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The possibility that hyperlipidemia and an increase of mononuclear cells in the glomeruli could participate in the pathogenesis of minimal change glomerulopathy was evaluated in puromycin aminonucleoside (PAN) nephrosis in rats. Significant increases in intraglomerular CD4-, IL-2-receptor (R)- and ED-l-positive cells were found in PAN rats. Urinary protein excretion and mononuclear cells in the glomeruli of 1% cholesterol diet-fed rats significantly increased, compared with standard diet feeding. Moreover, administration of a subnephrogenic dose of PAN in cholesterol diet-fed rats substantially increased urinary protein excretion and mononuclear cells in the glomeruli. Additionally, antihyperlipidemia agents and immunosuppressive agents prevented urinary protein excretion and increases of CD4-, IL-2R- and ED-l-positive cells in the glomeruli of PAN nephrotic rats. Monoclonal antibodies directed against these cells also prevented urinary protein excretion. These results suggest that CD4-, IL-2R- and ED-l-positive cells and hyperlipidemia are involved in the progression, but not the pathogenesis, of PAN.

In the initial stages of aminonucleoside nephrosis, functional alterations in the glomerular basement membrane occur, as evidenced by the development of proteinuria. However, it has not been possible to observe important ultrastructural modifications at the level of the basement membrane, probably because the changes are taking place at the molecular level. In this study, by the use of high-resolution electron microscopy, an attempt was made to evaluate such changes in rat glomerular basement membrane during acute aminonucleoside nephrosis. As previously reported, in control animals the glomerular basement membrane is composed of a network of 4-nm-wide irregular anastomosing strands, referred to as "cords," which are known to contain a core filament of type IV collagen surrounded by a "sheath" of other components, such as laminin and heparan sulfate proteoglycan (HSPG). The most conspicuous ultrastructural alteration of the nephrotic glomerular basement membrane, recognizable only at high magnification, is that the cords were denuded leaving only the core filament through the loss of the sheath material. Thus, the cord network was transformed, with the progress of pathological conditions, into a network of fine filaments. On the other hand, abundance and distribution of HSPG molecules known to be present in the form of 4.5- to 5-nm-wide ribbon-like "double tracks," were found to be similar in control and nephrotic tissues. Since HSPG is one of the charge proteins of the basement membrane, the little changes observed for HSPG are difficult to interpret in view of reported decreases in basement membrane anionic sites in nephrosis. In conclusion, the glomerular basement membrane in aminonucleoside nephrosis loses its cord network components and replaces them with a more perforated network, which could be a cause for the increased permeability of this basement membrane.

In vivo, glomerular visceral epithelial cells (GVEC), or podocytes, are morphologically highly differentiated cells which are in close contact with adjacent cells by complex interdigitating foot processes. In vitro, the dedifferentiated appearance of podocytes hampers investigations on podocyte structure and function. Cultured podocytes resemble simple epithelium in several ways with apical tight junctions and absence of foot processes. The morphological resemblances between GVEC early in embryonic development, in proteinuric diseases and in cultured cells are striking, but the mechanisms involved in these (de)differentiation processes are poorly understood. A common feature of GVEC in these various states of dedifferentiation is their altered exposure to or even total lack of hydrostatic pressure, suggesting that this may be one of the parameters involved in GVEC differentiation. In this study we investigated whether basolateral hydrostatic pressure could affect GVEC biology in vitro. We therefore exposed cultured GVEC grown on porous supports to basolateral hydrostatic pressure and investigated morphology with scanning and transmission electron microscopy, expression of specific podocyte markers and their biological responses to a model stimulus, the cytokine IFN-gamma. Morphologically, monolayers of pressurized GVEC contained large regions of whirl like, raised cell formations. Individual cells in these formations had a rounded morphology and pore-like indentations between adjacent cells were observed. Cell-cell contacts were often found more basally and intercellular spaces were widened. Moreover, protein expression of pressurized monolayers was altered, as demonstrated by regions of cells with decreased keratin expression. Finally, upon exposure to the model stimulus IFN-gamma, the pressurized as compared to the control GVEC demonstrated a 3-fold increased expression of MHC class II and a strongly decreased sensitivity to the toxic effects of IFN-gamma. In conclusion, we found several indications that hydrostatic pressure can affect podocyte biology in vitro and similar mechanisms may account for podocyte biology in vivo. The strikingly altered morphology and biology of pressurized GVEC suggest that this culture system can be quite relevant for future studies with cultured GVEC.

Mizoribine (MZR), a purine nucleoside antibiotic, is an effective immunosuppressive agent that prevents rejection reactions after kidney transplantation in humans. The present study was performed to examine the effect of MZR on nephrosis produced in rats given puromycin aminonucleoside (PAN). Urinary protein excretion in rats injected with PAN and MZR (PAN + MZR rats) was shown to be reduced significantly in comparison with rats given only PAN (PAN rats). Although mild hypoproteinemia persisted during the experimental period in PAN + MZR rats, no loss of body weight or state of malnutrition was observed. The reduction of serum IgG and C3 was reversed by administration of MZR. Polyethyleinamine (PEI) staining of renal sections showed increased numbers of anionic sites in PAN + MZR rats in comparison with PAN rats, suggesting that MZR improved the permselectivity of the glomerular basement membrane (GBM). Moreover, the production of thromboxane B2 (TxB2) was significantly inhibited in PAN + MZR rats compared with PAN rats. No serious adverse effects of MZR were observed after a large dose of the agent. It is possible that restoration of the charge barrier of the GBM damaged by PAN, or reduction of TxB2 production by the glomeruli may underlie the reduction of protein excretion induced by administration of MZR.

1. We investigated the glomerular expression of three types of myosin heavy-chain isoforms, including S-myosin heavy-chain 40 (SM1), S-myosin heavy-chain 29 (SM2) and FS-myosin heavy-chain 34 (SMemb) in puromycin aminonucleoside nephrosis. 2. There was little change in SM1 and SM2 mRNA levels throughout the experiment. In contrast, glomerular SMemb mRNA increased on days 2 and 4 (before and soon after the onset of proteinuria, respectively), but declined on day 8 (the peak of proteinuria). 3. Histological myosin heavy-chain expression was examined using three antibodies against SM1, SM2 and SMemb. Immunohistochemically, SM1 and SM2 were absent in the glomeruli associated with puromycin aminonucleoside nephrosis until day 20. The SMemb isoform was barely detectable in normal glomeruli, but substantial amounts of SMemb were demonstrated in the glomeruli of rats with puromycin aminonucleoside nephrosis. In the puromycin aminonucleoside-treated rats, the number of SMemb-positive glomerular cells increased on days 2 and 4. 4. We examined whether levels of α-smooth-muscle actin or proliferating cell nuclear antigen correlated with myosin heavy-chain levels in the glomeruli of rats with puromycin aminonucleoside nephrosis. None of the cellular components in the glomeruli was positive for either α-smooth-muscle actin or proliferating cell nuclear antigen in puromycin aminonucleoside nephrosis. 5. Administration of methylprednisolone to puromycin aminonucleoside-treated rats resulted in the rapid disappearance of proteinuria. However, methylprednisolone did not affect SMemb mRNA or immunostaining in the glomeruli of rats with puromycin aminonucleoside nephrosis. 6. These data suggest that SMemb may be a molecular marker for phenotypic change in early glomerular injury, and demonstrate that SMemb regulation differs from that of SM1, SM2, α-smooth-muscle actin and proliferating cell nuclear antigen in the glomeruli of rats with puromycin aminonucleoside nephrosis.

Chronic puromycin aminonucleoside nephropathy (PAN) is an experimental analog of focal segmental glomerulosclerosis. Progressive renal damage in this model is partly mediated by excessive production of oxidant species. Whether dietary supplementation with vitamin E, an endogenous lipophilic antioxidant, ameliorates the severity of chronic PAN was tested. PAN was induced by seven serial injections of the glomerular epithelial cell toxin puromycin aminonucleoside, 2 mg/100 g body wt per dose, over a 12-wk period. Experimental animals (N = 8) received vitamin E-enriched chow (100 IU/kg), whereas control PAN rats (N = 10) were fed standard rodent diet containing vitamin E (30 IU/kg of chow). The administration of vitamin E had no effect on somatic growth or blood pressure; however, rats with PAN fed the vitamin E-enriched diet had an increased hematocrit. In addition, the experimental diet resulted in a 50% reduction in urinary total protein and albumin excretion and stabilization of the serum albumin, cholesterol, and triglyceride concentrations (P0.01). The inulin clearance was 69% higher in the vitamin E-supplemented animals (P0.001). Tubular function, namely, phosphate reabsorption and beta 2-microglobulin excretion, was improved in rats with chronic PAN treated with the vitamin E-enriched diet. There was a significant decrease in glomerulosclerosis and glomerular planar area, and tubulointerstitial scarring was diminished in vitamin E-treated animals with chronic PAN (P0.01). These beneficial effects on renal structure and function were associated with reduced malondialdehyde content in the kidney and liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Menjívar M, Vilchis F, Cárdenas M, Cruz C, Merchant H, Pérez-Palacios G, Pedraza-Chaverri J. Pituitary–ovarian dysfunction in rats with induced nephrotic syndrome. Eur J Endocrinol 1995; 132:502–6. ISSN 0804–4643 The reproductive hormonal profile was evaluated in female rats with experimental nephrotic syndrome induced with a single subcutaneous dose of puromycin aminonucleoside (PAN). Serum concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), testosterone and 17β-estradiol (E2) were determined sequentially in control and experimental groups on days 1,3,7 and 10 after PAN administration. Prolactin levels were also assessed on day 10. In both groups, vaginal smears were taken daily throughout the study to evaluate cyclic histological changes. At the end of the experiment the histological appearance of the ovaries was evaluated by light microscopy. Nephrotic rats had a rapid loss of the estrous cycle starting on day 4, which set them at diestrus. At the same time the hormonal evaluation indicated a gradual decrease in E2, LH and P4 concentrations, starting from days 3, 7 and 10, respectively. No significant changes were noted in FSH or testosterone values. Besides, on day 10, prolactin concentrations remained unmodified. Even though most hormonal levels were found low on day 10, all values except E2 (undetectable) corresponded to the interval reported for the diestrus phase. Likewise, histological examination of ovarian tissue from nephrotic rats showed a considerable increase in the number of atretic follicles. These findings indicate that female rats with nephrotic syndrome develop an important endocrine dysfunction that probably involves steroidogenic tissues (ovary and/or adrenal gland), and suggest the existence of a hypothalamic–hypophyseal impairment. Marta Menjívar, Department of Reproductive Biology, Instituto Nacional de la Nutrición Salvador Zubirán, Vasco de Quiroga N.15, Tlalpan 14000, México, D.F.

Puromycin aminonucleoside (PAN)-induced proteinuria in rats may be mediated by reactive oxygen metabolites (ROM), which are injurious to several cell components including membrane lipids. Increased malondialdehyde (MDA) production is indicative of lipid peroxidation. We examined if MDA content of glomeruli and its urinary excretion were increased in rats administered PAN. Of three groups of 8 Sprague-Dawley rats each, group 1 served a control, group 2 animals received a single intravenous injection of PAN (5 mg/100 g body weight) and group 3 animals PAN with intraperitoneal injections of dimethylthiourea (DMTU), a free radical scavenger of oxidants such as hydroxyl radicals, for 4 days. The rats were sacrificed on day 8 after PAN injection. Increasing proteinuria, starting on day 4, developed in animals in group 2 but not in the others. The glomerular MDA (nmol/mg protein) in group 2 animals was 2.93 +/- 1.91, significantly higher than 0.87 +/- 0.63 and 1.26 +/- 0.76 in groups 1 and 3, respectively. Urinary levels of MDA markedly increased in group 2 rats on day 3 and remained high thereafter, but no such increase occurred in the control animals and those administered PAN with DMTU; the latter was thus protective against PAN toxicity. Our observations support the view that ROM are involved in PAN-induced glomerular injury and that increased urinary MDA excretion can be a marker of ROM-mediated lipid peroxidation.

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.

To establish models of proteinuria in the mouse, BALB/c mice were injected with puromycin aminonucleoside (PAN, 1.5 or 4.5 mg/10 g body weight), adriamycin (AD, 0.2 mg/10 g body weight) intravenously or bovine serum albumin (BSA, 100 mg/10 g body weight) intraperitoneally. Proteinuria was measured as the ratio of urinary albumin (micrograms/ml) to creatinine (mg/dl) and further characterized by isotyping the immunoglobulin. Although not obtained with PAN (followed for 4 weeks), proteinuria was readily attained in the mouse after treatment with AD or BSA. Most AD-treated mice (5/7) developed an abrupt increase of proteinuria at day 2 after injection, with the ratio of urinary albumin to creatinine in the range 0.28-0.45. The degree of proteinuria increased with time and all mice tested (7/7) showed overt proteinuria at day 4. These mice became anuric at day 5 and died at days 6 and 7. For BSA, 4 h after administration, four of seven mice showed enhanced proteinuria, lasting 8 h with urinary albumin and creatinine in a ratio0.05. Isotyping of urine samples collected at the time of heavy proteinuria (ratio of urinary albumin to creatinine:0.40 for AD-treated mice,0.15 for BSA-treated mice) showed that all of the mice (7/7) with AD-induced proteinuria (ADp) and three of four mice with BSA-induced proteinuria (BSAp) revealed urinary IgG2b and IgA, while only one of seven control mice showed IgG2b alone in urine. The mice were sacrificed at the time they presented with heavy proteinura for pathologic and anionic studies on renal tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Nephrotic syndrome is characterized by water and sodium retention, which leads to edema formation. The nonosmotic stimulation of arginine vasopressin (AVP) release from the pituitary gland has been implicated to be one of the important factors of abnormal water retention in patients with nephrotic syndrome. It is not known, however, whether nephrotic syndrome is associated with stimulation of hypothalamic vasopressin gene expression. Puromycin aminonucleoside is known to cause altered glomerular permeability, which results in experimental nephrotic syndrome in rats. In the present study, therefore, AVP gene expression has been studied in the hypothalamus of rats with puromycin aminonucleoside-induced nephrotic syndrome (PNS). Nephrotic syndrome was induced by a single intravenous injection of puromycin aminonucleoside (50 mg/kg body weight). Nephrotic syndrome was confirmed by urinary protein excretion (control 20.8 +/- 3.5 mg/24 hr v PNS 273.9 +/- 41.4 mg/24 hr; P0.0001, n = 6) and serum albumin concentrations (control 4.52 +/- 0.07 g/dL v PNS 2.96 +/- 0.22 g/dL; P0.001, n = 6). In PNS rats, plasma AVP was significantly higher than in control rats (control 0.77 +/- 0.10 pg/mL v PNS 2.13 +/- 0.42 pg/mL; P0.005, n = 12), even though there were no differences in plasma osmolality (control 292.0 +/- 2.0 mOsm/kg H2O v PNS 290.3 +/- 2.5 mOsm/kg H2O; P = NS, n = 12) or serum sodium concentration (control 142.7 +/- 0.7 v PNS 142.1 +/- 1.1; PNS, n = 12).(ABSTRACT TRUNCATED AT 250 WORDS)

1. The present study was designed to assess whether glomerular expression of mRNAs for endothelin-1 and endothelin-3, as well as endothelin receptors A and B is affected by a low-protein diet during the course of focal glomerular sclerosis. 2. Focal glomerular sclerosis was induced in rats by injection of puromycin aminonucleoside on days 0, 27, 34 and 41 in conjunction with unilateral nephrectomy on day 22. Control rats were subjected to nephrectomy or sham operation on day 22. 3. Animals were divided into six groups. In group 1, the puromycin aminonucleoside-injected rats were fed a standard diet containing 22% protein. In group 2, the puromycin aminonucleoside-injected rats were fed a low-protein diet containing 6% protein, which was initiated on the day of the first puromycin aminonucleoside injection. In group 3, the nephrectomized rats without puromycin aminonucleoside were fed a standard diet. In group 4, the nephrectomized rats without puromycin aminonucleoside were fed a low-protein diet. In group 5, the sham-operated rats were fed a standard diet. In group 6, the sham-operated rats were fed a low-protein diet. 4. The percentage of sclerotic glomeruli in group 1 rats increased markedly with time, reaching 77% on day 80. 5. The glomerular mRNA levels for endothelin-1 and endothelin receptors A and B increased significantly as glomerular sclerosis progressed, whereas no endothelin-3 mRNA was detected in the glomeruli of any group. 6. The endothelin-1 production in isolated glomeruli from group 1 increased significantly as glomerular sclerosis progressed. 7. In group 2, the low-protein diet reduced the prevalence of glomerular sclerosis, attenuated the rise in mRNA levels for endothelin-1 and endothelin receptors A and B and reduced endothelin-1 production in glomeruli from rats with focal glomerular sclerosis. 8. These data indicate that increases in glomerular endothelin-1 and endothelin receptor mRNA levels are associated with the development of puromycin aminonucleoside-induced glomerular sclerosis. These effects are blunted by administration of a low-protein diet.

We investigated the effects of KW-3902 (8-(noradamantan-3-yl)-1, 3-dipropylxanthine), a potent adenosine A1-receptor antagonist, on the nephrotic edema induced by puromycin aminonucleoside (PAN; 100 mg/kg, i.v.) in rats. The treatment with PAN decreased urine volume and urinary excretions of sodium and potassium, resulting in the ascites formation in 7 days. In rats with the nephrosis, KW-3902 (0.01-1 mg/kg/day for 3 days, p.o.) showed diuretic effects and reduced the volume of ascites, as was the case with furosemide (30 mg/kg/day) and trichlormethiazide (1 mg/kg/day). These results suggest that even in the nephrotic state, the adenosine A1-receptor antagonist can be an effective diuretic to ameliorate edema.

Microtubule-associated protein 1 light chain 3 (LC3) is a unique modifier protein. LC3-I, the cytosolic form, is modified to LC3-II, the membrane-bound form, by a mechanism similar to ubiquitylation by E1- and E2-like enzymes, Apg7p and Apg3p, respectively. In the present study, we found that LC3-I is processed to LC3-II during the differentiation and recovery from puromycin aminonucleoside-induced nephrosis of podocytes. LC3 is especially expressed in the podocytes of rat kidney as the membrane-bound form LC3-II. Biochemical analysis using a conditionally immortalized mouse podocyte clone (MPC) revealed that LC3-I is processed to LC3-II during the differentiation of cells into mature podocytes and accumulates in the membrane-rich fraction of the cell lysate. LC3-II-localized vesicles, which differ from lysosomes and endosomes, in differentiated MPC cells are morphologically similar to autophagic vacuoles during starvation-induced autophagy. During starvation-induced autophagy, autophagosomes fuses with lysosome and LC3-II on autophagosomes is finally degraded by lysosomal proteases. However, in differentiated MPC cells, little LC3-II on the vesicles is degraded by lysosomal proteases, suggesting that little LC3-II-localized vesicles in differentiated MPC cells fuse with lysosome. Furthermore, the LC3-II level in differentiated MPC cells increases with recovery from damage caused by experimental puromycin aminonucleoside-induced nephrosis. These results suggest that LC3-II-localized vesicles play an important role in the physiological function of podocytes.

Minimal-change nephrotic syndrome (MCNS) is a kidney disease defined by selective proteinuria and hypoalbuminemia occurring in the absence of cellular glomerular infiltrates or immunoglobulin deposits. Recent observations suggest that nuclear factor κB (NF-κB) of podocyte is strongly associated with the development of proteinuria in MCNS. Dehydroxymethylepoxyquinomicin (DHMEQ) is a novel NF-κB inhibitor that potently inhibits DNA-binding activity of NF-κB, resulting in several therapeutic effects in various pathological conditions. We conducted this study to ask whether DHMEQ may ameliorate the nephrosis in mice induced by puromycin aminonucleoside (PAN), which is considered to be an animal model for MCNS.Pretreatment with DHMEQ alleviated the proteinuria and reversed the serum abnormalities in mice nephrosis induced by 450 mg/kg of PAN. Increased serum interleukin-6 level in PAN-induced nephrosis was also completely suppressed by DHMEQ. Electron microscopic analyses of glo-meruli indicated that DHMEQ can inhibit the podocyte foot process effacement via blocking the translocation of podocyte NF-κB from cytoplasm to nucleus.These results suggest that DHMEQ can be a potential therapeutic agent for MCNS.

Proteinuria is considered to play an essential role in the progression of tubulointerstitial damage, which causes end-stage renal disease. Fatty acid-binding albumins are filtered through glomeruli and reabsorbed into proximal tubular epithelial cells (PTECs). However, the role of fatty acid metabolism associated with albuminuria in the development of tubulointerstitial damage remains unclear. Thus, the present study was designed to determine the changes of fatty acid metabolism in the nephrotic kidney. To induce nephrotic syndrome, Sprague-Dawley rats (SDRs) and Nagase analbuminemic rats (NARs) with inherited hypoalbuminemia were treated with a single injection of puromycin aminonucleoside (PAN). In SDRs, PAN treatment induced massive proteinuria and albuminuria and caused tubular damage, apoptosis, and lipid accumulation in PTECs. Among the enzymes of fatty acid metabolism, expressions of medium-chain acyl-CoA dehydrogenase (MCAD) and cytochrome P-450 (CYP)4A significantly decreased in PTECs of PAN-treated SDRs. Expressions of peroxisome proliferator-activated receptor (PPAR)-γ coactivator (PGC)-1α and estrogen-related receptor (ERR)α also significantly decreased, without changes in the expression of PPAR-α. In NARs, PAN treatment induced proteinuria but not albuminuria and did not cause tubular damage, apoptosis, or lipid accumulation. Expressions of MCAD, PGC-1α, or ERRα did not change in the kidney cortex of PAN-treated NARs, but the expression of CYP4A significantly decreased. These results indicate that massive albuminuria causes tubular damage and lipid accumulation with the reduction of MCAD, CYP4A, PGC-1α, and ERRα in PTECs.

Dietary salt restriction may reduce proteinuria although the mechanism is not clear. We investigated the effects of dietary salt restriction on rat kidneys in an animal model of glomerular proteinuria. Male Sprague-Dawley rats were divided into 3 groups: vehicle-treated normal-salt controls, puromycin aminonucleoside (PA)-treated normal-salt rats, and PA-treated low-salt rats. PA was given at a dose of 150 mg/kg BW at time 0, followed by 50 mg/kg BW on days 28, 35, and 42. Sodium-deficient rodent diet with and without additional NaCl (0.5%) were provided for normal-salt rats and low-salt rats, respectively. On day 63, kidneys were harvested for histopathologic examination. PA treatment produced overt proteinuria and renal damage. Dietary salt restriction insignificantly reduced proteinuria in PA-treated rats, and PA-treated low-salt rats had lower urine output and smaller creatinine clearance than vehicle-treated normal-salt controls. The tubulointerstitial injury score positively correlated with proteinuria. It was significantly increased by PA treatment and relieved by low-salt diet. ED1-positive infiltrating cells and immunostaining for interstitial collagen III were significantly increased by PA treatment. These changes appeared less in PA-treated low-salt rats although the differences in PA-treated normal-salt versus low-salt rats did not reach the statistical significance. Our results suggest that renal histopathology in PA nephrosis may potentially be improved by dietary salt restriction. The decreasing tendency in glomerular filtration rate induced by low-sodium diet might be beneficial in retarding renal progression.

Apoptosis, which is usually a response to the microenvironment, requires inactivation of prosurvival molecules. Apoptosis contributes to loss of podocytes in the course of renal injury, an event closely associated with the development of proteinuria. Dexamethasone (DEX) is the standard of care for most forms of nephrotic syndrome. However, the precise mechanisms of DEX action on podocytes are unknown. This study examined the hypothesis that cultured podocytes exposed to puromycin aminonucleoside (PAN) showed a reduced rate of apoptosis upon DEX exposure. Apoptotic podocytes seemed to be related to increased Bad mRNA and protein expressions. DEX reduced apoptosis by decreasing Bad mRNA and protein expressions, thereby protecting podocytes. These findings provided insights into the beneficial effects of DEX directly on podocytes. The present study illustrated the signal transduction mechanism of podocyte apoptosis induced by PAN.

Macrophage infiltration into the glomerular mesangium is a prominent feature of various glomerulopathies. Recent evidence suggests that infiltrating macrophages may play a role in propagating initial glomerular injury to the development of glomerulosclerosis via transforming growth factor-beta (TGF-beta)-stimulating matrix accumulation. Rats with the acute puromycin aminonucleoside (PA) nephrosis exhibit an elevated gene expression of glomerular TGF-beta 1; however, the cellular origin of this upregulation is unknown. Using polymerase chain reaction (PCR), we detected that the TGF-beta 1 isoform is expressed in glomerular macrophages isolated from experimental rats made hypercholesterolemic by either diet or by induction of PA nephrosis. Peritoneal macrophages from nephrotic or dietary-hypercholesterolemic animals also exhibited a significant increment in the expression of TGF-beta 1 mRNA on Northern analysis, in contrast to similar cells obtained from normal control rats. PCR analysis of glomerular RNA also detected the expression of the TGF-beta 2 mRNA isoform. TGF-beta 2 mRNA expression was not observed in isolated glomerular macrophages from either glomeruli of PA-nephrotic rats or from glomeruli of animals with dietary hypercholesterolemia. Expression of the TGF-beta 3 mRNA isoform was only observed by PCR in J774 A.1 cells. Thus the as a cellular source for the enhanced expression of TGF-beta 1 during the acute nephrotic phase of our toxic, progressive glomerulopathy model and within several days of inducing only hypercholesterolemia by dietary means.

Whether a reduction in urinary protein excretion in rats coadministered puromycin aminonucleoside and antioxidants was associated with a reduction in alterations to glomerular epithelial cell (podocyte) ultrastructure was examined. Daily urinary protein excretion was measured in rats that received a single i.v. injection of saline or puromycin aminonucleoside with or without coadministration of antioxidants. The coadministration of alpha-tocopherol/ascorbic acid, dimethyl thiourea, or superoxide dismutase to puromycin aminonucleoside-treated rats reduced proteinuria by approximately 90, 40, and 60%, respectively, over the 18-day period studied. For a second group of rats, daily urinary protein excretion was measured and kidneys were processed for light microscopy and transmission and scanning electron microscopy 4, 5, and 10 days after injection. Transmission electron microscopic morphometric analysis of glomeruli from puromycin aminonucleoside-treated rats coadministered antioxidants revealed significantly reduced foot process effacement on Days, 4, 5, and 10 compared with rats that received puromycin aminonucleoside alone. Thus, at Day 10, puromycin aminonucleoside-treated rats coadministered alpha-tocopherol/ascorbic acid, dimethyl thiourea, or superoxide dismutase contained 90, 74, and 88% (P < 0.01 in all cases) more glomerular epithelial cell filtration slits per unit length of glomerular basement membrane than rats treated with puromycin aminonucleoside alone. In contrast, by scanning electron microscopy, the antioxidants were found to provide no protection against the changes occurring in glomerular epithelial cell bodies and major processes. These results provide further evidence of a role for reactive oxygen species in puromycin aminonucleoside nephrosis and indicate that the antioxidants provide protection against the changes occurring in glomerular epithelial cell foot processes.

Added to these could be proteinuria. The abnormal passage of macromolecules through the glomerular basement membrane may itself set in train a series of events which leads to end-stage renal disease. Not only can one argue that proteinuria may contribute to intrinsic glomerular damage, but also that the presence of large quantities of protein within the tubules may have a damaging effect on the interstitium. This is particularly relevant since attention has recently focused on a long known but ignored fact that the progression of renal disease correlates best with interstitial damage and not with glomerulosclerosis per se [3, 4]. This review will examine the evidence in both humans and animals which purports to support the "proteinuria hypothesis." It will also address mechanisms whereby increased gbmerular permselectivity might contribute indirectly to a decline in renal function.

To clarify the pathogenesis of idiopathic iron deficiency anemia, small amounts of Puromycin aminonucleoside (PAN) which is known to be nephrotoxic, was administered repeatedly to induce continuous urinary iron excretion in rats. Urinary iron, serum iron, stored iron content and hemoglobin concentration were investigated. Urinary iron excretion was increased by PAN administration along with increased excretion of urinary transferrin. Consequently, a large amount of urinary iron seemed to be was observed with respect to continuous urinary iron excretion, manifesting normocytic hypochromic anemia at 13th week. These results suggest that prolonged loss of small amounts of iron through the urinary tract causes iron deficiency without alinical manifestation of ranal failure.

Puromycin aminonucleoside (PA) and Adriamycin (ADR) cause glomerular proteinuria associated with degenerative alterations of glomenular visceral epithelial cells (GVEC) and detachment from the glomenular basement membrane when administered to rats. This in vitro study was performed to define, in detail, the quantitative and qualitative changes of a number of adhesion-associated proteins (cytoskeletal, extracellular matrix and integrin proteins) upon exposure to PA and ADR. By immunofluorescence we observed: (1) dose- and incubation-time-dependent filament pattern changes and decreased staining of the cytoskeletal proteins actin, vimentin, keratin, and beta-tubulin; (2) an altered distribution, and decreased expression of the extracellular matrix proteins laminin and heparan sulfate and (3) a loss of the beta(1)-integrin focal adhesions upon exposure to PA and ADR. Using an ELISA, a concentration-dependent decrease was found (a 50% reduction with 50 mu g/ml PA for 48 h and with 2 mu g/ml ADR for 24 h) in the production of cytoskeletal and extracellular matrix proteins per cell. These general effects were suggestive of a disturbance of protein synthesis but, by metabolic labelling studies, no reduction in overall protein synthesis was found. Using two-dimensional PAGE on S-35-methionine steady-state labeled cells, no changes were found in intracellular protein patterns of PA- and ADR-treated cells (pH 5-7.5, MW 110-20 kD). We hypothesize that exposure of GVEC in vitro to PA and ADR might result, directly or indirectly, in perturbation of the macromolecular organization of cytoskeletal and extracellular matrix proteins with loss of GVEC adhesion.

The human nephrotic syndrome is accompanied by important alterations of the coagulation system related proteins. The purpose of the present study was to examine the activity of coagulation- and fibrinolysis-related proteins in plasma and urine of control and puromycin aminonucleoside injected rats on days 2 (prenephrotic stage) and 10 (nephrotic stage). We measured the prothrombin time (PT), the activated partial thromboplastin time (aPTT), and the activities of (1) the coagulation factors (CFs) I, II, V, and VII-XII; (2) the inhibitor of coagulation antithrombin III (ATIII), and (3) the component of the fibrinolytic system α2-antiplasmin (α2-APL). PT and aPTT and the activities of CF, ATIII, and α2-APL were not measurable in the urine of control and puromycin aminonucleoside injected rats on day 2. On this same day, plasma ATIII and CF VIII decreased. On day 10 (1) PT and aPTT decreased in plasma and were not measurable in urine; (2), plasma CFs I, II, V, VII, VIII, X, and XI increased; (3), plasma ATIII decreased; (4), plasma CFs IX and XII and α2-APL did not change, and (5) ATIII and CFs II, VII, VIII, IX, X, XI, and XII, but not CFs I and V and α2-APL, appeared in urine on day 10. ATIII deficiency was secondary probably to the urinary losses; however, the plasma activity of CFs II, VII, VIII, X, and XI increased and that of CFs IX and XII remained unchanged in spite of their urinary losses which suggests that other mechanisms such as deranged catabolism and altered hepatic synthesis may be involved. This model may be useful to study the pathophysiology of the abnormalities of coagulation in humans with nephrotic syndrome.

2'-Deoxy-2'-N-phthaloyl nucleosides were prepared from arabino nucleosides by triflate displacement with phthalimide in the presence of DBU. The corresponding phosphoramidites suitable for automated oligonucleotide synthesis were also synthesized. The scalability of described procedures was demonstrated on a 100-g scale preparation of 2'-deoxy-2'-amino-C phosphoramidite.

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.

Ribosomally mediated protein biosynthesis is limited to α-L-amino acids. A strong bias against β-L-amino acids precludes their incorporation into proteins in vivo and also in vitro in the presence of misacylated β-aminoacyl-tRNAs. Nonetheless, earlier studies provide some evidence that analogues of aminoacyl-tRNAs bearing β-amino acids can be accommodated in the ribosomal A-site. Both functional and X-ray crystallographic data make it clear that the exclusion of β-L-amino acids as participants in protein synthesis is a consequence of the architecture of the ribosomal peptidyltransferase center (PTC). To enable the reorganization of ribosomal PTC architecture through mutagenesis of 23S rRNA, a library of modified ribosomes having modifications in two regions of the 23S rRNA (2057-2063 and 2496-2507 or 2582-2588) was prepared. A dual selection procedure was used to obtain a set of modified ribosomes able to carry out protein synthesis in the presence β-L-amino acids and to provide evidence for the utilization of such amino acids, in addition to α-L-amino acids. β-Puromycin, a putative mimetic for β-aminoacyl-tRNAs, was used to select modified ribosome variants having altered PTC architectures, thus potentially enabling incorporation of β-L-amino acids. Eight types of modified ribosomes altered within the PTC have been selected by monitoring improved sensitivity to β-puromycin in vivo. Two of the modified ribosomes, having 2057AGCGUGA2063 and 2502UGGCAG2507 or 2502AGCCAG2507, were able to suppress UAG codons in E. coli dihydrofolate reductase (DHFR) and scorpion Opisthorcanthus madagascariensis peptide IsCT mRNAs in the presence of β-alanyl-tRNA(CUA).

Besides its primary function in maintaining systemic calcium homeostasis, the calcium-sensing receptor (CaSR) is expressed by many cell types, with different, sometimes opposite, regulatory functions. Novel work from Oh and collaborators shows that activation of CaSR in podocytes has prosurvival effects and protects the cell from puromycin aminonucleoside damage. Given that the cellular consequences of CaSR activation are largely context-dependent, further studies will be required to elucidate its precise role in podocyte physiology and pathophysiology.

BACKGROUND Changes in podocyte phenotype and function are characteristic of proteinuric glomerular diseases. Integrin-linked kinase (ILK) functions as a common downstream effector in proteinuric diseases. In addition, ILK was shown to interact with the Wnt signaling pathway. Here, we investigated ILK expression as well as its involvement with the Wnt signaling pathway in renal biopsies of patients with primary focal segmental glomerulosclerosis (FSGS), and in a correspondent in vivo model of podocyte lesion. METHODS Biopsies from 37 patients with primary FSGS were evaluated by immunohistochemistry for ILK, phosphorylated GSK-3s (pGSK-3s) and s-catenin expression. As experimental model, male Wistar rats received 5 injections of puromycin aminonucleoside (PAN) at 2-week intervals, and their kidneys were evaluated for ILK, P-cadherin and pAkt expression as well as s-catenin and LEF-1 colocalization. RESULTS Patients presented de novo ILK expression and pGSK-3s in podocytes. In animals, there was an increase in gene and protein expression of ILK, mainly detected in the podocytes, as well as increased protein expression of pAkt compared with controls. s-Catenin translocated to the nuclei of podocytes in animals and patients. s-Catenin colocalized with LEF-1 in the nuclei of podocytes of animals. Gene expression of s-catenin and P-cadherin in PAN rats was lower compared with controls. CONCLUSIONS Our findings suggest that activation of ILK activated the Wnt signaling pathway in damaged podocytes. This phenomenon could have an important role in development and/or progression of clinical and experimental FSGS.

This study was aimed to construct eukaryotic expression vectors carrying the small hairpin RNA (shRNA) targeting TRPC6 gene and investigate the effect of TRPC6 knockdown on puromucin aminonucleoside (PAN)-induced podocyte injury. Two DNA sequences containing the small hairpin structure targeting TRPC6 were designed, synthesized and then inserted into the green fluorescence protein (GFP)-contained plasmids (pGC) to establish the plasmids pGCsi-TRPC6A and pGCsi-TRPC6B. Plasmids expressing scrambled shRNA were used as negative control and named pGCsi-NC. These plasmids were transfected into a conditionally immortalized murine podocyte cell line by using liposome. Flow cytometry was used to examine the transfection efficiency. TRPC6 mRNA and protein expression levels were detected by RT-PCR and Western blotting. Cultured podocytes were divided into four groups: control group, PAN treatment group, PAN+TRPC6 shRNA transfected group and PAN+scrambled shRNA transfected group. The paracelluar permeability to BSA was evaluated by Millicell-PCF Inserts and cell viability was measured by the trypan blue assay. Immunofluorescent assay was used to observe the distribution of α-actinin-4 and α-tubulin. The results showed that the transfection efficiency of the shRNA expression vector was about 45%. Expression levels of TRPC6 mRNA and protein were downregulated after transfection with pGCsi-TRPC6A and pGCsi-TRPC6B. Knocking down TRPC6 gene could effectively reverse the PAN-induced increase in the paracelluar permeability to BSA. The distribution of α-actinin-4 and α-tubulin was disrupted after treatment with PAN, which was reversed by knocking down TRPC6 gene. It was concluded that knocking down TRPC6 gene could effectively prevent podocytes from the permeability increase induced by PAN, which may be related to the regulation of podocyte cytoskeleton.

Structural changes of podocytes and retraction of their foot processes are a critical factor in the pathogenesis of minimal change nephritis and glomerulosclerosis. Here we tested, if connective tissue growth factor (CTGF) is involved in podocyte injury during acute and chronic puromycin aminonucleoside nephrosis (PAN) as animal models of minimal change nephritis, and focal segmental glomerulosclerosis, respectively. Rats were treated once (acute PAN) or for 13 weeks (chronic PAN). In both experimental conditions, CTGF and its mRNA were found to be highly upregulated in podocytes. The upregulation correlated with onset and duration of proteinuria in acute PAN, and glomerulosclerosis and high expression of glomerular fibronectin, and collagens I, III, and IV in chronic PAN. In vitro, treatment of podocytes with recombinant CTGF increased amount and density of actin stress fibers, the expression of actin-associated molecules such as podocalyxin, synaptopodin, ezrin, and actinin-4, and activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Moreover, we observed increased podocyte expression of mRNA for transforming growth factor (TGF)-β2, TGF-β receptor II, fibronectin, and collagens I, III, and IV. Treatment of cultured podocytes with puromycin aminonucleoside resulted in loss of actin stress fibers and cell death, effects that were partially prevented when CTGF was added to the culture medium. Depletion of CTGF mRNA in cultured podocytes by RNA interference reduced both the number of actin stress fibers and the expression of actin-associated molecules. We propose that the expression of CTGF is acutely upregulated in podocytes as part of a cellular attempt to repair structural changes of the actin cytoskeleton. When the damaging effects on podocyte structure and function persist chronically, continuous CTGF expression in podocytes is a critical factor that promotes progressive accumulation of glomerular extracellular matrix and glomerulosclerosis.

Ethnopharmacological relevance Macrothelypteris torresiana is traditionally used in Chinese folk medicine for the treatment of edema for patients suffering from kidney/bladder problems due to its satisfactory therapeutic effectiveness. Aim of the study The aim of this study was to investigate the renoprotective nature of the total polyphenols fraction from Macrothelypteris torresiana (PMT). Materials and methods The biochemical criterions of plasma and kidney tissues were evaluated to study the effects of PMT on puromycin aminonucleoside-induced chronic nephrotic syndrome (NS) in hyperlipidemic mice. Results In this study, the NS and hyperlipidemia were ameliorated after 9 weeks administration of PMT. Besides, PMT was able to modulate the level of renal oxidative stress and vascular endothelial growth factor–nitric oxide (VEGF–NO) pathway. Conclusions It represented a potential resource of PMT for the treatment of NS involved in metabolic syndrome.

Background: Nestin is an intermediate filament protein widely used as a marker of stem cells or progenitor cells. Nestin is also highly expressed in the glomerular podocyte, a type of terminally differentiated epithelial cell. Little is known about the significance of nestin in podocytes. Methods: Puromycin aminonucleoside (PAN) was injected into the rats to produce a PAN nephrosis model. Transmission electronic microscopy and terminal dUTP nick end-labeling assay were used to examine the podocyte foot process (FP) effacement and apoptosis, respectively. A mouse podocyte cell line was cultured and incubated with PAN. Immunoblot was used to examine the level of nestin expression both in vivo and in vitro. Enhanced green fluorescence protein-tagged plasmids containing nestin shRNA were transfected into the cultured podocytes to silence nestin expression. F-actin arrangement within cultured podocytes was investigated by immunofluorescence, while the apoptosis rate was examined by both Hoechst stain and flow cytometry. Results: In the PAN-induced rat nephrosis model, podocyte nestin expression was increased in the absence of apparent podocyte apoptosis, even though the FP was significantly effaced. In the cultured mouse podocytes, PAN upregulated nestin expression in a time-dependent manner within 24 h of treatment. Notably, no significant apoptosis occurred, however knocking down nestin expression resulted in a remarkable derangement of actin cytoskeleton and an increase in apoptosis in the cultured podocytes 24 h after being incubated with PAN. Conclusions: Upregulation of nestin expression during PAN nephrosis could protect podocytes from apoptosis and that this process is mediated by maintaining the regular arrangement of actin cytoskeleton.

While mitogen-activated protein kinase (MAPK) activation has been implicated in the pathogenesis of various glomerular diseases, including nephrotic syndrome (NS), its specific role in podocyte injury is not known. We hypothesized that MK-2, a downstream substrate of p38 MAPK, mediates the adverse effects of this pathway and that inhibition of MK-2 would protect podocytes from NS-related injury. Using cultured podocytes, we analyzed 1) the roles of MK-2 and p38 MAPK in puromycin aminonucleoside (PAN)-induced podocyte injury; 2) the ability of specific MK-2 and p38 MAPK inhibitors to protect podocytes against injury; 3) the role of serum albumin, known to induce podocyte injury, in activating p38 MAPK/MK-2 signaling; and 4) the role of p38 MAPK/MK-2 signaling in the expression of Cox-2, an enzyme associated with podocyte injury. Treatment with protein kinase inhibitors specific for both MK-2 (C23, a pyrrolopyridine-type compound) or p38 MAPK (SB203580) reduced PAN-induced podocyte injury and actin cytoskeletal disruption. Both inhibitors reduced baseline podocyte p38 MAPK/MK-2 signaling, as measured by the degree of phosphorylation of HSPB1, a downstream substrate of MK-2, but exhibited disparate effects on upstream signaling. Serum albumin activated p38 MAPK/MK-2 signaling and induced Cox-2 expression, and these responses were blocked by both inhibitors. Given the critical importance of podocyte injury to both NS and other progressive glomerular diseases, these data suggest an important role for p38 MAPK/MK-2 signaling in podocyte injury and identify MK-2 inhibition as a promising potential therapeutic strategy to protect podocytes in various glomerular diseases.

Background. Peroxisome proliferator-activated receptor gamma (PPARc) agonists have beneficial effects on renal structure and function in models of diabetes and chronic kidney diseases. However, the increased incidence of weight gain and edema potentially limits their usefulness. We studied an acute minimal-change disease-like nephrotic syndrome model to assess effects of PPARc agonist on acute podocyte injury and effects on fluid homeostasis. Methods. Acute podocyte injury and nephrotic syndrome were induced by puromycin aminonucleoside (PAN) injection in rats. Results. PPARc agonist, given at the time or after, but not before PAN, reduced proteinuria, restored synaptopodin, decreased desmin and trended to improve foot process effacement. There was no significant difference in glomerular filtration, effective circulating volume, blood pressure or fractional sodium excretion. PAN-injured podocytes had decreased PPARc, less nephrin and a-actinin-4, more apoptosis and reduced phosphorylated Akt. In PAN-injured cultured podocytes, PPARc agonist also reversed abnormalities only when given simultaneously or after injury. Conclusions. These results show that PPARc agonist has protective effects on podocytes in acute nephrotic syndrome without deleterious effects on fluid homeostasis. PPARc agonist-induced decrease in proteinuria in acute nephrotic syndrome is dependent at least partially on regulation of peroxisome proliferator-response element-sensitive gene expression such as a-actinin-4 and nephrin and the restoration of podocyte structure.

Numb was originally discovered as an intrinsic cell fate determinant in Drosophila by antagonizing Notch signaling. The present study is to characterize the role of Numb in oxidative stress-induced apoptosis of renal proximal tubular cells. Exposure of NRK52E cells to puromycin aminonucleoside (PA) resulted in caspase 3-dependent apoptosis. Numb expression was downregulated by PA in a time- and dose-dependent manner. Knocking down endogenous Numb by siRNA sensitized NRK52E cells to PA-induced apoptosis, whereas overexpressing Numb protected NRK52E cells from PA-induced apoptosis. Moreover, PA activated Notch signaling in a time- and dose-dependent manner as indicated by increased expression of the intracellular domain of Notch and Hes-1. Notch signaling inhibitor DAPT significantly attenuated Numb siRNA-augmented apoptosis. On the other hand, overexpression of intracellular domain of Notch1 could reverse the protective effect of Numb on PA-induced apoptosis. Taken together, our data demonstrated that, in renal proximal tubular cells, Numb functions as a protective molecule on PA-induced apoptosis through antagonizing Notch signaling activity.

Taurine, a sulfur amino acid, is present in abundant amounts in cells throughout the body. The kidney regulates taurine balance by modulating proximal tubule reabsorption in response to fluctuations in dietary intake of this nutrient. There is no information about the localization of taurine within the kidney in normal and diseased renal parenchyma. Therefore, using an antibody to a taurine-glutaraldehyde-BSA conjugate, we examined the distribution of taurine in renal tissue. Normal rats, those with streptozocin diabetes, puromycin aminonucleoside nephropathy, bilateral ureteral ligation, and 5/6 nephrectomy were studied. In normal animals, taurine was found primarily in medullary tubules, with minimal staining of proximal tubules and glomeruli. There was increased taurine staining of all structures, especially medullary tubules, in rats with streptozocin diabetes and puromycin aminonucleoside nephropathy. These changes were more pronounced in diabetic rats and were unrelated to renal medullary osmolality. The distribution of taurine within the kidney was unchanged in the models of acute and chronic renal failure. Alterations in the immunohistochemical localization of taurine correlated with the beneficial effect of this amino acid to preserve renal function in the rats with chronic diabetes and puromycin aminonucleoside nephropathy. These results suggest that taurine is preferentially localized in the medullary regions of the kidney, where it exerts a protective effect against renal injury in select disease states.

SUMMARY 1. The effect of continuous intravenous administration of 1 UmUg/h atrial natriuretic peptide (ANP) for 4 days was studied in normal male Sprague-Dawley rats and rats made nephrotic with puromycin aminonucleoside (PA). 2. ANP infusion significantly increased urinary sodium and potassium excretion by 3 days of infusion in control rats but not in PA-treated rats. ANP infusion significantly increased glomerular filtration rate in PA-treated rats, while effective renal plasma flow was similarly decreased compared with non-infused nephrotic rats. 3. Plasma high density lipoproteins (HDL) were significantly decreased and low density lipoproteins (LDL) were increased in PA-treated rats that received ANP; HDL were increased in normal rats infused with ANP. 4. Competitive binding studies demonstrated a lower density of specific ANP receptors in glomerular membranes from rats injected with PA, while binding affinity was unchanged. 5. Infusion with exogenous ANP did not promote natriuresis in PA nephrosis despite an enhancement of glomerular filtration rate (GFR), thus suggesting that sodium retention in this model is due to a post-glomerular defect. Plasma lipoprotein composition in both normal and nephrotic rats may be affected by ANP.

Hypercholesterolemia aggravates experimental progressive glomerular injury. Evidence suggests the infiltrating glomerular macrophage (M phi) is a potential effector mechanism for the noxious effects of hypercholesterolemia. Because transforming growth factor (TGF)-beta 1 is secreted by activated M phi s and also stimulates fibronectin production by glomerular cells, we evaluated the kinetics of gene expression for these moieties in glomeruli isolated from nephrotic rats at 3, 7, 11, and 42 days after the delivery of puromycin aminonucleoside (PA). We also assessed whether cholesterol feeding, which raises the glomerular M phi number, alters the glomerular mRNA levels for TGF-beta 1 and fibronectin. Glomerular mRNA levels for TGF-beta 1 and fibronectin in nephrotic rats exhibited a biphasic temporal pattern, decreasing significantly below control at 3 and 7 days after PA but increasing significantly at 11 and 42 days after PA. The upregulated gene expression for TGF-beta 1 and fibronectin at 11 days after PA temporally corresponded to the phase of mesangial M phi infiltration in this model. Cholesterol feeding to both normal and nephrotic rats significantly increased glomerular TGF-beta 1 and fibronectin mRNA levels at 11 days after PA. Immunohistochemical labeling for M phi s and intracellular TGF-beta 1 demonstrated both mesangial and cortical interstitial localization with the TGF-beta1-positive cells possessing M phi nuclear morphology. These findings identify a novel interaction between hypercholesterolemia, augmented glomerular M phi accumulation, and upregulated glomerular TGF-beta 1 and fibronectin gene expression. These perturbations within the acutely injured glomerulus constitute an early pathobiological determinant for the later development of mesangial matrix expansion and glomerulosclerosis.

The effect of cyclosporin (CS) on intrinsic glomerular level of antioxidants in puromycin aminonucleoside (PAN) nephrosis was examined. A single intravenous dose of PAN (50 mg/kg body weight) given to Sprague-Dawley rats resulted in marked proteinuria. Ten days after PAN injection, the rats were treated with daily intraperitoneal injection of CS (10 mg/kg body weight/day) for 10 days. PAN-treated rats without CS treatment (PAN rats) had significantly lower activities of glomerular superoxide dismutase (SOD) and catalase (CAT) than normal rats (p0.05, respectively). When compared with PAN rats, CS-treated PAN rats had significantly less proteinuria and higher activities of glomerular SOD and CAT (p0.01 and p0.05, respectively). Significant elevation of glomerular malondialdehyde (MDA) level characteristic of PAN rats was absent in CS-treated PAN rats. Moreover, segmental sclerosis with capsular adhesion, hyalinosis, epithelial cell foot process fusion and microvillous transformation seen in PAN rats were apparently attenuated in CS-treated PAN rats. When compared with normal rats, rats receiving CS only had a significantly higher CAT activity and MDA level (p0.01 and p0.05, respectively). Assessment of glomerular reduced glutathione revealed no significant differences among PAN rats, CS-treated PAN rats, normal rats, and rats receiving only CS. These data indicate that glomerular antioxidant enzyme activities are modulated by CS.

In order to determine the activity of the renin-angiotensin system in the nephrotic syndrome, the plasma concentration of angiotensinogen was measured in rats with puromycin aminonucleoside (PA)-induced nephrosis using two different methods: a direct radioimmunoassay, which measures both angiotensinogen and des-angiotensin I-angiotensinogen, and an indirect assay, which measures angiotensin I liberated from angiotensinogen by excess renin. The plasma concentration of angiotensinogen as measured by the direct assay increased before the appearance of PA-induced hypoproteinemia or proteinuria and subsequently decreased to normal levels simultaneously with the appearance of proteinuria. The indirect assay of angiotensinogen also demonstrated an increased concentration of plasma angiotensinogen before the development of nephrosis, but the level decreased to below normal after the appearance of proteinuria. Both plasma renin concentration and renin activity also increased simultaneously with the increase in plasma angiotensinogen. The difference between the concentrations of plasma angiotensinogen determined by these methods increased before and during the early phase of PA-induced nephrosis, suggesting the increased consumption of angiotensinogen by renin during this period. Measurement of plasma corticosterone and serum interleukin-6 revealed that these circulating factors were not involved in the elevation of plasma angiotensinogen in rats with PA-induced nephrosis. These results indicate that the renin-angiotensin system is activated before the appearance of PA-induced nephrotic syndrome.

Rats treated with puromycin aminonucleoside (PAN) developed characteristics of the nephrotic syndrome, including albuminuria, hypoalbuminemia and hyperlipidemia. To study the regulation of apolipoprotein (apo) A-1 and apo E gene expression in nephrotic rats, we analyzed the steady-state levels (SSLs) of hepatic and intestinal apo A-1 and apo E mRNA using the Northern technique, and the plasma levels of high-density lipoprotein (HDL) by biochemical methods. Male Wistar rats were treated with PAN and compared with pair-fed and untreated control rats at different stages of disease. Nephrotic rats presented with marked hypoalbuminemia and albuminuria at between 6 and 11 days after PAN treatment. During this stage of disease, plasma levels of HDL were elevated in correlation with an increase of both hepatic and intestinal apo A-1 mRNA. In liver of nephrotic rats, high levels of apo A-1 mRNA together with low levels of apo E mRNA caused an increase in the ratio of apo A-1/apo E mRNA, reaching a maximum 6 days after treatment. Apo E mRNA was barely detected in small intestine of pair-fed controls and PAN-treated rats. However, contrary to nephrotic rats, the ratio apo A-1/apo E mRNA was inverted in liver of pair-fed rats due to an increase in apo E mRNA. In conclusion, in nephrotic rats, the SSL of apo A-1 mRNA is increased in liver and small intestine and appears to regulate the plasma levels of apo A-1. These results also suggest a coordinated regulation of the apo A-1 and apo E gene expression in liver of nephrotic and pair-fed rats.

Albumin; and alpha 1-, alpha 2-, beta-, and gamma-globulins were estimated by cellulose acetate electrophoresis in the serum and urine from rats with nephrotic syndrome (NS), 2, 4, 6, 8, 10, 12, 16, 20, and 30 days after a single injection of puromycin aminonucleoside (PAN). It was found that: (a) total serum protein level decreased on days 4-16, and total urine protein excretion rose on days 6-16; (b) serum albumin level fell on days 4-16, and urine albumin excretion increased on days 6-16; (c) serum alpha 1-globulin level rose on days 8-30, and urine alpha 1-globulin excretion increased on days 8-16; (d) serum alpha 2-globulin level remained essentially unchanged, and urine alpha 2-globulin excretion rose on days 4-10; (e) serum beta-globulin level decreased on days 4-20, and urine beta-globulin excretion increased on days 6-16, (f) serum gamma-globulin level diminished on days 6, 8, and 12, and urine gamma-globulin excretion rose on days 6-10. All serum protein fractions were excreted in the urine of nephrotic rats; these findings suggest that proteinuria is nonselective. The differences observed in the serum protein profiles, even when all protein fractions were lost in the urine, suggest an independent regulation of each protein fraction in PAN-nephrotic rats. In addition, the electrophoretic profile of serum proteins in PAN-nephrotic rats is different from previously reported patterns in human nephrosis and in rats with an acute-phase response.

The messenger ribonucleic acid (mRNA) levels of albumin, fibrinogen, alpha 1-acid glycoprotein (pAGP) and transferrin were analyzed in puromycin aminonucleoside (PAN)-treated (nephrotic) and in pair-fed (PF) rats with the Northern and dot blot hybridization techniques. Albumin mRNA levels in nephrotic and PF rats were 2- and 1.5-fold higher, respectively, than in ad-libitum-fed control (C) rats 6 days after PAN treatment. On day 11, this mRNA in PAN-treated rats was 2.5-fold higher than in PF and 4-fold higher than in C rats. A differential expression at the level of specific mRNAs was also detected for fibrinogen, pAGP, and transferrin in nephrotic and PF rats. On day 6, the fibrinogen and transferrin mRNA levels were significantly higher (p0.05) in nephrotic than in PF rats. In contrast, pAGP mRNA levels were normal or low in nephrotic rats and increased 2-fold in PF rats. These studies indicate the differential expression of hepatic genes in nephrotic and PF rats and show that albumin gene expression is only partially regulated by diet during the nephrotic stage of the disease.

This study was designed to assess how the expression of genes for components of the extracellular matrix is altered in a model of focal glomerular sclerosis. In this model, a unilateral nephrectomy combined with injections of puromycin aminonucleoside induces a much higher incidence of focal glomerular sclerosis. Rats received puromycin aminonucleoside on days 0, 27, 34, and 41 and underwent unilateral nephrectomy on day 22. Control rats received physiologic saline injections with and without unilateral nephrectomy. Rats from each group were killed on days 48, 60, and 80. The steady-state levels of glomerular mRNA encoding type IV collagen, the B1 and B2 chains of laminin, heparan sulfate proteoglycan, and type I and type III collagens were compared in both the puromycin aminonucleoside-treated and the control glomeruli. The mRNA levels encoding type IV collagen and laminin B1 and B2 were increased three-, two-, and twofold, respectively, on day 48 of focal glomerular sclerosis. These transcripts were further increased eight-, seven-, and eightfold, respectively, on day 80 compared with the control glomeruli (P0.01). In contrast, heparan sulfate proteoglycan mRNA levels were not increased on day 48 when the animals had marked proteinuria. However, the heparan sulfate proteoglycan mRNA levels did become elevated by day 60 and remained elevated thereafter. The expression of type I and type III collagen mRNA was increased 12- and 7-fold, respectively (P0.01), on day 80 in focal glomerular sclerosis rats compared with the controls. An immunofluorescence study revealed the accumulation of immunoglobulin M, C3, type IV collagen, laminin, heparan sulfate proteoglycan, and type I and type III collagens in the sclerotic area. These data indicate that changes in the mRNA levels for components of the basement membrane and interstitial collagen are associated with the development of glomerular sclerosis.

Sestrin 2, initially identified as a p53 target protein, accumulates in cells exposed to stress and inhibits mammalian target of rapamycin (mTOR) signaling. In normal rat kidneys, sestrin 2 was selectively expressed in parietal epithelial cells (PECs), identified by the marker protein gene product 9.5. In adriamycin nephropathy, sestrin 2 expression decreased in PECs on day 14, together with increased expression of phosphorylated S6 ribosomal protein (P-S6RP), a downstream target of mTOR. Sestrin 2 expression was markedly decreased on day 42, coinciding with glomerulosclerosis and severe periglomerular fibrosis. In puromycin aminonucleoside nephropathy, decreased sestrin 2 expression, increased P-S6RP expression, and periglomerular fibrosis were observed on day 9, when massive proteinuria developed. These changes were transient and nearly normalized by day 28. In crescentic glomerulonephritis, sestrin 2 expression was not detected in cellular crescents, whereas P-S6RP increased. In conditionally immortalized cultured PECs, the forced downregulation of sestrin 2 by short hairpin RNA resulted in increased expression of P-S6RP and increased apoptosis. These data suggest that sestrin 2 is involved in PEC homeostasis by regulating the activity of mTOR. In addition, sestrin 2 could be a novel marker of PECs, and decreased expression of sestrin 2 might be a marker of PEC injury.