Nicorandil protects podocytes via modulation of antioxidative capacity in acute puromycin aminonucleoside-induced nephrosis in rats.

Nephrotic syndrome, characterized by proteinuria and hypoalbuminemia, results from the dysregulation of glomerular podocytes and is a significant cause of end-stage kidney disease. Patients with idiopathic nephrotic syndrome are generally treated with immunosuppressive agents; however, these agents produce various adverse effects. Previously, we reported the renoprotective effects of a stimulator of the mitochondrial ATP-dependent K+ channel (MitKATP), nicorandil, in a remnant kidney model. Nonetheless, the cellular targets of these effects remain unknown. Here, we examined the effect of nicorandil on puromycin aminonucleoside-induced nephrosis (PAN) rats, a well-established model of podocyte injury and human nephrotic syndrome. PAN was induced using a single intraperitoneal injection. Nicorandil was administered orally at 30 mg/kg/day. We found that proteinuria and hypoalbuminemia in PAN rats were significantly ameliorated following nicorandil treatment. Immunostaining and ultrastructural analysis under electron microscopy demonstrated that podocyte injury in PAN rats showed a significant partial attenuation following nicorandil treatment. Nicorandil ameliorated the increase in the oxidative stress markers nitrotyrosine and 8-hydroxy-2-deoxyguanosine in glomeruli. Conversely, nicorandil prevented the decrease in levels of the antioxidant enzyme manganese superoxide dismutase in PAN rats. We found that mitochondrial Ca2+ uniporter levels in glomeruli were higher in PAN rats than in control rats, and this increase was significantly attenuated by nicorandil. We conclude that stimulation of MitKATP by nicorandil reduces proteinuria by attenuating podocyte injury in PAN nephrosis, which restores mitochondrial antioxidative capacity, possibly through mitochondrial Ca2+ uniporter modulation. These data indicate that MitKATP may represent a novel target for podocyte injury and nephrotic syndrome.NEW & NOTEWORTHY Our findings suggest that the mitochondrial Ca2+ uniporter may be an upstream regulator of manganese superoxide dismutase and indicate a biochemical basis for the interaction between the ATP-sensitive K+ channel and Ca2+ signaling. We believe that our study makes a significant contribution to the literature because our results indicate that the ATP-sensitive K+ channel may be a potential therapeutic target for podocyte injury and nephrotic syndrome.

Re-expression of Sall1 in podocytes protects against adriamycin-induced nephrosis.

The highly conserved spalt (sal) gene family members encode proteins characterized by multiple double zinc finger motifs of the C2H2 type. Humans and mice each have four known Sal-like genes (SALL1-4 in humans and Sall1-4 in mice). Sall1 is known to have a crucial role in kidney development. To explore the significance of Sall1 in differentiated podocytes, we investigated podocyte-specific Sall1-deficient mice (Sall1 KOp°d°/p°d°) using a podocin-Cre/loxP system and siRNA Sall1 knockdown (Sall1 KD) podocytes. Under physiological conditions, Sall1 KOp°d°/p°d° mice exhibited no proteinuria during their lifetime, but foot-process effacement was detected in some of the podocytes. To elucidate the role of Sall1 in injured podocytes, we used an adriamycin (ADR)-induced model of nephrosis and glomerulosclerosis. Surprisingly, the expression of Sall1 was elevated in control mice on day 14 after ADR injection. On day 28 after ADR injection, Sall1 KOp°d°/p°d° mice exhibited significantly higher levels of proteinuria and higher numbers of sclerotic glomeruli. Differentiated Sall1 KD podocytes showed a loss of synaptopodin, suppressed stress fiber formation, and, ultimately, impaired directed cell migration. In addition, the loss of Sall1 increased the number of apoptotic podocytes following ADR treatment. These results indicated that Sall1 has a protective role in podocytes; thus, we investigated the endoplasmic reticulum stress marker GRP78. GRP78 expression was higher in ADR-treated Sall1 KOp°d°/p°d° mice than in control mice. Sall1 appeared to influence the expression of GRP78 in injured podocytes. These results suggest that Sall1 is associated with actin reorganization, endoplasmic reticulum stress, and apoptosis in injured podocytes. These protective aspects of Sall1 re-expression in injured podocytes may have the potential to reduce apoptosis and possibly glomerulosclerosis.

Sodium Bicarbonate-Ascorbic Acid Combination for Prevention of Contrast-Induced Nephropathy in Chronic Kidney Disease Patients Undergoing Catheterization.

BACKGROUND: Sodium bicarbonate and ascorbic acid have been proposed to prevent contrast-induced nephropathy (CIN). The present study evaluated the effect of their combined use on CIN incidence.Methods and Results:We prospectively enrolled 429 patients with chronic kidney disease (CKD: baseline estimated glomerular filtration rate <60 mL/min/1.73 m2) prior to elective coronary catheterization. CIN was defined as absolute (≥0.5 mg/dL) or relative (≥25%) increase in serum creatinine within 72 h. In the saline hydration (n=218) and combined sodium bicarbonate+ascorbic acid (n=211) groups, a total of 1,500-2,500 mL 0.9% saline was given before and after the procedure. In addition, the combination group received 20 mEq sodium bicarbonate and 3 g ascorbic acid i.v. before the procedure, followed by 2 g ascorbic acid after the procedure and a further 2 g after 12 h. There were no significant differences between the basic characteristics and contrast volume in the 2 groups. CIN occurred in 19 patients (8.7%) in the saline group, and in 6 patients (2.8%) in the combined treatment group (P=0.008). CONCLUSIONS: Combined sodium bicarbonate and ascorbic acid could prevent CIN following catheterization in CKD patients.

Organic Anion Transporter 5 (Oat5) Urinary Excretion Is a Specific Biomarker of Kidney Injury: Evaluation of Urinary Excretion of Exosomal Oat5 after N-Acetylcysteine Prevention of Cisplatin Induced Nephrotoxicity.

Cisplatin is a commonly used chemotherapeutic agent. Its main side-effect is nephrotoxicity. It was reported that the organic anion transporter 5 (Oat5) urinary excretion is elevated, implying renal perturbation, when no modifications of traditional markers of renal damage are still observed in cisplatin-induced acute kidney injury (AKI). It was also demonstrated that Oat5 is excreted in urine by the exosomal pathway. This study was designated to demonstrate the specific response of the urinary excretion of exosomal Oat5 to kidney injury independently of other cisplatin toxic effects, in order to strengthen Oat5 urinary levels as a specific biomarker of AKI. To accomplish that aim, we evaluated if urinary excretion of exosomal Oat5 returns to its basal levels when cisplatin renal damage is prevented by the coadministration of the renoprotective compound N-acetylcysteine. Four days after cisplatin administration, AKI was induced in cisplatin-treated male Wistar rats (Cis group), as it was corroborated by increased urea and creatinine plasma levels. Tubular damage was also observed. In cotreated animals (Cis + NAC group), plasma urea and creatinine concentrations tended to return to their basal values, and tubular damage was improved. Urinary excretion of exosomal Oat5 was notably increased in the Cis group, but when renal injury was ameliorated by N-acetylcysteine coadministration, that increase was undetected. So, in this work we observed that urinary excretion of exosomal Oat5 was only increased if renal insult is produced, demonstrating its specificity as a renal injury biomarker.

Discrete functions of M2a and M2c macrophage subsets determine their relative efficacy in treating chronic kidney disease.

Two types of alternatively activated macrophages, M(2a) induced by IL-4/IL-13 and M(2c) by IL-10/TGF-ß, exhibit anti-inflammatory functions in vitro and protect against renal injury in vivo. Since their relative therapeutic efficacy is unclear, we compared the effects of these two macrophage subsets in murine adriamycin nephrosis. Both subsets significantly reduced renal inflammation and renal injury; however, M(2c) macrophages more effectively reduced glomerulosclerosis, tubular atrophy, interstitial expansion, and proteinuria than M(2a) macrophages. The M(2c) macrophages were also more effective than M(2a) in reduction of macrophage and CD4(+) T-cell infiltration in kidney. Moreover, nephrotic mice treated with M(2c) had a greater reduction in renal fibrosis than those treated with M(2a). M(2c) but not M(2a) macrophages induced regulatory T cells (Tregs) from CD4(+)CD25(-) T cells in vitro, and increased Treg numbers in local draining lymph nodes of nephrotic mice. To determine whether the greater protection with M(2c) was due to their capability to induce Tregs, the Tregs were depleted by PC61 antibody in nephrotic mice treated with M(2a) or M(2c). Treg depletion diminished the superior effects of M(2c) compared to M(2a) in protection against renal injury, inflammatory infiltrates, and renal fibrosis. Thus, M(2c) are more potent than M(2a) macrophages in protecting against renal injury due to their ability to induce Tregs.

A novel nuclear factor κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates puromycin aminonucleoside-induced nephrosis in mice.

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

IL-10/TGF-beta-modified macrophages induce regulatory T cells and protect against adriamycin nephrosis.

IL-10/TGF-beta-modified macrophages, a subset of activated macrophages, produce anti-inflammatory cytokines, suggesting that they may protect against inflammation-mediated injury. Here, macrophages modified ex vivo by IL-10/TGF-beta (IL-10/TGF-beta Mu2) significantly attenuated renal inflammation, structural injury, and functional decline in murine adriamycin nephrosis (AN). These cells deactivated effector macrophages and inhibited CD4+ T cell proliferation. IL-10/TGF-beta Mu2 expressed high levels of the regulatory co-stimulatory molecule B7-H4, induced regulatory T cells from CD4+CD25- T cells in vitro, and increased the number of regulatory T cells in lymph nodes draining the kidneys in AN. The phenotype of IL-10/TGF-beta Mu2 did not switch to that of effector macrophages in the inflamed kidney, and these cells did not promote fibrosis. Taken together, these data demonstrate that IL-10/TGF-beta-modified macrophages effectively protect against renal injury in AN and may become part of a therapeutic strategy for chronic inflammatory disease.

Renoprotective effect of the L/N-type calcium channel antagonist cilnidipine on puromycin aminonucleoside-induced nephrosis in rats.

The renoprotective effect of cilnidipine ((+/-)-2-methoxyethyl 3-phenyl-2(E)-propenyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylate, CAS 132203-70-4), a L/N-type calcium channel antagonist, on puromycin aminonucleoside (PAN)-induced nephrosis was investigated in rats. In the Experiment I, rats were given an intravenous injection of PAN (70 mg/kg). Cilnidipine (3 mg/kg/day) and enalapril (CAS 75847-73-3, 5 mg/kg/day) were administered orally from 6 days after treatment with PAN (day 6) to day 26, and urinary analysis was performed on days 9, 15, 20 and 27. In the Experiment II, nephrosis was also induced by intravenous injection of PAN (70 or 100 mg/kg) in rats which were treated with cilnidipine and enalapril from days 6 to 10. Systolic blood pressure was measured on day 7 and urinary analysis was performed on day 10. On day 11, serum was collected and the kidneys were removed for immunofluorescence staining for nephrin and podocin proteins. In PAN-treated rats, the daily urinary protein excretion was dramatically elevated on day 5, reached a peak on day 9 and gradually returned to a normal level from days 15 to 27. Cilnidipine (3 mg/kg/ day) significantly suppressed the increase in proteinuria on day 9 and also improved the decrease in creatinine clearance without evident effect on the blood pressure. Furthermore, the elevations in serum total cholesterol and triglyceride tended to be suppressed by cilnidipine. The expression of nephrin and podocin proteins in PAN-treated rats showed the granular pattern in the glomeruli, while the intensity of staining seemed to be dependent on the urinary protein excretion level in the cilnidipine-treated rats. The results obtained in this study suggest a renoprotective effect of cilnidipine in PAN-induced nephrosis in rats.

Inhibition of matrix metalloproteinases during chronic allograft nephropathy in rats.

BACKGROUND: Chronic allograft nephropathy (CAN) belongs to the major causes of long-term kidney allograft failure. One of the histologic hallmarks of CAN is interstitial fibrosis, influenced by matrix metalloproteinases (MMPs) that are controlling extracellular matrix (ECM) degradation. Whether MMPs affect the development and progression of CAN is not clear so far. To analyze the role of MMPs in CAN, we investigated the effects of an early and a late application of BAY 12-9566, an inhibitor of MMP-2, -3, and -9 on the development and progression of CAN in a rat kidney-transplantation model. METHODS: Fisher kidneys were orthotopically transplanted into Lewis recipients that were treated with BAY 12-9566 (15 mg/kg per day) or vehicle either for the first 10 days after transplantation (early treatment) or from week 12 to week 20 after transplantation (late treatment). Proteinuria was analyzed every 4 weeks up to week 20 after transplantation when kidney grafts were removed for further analysis. RESULTS: Early MMP-inhibition resulted in a significantly reduced 24-hour protein excretion that was paralleled by a lower grade of CAN after 20 weeks. However, late MMP inhibition starting at week 12 after transplantation resulted in significantly higher proteinuria and a higher grade of CAN as compared with controls. Furthermore, transforming growth factor-beta and platelet-derived growth factor-B chain mRNA levels were significantly increased in these animals. CONCLUSIONS: Inhibition of MMPs early after transplantation reduced the development and progression of CAN but promoted CAN if initiated at later stages. Thus, MMPs are involved in the development and progression of CAN.

Effect of DTPA on the nephrotoxicity induced by uranium in the rat.

The only treatment proposed after human contamination with MOX (mixed oxide of uranium and plutonium) is diethylenetriaminepentaacetic acid (DTPA), because plutonium is considered to be the major risk. However, both DTPA and uranium are nephrotoxic at high dosages and DTPA has been shown to increase in vitro the cytotoxicity induced by uranium on cultured epithelial tubular cells. This work aimed to test this effect in vivo. Rats were injected with subtoxic (57 microg kg(-1)) to toxic (639 microg kg(-1)) amounts of uranium as nitrate at 0 h, they received two DTPA injections (30 micromol kg(-1)) at 2 min and 24 h and were euthanased at 48 h. The nephrotoxic effects were evaluated by measurement of the body weight gain, food and water intake, measurement of biochemical parameters in urine and blood, and histological examination of one kidney. The main result was that DTPA did not increase the nephrotoxicity induced by uranium in the range of concentrations tested, which was inconsistent with the in vitro results.

Retinoids as a potential treatment for experimental puromycin-induced nephrosis.

1 Puromycin aminonucleoside (PAN)-induced nephrosis is a model of human minimal change disease. In rats, PAN induces nephrotic-range proteinuria, renal epithelial cell (podocyte) damage, infiltration of mononuclear leukocytes, and apoptosis of several renal cell types. 2 Retinoic acid (RA) modulates a wide range of biological processes, such as inflammation and apoptosis. Since renal damage by PAN is characterized by inflammatory infiltration and epithelial cell death, the effect of treatment with all-trans RA (tRA) was examined in the PAN nephrosis model and in the cultured differentiated podocyte. 3 Treatment with tRA 4 days after PAN injection did not inhibit the proteinuria peak but reversed it significantly. However, treatment with tRA both before and 2 days after the injection of PAN protected the glomerular epithelial cells, diminishing the cellular edema and diffuseness of the foot process effacement. Preservation of the podocyte architecture correlated with the inhibition of proteinuria. The anti-inflammatory effect of tRA was evidenced by the inhibition of PAN-induced interstitial mononuclear cell infiltration and the decreased renal expression of two molecules involved in monocyte infiltration: fibronectin and monocyte chemoattractant protein-1. TUNEL assays showed that tRA inhibited the PAN-induced apoptosis of cultured differentiated mouse podocytes. 4 We conclude that tRA treatment may prevent proteinuria by protecting the podocytes from injury and diminishing the interstitial mononuclear infiltrate in the model of PAN nephrosis. Retinoids are a potential new treatment for kidney diseases characterized by proteinuria and mononuclear cell infiltration.

Amelioration of glomerular injury in doxorubicin hydrochloride nephrosis by dimethylthiourea.

The hydroxyl radical scavengers dimethylthiourea (DMTU), sodium benzoate, and dimethylsulfoxide (DMSO) were administered to rats before doxorubicin hydrochloride (ADR) (5 mg/kg, IV) to probe the role of free radicals in mediating proteinuria in doxorubicin hydrochloride nephrosis (AN). Because ADR stimulates free radical production, the role of renal glutathione was also evaluated; glutathione metabolism is involved in tissue detoxification processes. DMTU administration to rats with AN caused a significant (p less than 0.01) reduction in their proteinuria after 7 days (52.84 +/- 13.21 mg/24 hours) when they were compared with ADR controls (155.81 +/- 20.16 mg/24 hours). In similar fashion, their urine albumin excretion was also significantly reduced when compared with that of ADR controls (11.13 +/- 2.75 mg/24 hours vs 32.08 +/- 4.14 mg/24 hours; p less than 0.01). DMTU-treated rats also had significantly (p less than 0.001) reduced urinary protein and albumin excretion at 14 days when compared with rats that received ADR alone. The urinary excretion of lysozyme and N-acetyl-glucosaminidase, markers of renal tubular injury, were significantly increased after 7 or 14 days in rats with AN, despite DMTU treatment. Creatinine clearance was significantly reduced (p less than 0.05) in rats receiving ADR alone (0.223 +/- 0.011 ml/min/100 gm) when compared with that in normal controls (0.331 +/- 0.027 ml/min/100 gm) or DMTU-treated rats (0.289 +/- 0.035 ml/min/100 gm). Unlike DMTU, neither sodium benzoate nor DMSO reduced proteinuria in rats with AN.(ABSTRACT TRUNCATED AT 250 WORDS)

Congenital nephrosis as a cause of elevated alpha-fetoprotein.

Two cases of congenital nephrosis were detected through routine maternal serum alpha-fetoprotein (MSAFP) screening of 95,135 patients. No other cases of congenital nephrosis from this group were reported, resulting in an incidence of approximately one in 47,500 in this low-risk population. In both of these cases, similar to other reported cases of congenital nephrosis having MSAFP screening, the protein concentrations were greater than or equal to 10 multiples of the median (MOM). Therefore, in the case of an MSAFP over 10 MOM and a normal ultrasound examination, congenital nephrosis should be included in counseling regarding the possibility of undetected malformations. Furthermore, in the case of a pregnancy with elevated amniotic fluid AFP with negative acetylcholinesterase and normal ultrasound, the possibility of congenital nephrosis should be mentioned, regardless of family history or ancestry. When a pregnancy is terminated because of these biochemical findings, special and immediate attention to the fetal kidneys using electron microscopy is necessary to evaluate properly the possibility of congenital nephrosis.