Puromycin Aminonucleoside Nephrosis Rats Research Articles

Differential Expression of Renal and Hepatic PCSK9 During Development of Hypercholesterolemia in the Puromycin Aminonucleoside Nephrosis Rat Model of Nephrotic Syndrome

Differential Expression of Renal and Hepatic PCSK9 During Development of Hypercholesterolemia in the Puromycin Aminonucleoside Nephrosis Rat Model of Nephrotic Syndrome

SUN-190 GFB-887, a small molecule inhibitor of TRPC5, protects against podocyte injury and attenuates proteinuria in models of FSGS

Focal segmental glomerulosclerosis (FSGS) is a rare podocytopathy with a high likelihood of progression to end stage renal disease. The Ca2+-permeable transient receptor potential canonical 5 (TRPC5) channel is a critical mediator of proteinuria in FSGS. Activation of the TRPC5 pathway in podocytes culminates in activation of Rac1, which is the primary driver of proteinuria in many forms of familial and sporadic FSGS. Inhibition of TRPC5 channel activity has been shown to protect against proteinuria and podocyte loss in AT1R transgenic and Dahl salt-sensitive rats. We performed high throughput screening of a structurally diverse compound collection followed by lead optimization for the development of several potent TRPC5 inhibitors. We characterized TRPC5 inhibitors in vitrousing a mouse podocyte cell line and podocyte injury models derived from human inducible pluripotent stem cells (hiPSC), and in two in vivomodels of FSGS, the unilaterally nephrectomized deoxycorticosterone acetate (DOCA)-salt rat model and the puromycin aminonucleoside nephrosis (PAN) rat model Here we report the identification of GFB-887, a potent, selective and orally bioavailable small molecule inhibitor of TRPC5. Potency and selectivity across TRP channels were determined using FLIPR based assays and electrophysiology. We show that TRPC5 inhibition protects against loss of stress fibers induced by protamine sulfate, a known activator of TRPC5, and restores stress fibers in podocytes after knockdown of synaptopodin. We further show that inhibition of TRPC5 suppresses pathogenic podocyte motility in a scratch assay. Finally, we demonstrate that inhibition of TRPC5 attenuates proteinuria in hypertension-induced FSGS in DOCA-salt rat model without altering blood pressure, and in non-hypertensive FSGS in the PAN rat model suggesting direct glomerular protection in vivo. We have identified GFB-887 as a new selective small molecule inhibitor of TRPC5 for the treatment of proteinuria in FSGS

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

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

Renoprotective effect of topiroxostat via antioxidant activity in puromycin aminonucleoside nephrosis rats.

Topiroxostat is a novel inhibitor of xanthine oxidase, and is postulated to exert a renoprotective effect. Puromycin aminonucleoside nephrosis (PAN) is a rat model of minimal change nephrotic syndrome. In this study, we examined whether topiroxostat ameliorates the kidney injury in PAN rats that was induced by a single intraperitoneal injection of PA (100 mg/kg body weight). Rats were divided into four groups: control rats, PAN rats, control rats treated with topiroxostat (1.0 mg/kg/day), and PAN rats treated with topiroxostat. Topiroxostat significantly reduced the amount of uric acid in the kidney cortex, while serum UA concentration remained unaffected by this treatment. Urinary protein excretion decreased significantly on day 10 in PAN rats upon topiroxostat treatment. Podocyte injury in PAN rats, as indicated by the reduction in WT‐1‐positive cell numbers and podocin immunoreactivity and foot process effacement, was partially yet significantly alleviated with topiroxostat treatment. In the kidney cortex, the increase in oxidative stress markers such as nitrotyrosine and 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) and the enhanced expressions of xanthine oxidase and NADPH oxidase 4 (NOX4) in PAN rats were significantly ameliorated by topiroxostat. Using cultured podocytes NOX4 expression was upregulated by adding 12 mg/dL UA into the culture medium. These results suggest that topiroxostat ameliorates proteinuria and kidney injury in PAN rats by lowering oxidative stress and tissue UA concentration. The renoprotective effects of topiroxostat could be attributed to its potential to inhibit xanthine oxidase and NOX4 in concert with suppression of intracellular UA production.

Adrenomedullin ameliorates podocyte injury induced by puromycin aminonucleoside in vitro and in vivo through modulation of Rho GTPases.

Podocyte injury is a key event in proteinuric kidney disease and eventually glomerular scarring. While adrenomedullin (AM), a potent vasodilatory peptide, has been reported to confer renoprotection in several experimental models of kidney diseases, its effect on injured podocytes and the related mechanism is still largely unknown. Employing Western blotting analysis, immunoprecipitation and immunofluorescence, we investigated the effects of AM on the expressions of podocyte cytoskeletal proteins and Rho-family small GTPases (Rho GTPases) in puromycin aminonucleoside (PAN)-induced podocyte injury, both in cultured podocytes and in PAN nephrosis rats. Urinary protein excretion and the morphologic changes of kidney in PAN nephrosis rats were evaluated. Glutathione-S-transferase pull-down assay was applied for Rho GTPases activity. PAN induced massive albuminuria and morphologic injury, which were significantly mitigated by AM administration. AM significantly antagonized not only the PAN-decreased expressions of synaptopodin, nephrin, CD2AP and podocin, but also the PAN-disrupted interactions between synaptopodin-RhoA, nephrin-CD2AP, and CD2AP-Rac1-cortactin. These effects of AM in cultured podocytes were mostly significantly blocked by H89, a PKA inhibitor. AM dramatically upregulated the PAN-induced Rho GTPases protein expressions and their activities. PAN increased the expressions of endogenous AM and its receptor RAMP2 which was furthermore upregulated by AM administration. AM alleviated podocyte injury induced by PAN both in cell culture and in PAN nephrosis. The beneficial effects of AM on podocytes can be attributable to direct modulation of podocyte cytoskeletal proteins and Rho GTPases, mainly via a PKA-dependent pathway.

Podocin is translocated to cytoplasm in puromycin aminonucleoside nephrosis rats and in poor-prognosis patients with IgA nephropathy.

Podocytes serve as the final barrier to urinary protein loss through a highly specialized structure called a slit membrane and maintain foot process and glomerular basement membranes. Podocyte injury results in progressive glomerular damage and accelerates sclerotic changes, although the exact mechanism of podocyte injury is still obscure. We focus on the staining gap (podocin gap) defined as the staining difference between podocin and synaptopodin, which are normally located in the foot process. In puromycin aminonucleoside nephrosis rats, the podocin gap is significantly increased (p < 0.05) and podocin is translocated to the cytoplasm on days 7 and 14 but not on day 28. Surprisingly, the gap is also significantly increased (p < 0.05) in human kidney biopsy specimens of poor-prognosis IgA nephropathy patients. This suggests that the podocin gap could be a useful marker for classifying the prognosis of IgA nephropathy and indicating the translocation of podocin to the cytoplasm. Next, we find more evidence of podocin trafficking in podocytes where podocin merges with Rab5 in puromycin aminonucleoside nephrosis rats at day 14. In immunoelectron microscopy, the podocin positive area was significantly translocated from the foot process areas to the cytoplasm (p< 0.05) on days 7 and 14 in puromycin aminonucleoside nephrosis rats. Interestingly, podocin is also translocated to the cytoplasm in poor-prognosis human IgA nephropathy. In this paper, we demonstrate that the translocation of podocin by endocytosis could be a key traffic event of critical podocyte injury and that the podocin gap could indicate the prognosis of IgA nephropathy.

Dynamics of absolute amount of nephrin in a single podocyte in puromycin aminonucleoside nephrosis rats calculated by quantitative glomerular proteomics approach with selected reaction monitoring mode

The slit diaphragm (SD) is a complex of podocyte-specific proteins and plays a significant role in glomerular filtration. To understand podocyte biology, it is important to determine the expression amount of the SD complex proteins. This study aimed to quantify the absolute amount of nephrin, which is believed to be a major component of SD, in podocytes and to apply that method to normal and puromycin aminonucleoside (PAN) nephrosis rats. The counting method for podocyte number in a glomerulus was developed by three-dimensional reconstruction imaging of Wilms tumor (WT-1) immunofluorescence on isolated glomeruli. Absolute amount of nephrin was quantified by mass spectrometry using the selected reaction monitoring (SRM) mode with a stable isotope-labeled peptide. The number of podocytes per glomerulus was 95.5±17.6 in the control rats, 90.7±19.2 on Day 4 and 90.7±26.2 on Day 7 in PAN nephrosis rats. The amount of nephrin per glomerulus in control rats was 1.02±0.11 fmol and those in PAN nephrosis rats were reduced to 0.46±0.06 fmol and 0.35±0.04 fmol on Day 4 and Day 7. The nephrin amount per podocyte was significantly decreased association with the development of proteinuria in PAN nephrosis rats. This study established the absolute quantification of nephrin and determined the amount of nephrin in a podocyte of normal and PAN nephrosis rat kidneys. This highly sensitive and selective quantification method for protein is a useful tool for the analysis of SD protein in a podocyte.

Tolvaptan, a selective oral vasopressin V2 receptor antagonist, ameliorates podocyte injury in puromycin aminonucleoside nephrotic rats

Proteinuria caused by glomerular disease is characterized by podocyte injury. Vasopressin V2 receptor antagonists are effective in reducing albuminuria, although their actions on glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of tolvaptan, a selective oral V2 receptor antagonist, on podocytes in a puromycin aminonucleoside (PAN)-induced nephrosis rat model. Rats were allocated to a control, PAN nephrosis, or tolvaptan-treated PAN nephrosis group (n = 9 per group). Urinary protein excretion and serum levels of total protein, albumin, creatinine, and total cholesterol were measured on day 10. The influence of tolvaptan on podocytes was examined in renal tissues by immunofluorescence and electron microscopy. PAN induced massive proteinuria and serum creatinine elevation on day 10, both of which were significantly ameliorated by tolvaptan. Immunofluorescence studies of the podocyte-associated proteins nephrin and podocin revealed granular staining patterns in PAN nephrosis rats. In tolvaptan-treated rats, nephrin and podocin expressions retained their normal linear pattern. Electron microscopy showed foot process effacement was ameliorated in tolvaptan-treated rats. Tolvaptan is protective against podocyte damage and proteinuria in PAN nephrosis. This study indicates that tolvaptan exerts a renoprotective effect by affecting podocyte morphology and probably function in PAN nephrosis. Tolvaptan is a promising pharmacological tool in the treatment of renal edema.

NADPH Oxidase-derived ROS Contributes to Upregulation of TRPC6 Expression in Puromycin Aminonucleoside-induced Podocyte Injury

Recent studies have demonstrated upregulation of transient receptor potential cation channel 6 (TRPC6) contributes to podocyte injury in acquired forms of proteinuric kidney diseases, such as focal segmental glomerulosclerosis (FSGS). However, under these pathophysiological conditions, the mechanisms of regulation of TRPC6 expression and activity remain unknown. The present study tested the hypothesis that NADPH oxidase-mediated redox signaling importantly participates in the development of podocyte injury by regulation of TRPC6 expression and activity. Injection of puromycin aminonucleoside (PAN) to rats produced severe proteinuria and mimics the lesions of FSGS. Podocyte effacement, NADPH oxidase subunit NOX4 expression, enzyme activity and TRPC6 expression were significant increased in glomeruli from PAN nephrosis rats. Inhibition of NADPH oxidase activity by apocynin ameliorated proteinuria and podocyte effacement and reduced TRPC6 expression. In in vitro study, PAN significantly increased NOX4 and TRPC6 expression levels in cultured podocytes. This increased TRPC6 expression was attenuated by apocynin or siRNA-NOX4. Our results provide direct evidence for the first time that NADPH oxidase-derived reactive oxygen species (ROS) is one of critical components of a signal transduction pathway that links PAN nephrosis to TRPC6-mediated Ca²⁺ signaling.

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

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

Cardiac remodeling and dysfunction in nephrotic syndrome

There is an increased incidence of heart disease in patients with chronic nephrotic syndrome (NS), which may be attributable to the malnutrition and activated inflammatory state accompanying the sustained proteinuria. In this study, we evaluated renal function, cardiac morphometry, contractile function, and myocardial gene expression in the established puromycin aminonucleoside nephrosis rat model of NS. Two weeks after aminonucleoside injection, there was massive proteinuria, decreased creatinine clearance, and a negative sodium balance. Skeletal and cardiac muscle atrophy was present and was accompanied by impaired left ventricular (LV) hemodynamic function along with decreased contractile properties of isolated LV muscle strips. The expression of selected cytokines and proteins involved in calcium handling in myocardial tissue was evaluated by real time polymerase chain reaction. This revealed that the expression of interleukin-1beta, tumor necrosis factor-alpha, and phospholamban were elevated, whereas that of cardiac sarco(endo)plasmic reticulum calcium pump protein was decreased. We suggest that protein wasting and systemic inflammatory activation during NS contribute to cardiac remodeling and dysfunction.

Snail, a transcriptional regulator, represses nephrin expression in glomerular epithelial cells of nephrotic rats

Snail is a DNA-binding molecule that plays a pivotal role in regulating cell adhesion and epithelial to mesenchymal transition. Visceral epithelial cells (podocytes) in kidney glomeruli form a sophisticated cell–cell junction called a slit diaphragm that prevents the loss of plasma protein during ultrafiltration. Nephrin, located in the slit diaphragm and critical for maintaining the integrity of this structure, belongs to the class of cell adhesion molecules of the immunoglobulin super-family. As previously reported, the transcriptional activity of nephrin is a determinant of the integrity of the slit diaphragm in puromycin aminonucleoside (PAN) nephrosis rats. Here, we examined the role of Snail in nephrin expression. In accordance with the downregulation of nephrin in PAN nephrosis rats, Snail was upregulated in vivo and its DNA-binding activity was stimulated in injured podocytes while normal podocytes did not express Snail. An in vitro study demonstrated that Snail bound to E-box motifs in a specific segment of the rat nephrin gene repressed the transcription of nephrin and downregulated nephrin protein. We also found that the expression level of Snail in injured podocytes was regulated by GSK3, which is known to phosphorylate Snail and induce its proteolysis. Pharmacological in vitro and in vivo inhibition studies of GSK3 suggested that GSK3 activity decreased in injured podocytes and this change partially contributed to the decrease in nephrin and increase in Snail and proteinuria. Concordantly, we found that Wnt-2 was upregulated in injured podocytes and activated the Wnt canonical pathway. As the Wnt canonical pathway inactivates GSK3, it is likely that Wnt-2 accounts for the accumulation of Snail in injured podocytes. In conclusion, Snail is a key molecule, which perturbs the integrity of the slit diaphragm through transcriptional repression of nephrin under pathological conditions. Wnt-GSK3 pathway participates in this mechanism.

The relationship among nephrin, podocin, CD2AP, and α-actinin might not be a true ‘interaction’ in podocyte

The abnormality of a single podocyte molecule, caused by a single gene mutation, such as NPHS1 , NPHS2 , CD2AP , and ACTN4 , can lead to the hereditary/congenital nephrotic syndromes (NS). Further studies suggested that more than one podocyte molecule were together involved in acquired or experimental NS. However, we do not know much on the relationship among these podocyte molecules, and the molecular response induced by the change of each podocyte protein to the remaining ones. We respectively knockdown the nephrin, podocin, CD2AP, or α -actinin-4 mRNA by using reconstructed RNA interference vector – psiRNA-hH1GFPzeo in mouse podocyte clone. The molecular behavior or response was revealed by the quantitative expression both at mRNA and protein levels with RT-PCR and Western blot, and by the molecular distribution detected with confocal microscopy. With nephrin knockdown, only CD2AP increased, whereas podocin showed no change. Contrarily, with podocin or CD2AP knockdown, nephrin decreased, while CD2AP or podocin increased. Nephrin, podocin, or CD2AP knockdown did not change the expression of α -actinin-4, whereas α -actinin-4 knockdown begetted the reduction of nephrin, and the increment of podocin and CD2AP. The redistributions of nephrin, podocin, and CD2AP were revealed around a predominant nuclear staining compared with the membrane surface staining in the control podocytes. Our data imply that the response between the four podocyte molecules is very complicated and evidently different. There is not always an interaction between podocyte molecules. The normal localization of podocyte molecules would depend on their normal expression quantity and the molecular reactions between them.

Expression and regulation of adrenomedullin in renal glomerular podocytes

Adrenomedullin (AM) is postulated to exert organ-protective effects. It is expressed in the renal glomeruli, but its roles in the glomerular podocytes have been poorly elucidated. In the present study, we investigated the expression and regulation of AM in recently established conditionally immortalized mouse podocyte cell line in vitro and podocyte injury model in vivo. The cultured differentiated podocytes expressed AM mRNA and secreted measurable amount of AM. AM secretion from the podocytes was increased by H 2O 2, hypoxia, puromycin aminonucleoside (PAN), albumin overload, and TNF-α. Real-time RT-PCR analysis revealed that AM mRNA expression in the podocytes was enhanced by PAN and TNF-α, both of which were suppressed by mitochondrial antioxidants. Furthermore, AM expression was upregulated in the glomerular podocytes of PAN nephrosis rats. These results indicated that AM expression in the podocytes was upregulated by stimuli or condition relevant to podocyte injury, suggesting its potential role in podocyte pathophysiology.

Effect of zelandopam, a dopamine D1-like receptor agonist, in puromycin aminonucleoside nephrosis rats

The present experiment was designed to investigate the role of peripheral dopamine D1-like receptors and to evaluate the prophylactic effect of zelandopam, a dopamine D1-like receptor agonist, on puromycin aminonucleoside (PA)-induced nephrosis in rats. Rats were divided into six groups ( n=10 per group): 0.9% saline-injected rats (control); PA-injected rats (PAN); PA-injected rats treated with the selective dopamine D1-like receptor agonist zelandopam (30, 100, 300 mg/kg p.o. twice a day); PA-injected rats treated with prednisolone (1 mg/kg p.o. once a day). Nephrosis was induced in rats with a single intravenous injection of PA at a dose of 50 mg/kg. The effects of zelandopam and prednisolone in PA nephrosis rats were evaluated before injection of PA and at 7 and 14 days after injection. PA-induced nephrosis was characterized by an increase in urinary protein excretion (proteinuria) and plasma total cholesterol. Zelandopam dose-dependently attenuated the increase in proteinuria and total cholesterol. Prednisolone significantly attenuated the increase in proteinuria and total cholesterol and resulted in a significant decrease in body weight. The present study demonstrates for the first time that zelandopam, a selective dopamine D1-like receptor agonist, is effective in blunting the development of PA-induced nephrosis, and that the effects of zelandopam are dose dependent.

Impact of Cyclosporin on Podocyte ZO-1 Expression in Puromycin Aminonucleoside Nephrosis Rats

Puromycin aminonucleoside (PAN)-induced nephrosis is a well-described model of human idiopathic nephrotic syndrome, but the mechanism of PAN's effect is not completely understood. To investigate whether proteinuria in the PAN model is associated with an alteration of zonula occludens-1 (ZO-1) expression within the glomeruli, and whether cyclosporin A (CsA) has an effect on proteinuria and ZO-1 expression in this model, eighteen Sprague Dawley (SD) rats were assigned into three groups. Twelve rats received a single intraperitoneal injection of PAN (15 mg/100 g). The other six rats received an equal volume of saline (normal control group; control). CsA solution was administered intraperitoneally once a day for 20 days after the PAN injection (n=6, PAN+CsA). The remaining six rats received PAN, but they didn't receive CsA (n=6, PAN). Compared to control rats (35.1 ± 5.4 mg/day), the 24-hour urinary protein excretion on day 18 was significantly higher in the PAN rats (1021.9 ± 128.9 mg/day, p<0.01), and the CsA treatment partly reversed the increase in proteinuria in the PAN rats (556.4 ± 102.3 mg/day, p<0.05). Glomerular ZO-1 protein expressions were significantly increased in the PAN rats as compared to the control group on day 20 (176%, p<0.01). CsA treatment for 20 days in the PAN rats inhibited the increase in ZO-1 protein expression by 71.1% (p<0.05). CsA treatment significantly diminished the glomerular ZO-1 expression in the PAN rats as assessed by immunohistochemistry. CsA treatment significantly reduced proteinuria and the diminished glomerular ZO-1 expression in a PAN nephrosis rat model. These findings suggest the potential role of the slit diaphragm associated proteins in the development of the nephrotic syndrome, and CsA decreased the proteinuria probably by a direct action on the expression of these proteins in podocytes. Further investigations are needed to clarify the role of slit diaphragm associated proteins in the development of PAN nephrosis.

Nephrin and Podocin Expression Around the Onset of Puromycin Aminonucleoside Nephrosis

Decreased expression levels of the glomerular slit membrane proteins, nephrin and podocin, have been reported after the onset of puromycin aminonucleoside (PA) nephrosis. We examined nephrin and podocin expressions prior to the onset of proteinuria of PA nephrosis to elucidate the proteinuria induction mechanism of PA. PA nephrosis was induced by a subcutaneous single injection of 120 mg kg(-1) PA. The mRNA levels of nephrin and podocin in whole kidney total RNA were quantified by the TaqMan real time PCR quantification system. The localization and levels of nephrin and podocin molecules were analyzed by immunofluorescence and Western blotting, respectively. Albuminuria and proteinuria were significant on days 3 and 4 in PA nephrosis rats. The protein levels of nephrin and podocin decreased significantly at day 3. The protein localization of nephrin and podocin changed at day 2 and day 1, respectively. The mRNA level of nephrin increased at day 2 and subsequently decreased at day 4. The podocin mRNA level did not change significantly. In conclusions, the protein level of nephrin and podocin decreased at the onset of albuminuria in the PA nephrosis. However, the first change induced by PA was the change of podocin localization from a linear pattern to a dot-like one prior to the onset of albuminuria.

Key molecular events in puromycin aminonucleoside nephrosis rats.

Nephrin, podocin and alpha-actinin are all involved in proteinuria, but it is unclear which molecular event plays a crucial role during the development of proteinuria. Immunofluorescence staining and real-time quantitative polymerase chain reaction were used to study the glomerular expression of these molecules in puromycin aminonucleoside (PAN) nephrosis. Morphometric methods were applied to evaluate the podocyte foot process (FP) morphology. Two days after PAN injection, nephrin and podocin staining became discontinuous, podocin intensity decreased and FP swelled. Nephrin protein and mRNA decreased at day 5. Both podocin and nephrin intensity decreased dramatically when heavy proteinuria occurred, but nephrin mRNA was regained. When proteinuria disappeared, podocin recovered whereas nephrin did not (P = 0.02); alpha-actinin intensity increased (P = 0.009) and the distribution changed. The podocyte FP volume density correlated negatively with nephrin (r = -0.78, P = 0.0001) and podocin immunofluorescence intensity (r = -0.76, P = 0.0001). We conclude that, before the onset of proteinuria, the first response was the nephrin and podocin distribution change, podocin protein decrease and swollen FP; the podocin quantitative change was earlier than nephrin. Podocin and nephrin distribution and the protein level was associated with proteinuria more closely than their mRNA level. The delayed alpha-actinin induction might be a reparative response.

A Study of VEGF and Its Receptors in Two Rat Models of Proteinuria

Background: The high level of expression of vascular endothelial growth factor (VEGF) in normal podocyte foot processes suggests that VEGF has an important role in maintaining normal glomerular function. While altered VEGF expression occurs in many glomerular diseases, a direct role for VEGF in the pathogenesis of proteinuria has not been demonstrated. Methods: Expression of VEGF and its receptors (VEGFR-1 and VEGFR-2) was examined in passive Heymann nephritis (PHN) and puromycin aminonucleoside nephrosis (PAN), by immunohistochemistry, in situ hybridization, Northern and Western blotting. Inhibition of VEGF in the PAN model was performed by administration of a blocking antibody. Results: In both models, glomeruli showed upregulation of VEGF and VEGF receptors compared to control animals. VEGF mRNA was increased most significantly (5-fold) at day 5 after induction of PHN, prior to the onset of proteinuria, with persistent upregulation (3-fold) at day 21. Increased VEGF mRNA was also seen in PAN, but it was less marked. In situ hybridization and immunohistochemistry localized VEGF predominantly to podocytes. Increased expression of VEGFR-1 and VEGFR-2 protein was seen in glomerular endothelial cells of PHN and PAN rats by immunohistochemistry, as was VEGFR-2 mRNA by in situ hybridization. Upregulation of VEGFR-1 by endothelial cells was more striking in the PAN model than PHN. Administration of a blocking antibody to rats with PAN did not affect proteinuria, creatinine clearance or sodium excretion. Conclusion: The expression of VEGF and its receptors is significantly increased in the PHN and PAN rat models of proteinuria suggesting a role for VEGF in the disease process. VEGF may have an important role in promoting glomerular repair in a variety of glomerular diseases.

Increased Expression of CD54, CD18, MHC Class II Molecules, and Proliferating Cell Nuclear Antigen in Acute Puromycin Aminonucleoside Nephrosis

Cellular infiltration to renal tissues is an important feature during acute puromycin aminonucleoside nephrosis (PAN) in rats. The mechanisms responsible for this infiltration are poorly understood. To elucidate the participation of adhesion molecules in PAN, nephrosis was induced in rats by intraperitoneal puromycin aminonucleoside injection. Controls represent animals injected with a 0.9% saline solution. ICAM-1 (intercellular adhesion molecule 1), CD18 (beta chain of lymphocyte-function-associated antigen), LCA (leukocyte common antigen), ED1 (monocyte/macrophage marker), and proliferating cell nuclear antigen expressions were evaluated in renal tissues 1, 2, and 7 weeks after injection. Frozen sections from PAN rat kidneys showed increased expressions of ICAM-1 and its ligand, and these findings were associated with increased levels of LCA+ and ED1+ cells in glomerulus and interstitium. The kinetics of leukocyte infiltration was similar to the kinetics of ICAM-1 expression: high values at week 2 which returned to normal values at week 7. Increased glomerular and interstitial proliferative activities (proliferating cell nulear antigen positive cells) were also found at week 2 of nephrosis. There was a correlation between ICAM-1 expression and numbers of LCA+ and ED1+ cells and between numbers of LCA+ cells and proliferating cells in glomerulus and interstitium. Correlations between glomerular and tubular ICAM-1 expression, interstitial leukocyte infiltration, and glomerular, interstitial, and tubular proliferative activities with the proteinuria were also observed during the nephrotic phase. In addition, increased lymphocyte binding to PAN renal tissues was observed, and this binding was diminished by anti-LFA-1β monoclonal antibody pretreatment of lymphocytes. A similar result was found with anti-ICAM-1 monoclonal antibody pretreatment of renal tissues. Our results suggest that increased expression of ICAM-1 and proliferative activity could be important determinants in the renal hypercellularity found in this experimental model.