 (IL-6), IL-1β, tumor necrosis factor α (TNF-α) levels were detected by ELISA kit; the morphological changes of kidney tissues were observed by HE staining; the levels of miR-16 and MDR1 mRNA in kidney tissues were detected by real-time RT-PCR; the expression levels of NF-κB p65, p-NF-κB p65, and P-gp170 protein in kidney tissues were detected by Western blotting; and dual luciferase was used to verify the relationship between miR-16 and NF-κB.@*RESULTS@#The renal tissue structure of rats in the control group was normal without inflammatory cell infiltration. The renal glomeruli of rats in the model group were mildly congested, capillary stenosis or occlusion, and inflammatory cell infiltration was obvious. The rats in the low-dose and high-dose APS groups had no obvious glomerular congestion, the proliferation of mesangial cells was significantly reduced, and the inflammatory cells were reduced. Compared with the high-dose APS group and the APS+miR-16 antagomir control group, there were more severe renal tissue structure damages in the APS + miR-16 antagomir group. Compared with the control group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 in the model group were significantly increased (all P<0.05); the levels of ALB and miR-16 were significantly decreased (both P<0.05). Compared with the model group, the levels of UP, CHOL, BUN, SCr, IL-6, IL-1β, TNF-α, and MDR1 mRNA, and the protein levels of pNF-κB p65 and P-gp170 in the low-dose and high-dose APS groups were significant decreased (all P<0.05); and the levels of ALB and miR-16 were significantly increased (both P<0.05). Compared with APS+miR-16 antagomir control group, the UP, CHOL, BUN, SCr, IL-6, IL-1β, and TNF-α levels, MDR1 mRNA, and the protein levels of p-NF-κB p65 and P-gp170 were significantly increased (all P<0.05). The levels of ALB and miR-16 were significantly decreased in the APS+miR-16 antagomir group compared with the APS+miR-16 antagomir control group (both P<0.05).@*CONCLUSIONS@#APS can regulate the miR-16/NF-κB signaling pathway, thereby affecting the levels of MDR1 and P-gp170, and reducing the inflammation in the kidney tissues in the adriamycin nephropathy rats.

Ultrasound-Guided Transplantation of Mesenchymal Stem Cells Improves Adriamycin Nephropathy in Rats Through the RIPK3/MLKL and TLR-4/NF-κB Signaling.

Bone marrow stromal cell (BMSC) treatment has been shown to be beneficial for Adriamycin nephropathy (ADR). However, the low transplantation rate is still the key factor that affects this strategy. This study is the first to investigate the efficacy and potential mechanism of ultrasound-guided transrenal arterial transfer of BMSCs for the treatment of ADR in rats. The ADR rat model was established by two injections of doxorubicin. In addition, the rats were randomly divided into four groups (10 rats per group): the normal group (no treatment), the medium control group (treated with medium), the Adriamycin group (treated with phosphate buffer), and the BMSC group (treated with BMSCs). After 4 weeks, the levels of serum creatinine (SCr), blood urea nitrogen (BUN), and urine albumin (ALb) were measured. In addition, pathological changes in kidney tissue were evaluated by pathological sectioning and electron microscopy. Western blotting was used to determine the levels of proteins in rat kidneys. Ultrasound-guided renal artery transplantation of BMSCs reduced the levels of SCr, BUN, and ALb and improved the pathological structure of rat kidneys compared with those in the Adriamycin group. This treatment inhibited renal cell necrosis by reducing the expression of receptor-interacting Serine/threonine Kinase 3 (RIPK3) and Mixed lineage kinase domain-like pseudokinase (MLKL) and inhibited renal inflammation and fibrosis by reducing the expression of Toll-Like receptor 4 (TLR4) and nuclear factor κB (NF-κB). Our study shows that ultrasound-guided transrenal artery transplantation of BMSCs can improve adriamycin-induced renal injury in rats by regulating the RIPK3/MLKL and TLR-4/NF-κB pathways and inhibiting renal necrosis, inflammation, and fibrosis.

Protective action of ultrasound-guided intraparenchymal transplantation of BMSCs in adriamycin nephropathy rats through the RIPK3/MLKL and NLRP3 pathways.

BACKGROUND: Bone marrow stromal cells (BMSCs) are an effective new strategy for the treatment of kidney diseases. At present, noninvasive and efficient transplantation approaches to homing BMSCs to the renal parenchyma is still a serious challenge. The aim of this study was to investigate the feasibility and potential mechanism of ultrasound-guided intraparenchymal transplantation of BMSCs for the treatment of adriamycin nephropathy (AN) in rats. MATERIALS AND METHODS: A rat AN model was induced by 2 injections of doxorubicin. The rats were randomly divided into 4 groups (n = 10 animals in each group) : normal group (N group, no treatment), control medium group (CM group, transplant medium 1.0 mL), adriamycin nephropathy group (ADR group, phosphate buffered saline 1.0 mL), or BMSCs group (BMSCs fluid 1.0 mL). Intraparenchymal injection was completed under ultrasound guidance. After 4 weeks of treatment, blood samples were collected for serum biochemical measurements and ELISAs. The kidneys were removed for histopathological examination, electron microscopy, terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), and western blot analysis. RESULTS: No deaths occurred in any group after BMSCs transplantation through the renal parenchyma under ultrasound guidance. Compared with the N and CM groups, in the ADR group, blood serum creatinine (SCr), blood urea nitrogen (BUN) and urine albumin (ALb) were higher, glomerular and tubular dilatation was observed, the number of apoptotic cells was higher, and the protein levels of receptor-interacting protein kinase 3 (RIPK3)/mixed lineage kinase domain-like protein (MLKL) and nucleotide leukin-rich polypeptide 3 (NLRP3), key components of pathways in rat kidney, were significantly higher. Compared with those in the ADR group, the levels of SCr, BUN, ALb and serum proinflammatory cytokines in the BMSCs group were lower, the pathological structure of the kidney was improved, the number of apoptotic cells was lower, and the levels of RIPK3/MLKL and NLRP3 were significantly lower. CONCLUSION: Ultrasound-guided intraparenchymal transplantation of BMSCs regulated the RIPK3/MLKL and NLRP3 pathways in a minimally invasive and safe manner, thereby inhibiting renal necrosis and inflammation and playing a protective role in rat AN.

A single amino acid substitution in PRKDC is a determinant of sensitivity to Adriamycin-induced renal injury in mouse.

Adriamycin (ADR)-induced nephropathy is frequently utilized in rodent models of podocytopathy. However, the application of this model in mice is limited to a few strains, such as BALB/c mice. The most commonly used mouse strain, C57BL/6 (B6), is resistant to ADR-induced nephropathy, as are all mouse strains with a B6 genetic background. Reportedly, the R2140C variant of the Prkdc gene is the cause of susceptibility to ADR-induced nephropathy in mice. To verify this hypothesis, we produced Prkdc mutant B6 mice, termed B6-PrkdcR2140C, that possess the R2140C mutation. After administration of ADR, B6-PrkdcR2140C mice exhibited massive proteinuria and glomerular and renal tubular injuries. In addition, there was no significant difference in the severity between B6-PrkdcR2140C and BALB/c. These findings demonstrated that B6-PrkdcR2140C show ADR-induced nephropathy susceptibility at a similar level to BALB/c, and that the PRKDC R2140C variant causes susceptibility to ADR-induced nephropathy. In future studies, ADR-induced nephropathy may become applicable to various kinds of genetically modified mice with a B6 background by mating with B6-PrkdcR2140C.

Circadian rhythm of the intrarenal renin-angiotensin system is caused by glomerular filtration of liver-derived angiotensinogen depending on glomerular capillary pressure in adriamycin nephropathy rats.

Circadian fluctuation disorder of the intrarenal renin-angiotensin system (RAS) causes that of blood pressure (BP) and renal damage. In renal damage with an impaired glomerular filtration barrier, liver-derived angiotensinogen (AGT) filtered through damaged glomeruli regulates intrarenal RAS activity. Furthermore, glomerular permeability is more strongly affected by glomerular hypertension than by systemic hypertension. Thus, we aimed to clarify whether the circadian rhythm of intrarenal RAS activity is influenced by AGT filtered through damaged glomeruli due to glomerular capillary pressure. Rats with adriamycin nephropathy and an impaired glomerular filtration barrier were compared with control rats. In adriamycin nephropathy rats, olmesartan medoxomil (an angiotensin II type 1 receptor blocker) or hydralazine (a vasodilator) was administered, and the levels of intrarenal RAS components in the active and rest phases were evaluated. Moreover, the diameter ratio of afferent to efferent arterioles (A/E ratio), an indicator of glomerular capillary pressure, and the glomerular sieving coefficient (GSC) based on multiphoton microscopy in vivo imaging, which reflects glomerular permeability, were determined. Mild renal dysfunction was induced, and the systemic BP increased, resulting in increased A/E ratios in the adriamycin nephropathy rats compared with the control rats. Fluctuations in intrarenal RAS activity occurred in parallel with circadian fluctuations in glomerular capillary pressure, which disappeared with olmesartan treatment and were maintained with hydralazine treatment. Furthermore, the GSCs for AGT also showed similar changes. In conclusion, intrarenal RAS activity is influenced by the filtration of liver-derived AGT from damaged glomeruli due to circadian fluctuation disorder of the glomerular capillary pressure.

Olive leaf extract attenuates adriamycin-induced focal segmental glomerulosclerosis in spontaneously hypertensive rats via suppression of oxidative stress, hyperlipidemia, and fibrosis.

Olive (Olea europaea L.) leaf extract (OLE) possesses powerful antioxidant, antihyperlipidemic, and anti-inflammatory properties. The aim was to investigated the effects of OLE on the hyperlipidemia, antioxidant defense, heme oxygenase/biliverdin reductase (HO/BVR) pathway, inflammation, and fibrosis in spontaneously hypertensive rats with focal segmental glomerulosclerosis (FSGS, a progressive form of chronic kidney disease) induced by adriamycin (2 mg/kg, i.v., twice in a 21-day period). Daily treatment of OLE (80 mg/kg, p.o.) for 6 weeks suppressed protein oxidation and lipid peroxidation (p < .01 and p < .001, respectively), significantly increased antioxidant enzymes activities and normalized antioxidant capacity, leading to the improvement of antioxidant defense independently of the HO/BVR pathway. Furthermore, the values of triglycerides (p < .01), total, and low-density lipoprotein cholesterol (p < .05, both) were improved by OLE. OLE strongly prevented glomerulosclerosis, interstitial inflammation, and fibrosis (renal injury score, FSGS: 8 ± 0.45 vs. FSGS+OLE: 4.20 ± 1.07; p < .01), as evidenced by normalized fibronectin content (p < .001), suppressed interstitial inflammatory cells infiltration and collagen deposition, without changing cytokines expressions. OLE decreased blood pressure with a tendency to reduce urine albumin loss. These data suggest that OLE may be effective in slowing down the progression of FSGS.

Regenerated nephrons in kidney cortices ameliorate exacerbated serum creatinine levels in rats with adriamycin nephropathy.

Kidney regeneration could be classified into 2 groups: kidney generation and kidney repair. We have attempted in vivo nephron generation for kidney repair, as a therapy for chronic renal failure (CRF), by exploiting cellular interactions via conditioned media. In the previous report, we demonstrated the generation of rich nephrons in rat intact kidney cortices through percapsular injection of mesenchymal stem cell (MSC)-differentiated tubular epithelial cells (TECs) after pretreatment of 3-dimensional culture using a small amount of gel complex and condensed medium. In this study, to verify the amelioration of serum creatinine (sCr) levels by regenerated nephrons in rats with CRF, we first created damaged kidneys through systemic administration of adriamycin, and implanted the pretreated MSC-differentiated TECs into unilateral kidney cortices 2 weeks after adriamycin administration (A-2W, that is I-0W). After recovery of acute kidney injury, the control rats without cell implantation showed re-exacerbation of sCr levels, resulting in death within A-12W. Alternatively, the cell-implanted rats had a formation of mature nephrons in I-3W, and showed significant amelioration of sCr levels in I-7W. As a result, these rats could live until euthanization in I-12W or I-16W, indicating the utility of cell injection therapy into a kidney (K-CIT) for CRF. We expect that our K-CIT or the refined methods will be applied to patients with CRF.

An integrated transcriptomics and network pharmacology approach to exploring the mechanism of adriamycin-induced kidney injury.

BACKGROUND AND AIMS: Adriamycin nephropathy model (AN), a rodent model of nephrotic syndrome disease that was caused by the nephrotoxicity of adriamycin, has been widely used for pharmacodynamic evaluation of traditional Chinese medicine (TCM) in the treatment of kidney injury. Although some studies have clearly shown the pathological process of AN, the mechanism of kidney injury have not been systematically investigated. METHODS: The reliability of AN was evaluated by weight, urinary protein quantitation, serum biochemical and histopathological examination. Transcriptomic sequencing combined with network pharmacology were used to elucidate the molecular mechanism of AN, and cell experiment combined with real-time quantitative PCR (RT-qPCR) and was used to validate the accuracy of transcriptomic sequencing result and KEGG pathways. RESULTS: Network analysis result showed that Mapk10 and Ptgs2 played important roles in the development of adriamycin-induced kidney injury. KEGG pathway analysis showed that the mechanism of kidney injury may be related to the regulation of biosynthesis of unsaturated fatty acids, complement and coagulation cascades, PPAR signaling pathway and PI3K-AKT signaling pathway. CONCLUSION: These results provide a new insight into the deep research on the mechanism of kidney injury, and provide an experimental basis for finding drug targets for the treatment of AN.

Exploration the active compounds of Astragali Radix in treatment of adriamycin nephropathy by network pharmacology combined with transcriptomic approach.

PURPOSE: This paper aimed to study the active compounds of Astragali Radix (AR) in the treatment of adriamycin nephropathy (AN) by a combination of network pharmacology and transcriptomics. METHODS: The chemical compounds of AR were screened out by text mining and database searching. Pharm Mapper was used to predict the targets of these chemical compounds. Potential targets of AN were screened by integrating the data from network pharmacology with known transcriptomics analysis results of kidney tissue. Compound-active target-potential target interactions networks were constructed so as to illustrate the relationship between compounds and targets, and obtain the chemical compounds directly related to potential targets of AN. The formula of compound contribution index (CI) based on algorithm was used to screen the active compounds of AR in the treatment of AN. In addition, we established an adriamycin-induced cell damage model with MPC5 cell, and used MTT assay, trypan blue dyeing and western blot analyses to validate the pharmacodynamic effect of the active compounds. RESULTS: 27 chemical compounds and 376 targets in AR were obtained by network pharmacology. Through Compound-active target-potential target interactions networks analysis, 22 compounds and 9 active targets as well as 130 potential targets were linked through 282 edges. The CI of every chemical compounds was further calculated by formula, the first four chemical compounds, including astragaloside IV, formononetin, quercetin and calycosin, whose cumulative contribution rate reached 87.28%, were considered to be active compounds. The results of MTT and trypan blue staining indicate that four active compounds had the significant protective effect on adriamycin-induced cell damage with MPC5 cell. Western blot result showed that four active compounds could significantly increase the expression of podocin protein in MPC5 cell. CONCLUSION: The active compounds of AR in the treatment of AN were successfully identified by using a network pharmacology and transcriptomics approach. This approach is expected to be beneficial to the study of the pharmacodynamic material basis of traditional Chinese medicine (TCM) in treating specific diseases.

C-C chemokine receptor type 2 mediates glomerular injury and interstitial fibrosis in focal segmental glomerulosclerosis.

BACKGROUND: Glomerulosclerosis and tubulointerstitial fibrosis are hallmarks of chronic kidney injury leading to end-stage renal disease. Inflammatory mechanisms contribute to glomerular and interstitial scarring, including chemokine-mediated recruitment of leucocytes. In particular, accumulation of C-C chemokine receptor type 2 (CCR2)-expressing macrophages promotes renal injury and fibrotic remodelling in diseases like glomerulonephritis and diabetic nephropathy. The functional role of CCR2 in the initiation and progression of primary glomerulosclerosis induced by podocyte injury remains to be characterized. METHODS: We analysed glomerular expression of CCR2 and its chemokine ligand C-C motif chemokine ligand 2 (CCL2) in human focal segmental glomerulosclerosis (FSGS). Additionally, CCL2 expression was determined in stimulated murine glomeruli and glomerular cells in vitro. To explore pro-inflammatory and profibrotic functions of CCR2 we induced adriamycin nephropathy, a murine model of FSGS, in BALB/c wild-type and Ccr2-deficient mice. RESULTS: Glomerular expression of CCR2 and CCL2 significantly increased in human FSGS. In adriamycin-induced FSGS, progressive glomerular scarring and reduced glomerular nephrin expression was paralleled by induced glomerular expression of CCL2. Adriamycin exposure stimulated secretion of CCL2 and tumour necrosis factor-α (TNF) in isolated glomeruli and mesangial cells and CCL2 in parietal epithelial cells. In addition, TNF induced CCL2 expression in all glomerular cell populations, most prominently in podocytes. In vivo, Ccr2-deficient mice with adriamycin nephropathy showed reduced injury, macrophage and fibrocyte infiltration and inflammation in glomeruli and the tubulointerstitium. Importantly, glomerulosclerosis and tubulointerstitial fibrosis were significantly ameliorated. CONCLUSIONS: Our data indicate that CCR2 is an important mediator of glomerular injury and progression of FSGS. CCR2- targeting therapies may represent a novel approach for its treatment.

Podocyte GSK3α is important for autophagy and its loss detrimental for glomerular function.

Podocytes are key cells in maintaining the integrity of the glomerular filtration barrier and preventing albuminuria. Glycogen synthase kinase 3 (GSK3) is a multi-functional serine/threonine kinase existing as two distinct but related isoforms (α and ß). In the podocyte it has previously been reported that inhibition of the ß isoform is beneficial in attenuating a variety of glomerular disease models but loss of both isoforms is catastrophic. However, it is not known what the role of GSK3α is in these cells. We now show that GSK3α is present and dynamically modulated in podocytes. When GSK3α is transgenically knocked down specifically in the podocytes of mice it causes mild but significant albuminuria by 6-weeks of life. Its loss also does not protect in models of diabetic or Adriamycin-induced nephropathy. In vitro deletion of podocyte GSK3α causes cell death and impaired autophagic flux suggesting it is important for this key cellular process. Collectively this work shows that GSK3α is important for podocyte health and that augmenting its function may be beneficial in treating glomerular disease.