Mizoribine Corrects Defective Nephrin Biogenesis by Restoring Intracellular Energy Balance
Aya Nakajo*,
Jamshid Khoshnoodi,
Hitoshi Takenaka,
Emi Hagiwara,
Takashi Watanabe,
Hayato Kawakami||,
Ryota Kurayama*,
Yuji Sekine*,
Fumio Bessho*,
Shori Takahashi¶,
Agnieszka Swiatecka-Urban**,
Karl Tryggvason and
Kunimasa Yan*
Departments of * Pediatrics, Biochemistry, Laboratory Medicine, and || Anatomy, Kyorin University School of Medicine, Tokyo, Japan; Department of Medicine, Division of Nephrology and Hypertension, Vanderbilt University, Nashville, Tennessee; ¶ Department of Pediatrics, Nihon University School of Medicine, Tokyo, Japan; ** Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; and Department of Medical Biochemistry and Biophysics, Division of Matrix Biology, Karolinska Institute, Stockholm, Sweden
Correspondence: Dr. Kunimasa Yan, Department of Pediatrics, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka, Tokyo 181-8611, Japan. Phone: +81-422-47-5511, ext. 3573; Fax: +81-422-47-8184; E-mail: kuniyan{at}kyorin-u.ac.jp
Received for publication July 13, 2006.
Accepted for publication May 29, 2007.
Proteins are modified and folded within the endoplasmic reticulum(ER). When the influx of proteins exceeds the capacity of theER to handle the load, the ER is "stressed" and protein biogenesisis affected. We have previously shown that the induction ofER stress by ATP depletion in podocytes leads to mislocalizationof nephrin and subsequent injury of podocytes. The aim of thepresent study was to determine whether ER stress is associatedwith proteinuria in vivo and whether the immunosuppressant mizoribinemay exert its antiproteinuric effect by restoring normal nephrinbiogenesis. Induction of nephrotic-range proteinuria with puromycinaminonucleoside in mice increased expression of the ER stressmarker GRP78 in podocytes, and led to the mislocalization ofnephrin to the cytoplasm. In vitro, mizoribine, through a mechanismlikely dependent on the inhibition of inosine 5'-monophosphatedehydrogenase (IMPDH) activity in podocytes, restored the intracellularenergy balance by increasing levels of ATP and corrected theposttranslational processing of nephrin. Therefore, we speculatethat mizoribine may induce remission of proteinuria, at leastin part, by restoring the biogenesis of slit diaphragm proteinsin injured podocytes. Further understanding of the ER microenvironmentmay lead to novel approaches to treat diseases in which abnormalhandling of proteins plays a role in pathogenesis.
Nephrotic syndrome (NS) is a common disorder with multiple etiologies.1The pathomechanism of proteinuria in NS is not completely understood,but recent cloning of the gene mutated in the congenital NS,NPHS12,3 and characterization of its protein product, nephrin,opened new avenues for research. Besides its central role inthe development of congenital NS, nephrin has been implicatedin the pathogenesis of acquired forms of NS.4–7 The roleof nephrin in the pathogenesis of NS is not surprising becausenephrin is a critical component of the podocyte slit diaphragm,where it is instrumental in maintaining the integrity of theglomerular filtration barrier.8,9 Nephrin is a transmembraneglycoprotein of the Ig superfamily, and, like other glycoproteins,nephrin biogenesis involves steps such as synthesis, folding,modifications, including N-glycosylation, and trafficking tothe plasma membrane.10–13 Modifications that lead to proteinfolding take place in the endoplasmic reticulum (ER).14 Undercertain pathologic conditions, the influx of unfolded proteinsexceeds the folding/processing capacity of the ER. This ER imbalance,termed ER stress, triggers signaling pathways to return theER to its physiologic state.14 Conditions that lead to ER stressperturb protein biogenesis. Altered nephrin biogenesis has beendocumented in several forms of acquired NS,4–7 and nephrinbiogenesis can be inhibited experimentally during ER stressthat is induced by glucose starvation.13 However, ER stresshas never been documented during NS in podocytes.
Remission of proteinuria in idiopathic NS can be induced byglucocorticoids with or without immunosuppressive agents. Thevast majority of patients with the minimal-change NS achievecomplete remission with glucocorticoids alone.15 Additionaltherapeutic options are available for patients who have minimal-changeNS and develop steroid-resistant or frequently relapsing, steroid-dependentNS. These therapeutic options are aimed at the control of NSand at minimizing the exposure to glucocorticoids and includelevamisole and alkylating agents such as chlorambucil and cyclophosphamide.16Other immunosuppressive agents, originally developed for theuse in kidney transplantation and more recently used for thetreatment of the intractable forms of NS, are cyclosporine,mycophenolate mofetil, and mizoribine. The mechanism of theantiproteinuric effect of these agents is not completely understood.Compelling evidence, including our recently published data,indicate that some therapeutic agents may exert the antiproteinuriceffect, at least in part, by restoring nephrin biogenesis.7,13,17,18
Mizoribine, an agent developed in Japan in 1971 from Eupenicilliumbrefeldianum, M-2166,19 has been well accepted in Japan forthe treatment of steroid-resistant or frequently relapsing,steroid-dependent NS.20 The immunosuppressive effect of mizoribineconsists of a selective and competitive inhibition of inosine5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzymein the de novo purine nucleotide biosynthesis21,22 (Figure 1).Because lymphocytes rely solely on the de novo purine biosynthesis,inhibition of IMPDH activity in these cells leads to depletionof the purine nucleotides, inhibition of activated lymphocyteproliferation, and, subsequently, immunosuppression. It is unknownwhether remission of proteinuria, induced by mizoribine in susceptibleindividuals with NS, is mediated exclusively by the effect onactivated lymphocytes or whether it may result, at least inpart, from a direct effect of mizoribine on the podocyte. Compellingevidence demonstrates that ER stress inhibits the biogenesisof proteins, including nephrin. We verified that ER stress waspresent in the rat glomeruli during puromycin aminonucleoside(PAN) nephrosis, an in vivo model of minimal-change NS. Studieswere conducted to elucidate the effect of mizoribine on nephrinbiogenesis during ER stress. We report that mizoribine restoredthe intracellular energy balance during ER stress by salvagingthe ATP levels and rescued the expression of mature, fully glycosylatednephrin in the plasma membrane by a mechanism that depends onthe mizoribine-mediated inhibition of IMPDH activity.
Figure 1. Metabolic pathways of purine nucleotide biosynthesis. IMPDH is the rate-limiting enzyme in purine nucleotide biosynthesis. The substrate for IMPDH, IMP, can be synthesized (1) from glucose via the de novo pathway, (2) from free purines via the salvage pathway, and (3) by recycling AMP or GMP. XMP, xanthosine 5'-monophosphate; SAMP, adenylsuccinate.
ER Stress Is Present in Rat Glomeruli during NS
Studies were conducted to examine whether ER stress is presentin vivo during NS. PAN nephrosis is a well-accepted and widelyused in vivo model of minimal-change NS. As demonstrated bythe increased expression of the stress-inducible chaperones,glucose-regulated protein 78 (GRP78),23 ER stress was presenton days 4 and 5 after injection of PAN, the time that coincideswith development of heavy proteinuria in this model of NS (Figure 2).The expression of GRP78 was partially increased in the glomerulion day 4 of PAN nephrosis, whereas it was globally increasedon day 5 (Figure 2B). The pattern of nephrin expression waschanged in parallel with the induction of ER stress. In thecontrol glomeruli (day 0), nephrin expression was seen in alinear pattern, as described previously. In contrast, on day4 of PAN nephrosis, nephrin expression displayed a fine granularand linear pattern in the capillary loops. Moreover, on day5, the capillary loop pattern was almost completely abolishedand a cytoplasmic pattern was prominently observed (Figure 2B).The expression of GRP78 and nephrin on days 1, 2, and 3 afterPAN injection was similar to that of the control (data not shown).Taken together, our data demonstrate altered intracellular localizationof nephrin after induction of ER stress in podocytes duringPAN nephrosis.
Figure 2. Representative experiments demonstrating presence of ER stress in rat glomeruli during PAN nephrosis. (A) Western blot. Thirty micrograms of glomerular protein was subjected to SDS-PAGE. As demonstrated by the increased expression of the stress-inducible chaperone GRP78, ER stress was present on days 4 and 5 after injection of PAN, the time that coincides with development of heavy proteinuria in this model of NS. -Actin was used as an internal control. (B) Confocal microscopy image of fixed rat glomerular cryostat sections immunostained with anti-GRP78 antibody and anti-nephrin antibody (pAb2). GRP78 and nephrin were visualized by Alexa Fluor 488 goat anti-mouse antibody and Texas Red goat anti-rabbit antibody, respectively. The experiment was repeated three times from separate kidneys.
Mizoribine Reduces ER Stress and Corrects Defective Nephrin Biogenesis
Studies were conducted to examine the effect of mizoribine onnephrin biogenesis during ER stress that was induced by glucosestarvation. To this end, HEK293 cells stably expressing humannephrin (293-NPH cells) were cultured in medium containing standard(25 mM) glucose, as described in the Concise Methods section.Under these conditions, nephrin was expressed predominantlyas a doublet with molecular weight of approximately 185 and175 kD (Figure 3), corresponding to the mature, fully glycosylated,plasma membrane form and the partially glycosylated, ER formof nephrin, respectively, as described previously.12,13 Forinhibition of nephrin biogenesis, 293-NPH cells were culturedin medium containing low glucose (5.5 mM), as described previously.13As illustrated in Figure 3, glucose starvation induced ER stress,as demonstrated by increased expression of the stress-induciblechaperone GRP78.23 Under such conditions, nephrin was expressedpredominantly as a 155-kD band (Figure 3), corresponding toan immature ER form of nephrin.13 For determination of the effectof mizoribine on nephrin biogenesis, the glucose-starved cellswere cultured in the presence of various concentrations of mizoribinefor 48 h. Mizoribine reduced expression of GRP78 and correcteddefective nephrin biogenesis in a concentration-dependent manner(Figure 3). Moreover, the pattern of nephrin expression in theglucose-starved cells that were treated with 50 µg/mlmizoribine was indistinguishable from that in cells that weregrown in the standard glucose medium (Figure 3).
Figure 3. Representative Western blot experiment demonstrating the effect of mizoribine (MZR) on nephrin biogenesis. 293-NPH cells were cultured in medium containing standard (25 mM) or low (5.5 mM) glucose. In the standard glucose medium, nephrin was expressed predominantly as a doublet with molecular weight of approximately 185 and 175 kD, corresponding to the mature, fully glycosylated plasma membrane form and the partially glycosylated ER form of nephrin, respectively, as described previously.12,13 Glucose starvation induced ER stress, as demonstrated by increased expression of the stress-inducible chaperone GRP78, and inhibited nephrin biogenesis, as demonstrated by expression of the immature ER form of nephrin with the molecular weight of approximately 155 kD.13 A 48-h treatment of the glucose-starved cells with MZR reduced expression of GRP78 and corrected defective nephrin biogenesis in a concentration-dependent manner. An equal amount of total cellular proteins (10 µg) was separated by SDS-PAGE using a 7.5% gel. The experiment was repeated three times from separate cultures.
For more direct examination of the effect of mizoribine on theexpression of nephrin in the plasma membrane, 293-NPH cellswere cultured in medium containing standard (25 mM) or low (5.5mM) glucose in the absence or presence of mizoribine (50 µg/mlfor 48 h), followed by surface immunostaining with anti-nephrinantibody, and analyzed by flow cytometry. As illustrated inFigure 4, glucose starvation inhibited nephrin expression inthe plasma membrane. Furthermore, mizoribine rescued the plasmamembrane expression of nephrin in the glucose-starved cells(Figure 4).
Figure 4. Summary of flow cytometry experiments demonstrating that MZR partially rescued nephrin expression in the plasma membrane. 293-NPH cells were cultured in medium containing standard (25 mM) or low (5.5 mM) glucose in the absence or presence of MZR (50 µg/ml for 48 h). Subsequently, cells were trypsinized, fixed, and stained with a monoclonal anti-nephrin antibody (mAb2) and a secondary Alexa Fluor 488 goat anti-mouse antibody, and the fluorescence intensities were analyzed, as described in the Concise Methods section.
To determine whether ER stress affects the biogenesis of otherproteins that are important for the integrity of podocyte footprocesses, we examined the effect of glucose starvation on 1-integrinbiogenesis. 1-Integrin is expressed in the basal membrane ofthe podocyte foot processes and plays an important role in theattachment of the podocyte to the glomerular basement membranethrough binding to laminin.24 Similar to nephrin, 1-integrinis a N-glycoprotein and demonstrates different molecular weightsdepending on the glycosylation status.25 Immortalized humanpodocytes were cultured in RPMI-1640 medium with low glucose(2.2 mM) or standard glucose (11 mM) in the absence or presenceof the N-glycosylation inhibitor tunicamycin for 48 h. In podocytescultured in the standard glucose medium, 1-integrin was expressedpredominantly as a 150-kD band, representing a fully glycosylatedform of 1-integrin (Figure 5). Tunicamycin induced expressionof a 100-kD band, likely an aglycoform of 1-integrin. The expressionof the 120-kD band, most probably a partially glycosylated formof 1-integrin, caused by glucose starvation, indicates thatER stress alters the biogenesis of 1-integrin (Figure 5). Thus,the effect of ER stress on protein biogenesis is not limitedto nephrin and may involve other members of the macromolecularslit diaphragm protein complex in podocytes.
Figure 5. Representative Western blot experiment demonstrating that ER stress that was induced by glucose starvation inhibits 1-integrin biogenesis. Human immortalized podocyte cells were cultured either in normal-glucose (11 mM) or low-glucose (2.2 mM) medium in the absence or presence of tunicamycin (5 µg/ml) for 48 h. In podocytes cultured in the standard glucose medium, 1-integrin was expressed predominantly as a 150-kD band, representing a fully glycosylated form of 1-integrin. Tunicamycin induced expression of a 100-kD band, likely an aglycoform of 1-integrin. The expression of the 120-kD band, most probably a partially glycosylated form of 1-integrin, caused by glucose starvation, indicates that ER stress alters the biogenesis of 1-integrin. An equal amount of total proteins were loaded. The experiments were performed three times from separate cultures.
Mizoribine Restores the Intracellular Energy Balance during ER Stress
We examined the effect of mizoribine on the intracellular ATPlevels during ER stress in 293-NPH cells using HPLC. In glucose-starvedcells, the ATP level was significantly decreased compared withcells in the standard glucose medium (Figure 6). These dataconfirm that glucose starvation caused intracellular energydepletion. Furthermore, treatment with mizoribine at a concentrationthat rescued nephrin biogenesis (50 µg/ml for 48 h) increasedthe level of intracellular ATP in the glucose-starved cells(Figure 6). Taken together, these data indicate that the mizoribine-mediatedrescue of nephrin biogenesis was mediated by restoration ofthe intracellular energy balance.
Figure 6. Summary of HPLC experiments performed to determine the effect of MZR on the intracellular ATP levels. 293-NPH cells were cultured in 25 or 5.5 mM glucose medium in the absence or presence of MZR (50 µg/ml for 48 h). Intracellular ATP level was measured by HPLC, as described in the Concise Methods section. In the glucose-starved cells, the ATP level was significantly decreased compared with the cells that were grown in the standard glucose medium. These data confirm that glucose starvation caused intracellular energy depletion. MZR increased the intracellular ATP level in the glucose-starved cells. *P < 0.01. Five measurements were performed per group.
Mizoribine Restores the Intracellular Energy Balance by a Mechanism that Likely Depends on the Inhibition of IMPDH Activity
Mizoribine is a selective and competitive inhibitor of IMPDH,the rate-limiting enzyme in purine nucleotide biosynthesis21(Figure 1). Thus, restoration of the intracellular energy balanceand rescue of nephrin biogenesis could be mediated by a mechanismthat depends on the inhibition of IMPDH activity. First, studieswere conducted to determine whether mizoribine, at concentrationsused in our study, inhibited IMPDH activity. As illustratedin Figure 7, glucose starvation alone had no effect on IMPDHactivity in the 293-NPH cells. However, mizoribine (50 µg/mlfor 48 h) significantly decreased IMPDH activity in the glucose-starvedcells (Figure 7).
Figure 7. Summary of experiments performed to determine the effect of MZR on IMPDH enzyme activity. MZR is a selective and competitive inhibitor of IMPDH, the rate-limiting enzyme in purine nucleotide biosynthesis. For determination of the effect of MZR on IMPDH activity during ER stress, 293-NPH cells were cultured in 25 or 5.5 mM glucose medium in the absence or presence of MZR (50 µg/ml for 48 h). IMPDH activity was studied by the enzyme activity assay as described in the Concise Methods section. Glucose starvation alone had no effect on IMPDH activity. MZR significantly decreased IMPDH activity in the glucose-starved cells. *P < 0.01. Three experiments were performed per group.
If the inhibition of IMPDH activity were responsible, at leastin part, for the mizoribine effect on the intracellular energybalance and nephrin biogenesis, then other IMPDH inhibitorswould be expected to have a similar effect. To test this prediction,we examined mycophenolic acid (MPA), a metabolite of mycophenolatemofetil and a selective and noncompetitive IMPDH inhibitor thatis structurally unrelated to mizoribine.21,26–31 As illustratedin Figure 8, MPA attenuated ER stress, as demonstrated by decreasedexpression of GRP78, and restored nephrin biogenesis in theglucose-starved 293-NPH cells in a concentration-dependent manner.The pattern of nephrin expression in the glucose-starved cellsthat were treated with 25 µg/ml MPA was indistinguishablefrom that in cells that were grown in the standard glucose medium(Figure 8). Taken together, these data indicate that inhibitionof IMPDH activity may play a role in the restoration of theintracellular energy balance and rescue of nephrin biogenesis.
Figure 8. Representative Western blot experiment demonstrating the effect of MPA on nephrin biogenesis. MPA, a metabolite of mycophenolate mofetil, is a selective and noncompetitive IMPDH inhibitor that is structurally unrelated to MZR.21,32–37 Studies were conducted to determine the effect of MPA on nephrin biogenesis. 293-NPH cells were cultured in medium containing standard (25 mM) or low (5.5 mM) glucose. A 48-h treatment with MPA attenuated ER stress, as demonstrated by decreased expression of GRP78 and corrected defective nephrin biogenesis in the glucose-starved cells. An equal amount of total cellular proteins (10 µg) was separated by SDS-PAGE using a 7.5% gel. The experiment was repeated three times from separate cultures.
Our recently published data demonstrated that rescue of nephrinexpression in the plasma membrane by dexamethasone is mediatedby the glucocorticoid receptor (GR).13 Mizoribine could interactwith the GR through binding to 14-3-3, a protein known to associatewith and accelerate the GR function.32 If rescue of nephrinbiogenesis by mizoribine were mediated by the GR, then inhibitionof the GR would be expected to attenuate this mizoribine effect.As demonstrated in Figure 9, a 30-min pretreatment of the glucose-starved293-NPH cells with the GR antagonist RU486 did not interferewith the rescue of nephrin biogenesis by mizoribine. Taken together,these data indicate that mizoribine rescued nephrin biogenesisby a mechanism likely mediated by inhibition of IMPDH activityand independent of the GR.
Figure 9. Representative Western blot experiment performed to determine the effect of the GR inhibitor RU486 on the rescue of nephrin biogenesis by MZR. For determination of whether rescue of nephrin biogenesis by MZR was mediated by the GR, 293-NPH cells cultured in low-glucose (5.5 mM) medium were treatment with the GR antagonist RU486 for 30 min before incubation with MZR (50 µg/ml for 48 h), as described in the Concise Methods section. RU486 did not inhibit the MZR-mediated rescue of nephrin biogenesis. The experiment was repeated three times from separate cultures.
Podocytes Express IMPDH mRNA
These experiments suggest that mizoribine may act directly onIMPDH in podocytes; however, the expression of IMPDH has neverbeen confirmed in podocytes. Two isoforms of IMPDH derived fromtwo different genes, designated type I and II, have been cloned.33,34For detection of whether these genes indeed exist in the glomerulusand podocytes in vivo, mouse glomeruli were isolated by usingmagnet beads and further cultured to obtain primary podocytes.As shown in Figure 10A, the glomerulus was found to expressboth transcripts, in which IMPDH I was more abundant comparedwith the rest of the kidney. Moreover, primary podocytes werefound to express distinctly both mRNA (Figure 10B); however,the level of relative expression of IMPDH I was much less thanthat of IMPDH II. Further experimentation will be required toexplore this meaning. Because primary podocytes are difficultto be propagated, we used immortalized murine podocytes to determinewhether mizoribine could directly affect IMPDH of podocytes.Differentiated murine podocytes were cultured in RPMI-1640 mediumcontaining a standard amount (11 mM) of glucose, as describedpreviously.35 As illustrated in Figure 11, A and B, IMPDH Iand II mRNA were expressed in the podocytes, and their expressionwas not affected by mizoribine (50 µg/ml for 48 h). Next,studies were conducted to determine the effect of mizoribineon IMPDH mRNA during ER stress. For induction of ER stress,podocytes were cultured in the low-glucose (2.2 mM) medium.Glucose starvation alone upregulated IMPDH I and GRP78 mRNAexpression (Figure 11, A and C) but had no effect on the expressionof IMPDH II mRNA (Figure 11B). These data indicate that theexpression of IMPDH I and II may be differentially regulatedin podocytes. These results are in general agreement with thepreviously published data that in other tissues, the expressionof IMPDH I and II is differentially regulated.36 It is interestingthat the same mizoribine treatment (50 µg/ml for 48 h),which dramatically reduced the IMPDH enzyme activity (Figure 7),significantly increased the expression of both IMPDH mRNA inthe glucose-starved podocytes (Figure 11, A and B). Thus, expressionof the IMPDH mRNA may be increased in response to the inhibitionof the IMPDH enzyme activity in podocytes. Taken together, thesedata indicate that the podocyte isoforms of IMPDH may be regulatedby mizoribine.
Figure 10. Summary of reverse transcriptase–PCR experiments performed to determine the expression of IMPDH I and IMPDH II mRNA in isolated mouse glomeruli and primary mouse podocytes. Mouse glomeruli were isolated using magnet beads and further cultured to obtain primary podocytes. Total RNA (1 µg) was amplified using specific primer sets for IMPDH I and IMPDH II with the samples from isolated glomeruli (A, 1), the rest of the kidney (A, 2), and primary podocytes (B).
Figure 11. Summary of reverse transcriptase–PCR experiments performed to determine the expression of IMPDH I and IMPDH II mRNA in differentiated immortalized mouse podocytes. Differentiated, immortalized mouse podocytes were cultured in medium containing standard (11 mM) or low (2.2 mM) glucose in the absence or presence of MZR (50 µg/ml for 48 h). Total RNA (1 µg) was amplified using specific primer sets for IMPDH I (A), IMPDH II (B), and GRP78 (C) as described in the Concise Methods section. The mRNA were normalized to the -actin mRNA and expressed as fractions of the corresponding mRNA from cells that were cultured in standard glucose medium without MZR. In cells that were cultured in the standard amount of glucose, MZR did not affect the expression of IMPDH I or II. Glucose starvation alone increased expression of IMPDH I but had no effect on the expression of IMPDH II, suggesting that expression of IMPDH I and II may be differentially regulated in podocytes. MZR increased expression of both IMPDH mRNA in the glucose-starved podocytes. *P < 0.01; **P < 0.05. Five experiments were performed in each group.
Glomeruli Express the CNT2 Protein
These data that mizoribine affects IMPDH in podocytes suggestthat mizoribine should be transported into the cells; however,the transport mechanism is unknown. Mizoribine is a water-solublenucleoside analog structurally similar to ribavirin.31 Recentdata demonstrate that the intracellular uptake of ribavirinis mediated by nucleoside transporters (NT).37 Mammalian cellscontain two major NT families: The equilibrative, which mediatenucleoside transport in both directions depending on the nucleosideconcentration gradient across the plasma membrane, and the concentrative(CNT) ones, which mediate nucleoside transport independent ofthe nucleoside concentration gradient across the plasma membrane.38,39The CNT mRNA are expressed in the kidney, and the CNT isoform2 (CNT2) mRNA is predominantly expressed in the glomeruli.38,40However, the glomerular expression and distribution of the CNT2protein remains unknown. Therefore, studies were conducted toexamine the expression of CNT2 in the human glomerulus. To thisend, we generated a polyclonal antibody against human CNT2 andcharacterized its specificity by Western blot analysis usinga human glomerular sample. As shown in Figure 12A, anti-CNT2antibody detected a single approximately 65-kD band correspondingto the predicted molecular weight of CNT2. The immunizing peptideabolished this band, indicating that the anti-CNT2 antibodyspecifically recognized CNT2 protein in human glomeruli. Thepositive signal of CNT2 immunostaining was clearly found inthe glomerulus (Figure 12B), suggesting that human glomeruluscontains a transport mechanism that may be used for the intracellularuptake of mizoribine.
Figure 12. The expression of the CNT2 protein in the glomerulus. (A) Characterization of anti-CNT2 antibody. Western blot experiment demonstrating that the anti-CNT2 antibody detected a single 65-kD band corresponding to the predicted molecular weight of CNT2. The immunizing peptide abolished the band, indicating that the antibody specifically recognized CNT2. (B) Confocal microscopy image. Frozen sections of normal human kidney cortex were stained with an anti-nephrin or anti-CNT2 antibody and an appropriate secondary fluorescence antibody. In the glomeruli, CNT2 demonstrated primarily a glomerular epithelial pattern. Pretreatment of the anti-CNT2 antibody with the immunizing peptide failed to produce any immunostaining.
The major novel observation in this study is that ER stresswas induced during NS and that mizoribine, an agent with a knownantiproteinuric effect in humans and rats,20,41 reduced ER stress,rescued nephrin biogenesis, and restored the expression of mature,fully glycosylated nephrin in the plasma membrane. These effectsof mizoribine were mediated by salvage of the intracellularATP levels. Our data indicate that inhibition of IMPDH activityby mizoribine plays a role in restoration of the intracellularenergy balance and reduction of ER stress. Moreover, our dataindicate that mizoribine may act directly on podocytes and thatthese cells contain a transport mechanism that may be used forthe intracellular uptake of mizoribine.
Compelling evidence highlights the critical role of podocyteinjury in the pathogenesis of proteinuria and NS.42,43 Podocyteinjury, similar to the injury of other cells, is closely associatedwith ER stress.44–47 In this study, we demonstrated forthe first time the presence of ER stress in the rat glomeruliduring NS induced by PAN. Furthermore, we demonstrated thatnephrin distribution was altered from the normal capillary looppattern to a cytoplasmic pattern, at the time of increased expressionof GRP78 and heavy proteinuria during PAN-induced nephrosis.Similar redistribution of nephrin into the cytoplasm of podocyteduring minimal-change NS was also found in the previously publishedwork.48,49 Thus, ER stress may play a critical role in the pathomechanismof proteinuria in minimal-change NS. Although we did not examinein this study whether mizoribine exerts the antiproteinuriceffect on PAN nephrosis, Shibasaki et al.41 already demonstratedthe antiproteinuric effect of mizoribine.
To examine the mechanism of the antiproteinuric effect of mizoribinein vitro, we used a previously characterized model whereby nephrinbiogenesis was inhibited by the intracellular energy depletion.13In this model, glucose starvation of cultured cells leads todepletion of the intracellular ATP levels (1) by suppressingthe mitochondrial oxidative phosphorylation and (2) by inhibitingthe synthesis of substrates for the de novo pathway of purinenucleotide (ATP and GTP) biosynthesis50 (Figure 1). During intracellularenergy depletion, induction of ER stress was demonstrated byincreased expression of the stress-inducible chaperone GRP78,and the inhibition of nephrin biogenesis was confirmed by expressionof the immature, 155-kD nephrin (Figure 3). Under these experimentalconditions, mizoribine rescued nephrin biogenesis, as demonstratedby expression of the mature, fully glycosylated, 185-kD formand the partially glycosylated, 175-kD form of nephrin and bythe rescue of nephrin targeting to the plasma membrane.
Data presented in this article indicate that ER stress may havea more global effect on protein biogenesis in podocytes becauseER stress also inhibited the biogenesis of another N-glycoprotein,1-integrin. Studies beyond the scope of this article are neededto determine whether ER stress affects the biogenesis of additionalproteins, besides nephrin and 1-integrin.
Our data demonstrate that mizoribine may rescue protein biogenesisby salvaging the intracellular ATP levels. Several lines ofevidence indicate that recovery of intracellular ATP levelsin the glucose-starved cells by mizoribine was mediated by inhibitionof the activity of IMPDH, the rate-limiting enzyme in the purinenucleotide biosynthesis. First, at concentrations used in ourstudy, mizoribine inhibited IMPDH activity. Second, rescue ofintracellular ATP in the glucose-starved cells was also achievedby MPA, another IMPDH inhibitor structurally unrelated to mizoribine.31Moreover, the mechanism of the ATP rescue by mizoribine wasindependent of the GR and therefore different from that of theglucocorticoids.13 Because of the existing interconnectionsbetween the ATP and GTP biosynthetic pathways (Figure 1), itis conceivable that the mizoribine-mediated inhibition of IMPDHactivity could salvage the intracellular ATP levels by shuttlingthe IMPDH substrate (IMP), depleted during glucose starvation,from the GTP to the ATP biosynthetic arm. Additional studiesbeyond the scope of this work are required to identify the exactmechanism.
Our data suggest that the remission of proteinuria induced bymizoribine in NS may be mediated, at least in part, by a directeffect on podocytes because (1) glomeruli contain a transportsystem that may be used for the intracellular uptake of mizoribine,as demonstrated by the expression of CNT2, and (2) podocytesexpress the IMPDH transcripts, which are regulated by mizoribineduring ER stress. Moreover, the effects of mizoribine on theintracellular energy balance and nephrin biogenesis were observedat clinically attainable concentrations.51–53 A betterunderstanding of the pathomechanism of NS and the mechanismsby which antiproteinuric agents lead to remission in NS mayallow development of new therapies.
Cell Culture
HEK293 cells stably expressing full-length human nephrin (293-NPH)were cultured in DMEM (Invitrogen, Carlsbad, CA) containingstandard (25 mM) glucose as described previously.12,13 Conditionallyimmortalized murine podocytes and cultured human podocytes werea gift from Dr. Peter Mundel35 and Dr. Jean-Daniel Sraer,54respectively. Podocytes were cultured in RPMI-1640 medium (Invitrogen)containing standard (11 mM) glucose.
Antibodies and Reagents
For generation of anti-human CNT2 polyclonal antibody, a rabbitwas immunized with 0.5 mg of the immunizing peptide (KEVEPEGSKRTDAQ,amino acids 29 to 42) conjugated to the carrier protein keyholelimpet hemocyanin. The monoclonal (mAb2) and polyclonal (pAb2)anti-nephrin antibodies were previously described.12,13 Theother antibodies used were monoclonal anti-GRP78/KDEL (StressgenBiotechnologies, Victoria, BC, Canada), monoclonal anti–human1 integrin (Chemicon Int., Temecula, CA), monoclonal anti–-actin(Sigma-Aldrich, St. Louis, MO), goat anti-mouse and goat anti-rabbithorseradish peroxidase secondary antibodies (Dako, Kyoto, Japan),and Texas Red goat anti-rabbit and Alexa Fluor 488 goat anti-mouseand goat anti-rabbit secondary antibodies (Molecular Probes,Eugene, OR). Mifepristone (RU486), tunicamycin, and MPA werepurchased from Sigma-Aldrich. Mizoribine was supplied by AsahiChemical Industry Co. (Tokyo, Japan).
PAN Nephrosis and Western Blotting
For determination of the presence of ER stress during NS, PANnephrosis was established in 30 male Sprague-Dawley strain ratsas described previously.55 Heavy proteinuria developed 4 d afterinjection of PAN. The kidneys were excised, minced, and subjectedto the sequential sieving to isolate the glomeruli as describedpreviously.55 Isolated glomeruli were lysed in ice-cold lysisbuffer (150 mM NaCl, 1 mM Na3VO4, 1% Triton-X 100, 0.2% saponin,0.5% NP-40, 20 mM NaF, 1 mM EDTA, 2 µg/ml aprotinin, 2µg/ml leupeptin, Complete Mini, 100 µg/ml PMSF,and 10 mM Tris-HCl [pH 7.4]). Proteins were separated by 7.5%SDS-PAGE under reducing conditions and analyzed by Western blottingwith a mouse anti-GRP78 antibody or anti–-actin antibody,followed by an anti-mouse horseradish peroxidase as secondaryantibody. Immunecomplex was developed by using the Western LightningChemiluminescence reagent (PerkinElmer Life Sciences, Boston,MA).
Immunofluorescence and Confocal Microscopy
For determination of the expression of CNT2 in the human kidney,the frozen section of normal human kidney cortex was obtainedas described previously.56–58 The slides were subjectedto immunostaining with anti-CNT2 antibody (5 µg/ml) orthe anti-CNT2 antibody preabsorbed by the immunizing peptideovernight at 4°C. Positive signal was visualized with AlexaFluor 488 goat anti-rabbit antibody. For determination of theER stress marker and nephrin localization in the glomeruli ofPAN nephrosis, the frozen sections were immediately fixed withcold methanol for 10 min, and each cryostat section was reactedto anti-GRP78 antibody (5 µg/ml) and anti-nephrin antibody(pAb2, 5 µg/ml) for 60 min at room temperature. Positivesignal was visualized with Alexa Fluor 488 goat anti-mouse antibodyand Texas Red 488 goat anti-rabbit antibody. Coverslips weremounted with 1 mg/ml p-phenylenediamine (Wako, Osaka, Japan)in PBS/glycerol (1:1) to prevent laser bleaching. Images wereacquired with a confocal laser-scanning microscope equippedwith a Krypton/Argon laser (MRC1024; Bio-Rad Laboratories, Hercules,CA).
Determination of the Effects of Mizoribine on Nephrin Biogenesis during ER Stress Induced by Glucose Starvation
Subconfluent 293-NPH cells were cultured in DMEM containing25 mM glucose supplemented with the charcoal/dextran-treated10% FBS (Perbio, Rockford, IL) for 24 h as described previously.13Subsequently, cells were cultured in medium containing standard(25 mM) or low (5.5 mM) glucose supplemented with the charcoal/dextran-treated10% FBS in the absence or presence of various concentrationsof mizoribine for 48 h. For determination of whether the effectsof mizoribine were mediated by the GR, cells were preincubatedwith the 0.01 µM RU486, a GR antagonist,59 for 30 minbefore the mizoribine treatment. Cellular proteins were obtainedby ice-cold lysis buffer and subjected to Western blotting withnephrin, GRP78, and -actin.
In additional experiments, immortalized human podocytes werecultured in RPMI-1640 medium containing standard (11 mM) orlow (2.2 mM) glucose for 48 h in the absence or presence ofN-glycosylation inhibitor tunicamycin (5 µg/ml). Cellularlysates were subjected to Western blotting with 1-integrin.
IMPDH Activity Assay
IMPDH activity assay was carried essentially as described previously60with the following modifications. 293-NPH cells were culturedin DMEM containing 25 or 5.5 mM glucose in the absence or presenceof 50 µg/ml mizoribine for 48 h at 37°C. Cells werewashed with PBS at room temperature and incubated with ice-coldlysis buffer. After an incubation at 4°C for 1 h, cellswere homogenized and centrifuged at 600 x g for 10 min at 4°C.The supernatant was centrifuged at 10,000 x g for 10 min at4°C, and the resultant supernatant was further centrifugedat 10,000 x g for 20 min at 4°C. The reaction mixture consistedof 1 mM IMP, 0.5 mM NAD+, 100 mM KCl, and 40 mM K+-phosphatebuffer (pH 7.4). The specific IMPDH activity was expressed innmol/mg protein per h. The protein was determined using BCAProtein Assay Reagent (Pierce Chemical Co., Rockford, IL) withBSA as the standard.
Reverse Transcriptase–PCR
Glomeruli were isolated from adult ICR mice by means of themagnet beads perfusion method.61 Total RNA was obtained fromboth of these glomeruli and the rest of the kidney. Primarypodocyte culture was further performed using these isolatedglomeruli and subjected to the isolation of total RNA as previouslyreported.62 Differentiated immortalized mouse podocytes werecultured in RPMI-1640 medium containing either 11 or 2.2 mMglucose in the absence or presence of mizoribine. The reversetranscriptase–PCR for mouse inosine 5'-monophosphate dehydrogenase-I(IMPDH-I) was carried out using the sense primer 5'-GTGGGTGATGTTCTGGAGGC-3'and the antisense primer 5'-GCAGATGGAACCACAGCCCA-3'. Mouse IMPDH-IIwas carried out using the sense primer 5'-CCCCATTGGTTTCCTCACCC-3'and the antisense primer 5'-ACATCCACACCAGCAAGGGC-3'. Mouse GRP78(Accession No. D78645) was carried out using the sense primer5'-GAAGTTCACTGTGGTGGCGG-3' and the antisense primer 5'-ATCTCCATTAGTGGCCACCC-3'.One microgram of total RNA was amplified under the followingconditions: 25 cycles at 94°C for 1 min, 58°C for 1min, and 72°C for 2 min. The PCR products were analyzedby electrophoresis on 1% agarose gels, followed by the directsequence analysis to confirm its correct product.
Determination of ATP Level by HPLC
293-NPH cells were harvested with a rubber policeman and pelletedby centrifugation at 600 x g for 10 min at 4°C. The cellularpellet was suspended in perchloric acid (final at approximately0.2 N) and thoroughly disrupted with a Hitachi HG30 homogenizer(Hitachi Co., Tokyo, Japan) at the maximal speed for 40 s. Thehomogenate was centrifuged at 10,000 x g for 10 min at 4°C.The resultant supernatant was neutralized with 2 M KHCO3 (0.4vol/vol), left on ice for 10 min, and centrifuged at 10,000x g for 15 min. The resultant supernatant was filtered withthe Millex-LH filter (Millipore Japan, Tokyo, Japan) and storedat –20°C until analysis. HPLC was performed usinga Supelcosil LC-18T column equipped with the Shimadzu LC-10ADchromatography system (Shimadzu Corp., Kyoto, Japan). 8-BromoATP was added to the sample as the internal standard. Nucleotideswere identified from their absorption spectra by a diode arrayspectrophotometer (Shimadzu SPD-M10AVP, Kyoto, Japan).
Flow Cytometry
Studies were conducted to determine the effects of mizoribineon the plasma membrane expression of nephrin by flow cytometry,as described previously.13 Briefly, 293-NPH cells were culturedin DMEM containing 25 or 5.5 mM glucose in the presence or absenceof 50 µg/ml mizoribine for 48 h. After trypsinization,cells were fixed with 3% formaldehyde, washed with PBS, andblocked with 3% BSA. Subsequently, cells were incubated witha monoclonal anti-nephrin antibody (0.5 µg/ml) at 4°Cfor 60 min, washed, and incubated with Alexa Fluor 488 goatanti-mouse secondary antibody. Fluorescence intensities wereanalyzed on a FACScan flow cytometer (Becton-Dickinson, MountainView, CA) using 488 nm.
Statistical Analysis
Statistical analysis of the data was performed using StatViewsoftware (Stat Corp., College Station, TX). Means were comparedusing a two-tailed t test. P < 0.05 was considered significant.Data are expressed as means ± SE.
This work was supported by a grant from the Promotion and MutualAid Corporation for Private Schools of Japan and from NovartisPharma K.K. It was also partly supported by a grant from Morinaga.
We thank Yuko Kimura for excellent technical assistance.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Introduction
RESULTS
1-integrin biogenesis. 
