Puromycin Aminonucleoside Group Research Articles

Effect of vitamin D analogues calcitriol and paricalcitol in a rat model of puromycin aminonucleoside-induced nephrotic syndrome

Background Renoprotective effects of vitamin D analogues have been shown in several experimental and clinical studies, the exact mechanism of the therapeutic effectiveness of these analogues in Nephrotic syndrome remains unclear, and these are relatively few studies on potential treatment roles for vitamin D analogues in nephrotic-range proteinuria. ?ndicate similar efficacy of the vitamin D analogues calcitriol and paricalcitol in time-limited amelioration of proteinuria in nephrotic syndrome, yet suggest the likelihood of mechanisms other than direct upregulation of nephrin and podocin in podocytes underlie the renoprotective effects of vitamin D analogues.
 Objective To investigate the effect of vitamin D (Vit D) analogues calcitriol and paricalcitol on urinary protein/creatinine ratio (UPCR) and renal podocin and nephrin expression in a rat model of puromycin aminonucleoside (PAN)-induced nephrotic syndrome (NS).
 Methods A total of 28 male Wistar Albino rats were separated into 4 groups (n=7 for each) including CON [control; intraperitoneal (IP) saline injection], PAN (NS + IP saline injection), PAN-C (NS + IP 0.4 µg/kg/day calcitriol injection), and PAN-P (NS + IP 240 ng/kg/day paricalcitol injection). Nephrotic syndrome was induced via intravenous (IV) administration of 10mg/100gr PAN. The UPCR as well as histopathological, immuno-histochemical, and real time PCR analyses of kidney tissue specimens were recorded and analyzed among the 4 groups.
 Results Median UPCR (Day 4) was significantly lower in both the PAN-C [1.45 (range 1.20-1.80)] and PAN-P [1.40 (range 1.10-1.80)] groups than in the PAN group [2.15 (range 2.00-2.40)] (P<0.01 for each). The PAN group had significantly higher mean UPCR than the CON group [1.75 (range 1.40-2.00); P<0.05]. No significant difference in UPCR was noted between groups on Day 7. Median podocin mRNA expression was significantly higher in the PAN-P group compared to the PAN group [22.55 (range 22.42-23.02) vs. 22.06 (range 21.81-22.06), respectively; (P<0.01)].
 Conclusion Seven-day calcitriol and paricalcitol supplementation in a rat model of PAN-induced nephrotic syndrome had similar efficacy, in terms of temporary amelioration of proteinuria.

Effect of tacrolimus on the expression of Park7 in glomerular podocytes injured by puromycin aminonucleoside.

To study the effect of puromycin aminonucleoside (PAN) on the apoptosis of mouse podocyte clone 5 (MPC-5) and the expression of recombinant human Parkinson's disease 7 (Park7) and to study the protective mechanism of tacrolimus (FK506) against MPC-5 injury. MPC-5 cells were cultured in vitro and then divided into three groups: blank control (control), PAN, and FK506. The cells in the PAN group were added with PAN (with a concentration of 50 mg/L) to establish a model of MPC-5 injury, and those in the FK506 group were added with PAN (with a concentration of 50 mg/L) and FK506 (with a concentration of 5 mg/L). An inverted microscope was used to observe the morphology and structure of MPC-5 cells at 12, 24, and 48 hours after treatment. Flow cytometry was used to measure cell apoptosis rate. Quantitative real-time PCR was used to measure the mRNA expression of Park7. Western blot and immunofluorescent staining were used to measure the protein expression of Park7. The control group had a large number of foot processes of the cell body at all time points, with tight connections between cells and a normal morphology. Compared with the control group, the PAN group had a significantly smaller cell volume at all time points, with loose connections between cells and the presence of ruptured cells. Compared with the PAN group, the FK506 group had an increased cell volume at all time points, with tighter connections between cells and a better morphology. The PAN group had a significantly higher apoptosis rate than the control group at all time points. Compared with the PAN group, the FK506 group had a significant reduction in the apoptosis rate at all time points (P<0.01). The PAN group had a significantly higher mRNA expression level of Park7 than the control group at all time points. Compared with the PAN group, the FK506 group had a significant reduction in the mRNA expression level of Park7 at all time points (P<0.01). Western blot showed that the PAN group had a significantly higher protein expression level of Park7 than the control group at all time points. Compared with the PAN group, the FK506 group had a significant reduction in the protein expression level of Park7 at all time points (P<0.01). Immunofluorescent staining showed that in the PAN group, there was a significantly lower expression of Park7 protein in cell membrane and cytoplasm, with a dense cluster distribution and increased fluorescence intensity. Compared with the PAN group, the FK506 group had a significant improvement in the distribution of Park7 protein. PAN can act on MPC-5 cells and cause morphological and structural damage and apoptosis of MPC-5 cells, as well as upregulated mRNA and protein expression of Park7. FK506 can downregulate the mRNA and protein expression of Park7 in the model of MPC-5 injury, maintain cellular homeostasis, reduce proteinuria, and delay glomerulosclerosis.

Decreased Excretion of Urinary Exosomal Aquaporin-2 in a Puromycin Aminonucleoside-Induced Nephrotic Syndrome Model.

Urinary exosomes, small extracellular vesicles present in urine, are secreted from all types of renal epithelial cells. Aquaporin-2 (AQP2), a vasopressin-regulated water channel protein, is known to be selectively excreted into the urine through exosomes (UE-AQP2), and its renal expression is decreased in nephrotic syndrome. However, it is still unclear whether excretion of UE-AQP2 is altered in nephrotic syndrome. In this study, we examined the excretion of UE-AQP2 in an experimental rat model of nephrotic syndrome induced by the administration of puromycin aminonucleoside (PAN). Rats were assigned to two groups: a control group administered saline and a PAN group given a single intraperitoneal injection of PAN (125 mg/kg) at day 0. The experiment was continued for 8 days, and samples of urine, blood, and tissue were collected on days 2, 5, and 8. The blood and urine parameters revealed that PAN induced nephrotic syndrome on days 5 and 8, and decreases in the excretion of UE-AQP2 were detected on days 2 through 8 in the PAN group. Immunohistochemistry showed that the renal expression of AQP2 was decreased on days 5 and 8. The release of exosomal marker proteins into the urine through UEs was decreased on day 5 and increased on day 8. These data suggest that UE-AQP2 is decreased in PAN-induced nephrotic syndrome and that this reflects its renal expression in the marked proteinuria phase after PAN treatment.

Efficacy of a mitochondrion-targeting agent for reducing the level of urinary protein in rats with puromycin aminonucleoside-induced minimal-change nephrotic syndrome.

BackgroundOxidative stress is a major factor responsible for minimal-change nephrotic syndrome (MCNS), which occurs most commonly in children. However, the influence of oxidative stress localized to mitochondria remains unclear. We examined the effect of a mitochondrion-targeting antioxidant, MitoTEMPO, in rats with puromycin aminonucleoside (PAN)-induced MCNS to clarify the degree to which mitochondrial oxidative stress affects MCNS.Materials and methodsThirty Wistar rats were divided into three groups: normal saline group (n = 7), PAN group (n = 12), and PAN + MitoTEMPO group (n = 11). Rats in the PAN and PAN + MitoTEMPO groups received PAN on day 1, and those in the PAN + MitoTEMPO group received MitoTEMPO on days 0 to 9. Whole-day urine samples were collected on days 3 and 9, and samples of glomeruli and blood were taken for measurement of lipid peroxidation products. We also estimated the mitochondrial damage score in podocytes in all 3 groups using electron microscopy.ResultsUrinary protein excretion on day 9 and the levels of lipid peroxidation products in urine, glomeruli, and blood were significantly lower in the PAN + MitoTEMPO group than in the PAN group (p = 0.0019, p = 0.011, p = 0.039, p = 0.030). The mitochondrial damage score in podocytes was significantly lower in the PAN + MitoTEMPO group than in the PAN group (p <0.0001).ConclusionsThis mitochondrion-targeting agent was shown to reduce oxidative stress and mitochondrial damage in a MCNS model. A radical scavenger targeting mitochondria could be a promising drug for treatment of MCNS.

MON-002 YI QI QING RE GAO, A TRADITIONAL CHINESE HERBAL FORMULA, ORCHESTRATES ACTIN CYTOSKELETON REORGANIZATION INDUCED BY PUROMYCIN AMINONUCLEOSIDE IN PODOCYTES VIA RHOA/ROCK SIGNALING PATHWAY

Podocyte injury is closely associated with the onset of proteinuria in many kidney diseases. A major mechanism of podocyte injury is the reorganization of actin cytoskeleton, which leads to increased podocyte motility and foot process effacement. Yi Qi Qing Re Gao (YQQRG), a traditional Chinese herbal formula, has been used clinically to treat chronic nephritis for decades with dramatic anti-proteinuria effects. In this study, we investigated the effects of YQQRG on actin cytoskeleton reorganization induced by puromycin aminonucleoside (PAN) and explored the possible mechanisms. Wistar rats were randomly divided into sham group, PAN group, PAN + YQQRG group, and PAN + Cyclosprin A (CsA) group. The PAN model was established by a single intravenous injection of PAN (40mg/kg body weight). Serum biochemical parameters and 24h urinary protein were assessed on days 5, 9, and 14, respectively. Renal tissues were harvested for observations of pathologic alterations and ultrastructural changes. Quantitative expressions of regulatory proteins of cytoskeleton were measured using Western-blot and real-time polymerase chain reaction. In vitro, the conditionally immortalized mouse podocytes (MPC5) were used to investigate the effects of Yi Qi Qing Re Gao-containing serum (YQ-S) on actin cytoskeleton reorganization. Podocytes injury were evaluated by released lactic dehydrogenase (LDH) activity assay. Podocytes apoptosis were determined by Annexin V assay. Wound healing assay and transwell migration assay were used to assess podocyte motility. Immunofluorescence was used to demonstrate the formation and distribution of stress fiber. Western Blot was performed to detect the expression of key proteins of RhoA/ROCK signaling pathway. YQQRG significantly decreased urinary protein level, lowered serum lipid level, elevated serum albumin, and ameliorated renal function in Wistar rats, similar to the effects of CsA. Renal pathological lesions, especially podocytes effacement were obviously attenuated. The experiments in vivo and in vitro both displayed that YQQRG could downregulate the mRNA and protein expressions of α-actinin-4, synaptopodin, vimentin, desmin, and nestin in renal tissues and podocytes. Besides, YQ-S alleviated PAN-induced podocytes injury and apoptosis, and reduced podocyte motility and migration. Disruption of F-actin and disorganization of cytoskeleton were largely recovered by YQ-S. Furthermore, YQ-S significantly antagonized PAN-induced downregulation of the protein expression of RhoA and ROCK, decreased the phosphorylation of MLC, LIMK and cofilin, and increased F-actin/G-actin levels. Addition of Y-27632 (a Rho-kinase inhibitor) could weaken the effects of YQ-S on F-actin reorganization and expressions of ROCK, p-MLC, and p-LIMK. YQQRG alleviates actin cytoskeleton reorganization and attenuates podocytes injury in PAN model in vivo and in vitro, and its mechanism may involve the upregulation of RhoA/ROCK signaling pathway. These findings suggest that YQQRG might be a promising natural drug for the treatment of kidney diseases.

In glomerular cells of puromycin aminonucleoside nephrosis rats both phosphorylated and total STAT3 levels increased during proteinuria

Recent studies showed that JAK/STAT pathway plays role in glomerular damages. The fact that STAT3 could be activated also by oxidative stress make Puromycin Aminonucleoside (PAN) Nephrosis model very appropriate for examination of STAT3 expression changes in glomerular pathology. Along with a control group, three PAN groups sacrificed on different days were formed by the i.p. injection of PAN for 5 consecutive days. Throughout the experiment, 24-hour-urines were collected on specific days and proteinuria levels were monitored. At the end of the experiments, tissue specimens were stained immunohistochemically for both total and phosphorylated STAT3 and evaluated subjectively. They were also examined ultrastructurally in transmission electron microscope. The proteinuria levels did not increase significantly on 5th day but showed a dramatic increase on 10th and 15th days. On 20th and 25th days, urinary protein levels gradually decreased. Ultrastructural examinations showed glomerular damages such as significant decrease in slit pore number, a significant gradual increase in glomerular basement membrane thickness and podocyte hypertrophy on 5th and 15th days; besides significant increase in mesangial matrix. The first significant increases in phosphorylated and total STAT3 levels occurred in 5th day and 15th day groups respectively. These increases diminished in 25th day group. Regarding all the findings, it was deduced that STAT3 is one of the active factors in glomerular pathologies.

Rapamycin Reduces Podocyte Apoptosis and is Involved in Autophagy and mTOR/ P70S6K/4EBP1 Signaling

Background/Aims: The purpose of this study was to investigate the impact of rapamycin (RAP) on autophagy in podocytes and the therapeutic effects of RAP on idiopathic membranous nephropathy (IMN). Methods: We established an in vitro model of IMN by preconditioning mouse podocytes with puromycin aminonucleoside (PAN). A Cell Counting Kit-8 was used to detect the proliferation of each group of podocytes. Podocyte apoptosis was analyzed by flow cytometry via annexin V/propidium iodide dual staining. Subsequently, we observed the number of autophagosomes by transmission electron microscopy. Western blotting was used to detect the levels of LC3, mTOR, p-mTOR, 4EBP1, p-4EBP1, P70S6K, and p-P70S6K in each group. Results: The number of podocytes in the PAN + 100 ng/mL RAP group, PAN + 200 ng/mL RAP group, and PAN + 300 ng/mL RAP group was significantly increased (P < 0.01). The apoptotic rate of podocytes was significantly different between the PAN group and the PAN + RAP group (P < 0.001). There were fewer autophagic corpuscles in the PAN group and more autophagosomes were observed in the PAN + RAP group. LC3 protein expression was down-regulated in the PAN group, while its expression was up-regulated in the PAN + RAP group. In the PAN group, the levels of phosphorylated mTOR, 4EBP1, and P70S6K were increased, while in the PAN + RAP group, protein phosphorylation was reduced. Conclusions: RAP can effectively inhibit the mTOR/P70S6K/4EBP1 signaling pathway, and activate podocyte autophagy, consequently reducing podocyte apoptosis. Therefore, RAP could be used for the treatment of idiopathic membranous nephropathy.

Usefulness of urinary biomarkers for nephrotoxicity in cynomolgus monkeys treated with gentamicin, cisplatin, and puromycin aminonucleoside.

The objective of this study was to investigate the availability of novel urinary biomarkers (BMs) such as total protein, albumin, β2-microglobulin, clusterin, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL) for the detection of acute nephrotoxicity in cynomolgus monkeys. Animals (total 9 males/3 groups) were administered gentamicin (GM) subcutaneously at 40 mg/kg for 7 days, cisplatin (CDDP) intravenously at 3 mg/kg once and puromycin aminonucleoside (PAN) intravenously at 20 mg/kg for 7 days. Two-hr urine on Days 0, 3, and 6, and 16-hr urine and blood on Days 1, 4, and 7 were collected. Novel urinary BMs and conventional clinical pathology parameters were evaluated in parallel to histopathological and electron microscopic examinations on the kidneys at termination. Urinary BMs and enzymes increased earlier than serum creatinine and blood urea nitrogen, particularly in 2-hr urine after dosing on Day 0, urinary albumin was increased in all groups and urinary NGAL with the highest magnitude of change rate among urinary BMs was observed in the GM and CDDP groups. Degeneration/necrosis and hyaline droplet of renal tubule, cellular cast and dilatation of renal tubule, and hypertrophy of podocytes were observed in the GEN, CDDP, and PAN groups, respectively. These results showed that the increases of urinary BMs reflected the agent-specific renal damages and these urinary BMs could be useful for the detection of segment-specific nephrotoxicity. Urinary albumin and NGAL are the most useful BMs to estimate glomerular and distal tubular damages, respectively, as well as proximal tubular damage in cynomolgus monkeys.

Antioxidative Stress Effects of Salvia przewalskii Extract in Experimentally Injured Podocytes

Background: Oxidative stress is a leading cause of puromycin aminonucleoside (PAN)-induced nephrosis. As the inhibition of oxidative stress may improve injury of podocyte, we aimed at examining the effect of total phenolic acid extract of Salvia przewalskii (SPE) on PAN-induced oxidative stress in vivo and in vitro. Methods: Seventy-two male Sprague-Dawley rats were randomly assigned into 6 groups (n = 12), PAN alone, tacrolimus (TAC), SPE (50, 100 and 200 mg/kg) and normal control group. Salvianolic acid B (SalB, 5.52%) and rosmarinic acid (RA, 31.58%) were isolated from SPE. The intensities of 8-oxo-2'-deoxyguanosine (8-OHdG) were evaluated by immunofluorescence. In vitro, the podocytes were assigned into groups of control, PAN alone, TAC (1 μg/ml), SPE (158, 316 μg/ml), SalB (8.5, 17 μg/ml) and RA (25, 50 μg/ml). The intracellular reactive oxygen species (ROS) production and cell apoptosis rate were measured by flow cytometry. Form factor and aspect ratio were calculated to assess mitochondrial morphology. Results: In vivo, PAN increased the intensity of 8-OHdG in the renal tissue in the PAN group (p < 0.05). The high-dose SPE reduced 8-OHdG significantly at levels comparable to TAC alone (p > 0.05) on day 15. The intracellular ROS production, podocytes apoptosis rate and mitochondrial fragmentation increased significantly following PAN exposure in podocytes (p < 0.05). Treatment with high-dose SalB significantly ameliorated the increase in the expression of ROS and revised the structure of mitochondria. The percentage of apoptotic cells was decreased compared with the PAN group after SPE, SalB, RA, and TAC treatment for 24 h (p < 0.05). Conclusion: These findings suggest that high-dose SPE significantly attenuated 8-OHdG in PAN nephrosis. Antioxidative stress effects of high-dose SPE, SalB against PAN-stimulated cultured podocyte via mechanisms include suppression of ROS expression and mitochondria fission. In addition, SPE, SalB and RA can suppress PAN-induced apoptosis.

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.

Yi Qi Qing Re Gao Attenuates Podocyte Injury and Inhibits Vascular Endothelial Growth Factor Overexpression in Puromycin Aminonucleoside Rat Model

Proteinuria is the hallmark of chronic kidney disease. Podocyte damage underlies the formation of proteinuria, and vascular endothelial growth factor (VEGF) functions as an autocrine/paracrine regulator. Yi Qi Qing Re Gao (YQQRG) has been used to treat proteinuria for more than two decades. The objective of this study was to investigate the protective effect and possible mechanisms of YQQRG on puromycin aminonucleoside (PAN) rat model. Eighty male Sprague-Dawley rats were randomized into sham group, PAN group, PAN + YQQRG group, and PAN + fosinopril group. Treatments were started 7 days before induction of nephrosis (a single intravenous injection of 40 mg/kg PAN) until day 15. 24 h urinary samples were collected on days 5, 9, and 14. The animals were sacrificed on days 3, 10, and 15, respectively. Blood samples and renal tissues were obtained for detection of biochemical and molecular biological parameters. YQQRG significantly reduced proteinuria, elevated serum albumin, and alleviated renal pathological lesions. YQQRG inhibited VEGF-A, nephrin, podocin, and CD2AP mRNA expression and elevated nephrin, podocin, and CD2AP protein levels starting on day 3. In conclusion, YQQRG attenuates podocyte injury in the rat PAN model through downregulation of VEGF-A and restoration of nephrin, podocin, and CD2AP protein expression.

Arginine and Asymmetric Dimethylarginine in Puromycin Aminonucleoside-Induced Chronic Kidney Disease in the Rat

Background/Aims: Reduced renal L-arginine (L-Arg) synthesis/transport, induction of arginases and increased endogenous NOS inhibitor, asymmetric dimethylarginine (ADMA) will inhibit NO production. This study investigated pathways of L-Arg synthesis/uptake/utilization, ADMA degradation and oxidant/antioxidants in puromycin aminonucleoside (PAN) chronic kidney disease (CKD). Methods: Rats were given low- (LD) or high-dose (HD) PAN and followed for 11 weeks for proteinuria. BP was measured and blood and tissues were harvested and analyzed for abundance of argininosuccinate synthase (ASS) and lyase (ASL), arginase, cationic amino acid transporter (CAT1) and dimethylargininedimethylaminohydrolase (DDAH) in kidney, cortex, aorta and liver. Arginase and DDAH activity, plasma L-Arg and ADMA, renal pathology and creatinine clearances were also measured. Results: PAN caused dose-dependent kidney damage and hypertension and creatinine clearance fell in HD-PAN. Renal ASS fell in HD-PAN, renal cortex and aortic ASL and membrane CAT1 fell in both PAN groups. There was no activation of renal arginase, but aortic arginase increased in LD-PAN. Renal DDAH activity fell moderately in LD-PAN and markedly in HD-PAN where hepatic DDAH activity also fell. Plasma L-Arg was unchanged while ADMA rose moderately and dose-dependently with PAN. There were several indices of oxidative stress which was most prominent in HD-PAN. Conclusion: Reduction in renal ASS/ASL and loss of renal cortex CAT1 compromises renal L-Arg synthesis and release. Loss of aortic CAT1 impairs L-Arg uptake. Increased plasma ADMA was associated with progressive loss of renal DDAH activity. However, loss of renal clearance and falls in hepatic DDAH activity in HD-PAN did not have additive effects on plasma ADMA.

Effects of mineralocorticoid and angiotensin II receptor blockers on proteinuria and glomerular podocyte protein expression in a model of minimal change nephrotic syndrome

Several proteins constituting the slit diaphragm are considered important for maintaining capillary wall permselectivity. Early intervention with blockers of angiotensin II receptors (AR) and mineralocorticoid receptors (MR) is effective against proteinuria in models of chronic hypertensive and protein-induced renal damage. However, the effects of AR and/or MR blockers in a model of acute nephrotic syndrome remain unknown. The effects of AR and MR blockers were examined in puromycin aminonucleoside (PAN)-treated rats. Six week old male Sprague-Dawley (SD) rats were injected with PAN or vehicle and assigned to groups as follows: vehicle (group C); PAN (group P); PAN followed 3 days later by administration of the MR blocker, eplerenone (group MR), and by the AR blocker, losartan (group AR). Blood pressure and urinary protein excretion were measured and all rats were killed for immunohistochemical investigation on day 14 after PAN administration. Blood pressure did not change throughout the study period. Proteinuria was decreased in groups MR and AR compared with group P (on day 14 after PAN administration, respectively; group P vs AR, P < 0.01; group P vs MR, P < 0.05). Nephrin, podocin and podocalyxin staining was preserved in the glomeruli of groups MR and AR compared with group P. The MR and AR blockers decreased proteinuria in the acute model of nephrotic syndrome with preserved expression of glomerular podocyte protein independently of blood pressure.

Effects of Celecoxib and Nordihydroguaiaretic Acid on Puromycin Aminonucleoside-Induced Nephrosis in the Rat

The selective cyclooxygenase-2 (COX-2) and 5-lipoxygenase (LOX) inhibitors might inhibit prostaglandin synthesis and reduce proteinuria. The present study was designed to investigate the anti-proteinuric effects of nordihydroguaiaretic acid (NDGA) as compared with celecoxib in puromycin aminonucleoside (PAN) nephrosis rats. Fifty five male Sprague-Dawley rats were divided into 4 groups; A, normal control; B, PAN group; C, PAN+COX-2 inhibitor (celecoxib) group; and D, PAN+5-LOX inhibitor (NDGA) group. After induction of PAN nephrosis through repeated injections of PAN (7.5 and 15 mg/100 g body weight), rats were treated with celecoxib, NDGA, or vehicle for 2 weeks. Twenty four hour urine protein excretions were significantly lower in PAN+celecoxib and PAN+NDGA groups than in PAN group. Serum creatinine (SCr) concentrations and 24 hr urine creatinine clearances (CCr) were not significantly different in the four groups. Electron microscopy showed that podocyte morphology was changed after the induction of PAN nephrosis and was recovered after celecoxib or NDGA administration. Celecoxib significantly recovered the expressions of nephrin, CD2AP, COX-2, and TGF-β. NDGA also recovered TGF-β expression, but did not alter the expressions of nephrin, CD2AP and COX-2. The present study suggested that celecoxib and NDGA might effectively reduce proteinuria in nephrotic syndrome without impairing renal function.

Low molecular weight heparin in the treatment of puromycin-induced nephrosis

Heparin may have a beneficial effect in proteinuric renal diseases, where negative charges of the glomerular capillary membrane are compromised. We evaluated the role of low molecular weight heparin (LMWH – 3000 Da) in puromycin aminonucleoside (PAN)-induced focal and segmental glomerulosclerosis in male Wistar rats: Controls ( C) n = 7 , LMWH-treated group, n = 9 , subcutaneously (SC), 6 mg/kg every day. The PAN group ( n = 7 ) received 7 doses on weeks 0, 1, 2, 4, 6, 8, 10 (SC – 2 mg/100 g), and a group PAN+LMWH ( n = 6 ). After 12 weeks, cholesterol and triglycerides were higher in nephrotic groups, as well as proteinuria and urinary IgG. Kidney weight, glomerular volume, and glomerular sclerosis index were higher in the PAN-treated groups. Glomerular capillary length density ( L Vcap) and glomerular capillary surface density ( S Vcap) were lower in the PAN group, and mesangial fractional volume was higher. Fibronectin immunostaining was more intense in the PAN group, and collagens I and III were absent in the studied glomeruli. Thus, LMWH prevented mesangial expansion and capillaries changes, showing antiproliferative properties, despite worsening glomerular permeability changes in the PAN model. In conclusion, LMWH interferes in the complications of PAN model, but not through inhibition of the proteinuria.

Beneficial effects of orosomucoid on the glomerular barrier in puromycin aminonucleoside-induced nephrosis

In a hitherto unconfirmed report, orosomucoid was reported to ameliorate the nephrotic syndrome induced by puromycin aminonucleoside (PAN) in rats. We wanted to test this hypothesis and extend the analysis of the effects on the glomerular barrier. Glomerular filtration rate (GFR), and fractional clearance for albumin (theta(albumin)) and for neutral Ficolls were estimated in cooled isolated perfused kidneys. Modern transport equations were used to estimate glomerular size selectivity and charge selectivity. Also, podocyte morphology was studied. Four groups of rats (4 x n = 8) were administered PAN intraperitoneally and treated daily for 5 days with orosomucoid in two different doses (groups A and B), albumin (group C) or saline (group D). Two additional groups of rats (2 x n = 8) were used as controls and these rats received either saline (group E) or orosomucoid (group F) but no PAN. Treatment with orosomucoid restored podocyte morphology and renal function from the damaging effects of PAN in a dose-dependent manner. GFR was significantly reduced by PAN (groups C and D) when compared with controls (groups E and F). This effect was partly (group A) or completely (group B) reversed by orosomucoid. The theta(albumin) was 0.002+/-0.001 (mean+/-SEM) in controls (group E) and was unaffected by orosomucoid per se (group F). PAN increased theta(albumin) to 0.020+/-0.001 in group C and to 0.021+/-0.001 in group D, while it was significantly less in group A, 0.014+/-0.001, P<0.05. The heterogeneous charged fibre model analysis revealed that PAN reduced the relative volume of negatively charged fibres, phi, from 7.1+/-0.08% (group E) to 48% of this value in groups C and D (P<0.001); phi was 4.5+/-0.04% in group A, 5.3+/-0.44% in group B, and 6.1+/-0.11% in group F. High doses of orosomucoid completely normalized the glomerular barrier in six out of eight animals with puromycin-induced nephrotic syndrome. Thus, orosomucoid has a promising therapeutic potential for certain kidney disorders.

Lack of a role for inducible nitric oxide synthase in an experimental model of nephrotic syndrome

Puromycin aminonucleoside (PAN) administration in rats produces an experimental model of nephrotic syndrome characterized by glomerular epithelial cell injury and proteinuria. The purpose of this study was to examine the role of nitric oxide (NO) in this model of minimal change glomerular disease. Aminoguanidine (AG) was used to inhibit inducible nitric oxide synthase (iNOS). Sprague–Dawley rats were divided into Control (N = 9), PAN (N = 14), AG (N = 2), and PAN + AG (N = 12) treatment groups. Control animals received saline (i.v.), PAN animals received PAN (75 mg/kg, i.v.), and PAN + AG animals received PAN plus AG (50 mg/kg, i.p., twice daily). AG animals received a saline injection (i.v.) on day 0 in the place of PAN and then AG on the same schedule as the PAN + AG group. Animals were kept in metabolic cages, and urinary protein excretion and nitrite (NO 2 −) excretion were measured daily. PAN administration increased urinary NO 2 − excretion by day 2, and levels remained elevated through day 7. AG prevented this PAN-induced increase in urinary NO 2 − excretion. Plasma nitrate (NO 3 −) and NO 2 − (NOx) concentrations were also increased in the PAN and PAN + AG groups. iNOS protein expression was not detected in either the glomeruli or the cortex at day 7. Proteinuria developed in PAN animals on day 4 and increased steadily through day 7. PAN + AG animals showed a pattern similar to that of the PAN group. These results indicated that in contrast to models of proliferative glomerulonephritis, NO formation during PAN-induced nephrotic syndrome is increased but does not participate in the development of glomerular injury as measured by proteinuria.

CAFFEINE AUGMENTS PROTEINURIA IN PUROMYCIN-AMINONUCLEOSIDE NEPHROTIC RATS*

Several studies indicate that increased intrarenal adenosine concentrations may attenuate puromycin-aminonucleoside (PAN)-induced nephropathy in rats. The purpose of this study was to investigate the chronic effects of caffeine, a nonselective adenosine receptor antagonist, on renal function and structure in PAN-induced nephropathy. Animals were randomized to receive drinking water or 0.1% caffeine solution. PAN was administered in two doses to a subset from each group at 1 week (100 mg/kg, sc.; Purom-1) and 15 wks (80 mg/kg, sc.; Purom-2) after initiating caffeine treatment (PAN and CAFF-PAN groups). The remaining animals served as time controls (CON and CAFF groups). Renal excretory function was followed for 23 wks. Caffeine consumption significantly augmented PAN-induced proteinuria after both PAN injections (Purom-1 and Purom-2, p < 0.05 and p < 0.001 respectively; CAFF-PAN vs. PAN). In addition, caffeine potentiated the transient reduction in creatinine clearance (CrCl) induced by PAN. Caffeine consumption for 23 wks significantly reduced CrCl in conscious nephrotic animals (4.76 ± 0.98 vs. 8.51 ± 1.55 L/kg/day, CAFF-PAN vs. PAN). Seven days after both PAN injections, increased plasma renin activity was detected in animals that were consuming caffeine as compared with corresponding control groups (CAFF and CAFF + PAN vs CON and PAN, respectively). Eight weeks after the second injection of PAN, acute measures of renal hemodynamic and excretory function were compared in anesthetized animals and renal samples were analyzed for histological changes. In PAN-rats, caffeine treatment for 23 weeks significantly reduced inulin clearance (0.28 ± 0.09 vs. 0.57 ± 0.12 mL/min/gr kidney, CAFF-PAN vs PAN, p < 0.05), tended to increase renal vascular resistance (59.0 ± 9.5 vs. 42.9 ± 5.5 mmHg/mL/min/gr kidney, CAFF-PAN vs. PAN, p < 0.06), potentiated the development of more severe tubulointerstitial damage (tubular atrophy, presence of proteinaceous material, tubular dilatation, interstitial inflammation, interstitial fibrosis), and tended to increase glomerulosclerosis. In conclusion, this study indicates that caffeine adversely affects renal function in PAN-nephrotic rats, and that this effect may be due, in part, to increased activity of the renin angiotensin system.

Effect of the in vivo catalase inhibition on aminonucleoside nephrosis

Reactive oxygen species have been involved in the pathophysiology of puromycin aminonucleoside (PAN)-nephrosis. The role of H 2O 2 in these rats may be studied modulating the amount or activity of catalase, which breakdowns H 2O 2 to water and oxygen. To explore the role of H 2O 2 in this experimental model, we studied the effect of the in vivo catalase inhibiton with 3-amino-1,2,4-triazole (ATZ) on the course of PAN-nephrosis. Four groups of rats were studied: control rats (CT group), PAN-injected rats (PAN group), ATZ-injected rats (ATZ group), and ATZ- and PAN-injected rats (ATZPAN group). Rats were placed in metabolic cages to collect 24 h urine along the study, ATZ (1 g/kg) was given 24 h before PAN injection (75 mg/kg), and the proteinuria was measured on days 0, 2, 4, 6, 8, and 10. Proteinuria started before (day 4) and was significantly higher on days 6, 8, and 10 in the ATZPAN group than in the PAN group. On day 10, hypercholesterolemia was significantly higher in the ATZPAN group than in the PAN group. These data indicate that the in vivo catalase inhibition magnifies PAN-nephrosis, suggesting that H 2O 2 is produced in vivo and involved in the renal damage in this experimental disease.

Heparin-binding epidermal growth factor-like growth factor is expressed in the adhesive lesions of experimental focal glomerular sclerosis

In this study, we attempted to determine whether heparin-binding epidermal growth factor-like growth factor (HB-EGF) was up-regulated in two chronic models of proteinuria. Chronic passive Heymann nephritis (PHN) and puromycin aminonucleoside (PAN) models were induced in Sprague-Dawley rats. HB-EGF expression was studied by Northern blotting, in situ hybridization, and immunohistochemistry. The chronic PAN model was associated with the development of glomerular lesions of focal glomerular sclerosis (FGS), severe interstitial fibrosis, and renal failure. Lesions of FGS were seen in approximately 80% of glomeruli at all time points, with a slight increase in the number of glomeruli showing extensive adhesion between 40 and 90 days. Northern blots of whole kidney tissue showed a 3- to 5.8-fold increased expression of HB-EGF mRNA in the chronic PAN group. Increased mRNA and protein were localized by in situ hybridization and immunohistochemistry to tubules, glomerular epithelial cells (GECs), and cells of Bowman's capsule. HB-EGF mRNA and protein were strongly expressed by epithelial cells involved in the formation of the lesions of FGS. By contrast, in chronic PHN, there was a small increase in HB-EGF, and the extensive lesions of FGS did not develop despite continued, heavy proteinuria. These data suggest that HB-EGF may contribute to formation of the lesions of FGS, perhaps through stimulation of abortive mitogenesis in GECs or an adhesive interaction between transmembrane HB-EGF and the exposed glomerular basement membrane.