Puromycin Aminonucleoside Nephrotic Rats Research Articles

Sialic acid supplementation ameliorates puromycin aminonucleoside nephrosis in rats

Defects in sialylation are known to have serious consequences on podocyte function leading to collapse of the glomerular filtration barrier and the development of proteinuria. However, the cellular processes underlying aberrant sialylation in renal disease are inadequately defined. We have shown in cultured human podocytes that puromycin aminonucleoside (PAN) downregulates enzymes involved in sialic acid metabolism and redox homeostasis and these can be rescued by co-treatment with free sialic acid. The aim of the current study was to ascertain whether sialic acid supplementation could improve renal function and attenuate desialylation in an in vivo model of proteinuria (PAN nephrosis) and to delineate the possible mechanisms involved. PAN nephrotic rats were supplemented with free sialic acid, its precursor N-acetyl mannosamine or the NADPH oxidase inhibitor apocynin. Glomeruli, urine, and sera were examined for evidence of kidney injury and therapeutic efficacy. Of the three treatment regimens, sialic acid had the broadest efficacy in attenuating PAN-induced injury. Proteinuria and urinary nephrin loss were reduced. Transmission electron microscopy revealed that podocyte ultrastructure, exhibited less severe foot process effacement. PAN-induced oxidative stress was ameliorated as evidenced by a reduction in glomerular NOX4 expression and a downregulation of urine xanthine oxidase levels. Sialylation dysfunction was improved as indicated by reduced urinary concentrations of free sialic acid, restored electrophoretic mobility of podocalyxin, and improved expression of a sialyltransferase. These data indicate that PAN induces alterations in the expression of enzymes involved in redox control and sialoglycoprotein metabolism, which can be ameliorated by sialic acid supplementation possibly via its properties as both an antioxidant and a substrate for sialylation.

Do large pores in the glomerular capillary wall account for albuminuria in nephrotic states?

Albuminuria in nephrotic states is thought to arise from the formation of large pores in the glomerular capillary wall as large hydrodynamic probes, like Ficoll, have increased fractional clearance. In the present study, we tested for large pore formation in a novel manner. We accounted for the rates of plasma elimination as determined for tritium-labeled tracers of uncharged polydisperse Ficoll (radii range: 35-85 Å) and two globular (14)C-labeled proteins, albumin (radius: 36 Å) and IgG (radius: 55 Å), in control and puromycin aminonucleoside nephrotic (PAN) Sprague-Dawley rats. The plasma elimination rates were then matched to the urinary excretion of these labeled materials (n = 7). Albumin and IgG plasma retention rates were identical and far enhanced compared with the retention rates of inert transport markers of equivalent hydrodynamic radius; their elimination rate corresponded to the elimination of a 75-Å radius Ficoll (n = 5) and >105-Å radius dextran (n = 5). In PAN, they were eliminated as ∼36- and ∼55-Å radii Ficoll, respectively, equivalent to their hydrodynamic radii. In contrast, there was no comparable increase in the elimination rate of Ficoll in PAN. The total plasma clearance of Ficoll in control and PAN rats and the urinary clearance in PAN rats were essentially the same for all radii. On the other hand, the urinary clearance of >45-Å radii Ficoll in controls was considerably lower with increasing radii, demonstrating a postfiltration cellular uptake in controls, which, when inhibited in nephrotic states, would account for apparent large pore formation.

Selective albuminuria via podocyte albumin transport in puromycin nephrotic rats is attenuated by an inhibitor of NADPH oxidase

The mechanism of selective albuminuria in minimal change nephrotic syndrome, in which glomerular capillaries are diffusely covered by effaced podocyte foot processes with reduced slit diaphragms, is unknown. Podocyte injury is due, in part, to NADPH-induced oxidative stress. Here we studied mechanism of selective albuminuria in puromycin aminonucleoside (PAN) nephrotic rats, a model of minimal change nephrotic syndrome. In these rats, Evans Blue-labeled human albumin was taken up by podocytes and its urinary excretion markedly increased, with retained selectivity for albumin. Immunogold scanning electron micrographic images found increased human albumin in podocyte vesicles and on the apical membrane in nephrotic compared with control rats. Apocynin, an inhibitor of NADPH oxidase, decreased superoxide production in podocytes, and inhibited endocytosis and urinary albumin excretion. Real-time confocal microscopy found an initial delay in the appearance of Evans Blue-labeled human albumin in the tubular lumen, reflecting the time needed for transcellular transport. Immunoprecipitation analysis indicated that FcRn, a receptor for albumin transport, mediated podocyte albumin transport, and treatment with anti-FcRn antibody reduced proteinuria in these nephrotic rats. Thus, podocyte albumin transport was enhanced in PAN nephrotic rats by means of FcRn, which may explain the mechanism of selective proteinuria. This was blocked by apocynin, suggesting a new therapeutic approach.

Phosphorylation of Podocalyxin (Ser415) Prevents RhoA and Ezrin Activation and Disrupts Its Interaction with the Actin Cytoskeleton

Podocalyxin (PC) is a polysialylated, anti-adhesin that is essential for maintaining foot process architecture and the integrity of the glomerular filtration barrier. We showed previously that PC is firmly attached to the actin cytoskeleton through ezrin, that in puromycin aminonucleoside (PAN)-mediated nephrosis the PC-ezrin-actin complex is disrupted, and that PC is uncoupled from actin. However, the precise mechanisms involved remained unknown. Here we show that detachment of PC from actin is regulated by phosphorylation of PC. PC is hyperphosphorylated at serines in PAN- and protamine sulfate (PS)-treated rat glomeruli. We determined that PC is a substrate of PKC and that the site of phosphorylation is Ser415, located within the juxtamembrane, ezrin-binding domain of the cytoplasmic tail of PC. Mutation of Ser415 to the phosphomimetic residues Glu (S415E) or Asp (S415D) interfered with direct binding of the PC cytoplasmic tail to ezrin in vitro. Moreover, stable expression of a phosphomimetic (S415E) PC mutant but not the WT or the phosphorylation-deficient (S415A) PC mutant, disrupted PC-ezrin-actin interaction, failed to activate RhoA, and the cytoskeletal linker, ezrin, remained inactive. Our data indicate that phosphorylation of PC at Ser415 prevents attachment of PC and ezrin to actin and highlights the strategic position of Ser415 and direct binding of PC to ezrin in regulating podocyte foot process architecture.

Silver-enhanced immunogold scanning electron microscopy using vibratome sections of rat kidneys: detection of albumin filtration and reabsorption

To show the three-dimensional distribution of proteins in renal cells, we applied the immunogold scanning electron microscopy method using vibratome slices. Kidney specimens from puromycin aminonucleoside (PAN) nephrotic rats and controls were obtained after intravenous infusion of human serum albumin and fixed in periodate-lysine-paraformaldehyde solution. Vibratome slices were incubated with anti-human albumin antibody and 25-nm gold-labeled secondary antibody. After silver enhancement, the immunogold particles were clearly observed by backscatter electron imaging, whereas they were ambiguous in the secondary electron image. The images showed a higher resolution of the tissues at an acceleration voltage of 5 mV than at 10 mV. Immunogold-labeled albumin was observed in the lumen and endocytotic vesicles of the proximal tubules, and on the podocyte surface in the PAN nephrotic rats, whereas only a few particles were observed in the controls. In conclusion, silver-enhanced immunogold scanning electron microscopy at low acceleration voltages using vibratome sections can be applicable for detecting the intracellular/extracellular localization of molecules in solid tissues. We succeeded in visualizing the enhanced albumin endocytosis of the proximal tubules and the exocytosis of albumin from podocytes in the nephrotic rats.

Tubular epithelial cells have the capacity to transdifferentiate into CD68-positive macrophage-like cells by oxidative stress

The present study was intended to assess transdifferentiation from tubular epithelial cells to macrophage- like cells. Puromycin aminonucleoside nephrotic rats were sacrificed at days 4, 8, 24 and 112. We immunohistochemically evaluated CD68, CD163, and cytokeratin AE1/AE3, known as markers for macrophages and tubular epithelial cells. Nitrotyrosine, gp91(phox) and Rac 1 expressions was also analyzed. CD68 expression in cultured murine proximal tubular epithelial cells (mProx) stimulated by crude and pure BSA was examined by flow cytometry and immunofluorescence. The tubular CD68-positive cells were observed on day 112. Immunoelectronmicroscopy revealed that some CD68-positive cells showed brush borders on the cell membrane and some of cytokeratin-positive tubular cells also expressed CD163 in mirror sections. The tubular CD68-positive cells were also positive for nitrotyrosine, gp91 (phox) and Rac 1. They contained lipid in their cytoplasm. Crude BSA, containing free fatty acid, induced CD68 expression in a dose- and time-dependent manner in mProx, but not pure BSA. The surface expression of CD68 was increased by high dose and long term stimulation with crude BSA as shown by immunofluorescence. We confirmed that tubular epithelial cells have the capacity to transdifferentiate to CD68-positive macrophage-like cells, which may be linked to oxidative stress.

Role of Fat1 in cell-cell contact formation of podocytes in puromycin aminonucleoside nephrosis and neonatal kidney

Fat1 is a member of the cadherin superfamily characterized by its 34 cadherin repeats in the extracellular domain. Fat1 was originally found as a component of the slit diaphragm of podocytes, but its function in podocytes remains obscure. To gain insight into its role in podocytes, we expanded our study of Fat1 expression to puromycin aminonucleoside (PAN) nephrosis, the neonatal kidney, and the primary podocyte culture, where slit diaphragms are absent or disappear. Expression of Fat1 was examined in isolated glomeruli of PAN nephrosis by the ribonuclease protection assay and Western blot analysis and in the neonatal kidney by in situ hybridization. Fat1 localization in glomeruli and in the primary culture was confirmed by immunofluorescence or immunoelectron microscopy. In PAN nephrotic rats, glomerular expression of Fat1 increased rather than decreased at both transcript and protein levels in comparison with normal rats. Immunofluorescence microscopy revealed distinct staining for Fat1 along the glomerular capillary wall, where nephrin staining was weakened or disappeared. Immunoelectron microscopy demonstrated significant accumulation of immunogold particles for Fat1 at intercellular junctions newly formed between podocytes in the nephrosis. In the primary culture of podocytes, Fat1 was mainly localized at cell-cell contact sites and in tips of cellular processes. In the neonatal kidney, immature podocytes expressed Fat1 more intensely than mature podocytes as shown by in situ hybridization. Double-labeled immunostaining using anti-pan cadherin antibody revealed that Fat1 in podocytes colocalized with cadherin in immature glomeruli, indicating that junctional complexes of developing podocytes contain Fat1. These findings suggest that Fat1 may be a fundamental component of intercellular junctions of podocytes, and may be involved in the initial step of cell contacts of podocytes.

Fate of Elevated L-Tryptophan in the Blood of Nephrotic Rats with Oral L-Tryptophan Administration in Their Body

We examined the changes in serum, liver, kidney, brain, and muscle L-tryptophan (Trp) levels in rats with puromycin aminonucleoside (PAN) nephrosis following oral Trp administration in order to elucidate the fate of elevated Trp in the blood of PAN nephrotic rats with oral Trp administration in their body. Before administration, nephrotic rats had higher liver and kidney Trp contents and lower serum Trp concentration than non-nephrotic rats. At 15 min after oral administration of Trp (100 micromol/kg body weight), PAN nephrotic rats had a much higher amount of liver Trp than non-nephrotic rats but there were no differences in increased amounts of serum, kidney, brain, and muscle Trp between the two groups. At 30 min after oral Trp administration, PAN nephrotic rats had a lower increased amount of serum Trp than non-nephrotic rats and had no increase in liver Trp content, although the increased amounts of Trp in other tissues were similar in both groups. At 30 min after oral Trp administration, liver tryptophan 2,3-dioxygenase activity increased approximately 2 fold in PAN nephrotic rats. These results indicate that elevated Trp in the blood of PAN nephrotic rats with oral Trp administration is transported mainly into the liver rapidly and then the transported Trp is actively metabolized by activated tryptophan 2,3-dioxygenase in the tissue.

Collecting duct is a site of sodium retention in PAN nephrosis: a rationale for amiloride therapy.

Micropuncture studies of the distal nephron and measurements of Na,K-ATPase activity in microdissected collecting tubules have suggested that renal retention of sodium in puromycin aminonucleoside (PAN) nephrotic rats originates in the collecting duct. The present study demonstrated this hypothesis by in vitro microperfusion and showed that amiloride was able to restore sodium balance. Indeed, isolated perfused cortical collecting ducts from PAN-treated rats exhibited an abnormally high transepithelial sodium reabsorption that was abolished by amiloride, and in vivo administration of amiloride fully prevented decreased urinary sodium excretion and positive sodium balance in nephrotic rats. As expected from the aldosterone independence of Na(+) retention in PAN nephrotic rats, blockade of aldosterone receptor by potassium canrenoate did not alter urinary Na(+) excretion, Na(+) balance, or ascites formation in PAN nephrotic rats.

Tissue distribution of alpha-tocopherol in nephrotic rats.

1. Reactive oxygen species are involved in the pathogenesis of puromycin aminonucleoside (PAN) nephrosis and alpha-tocopherol is one of the major anti-oxidants in the body. 2. In the present study, we measured the levels of alpha-tocopherol by high-performance liquid chromatography in the plasma and in nine tissues of control and nephrotic rats obtained 10 days after either 0.9% saline solution or PAN injection, respectively. 3. In nephrotic rats, alpha-tocopherol levels increased four-fold in plasma; however, the molar ratio of alpha-tocopherol/ cholesterol remained unchanged, suggesting that the increase in alpha-tocopherol content was attributable to an increase in plasma lipid concentration. 4. In nephrotic rats, the alpha-tocopherol/cholesterol ratio increased 1.33-fold in adrenal glands and 1.34-fold in the testis, but remained unchanged in heart, spleen, liver, kidney lung, brain and muscle. 5. These data suggest that, in PAN nephrotic rats, there are alterations in the distribution of alpha-tocopherol and there is no deficiency of alpha-tocopherol in plasma or tissues.

Size-selectivity of the glomerular barrier to high molecular weight proteins: Upper size limitations of shunt pathways

To evaluate the large pore radius of the glomerular capillary filter, plasma-to-urine fractional clearances of a number of endogenous proteins were assessed in normal and in nephrotic Wistar rats in which proximal tubular reabsorption had been inhibited using lysine. The proteins studied varied in radius from 16.2 A (Beta 2-microglobulin) to 90 A (alpha 2-macroglobulin). The nephrotic syndrome was induced by puromycin aminonucleoside (PAN). A marked restriction of the transport of large proteins across the glomerular capillary wall was found, indicating that there are no non-discriminatory 'shunt pathways' in the glomerular barrier. Rather, there seems to be large pores of radius 110 to 115 A accounting for the clearance of large proteins into the primary urine. This protein excretion pattern was almost the same for control and nephrotic rats, except that in the latter, the number of large pores was increased 170 times. The ratio between the number of large and small pores was calculated to be approximately equal to 7 x 10(-7) in normal rats and to 1.2 x 10(-4) in PAN nephrotic rats, assuming no classic shunt pathways. If classic shunt pathways had still existed, they would normally contribute to no more than approximately equal to 10(-5) of the total glomerular filtration rate. We postulate that very large macromolecules like IgM will not pass the glomerular filter at all under normal conditions, whereas the urine concentration of alpha2-macroglobulin will normally be extremely low.

The Possible Roles of Hyperlipidemia and Mononuclear Cells in Glomeruli in Puromycin Aminonucleoside Nephrosis in Rats

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-1-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-1-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-1-positive cells and hyperlipidemia are involved in the progression, but not the pathogenesis, of PAN.

Fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, suppresses very low-density lipoprotein secretion in puromycin aminonucleoside-nephrotic rats.

The effects of fluvastatin, a new inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase, on the hyperlipidemia associated with nephrosis were studied. Nephrotic rats, induced by a single intraperitoneal injection of puromycin aminonucleoside (100 mg/kg body weight), had significantly higher plasma triglyceride (TG), total cholesterol and apoprotein (apo) B concentrations than controls. Fluvastatin was administrated as a 0.01% solution in drinking water for 14 days to either normal control or nephrotic rats. Concentrations of TG and apo B in plasma, and very low-density lipoprotein (VLDL) in nephrosis were completely normalized by the treatment with fluvastatin, but concentrations of cholesterol in plasma and each lipoprotein fraction were not altered by the treatment. The ratio of apo E to C in VLDL was significantly decreased in nephrotic rats, but the fluvastatin treatment increased this ratio. TG secretion rate estimated by the Triton WR1339 method was significantly increased in nephrotic rats, but was normalized by fluvastatin. Percent composition of TG in newly secreted VLDL particles in post-Triton plasma was not decreased by fluvastatin treatment, suggesting that the number of newly secreted VLDL particles was reduced by the treatment. Postheparin plasma lipolytic activities were not affected by the fluvastatin treatment. These results demonstrate that fluvastatin can effectively ameliorate the high concentration of VLDL by suppressing the hepatic secretion in nephrotic rats, and suggest that an inhibition of cholesterol biosynthesis suppresses VLDL secretion from the liver.

Effects of low protein diet in puromycin aminonucleoside-induced nephrotic rats.

Wistar rats were injected with puromycin aminonucleoside (PAN) to induce massive proteinuria and were raised on a low protein diet to determine whether the urinary protein excretion might be reduced. Although PAN nephrotic rats fed with a normal protein diet containing 20% protein (group A) did not show any reduction of their urinary protein excretion, PAN rats fed with a low protein diet containing 6% protein (group B) revealed a marked reduction at day 9 of the present study in comparison with group A. The total serum protein levels were low in groups A and B as compared to group C, the non-treated control. However, group A recovered to normal levels at day 13. In terms of the absolute amount of oral protein intake, marked differences were observed between groups B and C, but little difference was observed between groups A and C. The body weight in all three groups was decreased at 8 days after the start of this study. The serum levels of creatinine were elevated to 1.1 +/- 0.3 mg/dl and 1.0 +/- 0.2 mg/dl in groups A and B, respectively, on day 7, but abruptly recovered to normal levels by day 12. These data indicate that the decreased urinary protein excretion in group B might be dependent on the total protein volume intake, and not on the renal function nor serum levels of total protein.

Hypolipidemic effect of beta, beta'-methyl-substituted hexadecanedioic acid (MEDICA 16) in normal and nephrotic rats.

Treatment of normal or puromycin aminonucleoside-nephrotic rats, kept on a balanced Purina chow diet, with beta, beta'-tetramethyl-substituted hexadecanedioic acid (MEDICA 16) (Bar-Tana, J., G. Rose-Kahn, and M. Srebnik. 1985. J. Biol. Chem. 260: 8404-8410) resulted in an acute reversible inhibition of liver lipogenesis and cholesterogenesis with a concomitant hypolipidemic effect which was sustained as long as the drug was administered. The hypolipidemic effect in normal and nephrotic rats consisted of 70-80% and 40-60% reduction in plasma VLDL-triacylglycerols and cholesterol, respectively, with a respective increase in the HDL-cholesterol/(VLDL + LDL)-cholesterol ratio. The observed hypolipidemic effect was accompanied by a 10-fold decrease in VLDL-apoC-III content with a concomitant enrichment of the VLDL fraction by VLDL remnants having an increased apoB-100/apoB-48 ratio. The pharmacological reduction of VLDL by MEDICA 16 may offer a treatment mode of choice for selected hyperlipidemic states.

The effect of captopril on urinary protein excretion in puromycin aminonucleoside nephrosis in rats.

We investigated the effect of captopril, an orally active angiotensin converting enzyme inhibitor, on urinary protein excretion in puromycin aminonucleoside nephrotic rats. The administration of captopril (10 mg/100 g body weight) decreased proteinuria on days 10-14 following the administration of puromycin aminonucleoside (73.0 versus 125.0 mg, p less than 0.01), without affecting glomerular filtration rate. The beneficial effect of captopril was not abolished by the continuous intravenous infusion of angiotensin II (10 micrograms/kg/h for 9 days) or subcutaneous injections of aprotinin (50,000 KIU/day for 3 days). Indomethacin, in moderate (5 mg/kg/day for 3 days) or high (10 mg/kg/day) doses, abolished the captopril attenuation in urinary protein excretion. The salutory effect of captopril was characterized by a reduction in the fractional excretion of protein without compromising the glomerular filtration rate. No difference in renal ultrastructure was noted in captopril-treated versus control animals. Captopril was ineffective in reducing urinary protein excretion in rats with adriamycin-induced glomerulopathy. We conclude that captopril acts to reduce proteinuria in renal disease states arising from depletion of the glomerular basement membrane polyanion. The mechanism of action is postulated to be an alteration in renal hemodynamics, namely increased blood flow and a decrease in the ultrafiltration coefficient, that are the consequence of increased intrarenal prostaglandin production.

Catabolism of apoprotein A-1 of HDL in normal and nephrotic rats

High density lipoprotein was isolated from the plasma of control and puromycin aminonucleoside nephrotic rats and labeled with 125I. It was injected into control and nephrotic rats and the plasma analyzed after 3 min, 5 hr, and 20 hr. High density lipoprotein was isolated and the specific activity of apo A-I determined following apoprotein separation using SDS-polyacrylamide gel electrophoresis. The distribution of labeled apoproteins was determined in whole plasma by SDS-gel filtration column chromatography and the plasma concentration of apo A-I was calculated from its specific activity and the total plasma apo A-I radioactivity. A 3 min to 5 or 20 hr fractional catabolic rate was calculated. When multiplied by the plasma concentration of apo A-I, an estimate of the absolute catabolic rate was obtained. When injected into normal animals, the high density lipoprotein apo A-I had similar catabolic rates whether derived from control or nephrotic rat plasma, averaging 65 and 48 μg of apoprotein per ml of plasma per hr at 5 or 20 hr, respectively. Labeled HDL was injected into nephrotic rats of varying degrees of severity. The moderately nephrotic rats with plasma cholesterol levels averaging 177 mg/dl had apo A-I levels that were 3.6 times that of controls (1799 ± 195 μg/ml) and 2.4-fold increases in apo A-I catabolic rates (134 ± 28.9 μg/ml plasma/hr). The severely nephrotic rats with cholesterol concentrations averaging 396 mg/dl had apo A-I levels 7.1 times that of the controls (3533 ± 220 μg/ml) while the catabolic rate was 2.7 times the control rate (153 ± 19.5 μg/ml/hr), which was not a significant increase beyond that of the moderately nephrotic group. It was concluded that compositional differences of HDL resulting in an increased proportion of apo A-I, as in nephrotic rat plasma, do not affect apo A-I metabolism. The high levels of apo A-I in the plasma of nephrotic rats is due to increased hepatic synthesis that results in an expansion of the pool size and saturation of catabolic pathways. Small increases in apo A-I synthesis lead to large increases in the plasma concentration, an observation that may be important in the regulation of HDL levels that are known to be correlated with a decreased incidence of atherosclerosis.