Nephrotic Rats Research Articles

#3314 DIURETIC AND RENOPROTECTIVE EFFECTS OF GOREISAN IN A RAT MODEL OF NEPHROTIC SYNDROME

Abstract Background and Aims Goreisan, a traditional Chinese medicine composed of five herbs, is commonly used for the purpose of control of edema and gastrointestinal symptoms. However, the detailed mechanisms have not been fully elucidated. In clinical settings, nephrotic syndrome often causes edema, mainly due to hypoalbuminemia and sodium retention. Thus, we investigated the effects of Goreisan on the kidney using a rat model of nephrotic syndrome. Method A model of nephrotic syndrome was created by a right nephrectomy and subsequent administration of adriamycin using Sprague Dawley rats. Nephrotic rats were randomly divided into three groups at 10 weeks of age as follows; vehicle (control), Goreisan 0.5 g/kg (GL), and Goreisan 1.0 g/kg (GH) groups. Goreisan was administered once a day for 4 weeks. At 14 weeks of age, the rats were sacrificed for the evaluation of blood, urine, mRNA expressions, and kidney histopathology. Results Urine volume was significantly increased in the GL and GH groups than in the control group (Δurine volume: control; −1.25±2.48 mL, GL group; 9.83±4.16 mL, GH group; 10.00±2.62 mL). The GH group showed significantly lower urine osmotic pressure compared to the control group. There were no significant differences in urinary sodium excretion and serum arginine-vasopressin levels among the three groups. Subsequently, we assessed the expression of aquaporin (AQP) in kidney tissue using real-time PCR and immunohistochemical staining. The mRNA expression of AQP1, AQP2, AQP3, and AQP4 did not differ among the three groups. However, immunohistochemical staining showed that the localization of AQP2 at the apical plasma membrane of the collecting ducts was decreased by treatment with Goreisan. Furthermore, deterioration of kidney function was prevented by treatment with Goreisan. Histological analysis revealed that glomerular and tubulointerstitial damages were ameliorated in the GL and GH groups compared to the control group (glomerular damage score: control; 2.19±0.05, GL group; 1.71±0.05, GH group; 1.10±0.05, tubulointerstitial damage score: control; 2.55±0.09, GL group; 2.28±0.10, GH group; 2.12±0.10). In addition, urinary 8-OHdG, which is an oxidative stress marker, was significantly lower in the GL and GH groups than in the control group. Conclusion Our results suggested that Goreisan may have diuretic and renoprotective effects by suppressing the trafficking of AQP2 to the apical plasma membrane of the collecting ducts and possibly reducing systemic oxidative stress.

Endothelial Progenitor Cells Affect the Growth and Apoptosis of Renal Cells by Secreting Microvesicles Carrying Dysregulated miR-205 and miR-206.

This study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs). Gene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry. A total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206. By upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.

P-Coumaric Acid Nanoparticles Ameliorate Diabetic Nephropathy via Regulating mRNA Expression of KIM-1 and GLUT-2 in Streptozotocin-Induced Diabetic Rats.

Diabetic nephropathy (DN) has become a leading cause of end-stage renal failure worldwide. The goal of the current study was to examine the protective effects of chitosan-loaded p-Coumaric acid nanoparticles (PCNPs) in nephrotoxicity induced by streptozotocin (STZ). Because of the antidiabetic, anti-inflammatory, and antioxidant properties of PCNPs, the development of DN may be considerably decreased. In this study, the rats received a single intraperitoneal injection (i.p.) of STZ (45 mg/kg) to induce DN. PCNPs were given orally 80 mg/kg b.w to the rats for a duration of four weeks. Body weight, kidney weight, blood glucose, and insulin levels were measured at the end of the experiment. Serum and urine parameters were also examined, along with the histological, immunobiological, and tumor necrosis factor (TNF) and interleukin-6 (IL-6) expression of the nephrotic rats. To comprehend the impact of PCNPs, the expression patterns of the kidney injury molecule (KIM-1) and glucose transporter-2 (GLUT-2) were evaluated. Administration of PCNPs significantly increased body weight, decreased kidney weight and also ameliorated blood glucose levels in the nephropathic rats. The administration of PCNPs also reverted the levels of urea, serum creatinine, urinary NAG, β-glucuronidase and albumin to near-normal levels. The administration of PCNPs also caused the levels of serum and urine parameters to return to near-normal levels. Additionally, the PCNP-treated rats had markedly reduced TNF-α, IL-6, and KIM-1 expressions as well as enhanced GLUT-2 mRNA expression. Our findings clearly showed that PCNP administration prevents the onset of DN in rats by lowering hyperglycemia, decreasing inflammation, and improving the expression of GLUT-2 mRNA in nephropathic rats.

Screening bioactive compounds from Danggui-shaoyao-san for treating sodium retention in nephrotic syndrome using bio-affinity ultrafiltration

Ethnopharmacological relevanceDanggui-shaoyao-san (DSS), a representative formula of Traditional Chinese Medicine (TCM) for promoting blood circulation and diuresis (Huo-Xue-Li-Shui) therapy, has been used to clinically nephrotic syndrome (NS) and relieve nephrotic edema. Aim of the studyTo explore the effects and mechanisms of DSS in improving sodium retention and to identify the bioactive compounds from DSS. Materials and methodsDSS prescriptions were disassembled into Yangxue-Huoxue (YXHX) and Jianpi-Lishui (JPLS). A nephrotic rat model was induced with puromycin aminonucleoside (PAN), and the effects on urinary sodium excretion, urinary plasmin(gen) content, and plasmin activity of DSS, YXHX, and JPLS extracts were assessed. The inhibitory effects on urokinase-type plasminogen activator (uPA) and plasmin activity of extracts were evaluated in vitro. Bio-affinity ultrafiltration and high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (BAU-UPLC-Q/TOF-MS) were used to rapidly screen and qualitatively analyze the uPA/plasmin affinity compounds from DSS extract. Additionally, uPA/plasmin inhibition assays and molecular docking were used to verify the activity and affinity mechanisms of the potential bioactive compounds. ResultsIn vivo, DSS, YXHX, and JPLS prevented sodium retention in nephrotic rats. DSS and YXHX treatment decreased urinary plasmin activity but did not alter urinary plasmin(ogen) concentration, and their extracts showed strong uPA and plasmin inhibitory activity in vitro. These results suggested that uPA and plasmin are direct targets of DSS and YXHX in intervening NS sodium retention. Using BAU-UPLC-Q/TOF-MS, gallic acids, methyl gallate, albiflorin, and 1,2,3,4,6-O-pentagalloylglucose (PGG) were screened as uPA or plasmin affinity compounds. Among them, PGG was found to be a uPA and plasmin dual inhibitor, with an IC50 of 6.861 μM against uPA and an IC50 of 149.0 μM against plasmin. The molecular docking results of PGG with uPA and plasmin were consistent with the verification results. ConclusionIntervening in sodium retention by inhibiting uPA-mediated plasmin generation and plasmin activity in the kidneys could be possible mechanisms for DSS, as indicated by the results in PAN-induced nephrotic rats. We conclude that PGG is a potential bioactive compound responsible for the effect of DSS on natriuresis.

A variant of ASIC2 mediates sodium retention in nephrotic syndrome.

Idiopathic nephrotic syndrome (INS) is characterized by proteinuria and renal sodium retention leading to edema. This sodium retention is usually attributed to epithelial sodium channel (ENaC) activation after plasma aldosterone increase. However, most nephrotic patients show normal aldosterone levels. Using a corticosteroid-clamped (CC) rat model of INS (CC-PAN), we showed that the observed electrogenic and amiloride-sensitive Na retention could not be attributed to ENaC. We then identified a truncated variant of acid-sensing ion channel 2b (ASIC2b) that induced sustained acid-stimulated sodium currents when coexpressed with ASIC2a. Interestingly, CC-PAN nephrotic ASIC2b-null rats did not develop sodium retention. We finally showed that the expression of the truncated ASIC2b in the kidney was dependent on the presence of albumin in the tubule lumen and activation of ERK in renal cells. Finally, the presence of ASIC2 mRNA was also detected in kidney biopsies from patients with INS but not in any of the patients with other renal diseases. We have therefore identified a variant of ASIC2b responsible for the renal Na retention in the pathological context of INS.

Shensu IV prevents glomerular podocyte injury in nephrotic rats via promoting lncRNA H19/DIRAS3-mediated autophagy.

Shensu IV is a Chinese prescription well-known for its function in treating chronic kidney diseases. However, the potential mechanisms underlying how Shensu IV exerts its effects remain unclear. In the present study, we investigated the effects of Shensu IV on glomerular podocyte injury in nephrotic rats and puromycin-induced injury in cultured podocytes, and assessed the associated molecular mechanisms. Liquid chromatography–mass spectrometry (LC–MS) results showed that the main components of Shensu IV were l-Carnitine, P-lysoPC (LPC) 16:0, Coumaroyl tyramine, Tetramethylpyrazine, LPC 18:1, Choline, (S,S)-Butane-2,3-diol, and Scopoletin. We further found that nephrotic rats displayed pathological alterations in kidney tissues and ultrastructural changes in glomerular podocytes; however, these effects were reversed with Shensu IV treatment. Compared with the control, the numbers of autophagosomes were markedly reduced in the model group, but not in the Shensu IV treatment group. Furthermore, the expression of p62 was significantly higher in the model group than in the controls, whereas the LC3-II/I ratio was significantly lower; however, these changes were not observed when Shensu IV was administered. The protective effects of Shensu IV were further confirmed in podocytes displaying puromycin-induced injury. Compared with control group, the expression of long non-coding RNA (lncRNA) H19, mTOR, p-mTOR, and p62 was significantly increased in the puromycin group, whereas that of distinct subgroup of the RAS family member 3 (DIRAS3) was significantly decreased, as was the LC3-II/I ratio. The opposite results were obtained for both shH19- and Shensu IV-treated cells. Collectively, our data demonstrated that Shensu IV can prevent glomerular podocyte injury in nephrotic rats and puromycin-treated podocytes, likely via promoting lncRNA H19/DIRAS3-regulated autophagy.

Kidney dysfunction and oxidative stress in doxorubicin-induced nephrotic rat: Protective role of sesame oil.

Doxorubicin (DOX) is an antineoplastic agent which it's clinical use has been limited due to its major side effects including cardiotoxicity and nephrotic syndrome. Sesame oil (SO) is an important edible oil with many pharmacologic effects. The aim of the present study was to investigate the effect of SO against DOX-induced nephropathy in the rat. In this study, two doses of SO (3 and 6 mL/kg) were administrated orally for six consecutive weeks and DOX (mg/kg) was intravenously injected on the 4th day of the experiment. Blood and urine samples were collected on days 1, 14, 30, and 42 for subsequent measurement of biochemical parameters. The left kidneys were removed for subsequent assessment of total thiol content, malondialdehyde (MDA) concentration, and renal activities of catalase and superoxide dismutase enzymes. DOX caused significant proteinuria, hypoalbuminemia, and hyperlipidemia compared to control group. Significant decrease in antioxidant enzyme activities and total thiol contents and significant increase in MDA levels were also observed following DOX injection when compared to control group. Oral administration of SO significantly reversed DOX-induced proteinuria, hypoalbuminemia, and hyperlipidemia compared to DOX group. Furthermore, compared to DOX group, SO significantly increased total thiols content. MDA concentration significantly decreased following SO administration when compared to DOX group. The current study suggests that SO is able to improve kidney function as well as kidney tissue oxidative damage in DOX-induced nephrotic the rat.

RNA Interference Targeting Liver Angiopoietin-Like Protein 3 Protects from Nephrotic Syndrome in a Rat Model Via Amelioration of Pathologic Hypertriglyceridemia.

Nephrotic syndrome (NS) is associated with metabolic perturbances including profound dyslipidemia characterized by hypercholesterolemia and hypertriglyceridemia. A major underlying mechanism of hypertriglyceridemia in NS is lipoprotein lipase (LPL) deficiency and dysfunction. There is emerging evidence that elevated angiopoietin-like protein 3 (ANGPTL3), an LPL inhibitor that is primarily expressed and secreted by hepatocytes, may be in part responsible for these findings. Furthermore, there is evidence pointing to the contribution of ANGPTL3 to the pathogenesis of proteinuria in NS. Therefore, we hypothesized that inhibition of hepatic ANGPTL3 by RNA interference will ameliorate dyslipidemia and other symptoms of NS and pave the way for a new therapeutic strategy. To this end, we used a subcutaneously delivered, GalNAc (N-Acetylgalactosamine)-conjugated small interfering RNA (siRNA) to selectively target and suppress liver Angptl3 in rats with puromycin-induced NS, which exhibits clinical features of NS including proteinuria, hypoalbuminemia, hyperlipidemia, and renal histologic abnormalities. The study demonstrated that siRNA-mediated knockdown of the liver Angptl3 relieved its inhibitory effect on LPL and significantly reduced hypertriglyceridemia in nephrotic rats. This was accompanied by diminished proteinuria and hypoalbuminemia, which are the hallmarks of NS, and significant attenuation of renal tissue inflammation and oxidative stress. Taken together, this study confirmed the hypothesis that suppression of Angptl3 is protective in NS and points to the possibility that the use of RNA interference to suppress hepatic Angptl3 can serve as a novel therapeutic strategy for NS. SIGNIFICANCE STATEMENT: The current standard of care for mitigating nephrotic dyslipidemia in nephrotic syndrome is statins therapy. However, the efficacy of statins and its safety in the context of impaired kidney function is not well established. Here, we present an alternate therapeutic approach by using siRNA targeting Angptl3 expressed in hepatocytes. As the liver is the major source of circulating Angptl3, siRNA treatment reduced the profound hypertriglyceridemia in a rat model of nephrotic syndrome and was also effective in improving kidney and cardiac function.

Combination therapy of phosphatidylserine liposome with cyclosporine A improves nephrotoxicity and attenuates delayed-type hypersensitivity response

This study aimed to evaluate the antioxidant capacity of phosphatidylserine liposome (PS) against oxidative stress due to cyclosporine A (CsA) and concurrent administration of PS and CsA on the attenuation of immune response. The effect of oral PS was evaluated on biochemical and oxidative renal markers and histopathology of nephrotic rats receiving CsA. The effect of co-administration of PS with CsA was also assessed on DTH (delayed-type hypersensitivity) reaction of immunized rats. The cytokines production level of IL-2 (Interleukin-2) and IFN-γ (Interferon gamma) was measured in immunized rat's splenocytes. PS treatment significantly (P < 0.05) reduced Cr and BUN of serum and MDA (malondialdehyde) in kidney tissue, and increased SOD (superoxide dismutase) and CAT (Catalase) of kidney tissue in CsA-nephrotic rats. Histopathology data indicated significantly (P < 0.05) nephrotoxicity improvement after 25-day treatment with PS. Furthermore, CsA plus PS administration significantly reduced DTH response and cytokines production of IL-2 and IFN-γ in immunized rats. In conclusion, coadministration of CsA plus PS may overcome oxidative stress and improve the performance of organ transplantation or autoimmune therapy.

Transcription factor 21 expression in injured podocytes of glomerular diseases

Transcription factor 21 (TCF21) is one of the essential transcription factors in kidney development. To elucidate its influence on glomerular disease, we have investigated TCF21 expression in human and rat kidney tissue, and its urinary concentration. Immunohistological analysis suggested the highest TCF21 expression in nephrotic syndrome along with the urinary protein level. Urinary TCF21 concentration in human showed a positive correlation with its podocyte expression level. In nephrotic rat models, TCF21 expression in podocytes increased along with the severity of nephrotic syndrome. Next, in vitro experiments using Tcf21-expressing murine podocyte cell line, we could observe some Tcf21-dependent effects, related with actin cytoskeleton dysregulation and apoptosis. Our study illustrated TCF21 expression changes in vivo and its in vitro-functional significance injured podocytes.

Protective effect of sulforaphane on kidney injury in rats with nephrotic syndrome

Objective To study the effects of sulforaphane on kidney injury and the expression levels of nitric oxide synthase in rats with nephrotic syndrome. Methods Five mg/kg of doxorubicin was used to construct the nephrotic syndrome model in rats. Rats were divided into control group, model group, sulforaphane treatment group and benazepril (positive drug) treatment group. After treatment with 30 mg/kg of sulforaphane and 6 mg/kg of benazepril for 6 weeks, HE staining was used to analyze pathological changes in kidney tissue in rats with nephrotic syndrome. Proteinuria level in 24 hours was measured by BCA assay. ELISA assay was used to detect the level of plasma albumin. NO content was detected by spectrophotometer. Western blot was used to determine the expression levels of endothelial NOS (eNOS), inducible NOS (iNOS) and neural NOS (nNOS). Results Compared with control group, the 24 hours proteinuria level was significantly increased in model group (P<0.05), and the pathological changes of the kidney were serious. The urine NO content of the model group was greatly decreased (P<0.05), the level of plasma albumin was greatly decreased (P<0.05), and the expression levels of eNOS, iNOS and nNOS were also significantly decreased (P<0.05). Compared with model group, the 24 hours proteinuria level was significantly increased in sulforaphane treatment group and benazepril treatment group group (P<0.05), the level of plasma albumin was greatly increased (P<0.05), and the pathological changes of the kidney were significantly improved. The urine NO content of the treatment groups was greatly increased (P<0.05), and the expression levels of eNOS, iNOS and nNOS were also significantly increased (P<0.05). Conclusions Sulforaphane can improve the expressions of eNOS, iNOS and nNOS in nephrotic syndrome rats, increase the content of NO in rats, reduce the content of urinary protein, and improve the pathological process of nephrotic syndrome. Key words: Models, Animal; Rats; Nephrosis, Lipoid; Sulforaphane

Nephrotic syndrome is associated with increased plasma K+ concentration, intestinal K+ losses, and attenuated urinary K+ excretion: a study in rats and humans.

The present study tested the hypotheses that nephrotic syndrome (NS) leads to renal K+ loss because of augmented epithelial Na+ channel (ENaC) activity followed by downregulation of renal K+ secretory pathways by suppressed aldosterone. The hypotheses were addressed by determining K+ balance and kidney abundance of K+ and Na+ transporter proteins in puromycin aminonucleoside (PAN)-induced rat nephrosis. The effects of amiloride and angiotensin II type 1 receptor and mineralocorticoid receptor (MR) antagonists were tested. Glucocorticoid-dependent MR activation was tested by suppression of endogenous glucocorticoid with dexamethasone. Urine and plasma samples were obtained from pediatric patients with NS in acute and remission phases. PAN-induced nephrotic rats had ENaC-dependent Na+ retention and displayed lower renal K+ excretion but elevated intestinal K+ secretion that resulted in less cumulated K+ in NS. Aldosterone was suppressed at day 8. The NS-associated changes in intestinal, but not renal, K+ handling responded to suppression of corticosterone, whereas angiotensin II type 1 receptor and MR blockers and amiloride had no effect on urine K+ excretion during NS. In PAN-induced nephrosis, kidney protein abundance of the renal outer medullary K+ channel and γ-ENaC were unchanged, whereas the Na+-Cl- cotransporter was suppressed and Na+-K+-ATPase increased. Pediatric patients with acute NS displayed suppressed urine Na+-to-K+ ratios compared with remission and elevated plasma K+ concentration, whereas fractional K+ excretion did not differ. Acute NS is associated with less cumulated K+ in a rat model, whereas patients with acute NS have elevated plasma K+ and normal renal fractional K+ excretion. In NS rats, K+ balance is not coupled to ENaC activity but results from opposite changes in renal and fecal K+ excretion with a contribution from corticosteroid MR-driven colonic secretion.

Zataria multiflora and its main ingredient, carvacrol, affect on the renal function, histopathological, biochemical and antioxidant parameters in adriamycin-induced nephrotic rats

Oxidative stress has a major role in the nephrosis. In the present study, the effects of hydroalcoholic extract of Zataria multiflora (ZM) and carvacrol (CAR) were evaluated on the renal damage induced by adriamycin (ADR). The animals accidentally divided into four groups including: Control, ADR, ZM + ADR and CAR + ADR. The renal tissue, urine, and blood samples subjected to biochemical markers and histopathological evaluation. ADR significantly decreased glomerular filtration rate (GFR) while escalated urine protein excretion as well as protein clearance (p < .01 to p < .001). Also, ADR significantly reduced the antioxidants and boosted the malondialdehyde (MDA) compared to the control (p < .05 to p < .01). In groups treated by ZM and CAR, GFR, and antioxidants significantly increased, whereas urine protein excretion and MDA decreased (p < .05 to p < .001). ZM and CAR induced an improvement in ADR-induced renal damage by improving renal function as well as antioxidant activity.

SUN-003 MECHANISM OF SELECTIVE ALBUMINURIA IN MINIMAL CHANGE NEPHROTIC SYNDROME

Water and electrolytes are filtered through slit membranes, however in minimal change nephrotic syndrome with foot process effacement it is not well understood whether albumin molecules will be filtered through the slit membrane. We reported that albumin endocytosis by FcRn was enhanced in the podocyte of puromycin aminonucleoside (PAN)-induced minimal change nephrotic syndrome rat, and treatment with anti-FcRn antibody decreased albumin endocytosis and urinary protein (Kidney Int 2011). Minimal change nephrotic syndrome model was induced by puromycin amino nucleoside, and podocytes were observed by electron microscope and low vacuum scanning electron microscope. Electron microscopy demonstrated many vesicles were observed in the podocytes of PAN rat with 3D observation. SDS-polyacrylamide electrophoresis of glomerular proteins isolated from PAN rats showed that several protein bands were increased than those from control rats, and those increased protein bands were identified as dynein -1, myosin 7 and myosin 9 by mass spectrometry. These motor molecules are capable of transporting vesicles containing albumin in the podocytes of PAN nephrotic rat. In renal biopsy, electron microscopic examination of human minimal change disease cases showed numerous vesicles and microtubules in podocytes, and a number of exocytotic holes were found on the surface of podocytes by low vacuum scanning electron microscopy. In conclusion, podocyte vesicle transport could play an important role in the selective albuminuria in the minimal change nephrotic syndrome.

A novel splicing variant of small nucleolar RNA host gene 4 is a podocyte-selective non-coding RNA upregulated in response to puromycin aminonucleoside-induced podocyte injury.

Podocytes are terminally differentiated cells that function as the glomerular filtration barrier in the kidney, and podocyte injury leads to serious proteinuria and podocyte leakage into urine. Recent studies have demonstrated that the number of urinary podocytes is correlated with the progression of glomerular diseases. Therefore, urinary podocytes may serve as an indicator of podocyte injury. In this study, to explore podocyte injury-related genes, we performed comprehensive transcriptome analysis of primary rat podocytes cultured in the presence or absence of puromycin aminonucleoside (PAN), an agent commonly used to induce podocyte injury. RNA-seq revealed that a transcript containing the intronic sequence of small nucleolar RNA host gene 4 (Snhg4) was expressed in podocytes and upregulated by PAN. RT-qPCR analysis demonstrated that this transcript, but not Snhg4, was selectively expressed in podocytes. Therefore, we designated the novel transcript Snhg4-pod. 5'- and 3'-RACE experiments revealed that Snhg4-pod is a novel splice variant of Snhg4 lacking a poly(A) tail. PAN induced Snhg4-pod expression in podocytes in a dose-dependent manner along with their mitochondria-mediated apoptotic cell death. Further, Snhg4-pod was detected in urinary sediments from PAN-induced nephrotic rats. Our findings suggest that Snhg4-pod may serve as a novel marker for the diagnosis of glomerular injury.

Modified Huangqi Chifeng decoction inhibits excessive autophagy to protect against Doxorubicin-induced nephrotic syndrome in rats via the PI3K/mTOR signaling pathway.

The aim of the present study was to investigate whether modified Huangqi Chifeng decoction (MHCD) could be an effective treatment against Doxorubicin-induced nephrosis in rats and whether it regulates autophagy via the phosphoinositide-3 kinase/mammalian target of rapamycin (PI3K/mTOR) signaling pathway. A total of 40 male Sprague-Dawley rats were randomly divided into blank, model, telmisartan and MHCD groups. The rat model of nephrosis was induced by intragastric administration of Doxorubicin for 8 weeks. Rats were housed in metabolic cages and urine was collected once every 2 weeks to measure 24-h protein levels. Blood samples were obtained from the abdominal aorta and levels of albumin (ALB), total cholesterol (TCH), triacylglyceride (TG) and serum creatinine (Scr) were assessed. Renal pathological changes were examined using hematoxylin-eosin, Masson's trichome and periodic acid-Schiff staining. Podocytes and autophagosomes were observed using an electron microscope. The expression and distribution of microtubule-associated proteins 1A/1B light chain 3B (LC3), LC3-I, LC3-II, beclin-1, PI3K and mTOR were determined using immunohistochemistry and western blotting. At weeks 6 and 8, 24-h proteinuria significantly decreased in the MHCD group compared with the model group (P<0.05). Compared with the model group, the MHCD group exhibited significantly reduced levels of TG, TCH and Scr, as well as significantly increased ALB levels (P<0.05). MHCD was demonstrated to prevent glomerular and podocyte injury. The number of autophagosomes was significantly decreased and the expression of beclin-1, LC3, LC3-I and LC3-II was inhibited following MHCD treatment compared with the model group (P<0.05). MHCD treatment significantly increased the expression of PI3K and mTOR in Doxorubicin nephrotic rats compared with the model group (P<0.05). In conclusion, MHCD was demonstrated to ameliorate proteinuria and protect against glomerular and podocyte injury by inhibiting excessive autophagy via the PI3K/mTOR signaling pathway.

A target for preventing kidney damage

Physiology Proteinuria, the appearance of protein in the urine, results when podocytes in the kidney are damaged. Rinschen et al. found that the activation of the transcriptional coactivator YAP and the expression of YAP target genes preceded proteinuria in rats with chemically induced nephrosis. YAP activity can be stimulated by mechanical stress, and activation of YAP in cultured podocytes depended on the stiffness of the substrate. A YAP inhibitor ameliorated proteinuria and damage-induced mechanosignaling in the nephrotic rat kidneys. Sci. Signal. 10 , eaaf8165 (2017).

Enhanced podocyte vesicle transport in the nephrotic rat.

Albumin endocytosis is enhanced in the podocytes of minimal change nephrotic syndrome. We investigated that the endocytic vesicle transport in the podocyte using three-dimensional observation in a rat model of puromycin aminonucleoside (PAN)-induced nephrotic syndrome. At day 7, Evans Blue-labeled albumin was intravenously injected in PAN rats, and one kidney was fixed for a morphological analysis; the other was used for the isolation of glomeruli through sieving and protein analyses. Evans Blue-labeled albumin was found to accumulate in an increased number of vesicles in the podocytes of PAN rat. Continuous sections and its three-dimensional observation demonstrated that vesicles may be transported from the cytoplasm to the apical membrane of the podocytes. The increased protein bands in the gel electrophoresis of the sieved glomeruli of nephrotic rats were analyzed by mass spectrometry in comparison to the control rats. The major proteins increased in the nephrotic rats were cytoplasmic dynein 1 heavy chain, myosin IX, and myosin VIIb. In conclusion, the podocyte endocytic vesicles carrying albumin increased with glomerular cytoplasmic dynein and myosin in minimal change nephrotic rats.

Effect of clofibrate on fatty acid metabolism in the kidney of puromycin-induced nephrotic rats.

Proteinuria plays an essential role in the progression of tubulointerstitial damage, which causes end-stage renal disease. An increased load of fatty acids bound to albumin reabsorbed into proximal tubular epithelial cells (PTECs) contributes to tubulointerstitial damage. Fibrates, agonists of peroxisome proliferator-activated receptor α (PPARα), have renoprotective effects against proteinuria whereas the effects of these compounds on fatty acid metabolism in the kidney are still unknown. Therefore, the present study examined whether the renoprotective effects of clofibrate were associated with improvement of fatty acid metabolism in puromycin aminonucleoside (PAN)-induced nephrotic rats. Rats were allocated to the control, PAN or clofibrate-treated PAN group. Biochemical parameters, renal injury and changes in fatty acid metabolism were studied on day14. PAN increased proteinuria, lipid accumulation in PTECs, excretions of N-acetyl-β-D-glucosaminidase (NAG) and 8-hydroxydeoxyguanosine (8OHdG) and the area of caspase 3-positive tubular cells. It decreased renal expressions of medium-chain acyl-CoA dehydrogenase (MCAD), cytochrome P450 (CYP)4A, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and estrogen-related receptor α (ERRα) without change of the expression of PPARα. Clofibrate reduced proteinuria, lipid accumulation, NAG excretion and the area of caspase 3-positive tubular cells. However, albumin excretion was not reduced and 8OHdG excretion was increased. Clofibrate minimized changes in MCAD, CYP4A, PGC-1α and ERRα expressions with increased PPARα, very long-chain acyl-CoA dehydrogenase (VLCAD) and long-chain acyl-CoA dehydrogenase (LCAD) expressions. Clofibrate is protective against renal lipotoxicity in PAN nephrosis. This study indicates that clofibrate has renoprotective effects through maintaining fatty acid metabolism in the kidney of PAN-induced nephrotic rats.

Urine and serum ghrelin, sCD80 and sCTLA-4 levels in doxorubicin-induced experimental nephrotic syndrome.

Nephrotic syndrome (NS) is an immune-mediated disorder associated with hyperlipidemia. NS has been proposed to be mediated through CD80-related T cell immune response, which could be blocked using soluble cytotoxic T lymphocyte-associated s(CTLA)-4. Although ghrelin is a hormone-modulating lipid metabolism and suppressing immune system, the precise role of ghrelin in NS is not well established. We evaluated the levels of ghrelin, soluble CD80 (sCD80) and sCTLA4 in serum and urine in doxorubicin-induced NS in rats. We also investigated the relation between their levels and the levels of serum total cholesterol (TC), triglyceride, albumin and urine protein. While urinary ghrelin levels were significantly lower in the nephrotic rats compared to the control group, serum ghrelin levels were comparable in the nephrotic and control rats. In contrast, serum and urinary sCD80 and sCTLA4 levels were higher in the nephrotic rats than the controls. The urinary ghrelin levels were negatively correlated with the levels of serum triglyceride, TC and urine protein, sCD80 and sCTLA4. The urine sCD80 levels were positively correlated with the TC, urine protein and urine sCTLA4 levels, and negatively correlated with the serum albumin. The urine sCTLA4 levels were positively correlated with the TC and urine protein levels and negatively correlated with the serum albumin levels. In regression analysis, the urine ghrelin levels significantly relate to urine sCD80 levels. Besides, hyperlipidemia in NS did not appear to be related to serum ghrelin levels. Low urine ghrelin levels might be relevant to pathogenesis of doxorubicin-induced NS. The reduction in urine ghrelin levels might also be associated with increased levels of urine sCTLA4 and sCD80 which reflect proteinuria.