Experimental Nephrosis Research Articles

Contribution of Proteoglycans Towards the Integrated Functions of Renal Glomerular Capillaries: A Historical Perspective

Since the original colloidal iron tracer study by Farquhar and Palade almost 50 years ago,1 the permeability characteristics of mammalian glomerular capillary wall have been the subject of extensive investigations.2,3 In the ensuing 2 to 3 decades, various studies were performed to delineate whether glomerular basement membrane or the slit diaphragm of the capillary wall is the primary filtration barrier.4,5 Amid this contentious issue, in the mid to late 1970s various clearance and additional tracer studies indicated that the glomerular capillary wall behaves as a size- as well as charge-selective barrier, and the latter biophysical properties were attributed to the anionic sites within the basement membrane that are enriched with sulfated proteoglycans (PGs).6,7,8,9 The article by Harvey et al10 in this issue of The American Journal of Pathology suggests that agrin, a basement sulfated proteoglycan, imparts an electronegative charge to the glomerular basement membrane (GBM), but it may not be responsible for its charge-selective permeability properties. In light of these findings, we deemed it necessary to revisit the tracer work performed during the past few decades and reconcile with recent observations made in various mutant mice keeping in perspective the complex ultrastructure and integrated functions of the glomerular capillary wall.11

Treatment of Experimental Nephrotic Syndrome with Artesunate

The present study was designed to test the therapeutic effect of a new antimalarial drug, artesunate in experimental model of nephrotic syndrome. To induce this experimental model, Adriamycin was given once by a single intravenous injection (7.5 mg/kg) through the tail vein. Six days after injection of Adriamycin, therapeutic protocol was developed by intraperitoneally (IP) administration of 5 mg/kg artesunate (ARS). Total of IP injections were 14, of which 5 injections were made every day and 9 injections were carried out at regular 48-h intervals. Therapeutic protocol was terminated on day 28 and animals were killed on day 49. The results showed that treatment with ARS caused a significant reduction in the level of proteinuria, urine urea and urine sodium compared with nontreated controls. In addition, decrease in serum triglyceride and increase in the level of serum albumin was significant in treated group with ARS compared with nontreated controls. Moreover, treatment with ARS significantly reduced glomerular polymorphonuclear (PMN) and mononuclear cells infiltration, hypercellularity, karyorrhexis, wire loops, and hydropic change in capillary network within the renal cortex, as well as decreased hyalin casts. On the other hand, healthy controls receiving ARS showed a significant decrease in amounts of serum triglyceride, urine urea, and urine sodium and potassium compared with normal group. These data suggest that artesunate therapy can ameliorate proteinuria, and suppress the progression of glomerular lesions in experimental model of nephrotic syndrome; it may also be recommended as a lipid-lowering drug.

Increased cyclosporine bioavailability induced by experimental nephrotic syndrome in rats

Components of whole blood and plasma are highly altered during the presentation of nephrotic syndrome. The present study was aimed to explore the influence of nephrotic syndrome on the pharmacokinetics of cyclosporine (CsA) (10 mg/kg) administered i.v. to control or puromycin-induced nephrotic rats (P-NS). We found an increase in CsA bioavailability in the nephrotic group compared with controls. The area under the curve of blood CsA versus time (AUCiv) increased from 27.7 +/- 5.3 to 60.6 +/- 13.8 mug.h.mL-1 in control and P-NS rats, respectively. The AUCiv augmentation was positively correlated with cholesterol levels. On the other hand, the total body clearance was significantly lower (0.38 +/- 0.06 vs. 0.17 +/- 0.03 L.(kg body mass)-1.h-1) and the volume of distribution at steady state (3.70 +/- 0.52 vs. 2.85 +/- 0.32 L/kg) was significantly smaller in nephrotic rats as compared with control. These pharmacokinetic changes lead to a longer terminal half-life of CsA in P-NS rats (11.8 +/- 1.6 vs. 6.9 +/- 0.91 h). We conclude that the physiopathologic changes induced by the nephrotic syndrome in P-NS animals result in a significant increase in CsA blood exposure by both the decrease in drug distribution and the reduction in elimination rate of CsA.

Effect of combining ACE inhibition with aldosterone blockade on proteinuria and renal damage in experimental nephrosis

Aldosterone has pro-fibrotic properties and is a potential target for additional intervention in patients with chronic renal disease showing resistance to therapy during treatment with angiotensin-converting enzyme inhibitors (ACEi). Combining ACEi and aldosterone receptor blockade (aldoRB) in proteinuric renal disease reduces proteinuria, but effects on proteinuria-induced renal damage are unknown. We studied the effect of ACEi/aldoRB in adriamycin nephrosis (AN). Six weeks after injection of adriamycin in Wistar rats, randomized treatment with vehicle (VEH, n=8), aldoRB (n=12), ACEi (n=10), or a combination of ACEi/aldoRB (n=14) was given for 12 weeks. Healthy rats served as controls (n=6). Renal damage was quantified by markers of tubular injury (osteopontin (OPN) and kidney injury molecule-1 (Kim-1)), pre-fibrotic lesions (alpha-smooth muscle actin (SMA)), interstitial fibrosis (IF), and focal glomerulosclerosis (FGS). In AN animals, proteinuria was increased compared with controls. ACEi and ACEi/aldoRB significantly reduced proteinuria compared with VEH, whereas aldoRB monotherapy was without effect. Blood pressure was reduced in ACEi and ACEi/aldoRB compared with VEH and aldoRB. OPN and Kim-1 were increased in AN animals, but significantly reduced by ACEi/aldoRB. Treatment with ACEi and ACEi/aldoRB prevented an increase of SMA, IF, and FGS. In conclusion, ACEi/aldoRB effectively reduced proteinuria and markers of tubular injury and prevented renal damage in this rat model of chronic proteinuria-induced renal damage.

Atorvastatin Preserves Renal Function in Chronic Complete Unilateral Ureteral Obstruction

The pleiotropic effects of hMG-CoA (3-hydroxy-3-metylglutaryl coenzyme A) reductase inhibitors may provide renal protection in chronic kidney disease. We examined whether atorvastatin administration preserved renal function in rats with chronic unilateral ureteral obstruction. Renal clearance experiments were performed in sham operated rats and rats subjected to 3 or 12-day unilateral ureteral obstruction. Hemodynamics parameters and urinary microalbumin levels from the obstructed kidney were also measured. The rats were maintained on a regular diet or the same diet but supplemented with atorvastatin (50 mg/kg daily). Atorvastatin administration did not alter plasma total cholesterol but it significantly decreased triglyceride levels. In sham operated and 3-day unilateral ureteral obstruction rats atorvastatin treatment did not have effects on the glomerular filtration rate or effective renal plasma flow and it also did not affect urinary microalbumin levels. In rats with 12-day unilateral ureteral obstruction the glomerular filtration rate but not effective renal plasma flow was significantly higher and urinary microalbumin was significantly lower in atorvastatin treated rats than in those without atorvastatin treatment. Atorvastatin treatment decreased microalbuminuria and helped preserve filtration function in chronic unilateral ureteral obstruction without altering plasma cholesterol levels, suggesting that pleiotropic renal protection is offered by this statin.

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.

Hyperbaric Oxygen Treatment Augments the Efficacy of a Losartan Regime in an Experimental Nephrotic Syndrome Model

Background/Aims: Proteinuria is associated with oxidant stress and inflammation. Hyperbaric oxygen (HBO) treatment has anti-inflammatory and anti-oxidant effects. The aim of the study was to investigate the benefits of HBO treatment on an experimental nephrotic syndrome model. Methods: 50 male Sprague-Dawley rats weighing 255 ± 39 g were housed. Forty rats were injected 6 mg/kg adriamycin into tail veins under anesthesia to induce nephrosis, while 10 rats were spared as sham control. After the stabilization of proteinuria at the sixth week, the rats were treated for 6 weeks by losartan (n = 10, 30 mg/kg/day), HBO (n = 10, 2.8 atmosphere absolute, 90 min/day), HBO + losartan (n = 10) and vehicle (n = 10). Protein carbonyl (PCO), superoxide dismutase (SOD) and glutathione peroxidase (GPx) were analyzed from tissue specimens. Biochemical markers were studied from venous samples and 24-hour urine was collected for proteinuria. The surviving animals at 12 weeks (vehicle group (n = 6), HBO (n = 6), losartan (n = 8), HBO + losartan (n = 10) were sacrificed. Glomerular sclerosis, tubulointerstitial and blood vessel changes were determined by semiquantitative scoring. Results: The PCO levels increased (p < 0.001), and the GPx and SOD levels decreased (p < 0.001 for both) in the nephrotic rats. In losartan and HBO groups GPx levels increased (p = 0.001, p = 0.002 respectively), but PCO and SOD levels did not change. The combination of HBO with losartan significantly increased the GPx and SOD levels (p = 0.001 for both) and decreased PCO levels (p = 0.005). HBO but not losartan significantly reduced proteinuria (p < 0.001). The combination of HBO and losartan reduced proteinuria better than the single losartan regime (p < 0.001). The effect of the combination was also noticed on the histological examination of the kidneys. The activities, appetites, weight gains, and improvement of edema were better in the HBO combined with losartan regime. Conclusions: These results indicate that the addition of HBO therapy to a conventional regime, angiotensin receptor blockers, has significant benefits in the management of proteinuria. Future clinical studies are needed to elucidate the role of HBO and other antioxidant strategies in the treatment of proteinuria.

Steroidogenic impairment due to reduced ovarian transcription of cytochrome P450 side-chain-cleavage (P450scc) and steroidogenic acute regulatory protein (StAR) during experimental nephrotic syndrome

The nephrotic syndrome is a renal disease characterized by proteinuria, hypoproteinemia, edema and hyperlipidemia. It has been reported that female nephrotic rats are characterized by loss of the oestrus cycle, follicle atresia, low gonadotropin and steroid concentrations; particularly, undetectable estradiol levels. Therefore, to determine the mechanisms involved in the ovarian steroidogenesis impairment, in this present study we evaluated the ovarian expression of the essential steroidogenesis components: cytochrome P450 side cholesterol chain cleavage enzyme (P450scc) and steroidogenic acute regulatory protein (StAR). The experiments were conducted in the rat experimental model of nephrosis induced by puromycin aminonucleoside (PAN) and in control groups. The evaluation of the expression of P450scc and StAR mRNA were performed during the acute phase of nephrosis as well as after the exogenous administration of 1 or 4 doses of human chorionic gonadotrophin (hCG), or a daily dose of FSH or FSH + hCG for 10 days. In addition, serum hormone concentrations, intra-ovarian steroid content, and the reproductive capacity were determined. The results revealed a decreased expression of mRNA of P450scc enzyme and StAR during nephrosis, and eventhough they increased after gonadotropins treatment, they did not conduce to a normal cycling rat period or fertility recovery. This study demonstrates that the mechanism by which ovarian steroid biosynthesis is altered during acute nephrosis involves damage at the P450scc and StAR mRNA synthesis and processing.

Dysregulation of ENaC in Animal Models of Nephrotic Syndrome and Liver Cirrhosis

Nephrotic syndrome and liver cirrhosis are common clinical manifestations, and are associated with avid sodium retention leading to the development of edema and ascites. However, the mechanism for the sodium retention is still incompletely understood and the molecular basis remains undefined. We examined the changes of sodium (co)transporters and epithelial sodium channels (ENaCs) in the kidneys of experimental nephrotic syndrome and liver cirrhosis. The results demonstrated that puromycin- or HgCl2-induced nephrotic syndrome was associated with 1) sodium retention, decreased urinary sodium excretion, development of ascites, and increased plasma aldosterone level; 2) increased apical targeting of ENaC subunits in connecting tubule and collecting duct segments; and 3) decreased protein abundance of type 2 11β-hydroxysteroid dehydrogenase (11βHSD2). Experimental liver cirrhosis was induced in rats by CCl4 treatment or common bile duct ligation. An increased apical targeting of alpha-, beta-, and gamma-ENaC subunits in connecting tubule, and cortical and medullary collecting duct segments in sodium retaining phase of liver cirhosis but not in escape phase of sodium retention. Immunolabeling intensity of 11βHSD2 in the connecting tubule and cortical collecting duct was significantly reduced in sodium retaining phase of liver cirrhosis, and this was confirmed by immunoblotting. These observations therefore strongly support the view that the renal sodium retention associated with nephrotic syndrome and liver cirrhosis is caused by increased sodium reabsorption in the aldosterone sensitive distal nephron including the connecting tubule and collecting duct, and increased apical targeting of ENaC subunits plays a role in the development of sodium retention in nephrotic syndrome and liver cirrhosis.

The efficiency of intraperitoneal high-dose immunoglobulin in experimental nephrotic syndrome

Although it has been reported that high-dose immunoglobulin has beneficial effects in chronic glomerulonephritis, it is not known whether it is effective in the treatment of idiopathic nephrotic syndrome. We have investigated the effects of intraperitoneal immunoglobulin in adriamycin-induced nephrotic syndrome. Adriamycin (2 mg kg(-1) per dose) was given intravenously to sixteen Wistar albino rats (eight control and eight treatment rats) on day 1 and at week 3. At week 5 intraperitoneal immunoglobulin (1 g kg(-1) per dose) was given to the treatment group on two consecutive days whereas the control group received intraperitoneal saline solution. In both treatment and control groups urinary protein excretion was significantly elevated after administration of adriamycin (P=0.018). Urinary protein excretion, serum albumin, and triglyceride levels in the two groups were not significantly different after 5, 8, 12, and 16 weeks. Serum creatinine levels were higher and creatinine clearance was significantly lower in the control group in week 16 (P=0.001 and P=0.049, respectively). Glomerular sclerosis index was significantly lower in the treatment group (P=0.012). Although intraperitoneal high-dose immunoglobulin did not reverse biochemical results, it is encouraging that glomerular sclerosis index was significantly lower in the treatment group.

Regulation of Epithelial Sodium Channel in Puromycin Aminonucleoside-Induced Unilateral Experimental Nephrotic Syndrome in Normal and Analbuminemic Nagase Rats

Background: Nephrotic syndrome (NS) is characterized by renal sodium retention and edema formation. In nephrotic rats the site of enhanced sodium retention has been localized in the cortical collecting duct (CCD). The epithelial sodium channel (ENaC) is the rate-limiting constituent of sodium transport in CCD. Amiloride, an ENaC-blocking drug, corrects the abnormal rate of sodium transport in isolated perfused CCD from puromycin aminonucleoside (PAN)-treated rats. Therefore, we hypothesized that ENaC functional expression is increased in NS. Methods: Unilateral NS was induced by PAN in Wistar rats and analbuminemic Nagase rats (NAR). Urinary protein excretion, renal abundance of mRNA and protein of ENaC subunits, as well as the ENaC regulatory serum glucocorticoid-inducible kinase (Sgk1) and Nedd4-2, were assessed. Results: Proteinuria appeared at day 2 in the Wistar rats and NAR. Surprisingly a downregulation rather than the expected upregulation of α-, β- and γ-ENaC mRNA abundance was observed in both Wistar rats and NAR, when the treated kidney was compared with the untreated kidney. The amount of protein of α-, β- and γ-ENaC was not affected by the NS. Sgk1 mRNA expression did not change and Nedd4-2 protein expression was only decreased at days 1 and 2 in Wistar rats. Conclusion: ENaC mRNA and protein expression are not increased in the early phase of unilateral PAN-induced NS. Sgk1, Nedd4-2 and analbuminemia are not important regulatory factors of ENaC protein expression in experimental NS.

Role of p38 Mitogen-Activated Protein Kinase Activation in Podocyte Injury and Proteinuria in Experimental Nephrotic Syndrome

Podocytes play an important role in maintaining normal glomerular function and structure, and podocyte injury leads to proteinuria and glomerulosclerosis. The family of mitogen-activated protein kinases (MAPK; extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase, and p38) may be implicated in the progression of various glomerulopathies, but the role of MAPK in podocyte injury remains elusive. This study examined phosphorylation of p38 MAPK in clinical glomerulopathies with podocyte injury, as well as in rat puromycin aminonucleoside (PAN) nephropathy and mouse adriamycin (ADR) nephropathy. The effect of treatment with FR167653, an inhibitor of p38 MAPK, was also investigated in rodent models. In human podocyte injury diseases, the increased phosphorylation of p38 MAPK was observed at podocytes. In PAN and ADR nephropathy, the phosphorylation of p38 MAPK and ERK was marked but transient, preceding overt proteinuria. Pretreatment with FR167653 (day -2 to day 14, subcutaneously) to PAN or ADR nephropathy completely inhibited p38 MAPK activation and attenuated ERK phosphorylation, with complete suppression of proteinuria. Electron microscopy and immunohistochemistry for nephrin and connexin43 revealed that podocyte injury was markedly ameliorated by FR167653. Furthermore, early treatment with FR167653 effectively prevented glomerulosclerosis and renal dysfunction in the chronic phase of ADR nephropathy. In cultured podocytes, PAN or oxidative stress induced the phosphorylation of p38 MAPK along with actin reorganization, and FR167653 inhibited such changes. These findings indicate that the activation of MAPK is necessary for podocyte injury, suggesting that p38 MAPK and, possibly, ERK should become a potential target for therapeutic intervention in proteinuric glomerulopathies.

Glomerular abundance of nephrin and podocin in experimental nephrotic syndrome: different effects of antiproteinuric therapies

Nephrotic syndrome (NS) is a clinical state characterized by massive proteinuria, hypoalbuminemia, and eventual edema formation. Although the mechanisms underlying this phenomenon are not yet fully clarified, it is well accepted that nephrin and podocin are involved in the development of proteinuria. The effects of early treatment with various antiproteinuric therapies on proteinuria and glomerular staining of nephrin and podocin in rats with experimental NS have not been previously studied. Proteinuria and glomerular nephrin and podocin immunofluorescence were examined in rat kidneys with adriamycin-induced NS and the effects of antiproteinuric drug therapies during 5 wk with enalapril, losartan, alone or in combination, omapatrilat, and mycophenolate mofetil on these parameters were assessed. Injection of adriamycin caused a significant increase in daily (from 21.8 +/- 1.4 to 983.1 +/- 45.8 mg/day, P < 0.01) and cumulative protein excretion (from negligible values to 22,490 +/- 931 mg, P < 0.001) during 5 wk. Early treatment with enalapril significantly decreased the daily (641.7 +/- 82.4 mg/day, P < 0.0023) and cumulative proteinuria (15,727 +/- 2,204 mg, P < 0.001). A similar effect, although to a lesser extent, was obtained after omapatrilat treatment: cumulative proteinuria was reduced to 18,706 +/- 1,042 mg, P < 0.001. In contrast, losartan treatment did not significantly influence the cumulative proteinuria that remained comparable (20,351 +/- 1,360 mg, P > 0.05) to that observed in untreated NS rats. Unexpectedly, when losartan was given in combination with enalapril, it abolished the beneficial effects of the latter. Pretreatment with mycophenolate mofetil exerted a moderate antiproteinuric effect, which appeared only during the last week of the experimental treatment. Nephrotic rats exhibited severe disruption of slit diaphragm structure as seen by rapid and profound loss of nephrin and podocin. Beneficial effects of enalapril, omapatrilat, and mycophenolate mofetil paralleled the preservation of nephrin, as determined immunohistochemically, and enabled prediction of significant antiproteinuric responses. Enalapril alone or in combination with losartan resulted in significant preservation of podocin. Pretreatment with enalapril, and to a lesser extent omapatrilat, is superior to losartan in reducing proteinuria in NS rats. A combination of ACE inhibitors with ANG II receptor blockers does not provide any advantageous antiproteinuric therapy in these animals. Nephrin loss is an indication of proteinuria in NS and the antiproteinuric effects of ACE inhibitors, vasopeptidase inhibitors, and mycophenolate mofetil attenuate this reduction. Not all the drugs which restore podocin reduce urinary protein in NS.

Reduced activity of 11β‐hydroxysteroid dehydrogenase type 2 is not responsible for sodium retention in nephrotic rats

In mineralocorticoid target cells 11-beta-hydroxysteroid dehydrogenase type 2 (11betaHSD2) converts glucocorticoids into non-active metabolites thereby protecting the mineralocorticoid receptor (MR) from stimulation by glucocorticoids. In nephrotic syndrome, a decreased activity of 11betaHSD2 has been suggested to allow glucocorticoids to stimulate MR, thereby contributing to sodium retention. We tested this hypothesis in the puromycin aminonucleoside model of nephrotic syndrome in rats. Complete sodium and potassium intakes and excretions (faeces and urine) were measured in rats in metabolic cages. RNase protection assay of mRNA and Western blotting of protein were used to estimate renocortical expression of 11betaHSD2 and of the MR downstream effector serum and glucocorticoid induced kinase (SGK). In an intervention series, dexamethasone was given [10 microg (100 g bw)(-1)] to suppress endogenous glucocorticoids in the proteinuric stage during active sodium retention. Nephrotic rats developed proteinuria, positive sodium balance, decreased plasma aldosterone concentration, and decreased urinary Na(+)/K(+) ratio. 11betaHSD2 mRNA expression was down-regulated but protein expression was unchanged. SGK mRNA and phosphorylated SGK protein were up-regulated while total SGK protein expression was unchanged. Dexamethasone treatment, which suppressed plasma corticosterone concentration, did not correct sodium balance or fluid retention in nephrotic rats. Our results do not support the hypothesis that stimulation of the MR by endogenous glucocorticoids induces sodium and fluid retention in experimental nephrotic syndrome in rats.

Complement Inhibitors Targeted to the Proximal Tubule Prevent Injury in Experimental Nephrotic Syndrome and Demonstrate a Key Role for C5b-9

In glomerular diseases of diverse etiologies, dysfunction of the glomerular barrier to protein passage results in proteinuria, and proteinuria is considered an independent risk factor that plays a direct role in inflammation, interstitial fibrosis, and renal failure. The mechanism by which proteinuria leads to nephrotoxic injury is unclear, but a role for complement in mediating interstitial damage appears likely. We describe a strategy for Ag-specific targeting of complement inhibitors using a single chain Ab fragment and show that complement inhibitors targeted to the tubular epithelium protect against tubulointerstitial injury and renal dysfunction in a rat model of puromycin-induced nephrosis. The targeting of systemically administered complement inhibitors markedly enhanced their efficacy and obviated the need to systemically inhibit complement, thus reducing the risk of compromising host defense and immune homeostasis. Targeted inhibition of complement activation by Crry, and of membrane attack complex (MAC) formation by CD59 was equally therapeutic, demonstrating that the MAC plays a key role in proteinuria-induced tubulointerstitial injury. CD59 activity was dependent on its being targeted to the site of complement activation, and this is the first report of specific inhibition of the MAC in vivo after systemic administration of inhibitor. The data establish the MAC is a valid target for pharmaceutical intervention in proteinuric disorders and provide an approach to investigate the role of the MAC in complement-dependent disease under clinically relevant conditions.

Differential Induction of Podocyte Heat Shock Proteins by Prolonged Single and Combination Toxic Metal Exposure

Cadmium, mercury, and arsenite are among the most abundant toxic metals (TM) in our environment, and chronic TM exposure leads to injury to the kidney's glomerular filtration barrier. The small heat shock protein hsp25, highly expressed in glomerular podocytes, is induced during development of experimental nephrotic syndrome, and hsp25 overexpression can protect cultured podocytes from injury. Because little is known about the effect of multiple TM on podocytes, we measured the response of cultured podocytes to prolonged exposures to single and multiple TM. Podocyte viability declined by approximately 50% after 3 days of treatment with 20 microM cadmium, mercury, or arsenite, and 40 microM of any of these metals was lethal. The toxicity of equimolar concentrations of two or all three metals in combination was significantly altered compared to individual metal treatments. Single TM treatments induced only modest increases in the amounts of hsp25, alphaB-crystallin, and inducible hsp70. Toxic metal combinations induced greater stress protein accumulation, especially arsenite + cadmium or arsenite + cadmium + mercury treatments, the TM mixtures with the lowest toxicity. All TM treatments caused a rapid and sustained increase in hsp25 phosphorylation. The intracellular accumulation of cadmium was greater and that of mercury was less in cells treated with TM combinations than in cells treated with a single TM. Our results showed that multiple TM effects on podocyte viability were neither additive nor synergistic and that induction of heat shock proteins correlated with increased resistance to TM injury, suggesting that induction of small heat shock proteins may play an important role in preventing TM-induced podocyte injury.

Role of Uroguanylin, a Peptide with Natriuretic Activity, in Rats with Experimental Nephrotic Syndrome

Uroguanylin induces natriuresis and diuresis in vivo as well as in vitro and is found mainly in the intestine and the kidney. However, the roles of uroguanylin in nephrotic syndrome, which is associated with sodium and water retention, have not been determined. Therefore, changes in the urine and plasma concentration of immunoreactive uroguanylin (ir-uroguanylin) and its mRNA expression in the kidney and intestine were examined using rats with puromycin aminonucleoside (PAN)-induced nephrosis. Male Sprague-Dawley rats were separated into control and nephrotic groups, and then the urinary excretion of sodium, protein, and ir-uroguanylin was examined over time. The plasma levels and renal and intestinal mRNA expression of uroguanylin at the periods of sodium retention and remarkable natriuresis also were evaluated. The sequential changes of urinary ir-uroguanylin excretion in the nephrotic group were similar to those of urinary sodium excretion. When the urinary excretion of ir-uroguanylin and sodium peaked, the plasma level of ir-uroguanylin also increased compared with that of the control group. Uroguanylin mRNA expression in the kidney increased during the period of sodium retention and then decreased during the period of remarkable natriuresis. Uroguanylin mRNA expression in the small intestines of control and nephrotic rats were identical. However, in a unilateral PAN-induced proteinuria, uroguanylin expression significantly increased in the PAN-perfused kidney compared with that in the opposite kidney. Considering the natriuretic effect of uroguanylin, these results suggested that uroguanylin plays an important role as a natriuretic factor in nephrotic syndrome via both the circulation and the kidney itself.

Induction of antioxidant enzymes in murine podocytes precedes injury by puromycin aminonucleoside

An imbalance between the generation of reactive oxygen species (ROS) and antioxidant defense mechanisms has been suggested to play an important role in podocyte injury in nephrotic syndrome. Experimental nephrotic syndrome induced by injection of puromycin aminonucleoside (PAN) into rats is a well-established model of nephrotic syndrome, and can be largely prevented by pretreatment with antioxidant enzymes (AOE), suggesting that podocyte injury may be mediated by ROS. To test the hypothesis that PAN-induced podocyte injury is modulated in part by podocyte antioxidant defenses, we analyzed AOE activities, lipid peroxidation products, and relative ROS levels in podocytes using our recently reported in vitro model of PAN-induced podocyte injury. PAN treatment induced early increases in both podocyte hydrogen peroxide and superoxide and later increases in lipid peroxidation products. Compared to baseline activities, PAN also induced significant changes in the major cellular AOE activities (maximum increases of 151% for catalase, 134% for superoxide dismutase, and 220% for glutathione peroxidase vs. time-matched controls). These changes largely preceded the development of extensive podocyte process retraction and actin filament disruption, which was maximal at 7 days. These results demonstrate that (1) PAN treatment induces significant early changes in podocyte ROS, (2) podocytes can mount an antioxidant defense against oxidant stress, and (3) this protective response is initiated prior to the development of extensive oxidant-induced podocyte structural injury. These findings suggest that enhancement of podocyte AOE activities represent a potential therapeutic target to protect from or ameliorate podocyte injury during nephrotic syndrome.

Increased Activity of cGMP-Specific Phosphodiesterase (PDE5) Contributes to Resistance to Atrial Natriuretic Peptide Natriuresis in the Pregnant Rat

Increased cGMP-specific phosphodiesterase (PDE5) activity in renal inner medullary collecting duct (IMCD) cells contributes to resistance to atrial natriuretic peptide (ANP) and the excessive sodium retention seen in experimental nephrotic syndrome and liver cirrhosis. Normal pregnancy is also accompanied by sodium retention and plasma volume expansion, and pregnant rats are resistant to the natriuretic action of ANP. The authors investigated a possible role of increased renal PDE5 activity in the physiologic sodium retention of normal rat pregnancy. The natriuresis and increased urinary cGMP excretion (U(cGMP)V) evoked by acute volume expansion (a measure of renal responsiveness to endogeneous ANP) was blunted in 16-d pregnant versus virgin rats, despite equivalent increases in circulating ANP in pregnants and virgins. The ANP-dependent cGMP accumulation in isolated IMCD cells from pregnants was blunted versus virgins and restored by the PDE5-selective antagonist DMPPO (10(-7) mol/L). PDE5 activity in vitro and PDE5 protein abundance in IMCD were greater in pregnants. Four days postpartum, volume expansion natriuresis, U(cGMP)V, and PDE5 protein levels in IMCD cell homogenates had returned to virgin values. These results demonstrate that normal rat pregnancy leads to in vivo and in vitro renal resistance to ANP, in association with heightened activity of the cGMP-specific PDE5 in IMCD. This may contribute to the physiologic sodium retention of normal pregnancy.

Novel expression of sodium/myo-inositol co-transporter in podocytes in puromycin aminonucleoside nephrosis

How podocytes respond to injury is poorly understood, although podocyte injury in the glomerulus has been proposed as the crucial mechanism in the pathogenesis of proteinuria and focal segmental glomerulosclerosis. An increase in sodium/myo-inositol co-transporter (SMIT) transcripts, an osmoprotective gene, has been demonstrated in a variety of brain injury models. In the present study, we investigated SMIT expression in podocytes in experimental nephrosis. Two types of nephrosis were induced in rats: puromycin aminonucleoside (PAN) nephrosis and monoclonal antibody (mAb) 5-1-6 nephropathy. Podocyte injury was morphologically distinct in the former type of nephrosis and limited to a minimum in the latter. SMIT expression in isolated glomeruli was estimated by ribonuclease protection assay. Localization of SMIT-expressing cells in glomeruli was examined by in situ hybridization. SMIT transcripts in glomeruli increased conspicuously in the nephrotic stage of PAN nephrosis, whereas the transcripts in cortices and medullae did not show significant changes. In situ hybridization revealed that podocytes were predominant cells expressing SMIT in the glomerulus. Significant increase of SMIT mRNA in the glomeruli was detected before the onset of massive proteinuria. In contrast, up-regulation of SMIT expression was not observed in mAb 5-1-6 nephropathy, whose urinary protein levels were comparable with those in the nephrotic stage of PAN nephrosis. These findings suggest that SMIT expression in podocytes is not provoked by an effect of massive proteinuria but by extensive cellular injury.