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¹ Department of Pediatrics, Peking University First Hospital, Beijing 100034, China
² Department of Pediatrics, Peking University Third Hospital, Beijing 100083, China
³ Department of Pediatrics, Fuzhou General Hospital, Fuzhou 350025, China

	Abstract

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A lot of mutations of podocin, a key protein of podocyte slit diaphragm (SD), have been found both in hereditary and sporadic focal segmental glomeruloscleorosis (FSGS). Nevertheless, the mechanisms of podocyte injury induced by mutant podocins are still unclear. A compound heterozygous podocin mutation was identified in our FSGS patient, leading to a truncated (podocin ^V165X) and a missense mutant protein (podocin ^R168H), respectively. Here, it was explored whether and how both mutant podocins induce podocyte injury in the in vitro cultured podocyte cell line. Our results showed that podocin ^R168H induced more significant podocyte apoptosis and expression changes in more podocyte molecules than podocin ^V165X. Podocyte injury caused by the normal localized podocin^V165X was effectively inhibited by TRPC6 knockdown. The abnormal retention of podocin^R168H in endoplasmic reticulum (ER) resulted in the mis-localizations of other critical SD molecules nephrin, CD2AP and TRPC6, and significantly up-regulated ER stress markers Bip/grp78, p-PERK and caspase-12. These results implicated that podocin ^R168H and podocin ^V165X induced different degrees of podocyte injury, which might be resulted from different molecular mechanisms. Our findings provided some possible clues for further exploring the pharmacological targets to the proteinuria induced by different mutant podocins.

	Introduction

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Focal segmental glomerulosclerosis (FSGS) is one of the most common pathologic types of glomerulonephritis both in children and adult, and often leads to end-stage renal failure (Srivastava et al. 1999). NPHS2, encoding a hairpin scaffold protein podocin with 383 amino acids, has been demonstrated as a causative gene for some familial forms of FSGS (Boute et al. 2000). Podocin, localizes specifically to the insertion of the slit diaphragm (SD) to the podocyte cytoplasm (Boute et al. 2000). Data suggested that podocin might act as an ‘organizer’ to maintain its structural and functional integrity (Schwarz et al. 2001; Roselli et al. 2002), by interacting with other important SD molecules nephrin, CD2-associated protein (CD2AP) and the transient receptor potential C channel 6 (TRPC6) (Winn et al. 2005).

To date, a lot of podocin mutations have been reported both in hereditary and sporadic FSGS (Caridi et al. 2005). Nevertheless, the molecular mechanisms of podocyte injuries and proteinuria development induced by different mutant podocins are still unclear. A compound heterozygous NPHS2 mutations were identified for the first time in a Chinese kindred with FSGS, which resulted in a C-terminal truncated podocin at the 165th valine (podocin^V165X) and a substitution of histidine for the 168th arginine (podocin^R168H), respectively (Yu et al. 2004). In this study, it was investigated whether and how the two different mutant podocins induce podocyte injury in the in vitro cultured podocyte. We explored the distribution change in both mutant podocins, and their effects on podocyte apoptosis, cytoskeleton arrangement and the expressions of other important SD molecules nephrin, CD2AP and TRPC6. TRPC6, the first identified podocyte ion channel related to proteinuria occurrence, was thought to be Ca²⁺ permeable. Some mutations of TRPC6 induced a significant increased and prolonged Ca²⁺ influx (Reiser et al. 2005). The expressions of TRPC6 increased in some acquired proteinuric renal diseases, and proteinuria was developed in TRPC6 over-expressed transgenic mice (Moller et al. 2007). These findings emphasized the important roles of the cytosolic free Ca²⁺ induced by TRPC6 in maintaining the normal podcocyte biology, and shed new light on the pathogenesis of FSGS (Kriz 2005; Hsu et al. 2007). Importantly, it has been proved that TRPC6 was tightly interacted with podocin (Winn et al. 2005; Huber et al. 2007). Therefore, the effects of both different podocin mutations were also investigated on the cytosolic free Ca²⁺ by the specific knockdown of TRPC6.

Our results showed that podocin^V165X and podocin^wild caused similar podocyte injury, and that podocin^R168H induced more significant podocyte injury and expression changes of more podocyte molecules. In addition, podocin^V165X and podocin^R168H led to the transient and persistent activation of extracellular signal-regulated kinase (ERK) pathway, respectively.

	Results

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Effects of wild and mutant podocins on podocyte injury

When compared with the control (Con)- and blank vector (BV)-transfected cells, the percentages of podocyte injury increased evidently (podocin^wild 13.91 ± 2.72, podocin^V165X 15.4 ± 3.23 vs Con 5.32 ± 1.17, BV 4.24 ± 1.22; P < 0.05) in podocin^wild- and podocin^V165X-transfected podocytes. Podocin^R168H caused more significant podocyte injury (podocin^R168H 23.24 ± 1.36 vs podocin^wild 13.91 ± 2.72, podocin^V165X 15.4 ± 3.23; P < 0.05) than podocin^V165X and podocin^wild.

The knockdown of TRPC6 (KdTrp) alone did not show any effects on podocyte injury. TRPC6 knockdown effectively inhibited podocyte injury induced by podocin^wild and podocin^V165X (podocin^wild + KdTrp: 6.50 ± 1.68, podocin^V165X + KdTrp: 8.28 ± 1.41 vs podocin^wild: 13.91 ± 2.72, podocin^V165X: 15.4 ± 3.23; P < 0.05). Nevertheless, TRPC6 knockdown did not inhibit podocyte injury induced by podocin^R168H (podocin^R168H + KdTrp: 21.06 ± 2.47 vs podocin^R168H: 23.24 ± 1.36; P > 0.05) (Fig. 1).

View larger version (17K): [in this window] [in a new window]   Figure 1  Effects of wild and mutant podocins on podocyte injuries. Annexin V-FITC was used to assay podocyte apoptosis using flow cytometry. When compared with the cells transfected with blank vector (BV) and the un-transfected cells (Con), the percentages of the injured podocytes transfected with podocinwild and podocinV165X were significantly increased, which was effectively inhibited by the TRPC6 knockdown (KdTrp). When compared with the podocinwild and podocinV165X cells, podocyte injuries increased much more significantly in the cells transfected with podocinR168H, which was not inhibited by the TRPC6 knockdown. TRPC6 knockdown alone did not show any effects on podocyte injuries. Data are represented as mean ± SD. n = 4 independent experiments. *P < 0.05 vs Con and BV; #P < 0.05 vs podocinwild; P < 0.05 vs podocinV165X.

To reveal the effects of wild and mutant podocins on the arrangement of podocyte cytoskeleton, actin filaments were directly labeled with TRITC-conjugated phalloidin. Podocytes were co-transfected with pEGFP-N1 and wild or mutant podocins, respectively. Podocytes transfected with pEGFP-N1 alone displayed a clear and filamentous actin cytoskeleton (Fig. 2b). Similar to podocin^wild (Fig. 2d), podocin^V165X changed the cytoskeletal arrangement and showed a cortical actin filament, presenting with a loss in cytoplasm and main localization on the cell membrane (Fig. 2f). Podocin^R168H induced a marked loss and aggregation of actin filaments in podocytes (Fig. 2h).

View larger version (42K): [in this window] [in a new window]   Figure 2  Effects of wild and mutant podocins on the arrangement of podocyte cytoskeleton. Podocytes transfected with pEGFP-N1 alone, or co-transfected with pEGFP-N1 and wild or mutant podocins, were indicated in green color (a,c,e,g). The actin filaments were labeled with TRITC-pholloidin and shown in red color (b,d,f,h). Podocytes transfected with pEGFP-N1 alone displayed a normally and clearly filamentous actin cytoskeleton (b). A cortical distribution of the actin filaments was observed in the podocytes transfected with the wild podocin (d) or V165X mutant podocin (f). A significant loss and aggregation of the actin filaments was shown in the podocytes transfected with the R168H mutant podocin (h). Bar: 50 µm.

The overexpressed wild and mutant podocins were tagged with red fluorescence protein (RFP). RFP-podocin^wildwas localized on the cell membrane in a linear pattern. RFP-podocin^V165X was partially targeted to the cell membrane. RFP-podocin^R168H was only localized around the nuclei in a granular pattern. As wild podocin, both mutant podocins still co-localized well with TRPC6, nephrin and CD2AP (Fig. 3).

View larger version (44K): [in this window] [in a new window]   Figure 3  Wild and mutant podocins colocalized with TRPC6, nephrin and CD2AP. The nuclei were stained in blue color (a1–a3, e1–e3 and i1–i3). The podocytes transfected with the pDsRed2_N1-podocinwild, podocinV165X or podocinR168H were indicated in red color (b1–b3, f1–f3 and j1–j3). Wild and mutant podocins were tagged with the red fluorescence protein (RFP). TRPC6, nephrin and CD2AP were stained in green color in (c1–c3), (g1–g3) and (k1–k3), respectively. (d1–d3), (h1–h3) and (l1–l3) were the merged photos of (a–c), (e–g) and (i–k), respectively. RFP-wild podocin was localized on the cell membrane in a linear pattern (b1–b3). RFP-V165X was partially targeted to the cell membrane (f1–f3), in which the asterisk indicated the loss of V165X. RFP-R168H was only localized around the nuclei in a granular pattern indicated with pound (j1–j3). TRPC6, nephrin and CD2AP completely co-localized with wild podocin in the cell membrane, which was indicated with yellow color in (d1–d3), respectively. TRPC6, nephrin and CD2AP partially co-localized with V165X in the cell membrane, and the arrows in (h1–h3) indicated their loss in membrane. TRPC6, nephrin and CD2AP co-localized with R168H around the nuclei in a granular pattern and were marked with arrowhead in (l1–l3). Bar: 50 µm.

HEK293 cells were co-transfected with FLAG-podocin^wild, podocin^V165X or podocin^R168H and TRPC6 plasmid, respectively. TRPC6 and the FLAG-tagged podocin were detected in the lysates by using anti-TRPC6, anti-podocin and anti-FLAG antibody, respectively. Immunoprecipitation assay revealed that as the podocin^wild, both podocin^V165X and podocin^R168H still co-immunoprecipitated with TRPC6 (Fig. 4a).

View larger version (25K): [in this window] [in a new window]   Figure 4  Interaction of mutant podocin and TRPC6 and their effects on the protein expressions of nephrin, CD2AP and TRPC6. (a) In the co-transfected HEK293 cells, TRPC6- and FLAG-tagged fusion podocin were detected in total cell lysates using Western blot (lower panel). FLAG-tagged fusion podocin was immunoprecipitated, eluted and visualized with anti-TRPC6 antibody. Co-immunoprecipitated TRPC6 was detected in eluted fractions (upper panel) but not in the immunoprecipitation supernatants (middle panel). Both mutant podocins and the wild-type podocin immunoprecipitated with TRPC6. (b) Wild and mutant podocins tagged with the red fluorescence protein (RFP) were used to transfect the in vitro cultured podocytes. RFP-wild podocin, RFP-V165X and RFP-R168H were detected with the size of 67, 47 and 67 kDa, respectively. Nephrin, CD2AP, TRPC6 and the endogenous podocin (Endo-pod) were detected with the size of 180, 80, 106 and 42 kDa, respectively. (c) Compared with the cells transfected with blank vector (BV) and the un-transfected cells (Con), TRPC6 increased evidently in the podocytes transfected with the wild podocin (podocinwild), and CD2AP increased markedly in the cells transfected with V165X (podocinV165X). The three slit diaphragm molecules, nephrin, CD2AP and TRPC6, all increased significantly in the podocytes transfected with R168H (podocinR168H). TRPC6 knockdown (KdTrp) did not affect the expressions of nephrin, CD2AP and TRPC6. Expressions of the endogenous podocin did not change in each group. Data are represented as mean ± SD (n = 4 independent experiments). *,#,P < 0.05 vs Con and BV.

In the podocytes transfected with pDsRed2_N1-podocin^wild, podocin^V165X and podocin^R168H, podocin^wild, podocin^V165X and podocin^R168H were detected with sizes of 67, 47 and 67 kDa, respectively. The endogenous podocin, TRPC6, nephrin and CD2AP were also detected with sizes of 42, 106, 180 and 80 kDa, respectively (Fig. 4b).

When compared with the Con- and BV-transfected cells, podocin^wild evidently increased TRPC6 expression (podocin^wild: 0.460 ± 0.089 vs Con: 0.283 ± 0.090 and BV: 0.300 ± 0.085; P < 0.05), whereas podocin^V165X increased CD2AP protein markedly (podocin^V165X: 0.993 ± 0.095 vs Con: 0.648 ± 0.101 and BV: 0.698 ± 0.066, P < 0.05). Podocin^R168H significantly increased the expressions of TRPC6 (podocin^R168H: 0.518 ± 0.119 vs Con: 0.283 ± 0.090 and BV: 0.300 ± 0.085; P < 0.05), CD2AP (podocin^R168H: 1.095 ± 0.091 vs Con: 0.648 ± 0.101 and BV: 0.698 ± 0.066; P < 0.05) and nephrin (podocin^R168H: 1.380 ± 0.256 vs Con: 0.845 ± 0.100 and BV: 0.908 ± 0.142; P < 0.05) compared with Con and BV cells (Fig. 4c).

The knockdown of TRPC6 (KdTrp: 0.078 ± 0.017; podocin^wild + KdTrp: 0.075 ± 0.034; podocin^V165X + KdTrp: 0.085 ± 0.037; podocin^R168H + KdTrp: 0.090 ± 0.022) was successfully accomplished (P < 0.05) compared with the Con- (0.283 ± 0.090), BV- (0.300 ± 0.085) and the Silencer^® Negative Control siRNA (Ambion, Austin, TX, USA) (siCon: 0.275 ± 0.034)-transfected cells. The knockdown of TRPC6 alone showed no effects on the expressions of nephrin and CD2AP compared with the Con, BV and siCon. The expressions of the endogenous podocin did not change in each group (Fig. 4c).

Effects of wild and mutant podocins on the cytosolic free Ca²⁺

Forty-eight hours after transfection, the average fluorescence intensity of the cytosolic free Ca²⁺ ([Ca²⁺]Fi) of individual cells was quantitatively measured by using the fluorescent indicator fluo-3AM and FLX800 spectrophotofluorometer.

Podocin^wild, podocin^V165X and podocin^R168H increased obviously (P < 0.05) the basal [Ca²⁺]Fi compared with the Con- and BV-transfected cells. Notably, the increase in the basal [Ca²⁺]Fi in podocin^R168H podocytes was much more significant (P < 0.05) compared with the podocin^V165X cells. After the stimulation of 100 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG) followed by 2 mM Ca²⁺, podocin^wild and podocin^V165X induced a rapid and marked (P < 0.05) increment in the [Ca²⁺]Fi, which was significantly inhibited (P < 0.05) by the knockdown of TRPC6. Compared with podocin^wild and podocin^V165X, podocin^R168H caused much more significant (P < 0.05) increment in the [Ca²⁺]Fi, which was not inhibited by the knockdown of TRPC6. TRPC6 knockdown alone showed no effects on the [Ca²⁺]Fi (Fig. 5).

View larger version (23K): [in this window] [in a new window]   Figure 5  Effects of wild and mutant podocins on the cytosolic free Ca2+. The fluorescence intensity of the cytosolic free Ca2+ ([Ca2+]Fi) labeled with fluo-3 AM was detected using FLX 800 spectrophotofluorometer. Compared with the un-transfected cells (Con) and the cells transfected with blank vector (BV), the basal [Ca2+]Fi increased obviously in the podocytes transfected with the wild podocin (podocinwild), V165X (podocinV165X) or R168H (podocinR168H) mutant podocin. The basal [Ca2+]Fi in the podocinR168H podocytes increased much more significantly compared with the podocinV165X cells. After the stimulation of 100 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG) and 2 mM CaCl2, podocinwild and podocinV165X induced a rapid and marked increment in the [Ca2+]Fi, which was effectively inhibited by TRPC6 knockdown (KdTrp). Compared with podocinwild and podocinV165X, podocinR168H caused a much more significant increment in the [Ca2+]Fi, which was not inhibited by TRPC6 knockdown. The [Ca2+]Fi was not affected by TRPC6 knockdown alone. Data are represented as mean ± SD. n = 4 independent experiments. *P < 0.05 vs Con and BV; #P < 0.05 vs podocinV165X; P < 0.05 vs podocinwild.

The distribution of podocin^R168H changed obviously and was localized around nuclei in a granular pattern (Fig. 3j1–j3). It was further verified that podocin^R168H completely co-localized with the ER marker calreticulin (Fig. 6a–c). Compared with the Con and BV cells, expressions of the ER stress markers including Bip/grp78, p-PERK and caspase-12 increased significantly (P < 0.05) only in podocin^R168H podocytes (Fig. 6d).

View larger version (49K): [in this window] [in a new window]   Figure 6  Activation of endoplasmic reticulum (ER) stress pathway induced by the retention of R168H mutant. (a) The localization of the ER was labeled with anti-calreticulin, and indicated with arrowhead. (b) R168H tagged with red fluorescence protein (RFP) was also indicated with arrowhead. (c) The merged yellow color from (a) and (b) demonstrated that R168H was retained in the ER, which was indicated with arrow. (d) The ER stress markers Bip/grp78, p-PERK and caspase-12 were detected in the un-transfected podocytes (Con), the cells transfected with blank vector (BV), podocinwild, podocinV165X and podocinR168H, respectively. (e) The densitometric quantitation results showed that Bip/grp78, p-PERK and caspase-12 increased significantly in the podocinR168H podocytes. Data are represented as mean ± SD. n = 4 independent experiments. *P < 0.05 vs Con and BV. Bar: 100 µm.

The family of mitogen-activated protein kinase (MAPK) consists of ERK, c-Jun N-terminal kinase (JNK) and p38 MAPK. The serine–threonine kinase AKT is a major downstream effector of phosphoinositide 3-OH kinase (PI3K). The critical molecules of PI3K/AKT and MAPK pathway were screened at different time points in the podocin mutant podocytes stimulated by angiotensin II (AngII).

Our results showed that ERK1/2 was obviously activated in each group after AngII stimulation. In the Con and BV cells, as well as podocin^wild and podocin^V165X podocytes, the phosphorylated ERK1/2 increased significantly (P < 0.05) at 1 min. In the podocin^R168H podocytes, more significant activation of ERK1/2 was detected (P < 0.05) at 1 min, and persisted to 10 min compared with other groups (Fig. 7).

View larger version (28K): [in this window] [in a new window]   Figure 7  Activation of ERK1/2 in wild and mutant podocin-transfected podocytes induced by angiotensin II. (a) At 0, 1, 5 and 10 min after angiotensin II stimulation, the phosphorylated ERK1/2 and the total ERK1/2 were detected in the un-transfected podocytes (Con), the cells transfected with blank vector (BV), podocinwild, podocinV165X and podocinR168H, respectively. (b) The densitometric quantitation results showed that ERK1/2 was obviously activated in each group. PodocinR168H induced a persistent and more significant activation of ERK1/2 compared with other groups. Data are represented as mean ± SD. n = 4 independent experiments. *P < 0.05 vs the corresponding Con, BV, podocinwild and podocinV165X at 0'; #P < 0.05 vs podocinR168H at 0'; P < 0.05 vs other groups at 1', 5' and 10', respectively.

	Discussion

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The distribution change in both different mutant podocins was also observed. Podocin^wild was localized on the plasma membrane in a linear pattern, whereas podocin^V165X was partially targeted to the cell membrane. The distribution of podocin^R168H changed more evidently, which was completely localized around nuclei in a granular manner. Further co-localization analysis revealed that nephrin, CD2AP and TRPC6 co-localized with podocin^V165X on the cell membrane, and still co-localized with podocin^R168H around nuclei. It was also reported that some mutant podocins also showed an abnormal subcellular localization and failed to recruit nephrin to the plasma membrane in HEK293 cells (Huber et al. 2004; Nishibori et al. 2004; Roselli et al. 2004).

The effects of the two different mutant podocins on the protein expressions of other important SD molecules were further analyzed in this study. Podocin^wild and podocin^V165X obviously increased the expressions of TRPC6 and CD2AP, respectively. In addition to TRPC6 and CD2AP, podocin^R168H significantly increased nephrin expression. Therefore, our results elucidated that podocin^R168H induced expression changes of more SD molecules than podocin^wild and podocin^V165X. These findings also showed that both mutant podocins affected the expressions of the ion channel TRPC6. In HEK293 cells, our studies further displayed that both podocin^V165X and podocin^R168H still co-immunoprecipitated with TRPC6, implying that TRPC6 was possibly involved into podocyte injury induced by mutant podocins.

Recent studies have shown that in podocytes the cytosolic free Ca²⁺ level was tightly regulated by TRPC6, which can be excited by the exogenous application of OAG (Walz 2005; Goel et al. 2006; Winn et al. 2006). The enhanced increment in Ca²⁺ activates the Ca²⁺-dependent phosphatase calcineurin linked to the induction of apoptosis through de-phosphorylation of the protein BAD (Wang et al. 1999). Here, the effects of both mutant podocins were explored on the [Ca²⁺]Fi. We found that the basal [Ca²⁺]Fi increased obviously both in the wild-type podocin and in the mutant podocin-expressed podocytes. After the stimulation of OAG and Ca²⁺, podoicn^wild and podocin^V165X induced a rapid and marked increment in the [Ca²⁺]Fi, which was effectively inhibited by the specific knockdown of TRPC6. In Xenopus oocytes, podocin^wild augmented the effects of OAG on the conductance of TRPC6 channels (Huber et al. 2006, 2007). It was also observed that podocyte apoptosis induced by podocin^wild and podocin^V165X was obviously ameliorated by the knockdown of TRPC6. However, the precise relationship and molecular pathway among the cytosolic Ca²⁺ and TRPC6 as well as podocyte injury induced by podocin^wild and podocin^V165X should be further explored and demonstrated.

Our results showed that podocin^R168H caused a more significant increment in the [Ca²⁺]Fi both at basal and after the stimulation of OAG and Ca²⁺ than podocin^wild and podocin^V165X. Although podocin^R168H markedly increased the expression of TRPC6 protein, the knockdown of TRPC6 did not inhibit the Ca²⁺ increment and podocyte injury induced by podocin^R168H, which might be associated with the mis-localization of TRPC6. It was reported that the mutant TRPC6 targeted only to the cell membrane could induce a large Ca²⁺ influx in HEK293 cells (Winn et al. 2005). In addition, the mutant podocins, podocin^P120S and podocin^C120/160A, could not enhance the currents induced by TRPC6 in Xenopus oocytes (Huber et al. 2006). These findings implied that there might be another molecular pathway involved into podocyte injury induced by podocin^R168H. We found that the distribution of podocin^R168H changed obviously from the normal cell membrane to the cytoplasm, and co-localized with the endoplasmic reticulum (ER) marker calreticulin, suggesting that R168H mutant podocin might be retained in the ER. Some studies found that most of mutant podocins exhibited an accumulation in instead of a plasma membrane distribution (Ohashi et al. 2003; Roselli et al. 2004). The retention of misfolding or unfolding protein in the ER could cause podocyte injury by activating the ER stress pathway (Bijian & Cybulsky 2005; Cybulsky et al. 2005; Kitamura 2008). In this study, the ER stress markers were further detected. Bip/grp78, as chaperones for the exocytosis from ER, forms a complex with defective proteins and targets them for degradation. Accumulation of misfolded proteins in the ER activates PKR-like ER kinase (PERK), which phosphorylates the eukaryotic translation initiation factor-2 (eIF2), reducing the initiation codon recognition and thus decreasing the general rate of translation. Substantial or prolonged ER stress leads to apoptosis via induction of specific genes, e.g. C/BEP homologous protein-10 (CHOP), and or activation of caspase-12 (Ron 2002; Xu et al. 2005). Our results showed that only podocin^R168H significantly increased the expressions of Bip/grp78, p-PERK and caspase-12. Nevertheless, the relationship between ER stress and podocyte injury induced by podocin^R168H should be investigated in detail.

Accumulating lines of evidence suggest that the SD is a signaling platform in podocyte (Huber & Benzing 2005) and that PI3K/AKT and MAPK pathways might be associated with podocyte injury (Huber et al. 2003; Koshikawa et al. 2005). It has been proved that angiotensin II (AngII) can induce podocyte injury and play an important role in the pathophysiologic process of proteinuric renal diseases (Hunt et al. 2005; Suzuki et al. 2007). Here, we found that podocin^V165X and podocin^wild induced a transient ERK activation, whereas podocin^R168H caused a persistent and more significant ERK activation. These results implied that the ERK pathway might be, at least partially, involved into podocyte injury induced by mutant podocins.

Taken together, podocin^V165X and podocin^wild presented with some similar behaviors. Podocin^R168H and podocin^V165X induced different degrees of podocyte injury, which might be resulted from different molecular mechanisms. These findings provided some possible clues for further exploring the pharmacological corrections of the defective protein processing.

	Experimental procedures

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Plasmids

Total RNA from the human kidney tissue, which was obtained from an informed-consent patient with renal cancer undergoing nephrectomy, was extracted with Trizol (Invitrogen, Carlsbad, CA, USA) and reversed with reverse transcription (RT) kit (Promega, Madison, WI, USA). The whole coding sequences of NPHS2 (NM_014625 [GenBank] ) were generated using a polymerase chain reaction (PCR) kit (Promega) using the following primers with the underlined restriction sites BamHI and XhoI (forward: 5'-ACGTGGATCCGCTCTGAG GATGGAGAGGAG-3'; reverse: 5'-ACGTCTCGAGTCACATTATGCCCCATCCTT-3'), and cloned into pBluescript SK⁺ vector (Stratagene, La Jolla, CA, USA). A point mutation at 467_468insT (V165X) or 503G>A (R168H) was generated using a site-directed mutagenesis kit (TaKaRa, Shiga, Japan).

pcDNA3.0 (Invitrogen), pDsRed2_N1 (Clontech, Mountain View, CA, USA) and pCMV-Tag2B (Stratagene) with podocin^wild, podocin^V165X or podocin^R168H were generated using standard cloning procedures, respectively. All constructs were verified by sequencing and extracted with the EndoFree^® Plasmid Max kit (Qiagen, Hilden, Germany). pReceiver-M29 with the whole coding sequences of TRPC6 was obtained from FulenGen company (Guangzhou, China). pEGFP-N1 (Clontech) was used as the control to indicate the transfected cells when evaluating the effects of pcDNA3.0-podocin^wild, podocin^V165X or podocin^R168H on the arrangement of podocyte cytoskeleton.

Antibodies

The following primary antibodies were used: rabbit polyclonal antibody against mouse nephrin (Prof. Karl Tryggvason, Sweden), rabbit polyclonal antibody against t-human podocin (Prof. Corinne Antignac, France), rabbit polyclonal antibody against human CD2AP (Santa Cruz, Santa Cruz, CA, USA), rabbit polyclonal antibody against mouse TRPC6 (Chemicon, Temecula, CA, USA), and mouse monoclonal antibody against GAPDH (Chemicon).

Mouse anti-calreticulin monoclonal antibody (Stressgen, Ann Arbor, Michigan, USA) was used to label the localization of the ER. Rabbit anti-phospho-PERK monoclonal antibody (Cell Signaling, Danvers, MA, USA), rabbit anti-Bip/grp78 polyclonal antibody (Cell Signaling) and rabbit anti-caspase12 polyclonal antibody (Biovision, Mountain View, CA, USA) were used to evaluate the activation of the ER stress.

Mouse monoclonal antibody against phospho-ERK1/2 (Kangcheng, Shanghai, China), and rabbit polyclonal antibody against ERK1/2 (Kangcheng) was used to detect the activation of the ERK/MAPK pathway.

TRPC6 knockdown

RNA interference (RNAi) was used to evaluate the function of TRPC6 in podocyte injury induced by mutant podocins. Three pairs of the short interference RNA (siRNA), specifically targeted to TRPC6, were obtained from Ambion (Austin, TX, USA) (siRNA ID: 188581, 188582 and 71986). Preliminary experiments demonstrated that the most effective for the knockdown of TRPC6 is the siRNA 188581: 5'-GGUUAUGUACGGAUUGUGGtt-3' (sense) and 5'-CCACAAUCCGUACAUAACCtt-3' (antisense). Silencer^® Negative Control siRNAs were also obtained from Ambion, which have no significant sequence similarity to mouse, rat or human transcript sequences and have no significant impact on cell proliferation, apoptosis or cell morphology in multiple cell lines.

Podocyte culture

Conditionally immortalized mouse podocyte clone (a kindly gift from Prof. Peter Mundel, USA) was cultured at 33 °C in RPMI-1640 containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), 100 U/mL Penicillin/Streptomycin and 10 U/mL of mouse recombinant -interferon (PEPRO Tech, London, UK), then shifted to 37 °C for differentiation by removal of -interferon (Mundel et al. 1997). When they grew to about 80% confluence, the podocytes were transfected with the verified recombinant plasmids by using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions, and harvested after 48 hours.

For the knockdown of TRPC6, TRPC6 siRNA was transfected into podocytes with the final concentration 30 nM using siPORT^TM NeoFX^TM (Ambion). To evaluate the indispensable role of TRPC6 in podocyte injury, podocytes were transfected with TRPC6 siRNA for 8 h, and then followed by the transfection with wild or mutant podocins, respectively.

To detect the signal pathway involved in podocyte injury, podocytes transfected with wild or mutant podocins for 48 h were treated with 10^–7 M angiotensin II (Sigma, St Louis, MO, USA) and then harvested at 0, 1, 5 and 10 min, respectively.

Flow cytometry

Annexin V-FITC Apoptosis Detection Kit was obtained from BD Biosciences Company (San Jose, CA, USA). The percentages of the apoptotic and died podocytes were applied to evaluate podocyte injury. Briefly, podocytes transfected with pcDNA3.0-podocin^wild, podocin^V165X or podocin^R168H were harvested and washed two times with pre-cold phosphate-buffered saline (PBS). Cells (1 x 10⁵) were resuspended in 1 µg/mL FITC-Annexin V for 30 min at 4 °C followed by 5 µL of 50 µg/mL PI immediately prior to detection with flow cytometry (FACScan (BD Biosciences)).

Immunofluorescence staining

Cells transfected with pDsRed2_N1-podocin^wild, podocin^V165X or podocin^R168H were fixed with 4% paraformaldehyde, then permeabilized and blocked with 0.3% Triton X-100 and 5% bovine serum albumin. The primary antibody, rabbit anti-nephrin, CD2AP or TRPC6 antibody, and mouse anti-calreticulin monoclonal antibody was applied for overnight at 4 °C. FITC-conjugated goat anti-rabbit or mouse IgG and the nuclei dye Hoechst were used for 45 min at room temperature. TRITC-phalloidin (Sigma-Aldrich) was directly used to label the actin filaments of the podocytes co-transfected with pEGFP-N1 and pcDNA3.0-podocin^wild, podocin^V165X or podocin^R168H. Finally, the coverslips were mounted and images were taken by using a Bio-Rad Radiance 2100 TM confocal laser-scanning system attached to a Nikon TE 300 microscope (Nikon, Tokyo, Japan).

Western blot

Cells transfected with pDsRed2_N1-podocin^wild, podocin^V165X and podocin^R168H were lyzed in the buffer containing 1% Tritonx-100, 150 mM NaCl, 1 mM EDTA, 50 mM Tris–HCl (pH7.7), 1 mM NaF, 1 mM NaVO₃ and a protease inhibitor cocktail (Roche, Nutley, NJ, USA). Seventy-five micrograms of total protein was loaded to run 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the gel was set up for transfer protein to nitrocellulose membranes (Amersham Life Science, Piscataway, NJ, USA). Then, the membranes were rinsed in a Tris-buffered saline with 0.02% Tween-20 (TTBS), followed by immersing in 5% low-fat milk. Subsequently, the membranes were incubated with rabbit anti-podocin, nephrin, CD2AP and TRPC6 antibody; mouse anti-GAPDH antibody; rabbit anti-phospho-PERK, Bip/grp78 and anti-caspase12 antibody; mouse anti-phospho ERK1/2 and rabbit anti-ERK1/2 antibody. After rinsing three times with TTBS, the membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG (Santa Cruz) for 45 min at room temperature, and then developed using ECL chemiluminescence reagent (Santa Cruz). The specific protein bands were scanned and quantitated using densitometry in relation to the GAPDH.

Immunoprecipitation assay

To evaluate the interaction of mutant podocins and TRPC6, HEK293 cells co-transfected with pCMV-Tag2B-podocin^wild, podocin^V165X or podocin^R168H and pRerceiver-M29 TRPC6 were lyzed in RIPA buffer containing a protease inhibitor cocktail on ice. Five hundred micrograms of total protein was precipitated with 20 µL of anti-FLAG^® M2 Affinity Gel (Sigma) overnight at 4 °C. The gel was washed three times with 0.5 mL of TBS and the supernatant removed. Then, 20 µL of 2x sample buffer [125 mM Tris–HCl (pH 6.8), 4% SDS, 20% (v/v) glycerol, 0.004% bromphenol blue] was added and the sample was boiled for 5 min. Finally, the samples were centrifuged to pellet any undissolved gel, and the supernatants were transferred to fresh tubes for loading on 8% SDS-PAGE and immunoblotting using anti-TRPC6 antibody.

Measurement of the cytosolic free Ca²⁺

The [Ca²⁺]Fi was measured with the specific fluorescent indicator, fluo-3AM (Invitrogen) as previously described (Estacion et al. 2006). Cells transfected with pcDNA3.0-podocin^wild, podocin^V165X or podocin^R168H were harvested, and 5 x 10⁵ cells were resuspended in Ca²⁺/Mg^2+-free PBS containing 10 µM fluo-3AM at 37 °C and incubated for 30 min. The cell suspension was washed three times and resuspended again in fresh Ca²⁺/Mg²⁺-free PBS immediately prior to fluorescence measurement using FLX 800 spectrophotofluorometer (BioTek, Winooski, VT, USA) with a filter of 480 nm excitation and 510 nm emission wavelength. After the basal [Ca²⁺]Fi was recorded, the cells were stimulated by 100 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG) (Sigma) for three time points followed by 2 mM CaCl₂ for six time points at 1-min intervals. For statistical analysis, the [Ca²⁺]Fi from individual cells were averaged on each group.

Statistical analysis

Data were reported as mean ± SD with n equal to the number of experiments. Statistical evaluation was performed using a one-way ANOVA (two-sided test), followed by LSD (equal variances assumed) or Dunnett’s T3 (equal variances not assumed) for post hoc test between two groups, and also using the nonparametric tests (Mann–Whitney U-test) as a post-test. Values of P < 0.05 were considered as statistic significance.

	Acknowledgements

 
We thank Professor Peter Mundel (America) for the podocyte clone, Professor Karl Tryggvason (Sweden) and Professor Corinne Antignac (France) for the kind gifts of nephrin and podocin antibodies, respectively. We thank the Institute of Space Medico-Engineering of China for the FLX 800 spectrophotofluorometer, and thank the Central Laboratory of Peking University First Hospital for technique helps.

This work was supported by the National Nature Science Foundation of China (30170992, 30672259 and 30801250), Beijing Nature Science Foundation (7072080) and New Teacher Foundation from Chinese Ministry of Education (20070001764).

	Footnotes

 
Communicated by: Moshe Yaniv

^* djnc_5855{at}126.com

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Received: 13 January 2009
Accepted: 15 June 2009

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