Puromycin aminonucleoside-induced podocyte injury is ameliorated by the Smad3 inhibitor SIS3.

Smad3 signaling and transgelin expression are often activated during puromycin aminonucleoside (PAN)-induced podocyte injury. Here, we investigated whether the Smad3 inhibitor SIS3 can ameliorate damage to injured podocytes. A model of PAN-induced podocyte injury was constructed using the MPC5 cell line. The effects of SIS3 on the expression of the podocyte cytoskeletal proteins transgelin, p15INK4B , phosphor-smad3, phosphor-JAK/stat3, the apoptotic marker cleaved caspase 3, and c-myc were investigated using western blot. The distribution of F-actin in PAN-induced podocyte injury was observed under an immunofluorescence microscope. PAN-induced podocyte injury altered the distribution of F-actin and transgelin, and colocalization of these two proteins was observed. Transgelin expression and Smad3 phosphorylation were increased in the MPC5 cell line with prolonged PAN treatment. In addition, c-myc expression, p15INK4B , and JAK phosphorylation were all increased after treatment with PAN. Treatment with the Smad3 inhibitor SIS3 reversed these phenomena and protected against PAN-induced podocyte injury. Moreover, stimulating podocytes directly with TGFß-1 also led to enhanced expression of transgelin or phosphor-JAK/stat3, and this could be inhibited by SIS3. In conclusion, transgelin expression was induced through the Smad3 signaling pathway during PAN-induced podocyte injury, and the resulting abnormal distribution of F-actin and the enhanced expression of transgelin could be reversed by blockade of this pathway.

Glucocorticoid therapy regulates podocyte motility by inhibition of Rac1.

Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes excessively permeable leading to massive proteinuria. In childhood NS, immune system dysregulation has been implicated and increasing evidence points to the central role of podocytes in the pathogenesis. Children with NS are typically treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids that are activating ligands for the glucocorticoid receptor (GR) transcription factor. Although Gc-therapy has been the cornerstone of NS management for decades, the mechanism of action, and target cell, remain poorly understood. We tested the hypothesis that Gc acts directly on the podocyte to produce clinically useful effects without involvement of the immune system. In human podocytes, we demonstrated that the basic GR-signalling mechanism is intact and that Gc induced an increase in podocyte barrier function. Defining the GR-cistrome identified Gc regulation of motility genes. These findings were functionally validated with live-cell imaging. We demonstrated that treatment with Gc reduced the activity of the pro-migratory small GTPase regulator Rac1. Furthermore, Rac1 inhibition had a direct, protective effect on podocyte barrier function. Our studies reveal a new mechanism for Gc action directly on the podocyte, with translational relevance to designing new selective synthetic Gc molecules.

Levamisole in steroid-sensitive nephrotic syndrome: usefulness in adult patients and laboratory insights into mechanisms of action via direct action on the kidney podocyte.

Minimal change nephropathy (MCN) is the third most common cause of primary nephrotic syndrome in adults. Most patients with MCN respond to corticosteroid therapy, but relapse is common. In children, steroid-dependent patients are often given alternative agents to spare the use of steroids and to avoid the cumulative steroid toxicity. In this respect, levamisole has shown promise due to its ability to effectively maintain remission in children with steroid-sensitive or steroid-dependent nephrotic syndrome. Despite clinical effectiveness, there is a complete lack of molecular evidence to explain its mode of action and there are no published reports on the use of this compound in adult patients. We studied the effectiveness of levamisole in a small cohort of adult patients and also tested the hypothesis that levamisole's mode of action is attributable to its direct effects on podocytes. In the clinic, we demonstrate that in our adult patients, cohort levamisole is generally well tolerated and clinically useful. Using conditionally immortalized human podocytes, we show that levamisole is able to induce expression of glucocorticoid receptor (GR) and to activate GR signalling. Furthermore, levamisole is able to protect against podocyte injury in a puromycin aminonucleoside (PAN)-treated cell model. In this model the effects of levamisole are blocked by the GR antagonist mifepristone (RU486), suggesting that GR signalling is a critical target of levamisole's action. These results indicate that levamisole is effective in nephrotic syndrome in adults, as well as in children, and point to molecular mechanisms for this drug's actions in podocyte diseases.

The immunosuppressive drug mizoribine directly prevents podocyte injury in puromycin aminonucleoside nephrosis.

BACKGROUND/AIMS: Mizoribine (MZR) is an imidazole nucleoside used as a therapeutic immunosuppressive agent. Though a previous report showed that MZR ameliorates proteinuria in puromycin aminonucleoside (PAN) nephropathy, the effect of MZR on podocytes has not been clarified. In this study, we determined whether MZR directly prevents PAN-induced podocyte injury. METHODS: Rats were intravenously injected once on day 0 with 100 mg/kg of PAN and received daily subcutaneous injections of MZR at a dose of 10 mg/kg from days 0 to 14. Cultured podocytes were pretreated with 50 microg/ml of MZR and then treated with 30 microg/ml of PAN. RESULTS: In rat PAN nephrosis, treatment with MZR from days 0 to 14 almost completely inhibited proteinuria. Immunofluorescence staining of nephrin was diminished, showing a discontinuous pattern in saline-treated PAN rats. In contrast, MZR treatment resulted in maintenance of a normal linear pattern. In cultured podocytes exposed to PAN, the percentages of viable cells were significantly increased with MZR treatment. The protective effect of MZR on PAN-induced podocyte injury was independent of inosine 5'-monophosphate dehydrogenase that is a known target enzyme of MZR as an immunosuppressant. MZR reduced PAN-induced integrin-linked kinase activation (ILK) and phosphorylation of glycogen synthase kinase-3beta (GSK3beta) in vivo and in vitro. CONCLUSION: MZR directly prevents PAN-induced podocyte injury, possibly by affecting signaling cascades involving ILK and GSK3beta.

Triptolide protects podocytes from puromycin aminonucleoside induced injury in vivo and in vitro.

Extracts of Tripterygium wilfordii Hook F have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. Triptolide, a diterpene triepoxide, is one of the major active components of these extracts. To clarify its antiproteinuric effects we induced podocyte injury by puromycin aminonucleoside. Triptolide effectively reduced the proteinuria induced by puromycin in nephrotic rats without reducing the glomerular filtration rate. The antiproteinuric effect was associated with improvement in the foot process effacement, a decrease in the podocyte injury marker desmin as well as the restoration of nephrin and podocin expression and distribution. In cultured mouse podocytes triptolide pretreatment prevented the puromycin-induced disruption of the actin cytoskeleton and microfilament-associated synaptopodin while protecting nephrin and podocin expression. Triptolide suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase activation while restoring RhoA signaling activity. These results show that triptolide ameliorates puromycin aminonucleoside-mediated podocyte injury in vivo and in vitro.

An adenosine deaminase inhibitor prevents puromycin aminonucleoside nephrotoxicity.

Puromycin aminonucleoside (PAN) toxicity was totally inhibited in the rat in vivo and in cultured glomerular epithelial cells (GECs) in vitro using the adenosine deaminase (ADA) inhibitor, 2'-deoxycoformycin (DCF). DCF completely inhibited ADA activity in glomeruli and protected against the development of PAN nephrosis; the 24-h urinary protein excretion of treated rats compared with controls (PAN rats) 9 days after PAN injection was 16 +/- 2 mg and 524 +/- 55 mg, respectively (p < .01). Morphological examination also demonstrated that the glomerular epithelial cells were protected against PAN-induced damage. Furthermore, when DCF was added to the first passage of GECs simultaneously with PAN, the adenosine triphosphate contents of remnant GECs on culture substrata increased in a dose-dependent manner, and PA toxicity was completely inhibited by 10(-4) M DCF. The order of ADA activity in glomeruli from various species was as follows: rat > monkey > guinea pig > dog > rabbit > mouse. High activity of ADA in the glomerulus was limited to species in which PAN induced nephrosis. Additionally, DCF increased glomerular cyclic AMP contents, resulting from enhanced adenosine accumulation in the pericellular space. These results indicate that the pathogenesis of PAN toxicity is closely related to adenosine metabolism and that ADA plays a key role in this model. Furthermore, we speculate that DCF contributes to the inhibition of reactive oxygen metabolites by decreasing the substrate of xanthine oxidase and/or increasing pericellular adenosine accumulation.

Inhibition of puromycin-induced renal injury by a superoxide dismutase derivative with prolonged in vivo half-life.

To know the possible involvement of reactive oxygen species and the site(s) of their action in puromycin aminonucleoside (PAN)-induced renal injury, two types of superoxide dismutase (SOD) derivatives were synthesized: one (SM-SOD) circulates bound to albumin with a half-life of 6 h and the other (AH-SOD) linked with hexamethylenediamines rapidly undergoes glomerular filtration and accumulates in renal proximal tubule cells without being excreted in urine. When injected intravenously to the rat, PAN induced a marked proteinuria, increased plasma levels of cholesterol and triglyceride, and suppressed the growth of animals. Intravenously administered SM-SOD significantly inhibited such changes induced by PAN. However, native SOD which rapidly undergoes urinary excretion failed to inhibit the renal injury caused by PAN. Though AH-SOD markedly accumulated in renal proximal tubule cells, it also failed to inhibit the renal injury. These results suggested that superoxide and/or its hazardous metabolite(s) in and around the renal glomerulus, but not in tubule cells, may play critical roles in the pathogenesis of PAN-induced renal injury.

Dup 753 prevents the development of puromycin aminonucleoside-induced nephrosis.

The appearance of nephrotic syndromes such as proteinuria, hypoalbuminemia, hypercholesterolemia and increase in blood nitrogen urea, induced in rats by injection of puromycin aminonucleoside was markedly inhibited by oral administration of Dup 753 (losartan), a novel angiotensin II receptor antagonist, at a dose of 1 or 2 mg/kg per day. The results suggest a possible involvement of the renin-angiotensin system in the development of puromycin aminonucleoside-induced nephrosis.

Induction of differentiation of murine erythroleukemia cells by aminonucleoside of puromycin and inhibition of this induction by purines and purine derivatives.

The effect of the aminonucleoside of puromycin (AMS) on Friend erythroleukemia cells in culture was investigated, because purines and purine analogues are known to act as inducers of differentiation. After treatment with 20-30 micro M AMS for 4 days, the cultures contained between 80 and 90% benzidine-positive cells. Stimulation of hemoglobin synthesis was dose and time dependent. Inosine had no stimulatory activity; however, when it was added to the medium together with AMS, erythroid differentiation was almost completely inhibited. The inhibitory effect of inosine on this potent inducer was also dose and time dependent. No cytotoxicity was observed with either compound, alone or in combination. Inhibition of AMS stimulation of erythroid differentiation was also observed in the presence of inosine monophosphate and poly(inosinic acid). Hypoxanthine had a dual effect. At high concentrations (500 microgram/ml) it acted as an inducer, but when added at low concentrations (20 microgram/ml) together with AMS it inhibited differentiation. These findings suggest there is a link between purine biosynthesis and the event(s) required to trigger differentiation. Agonist-antagonist activity of closely related biological compounds has thus been revealed in the erythroleukemia cells.