http://informahealthcare.com/rst ISSN: 1079-9893 (print), 1532-4281 (electronic) J Recept Signal Transduct Res, Early Online: 1–5 ! 2014 Informa Healthcare USA, Inc.. DOI: 10.3109/10799893.2014.963872
RESEARCH ARTICLE
Role of nephrin in podocyte injury induced by angiotension II Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
Shengyou Yu Guangzhou Medical University, Guangzhou First People’s Hospital, Guangdong Province, China
Abstract
Keywords
Objective: To investigate the function of nephrin in podocytes and its relation to proteinuria in kidney diseases, and to study more clearly theoretical basis for the molecular mechanism of losartan anti-proteinuria and the special beneficial effects of losartan on podocyte injury. Methods: Experiment set up control, Ang II and losartan group. Cell morphology was observed perturbation, and using image processing software to analyze the cell body of cell morphology and size of the difference after 8 h, 24 h and 48 h. Detecting nephrin mRNA and protein expression changes by real time PCR (RT-PCR) and western blotting at different time points. Results: Podocyte cell bodies were significantly reduced after Ang II injury (p50.01), losartan directly reduces the rate of apoptotic podocytes induced by Ang. Apoptotic podocytes may related to the decrease of nephrin mRNA and protein expressions, losartan reduced the apoptosis and proteinuria by declining nephrin mRNA and protein expressions. Conclusion: Ang II induced podocyte injury caused abnormal expression and distribution of nephrin in podocytes, losartan maybe maintain the stability of nephrin expression and the integrity of hole diaphragm (SD) structure and function by blocking the signal path, playing a important role in protection mechanisms of anti-proteinuria. Our findings provide some possible clues for further exploring the pharmacological targets to the proteinuria. These novel findings provide new insights into the beneficial effects of losartan on podocytes directly.
Losartan, nephrin, podocyte, molecular mechanisms
Introduction Nephrin is the key structural and signaling molecule, which regulates a number of podocyte signaling pathways, activation of the phosphoinositide 3-OH kinase–PKB pathway leads to a reduction in cell death (1). These effects require interaction with podocin, which targets nephrin to lipid rafts at the plasma membrane and facilitates nephrin signaling (2,3). Ang II is used to induce a well established cell model for podocytes injury (4). Losartan is widely used for the treatment of a variety of glomerular diseases. However, the signaling mechanisms underlying the anti-proteinuria effects of losartan have not been well defined. In this study, we further investigated the role of nephrin in podocytes and its relation to proteinuria in kidney diseases. To study more clearly theoretical basis for the molecular mechanism of losartan anti-proteinuria and the special beneficial effects of losartan on podocyte injury.
Materials and methods Cells in culture Podocytes were cultured at 33 C in RPMI-1640 containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), Address for correspondence: Shengyou Yu, Guangzhou Medical University, Guangzhou First People’s Hospital, Guangdong Province, China. E-mail: [email protected]
History Received 12 August 2014 Revised 31 August 2014 Accepted 07 September 2014 Published online 29 September 2014
and then shifted to 37 C for differentiation. In the studies described below, all experiments were performed in growthrestricted podocytes, experiment set up control, Ang II and losartan group. Cell morphology was observed perturbation, and using image processing software to analyze the cell body of cell morphology and size of the difference after 8 h, 24 h and 48 h. The experiments were all repeated three times. RT-PCR analysis We used RT-PCR assay to confirm the expression of nephrin, total RNA was extracted from podocyte using trizol reagent according to the manufacturer’s instruction, and the RNA concentration was determined after the sample was dissolved in diethylpyrocarbonate-treated water. The assay was also run on an Applied Biosystems Model 7900 Genetic Analyzer (Lingley Ho 120, Warrington, UK) and the resulting data again were analyzed using the Applied Biosystems StatMiner software (GuangZhou, GuangDong, China). Independent experiment was repeated three times. Western blot analysis The podocytes were prepared for western blot analysis according to the method described (5,6). An analytical 10% SDS-PAGE was performed, and 30 mg of protein of each was analyzed, proteins in the SDS gels were transferred
2
S. Y. Yu
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
to a polyvinylidene difluoride (PVDF) membrane by an electroblot apparatus. The membranes were incubated with rabbit anti-nephrin antibody (Sigma Chemical Co, St Louis, MO); mouse anti-GAPDH antibody (Sigma Chemical Co). After rinsing three times with TTBS, the membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG (Sigma Chemical Co) for 45 min at room temperature and then developed using ECL chemiluminescence reagent (Sigma Chemical Co). The specific protein bands were scanned and quantitated using densitometry in relation to the GAPDH, we repeated each western blot analysis using protein from three different and separate experiments. Assessment of apoptosis Podocytes were plated at a density of 7.5 104/cm2 and were allowed to attach to tissue culture plates for 24 h. The cells were then incubated with medium that contained 10% FBS in the presence or absence of losartan (Sigma Chemical Co). Ang II was added to the medium after 1 h. The percentage of apoptotic cells was assessed. Apoptosis was measured by staining with FITC-annexin V and PI (Sigma Chemical Co). Five hundred microliters of cell suspension liquid was taken and mixed with 5 ll FITC-annexin V and 5 ll PI (concentration 250 mg/l), avoiding light at 4 C for 10 min, excitation wavelength for 488 nm, each specimen collected 10 000 cells, and then relevant software analyzed and calculated the cell apoptosis rate. Statistical analysis Data were reported as mean ± SD with an equal to the number of experiments. Statistical evaluation was performed using a one-way ANOVA (two sided test), followed by LSD (equal variances assumed) for post hoc test between two groups, and also using the nonparametric tests (Mann–Whitney U-test) as a posttest. Values of p50.05 were considered as statistically significance.
Results Effect of losartan on Ang II-induced podocytes changes Podocytes were observed and photographed under inverted microscope. The cell bodies and nucleus of Ang II-induced podocytes were significantly decreased. Foot processes appeared retraction and the area of Ang II-inducted podocytes was significantly reduced (p50.05); foot processes was obviously retracted. However, after losartan treated, the area of podocytes was significantly greater at 8 h, 24 h and 48 h, the difference was significant (p50.05) (Figure 1). Effect of losartan on Ang II-induced nephrin mRNA expression We performed RT-PCR to measure the nephrin mRNA expression. Nephrin plays a central role in Ang II-induced apoptosis, but the role of nephrin is not well understood in podocyte apoptosis. RT-PCR showed that in the control, Ang II and losartan group, nephrin mRNA expression had no changes at 8 h (p40.05), incubation of the podocytes with Ang II markedly decreased nephrin mRNA levels at 24 and
J Recept Signal Transduct Res, Early Online: 1–5
48 h. In contrast, losartan completely prevented the mRNA decrease in nephrin induced by Ang II. These results showed that losartan significantly increased the nephrin mRNA levels (p50.05 versus Ang II). This suggests that losartan may inhibit podocyte apoptosis induced by Ang II by preventing the down-regulation of nephrin mRNA levels (Table 1). Effect of losartan on Ang II-induced nephrin protein expression We performed western blot analysis to measure the protein expression of nephrin. Nephrin plays a central role in Ang IIinduced apoptosis, but the role of nephrin is not well understood in podocyte apoptosis. Western blot analysis showed that in the control, Ang II and losartan group, nephrin protein expression had no changes at 8 h (p40.05), incubation of the podocytes with Ang II markedly decreased nephrin protein levels at 24 and 48 h. In contrast, losartan completely prevented the decrease in nephrin induced by Ang II. These results demonstrate that incubation of cultured podocytes with Ang II decreased the protein levels of nephrin and that addition of losartan prevented podocytes from decreasing nephrin levels in response to Ang II. Our results showed that losartan significantly increased the nephrin (p50.05 versus Ang II). This suggests that losartan may inhibit podocyte apoptosis induced by Ang II by preventing the downregulation of nephrin (Figure 2 and Table 2). Assessment of apoptosis We quantified podocyte apoptosis and measured viable cell number in cultured immortalized podocytes that were grown under restrictive conditions for 14 d and then were injured by exposure to Ang II. We first measured the percentage of apoptotic cells. Our results showed that losartan significantly decreased the number of apoptotic podocytes induced by Ang II (p50.05 versus control). The results confirmed that podocyte apoptosis was a dose-dependent manner. There was an increase in apoptotic cell number after 48 h of exposure to Ang II. In contrast, losartan significantly reduced Ang IIinduced apoptosis at 48 h. These results show that Ang IIinduced podocyte apoptosis is Ang II dependent and supports the notion that losartan exerts an antiapoptotic effect on cells that are exposed to Ang II through the regulation of losartan.
Discussion Nephrin is expressed in the glomerular podocytes, which regulates the structure, function of podocytes and maintain normal glomerular ultrafiltration. Its role in cell biology to date is limited to the podocyte, it appears likely that many glomerular disease pathways are directed via the slit diaphragm–protein complex, and the loss or dysregulation of nephrin will initiate potentially catastrophic downstream events within the podocyte (7). Podocyte injury is closely associated with the development of glomerulosclerosis in glomerular disease. Podocyte depletion as a result of apoptosis and the inability of podocytes to replicate causes glomerulosclerosis. Podocyte apoptosis is a major factor causing a decrease in podocyte number (8–11). In diabetic nephropathy, nephrin expression decreases as albuminuria
Role of nephrin
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
DOI: 10.3109/10799893.2014.963872
3
Figure 1. Ang II -induced podocytes changes at different time points (inverted-phase contrast microscope 200). Note: (a, d and g) the control group, 8 h, 24 h and 48 h, respectively (foot processes and the connection between podocytes are intact); (b): Ang II -induced 8 h; (c) losartan -treated 8 h; (e) Ang II -induced 24 h; (f) losartan-treated 24 h; (h) Ang II -induced 48 h (foot process retracted and lost, cell interconnected disappeared) and (i) losartan-treated 48 h.
Table 1. Nephrin mRNA levels at different time.
8h 24 h 48 h
The control
Ang II
Losartan
1.78 ± 0.11 1.82 ± 0.17 1.85 ± 0.20
1.57 ± 0.10 0.93 ± 0.06* 0.62 ± 0.03*
1.51 ± 0.10 1.62 ± 0.12 1.74 ± 0.14
Losartan prevents podocyte apoptosis induced by Ang II by preventing the down-regulation of nephrin mRNA levels. The nephrin mRNA levels increased at 8 h in the cells that were exposed to Ang II without losartan, whereas cells that were treated with losartan were resistant to Ang II-induced apoptosis.
progresses (12–14), and that this occurs independent of expression of other molecules located at the slit diaphragm of the podocyte, such as podocin and CD2AP, suggesting that decreased nephrin expression is not a non-specific marker of generalized podocyte dysfunction (15).
In this study, we found that nephrin plays an important role in Ang II-induced apoptosis, but the role of nephrin is not well understood in podocyte apoptosis. Western blot analysis showed that in the control, Ang II and losartan group, nephrin protein expression had no changes at 8 h (p40.05), incubation of the podocytes with Ang II markedly decreased nephrin protein levels at 24 and 48 h. Our results showed that losartan significantly decreased the nephrin (p50.05 versus Ang II). This suggests that losartan may inhibit podocyte apoptosis induced by Ang II by preventing the down-regulation of nephrin. We first tested the hypothesis that losartan has an inhibitory effect on Ang II-induced apoptosis in podocytes. To test this hypothesis, we quantified podocyte apoptosis and measured viable cell number in cultured immortalized podocytes that were grown under restrictive conditions for 14 d and then were injured by exposure to Ang II. We first measured the percentage of apoptotic cells. Our results
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
4
S. Y. Yu
J Recept Signal Transduct Res, Early Online: 1–5
Figure 2. The percentage of apoptosis measured by FITC-annexin V and PI staining. (a) The control; (b) Ang II and (c) losartan. Losartan prevents Ang II -induced apoptosis. The percentage of apoptotic cells, measured by FITC-annexin V and PI staining, increased at 24 h in the cells that were exposed to Ang II without losartan, whereas cells that were treated with losartan were resistant to Ang II -induced apoptosis. *p50.05 versus losartan ( ) Ang II (+).
Table 2. Nephrin protein levels at different time.
8h 24 h 48 h
The control
Ang II
0.164 ± 0.010 0.176 ± 0.011 0.179 ± 0.012
0.134 ± 0.005 0.087 ± 0.003* 0.053 ± 0.001*
Losartan
0.134 ± 0.003 0.146 ± 0.018 0.173 ± 0.021
Losartan may inhibit podocyte apoptosis induced by Ang II by preventing the downregulation of nephrin protein levels. Nephrin protein levels decreased at 24 h in the cells that were exposed to Ang II without losartan, whereas cells that were treated with losartan were resistant to Ang II -induced apoptosis. *p50.05 versus losartan ( )Ang II (+).
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
DOI: 10.3109/10799893.2014.963872
showed that losartan significantly decreased the number of apoptotic podocytes induced by Ang II (p50.05 versus control). The results confirmed that podocyte apoptosis was a dose-dependent manner. There was a increase in apoptotic cell number after 48 h of exposure to Ang II. In contrast, losartan significantly reduced Ang II-induced apoptosis at 48 h. These results show that Ang II-induced podocyte apoptosis is Ang II dependent and supports the notion that losartan exerts an antiapoptotic effect on cells that are exposed to Ang II through the regulation of losartan. In summary, the major goal of this study was to explore the role of nephrin in Ang II-induced podocyte injury and proteinuria occurrence. Our present study revealed a novel molecular mechanism of Ang II-induced podocyte injury and proteinuria, which was associated with nephrin downregulation. We found that Ang II caused nephrin lower expression, and increased apoptosis. These results demonstrated that the direct harmful effect of Ang II on podocytes was mainly mediated by a mechanism that involved downregulation of nephrin. These novel findings provide new insights into the podocyte injury and proteinuria occurrence.
Declaration of interests The authors declare that they have no conflict of interests.
References 1. Foster RR, Saleem MA, Mathieson PW, et al. Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol 2005;288:48–57. 2. Huber TB, Kottgen M, Schilling B, et al. Interaction with podocin facilitates nephrin signaling. J Biol Chem 2001;276:41543–6.
Role of nephrin
5
3. Schwarz K, Simons M, Reiser J, et al. Podocin, a raft-associated component of the glomerular slit diaphragm, interacts with CD2AP and nephrin. J Clin Invest 2001;108:1621–9. 4. Hughes J, Savill JS. Apoptosis in glomerulonephritis. Curr Opin Nephrol Hypertens 2005;14:389–95. 5. Yu SY, Qi R, Zhao H. Losartan reverses glomerular podocytes injury induced by AngII via stabilizing the expression of GLUT1. Mol Biol Rep 2013;40:6295–301. 6. Qi R, Li W. FK506 inhibits the mice glomerular mesangial cells proliferation by affecting the transforming growth factor-b and smads signal pathways. Ren Fail 2014;36:589–92. 7. Gavin I Welsh, Moin A Saleem. Nephrin-signature molecule of the glomerular podocyte? J Pathol 2010;220:328–37 8. Yu SY, Qi R. Role of bad in podocyte apoptosis induced by puromycin aminonucleoside. Transplant Proceed 2013;45:569–73. 9. Facemire CS, Mohler PJ, Arendshorst WJ, et al. Expression and relative abundance of short transient receptor potential channels in the rat renal microcirculation. Am J Physiol Renal Physiol 2004;28: 546–51. 10. Limbourg FP, Huang Z, Plumier JC, et al. Rapid nontranscriptional activation of endothelial nitric oxide synthase mediates increased cerebral blood flow and stroke protection by corticosteroids. J Clin Invest 2002;110:1729–38. 11. Huang L, You YS, Wu W. Role of CD2-associated protein in podocyte apoptosis and proteinuria induced by angiotensin II. Ren Fail 2014;36:1328–32. 12. Bonnet F, Cooper ME, Kawachi H, et al. Irbesartan normalizes the deficiency in glomerular nephrin expression in a model of diabetes and hypertension. Diabetologia 2001;44:874–7. 13. Doublier S, Salvidio G, Lupia E, et al. Nephrin expression is reduced in human diabetic nephropathy: evidence for a distinct role for glycated albumin and angiotensin II. Diabetes 2003;52: 1023–30. 14. Davis BJ, Cao Z, de Gasparo M, et al. Disparate effects of angiotensin II antagonists and calcium channel blockers on albuminuria in experimental diabetes and hypertension: potential role of nephrin. J Hypertens 2003;21:209–16. 15. Benigni A, Gagliardini E, Tomasoni S, et al. Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy. Kidney Int 2004;65:2193–200.
RESEARCH ARTICLE
Role of nephrin in podocyte injury induced by angiotension II Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
Shengyou Yu Guangzhou Medical University, Guangzhou First People’s Hospital, Guangdong Province, China
Abstract
Keywords
Objective: To investigate the function of nephrin in podocytes and its relation to proteinuria in kidney diseases, and to study more clearly theoretical basis for the molecular mechanism of losartan anti-proteinuria and the special beneficial effects of losartan on podocyte injury. Methods: Experiment set up control, Ang II and losartan group. Cell morphology was observed perturbation, and using image processing software to analyze the cell body of cell morphology and size of the difference after 8 h, 24 h and 48 h. Detecting nephrin mRNA and protein expression changes by real time PCR (RT-PCR) and western blotting at different time points. Results: Podocyte cell bodies were significantly reduced after Ang II injury (p50.01), losartan directly reduces the rate of apoptotic podocytes induced by Ang. Apoptotic podocytes may related to the decrease of nephrin mRNA and protein expressions, losartan reduced the apoptosis and proteinuria by declining nephrin mRNA and protein expressions. Conclusion: Ang II induced podocyte injury caused abnormal expression and distribution of nephrin in podocytes, losartan maybe maintain the stability of nephrin expression and the integrity of hole diaphragm (SD) structure and function by blocking the signal path, playing a important role in protection mechanisms of anti-proteinuria. Our findings provide some possible clues for further exploring the pharmacological targets to the proteinuria. These novel findings provide new insights into the beneficial effects of losartan on podocytes directly.
Losartan, nephrin, podocyte, molecular mechanisms
Introduction Nephrin is the key structural and signaling molecule, which regulates a number of podocyte signaling pathways, activation of the phosphoinositide 3-OH kinase–PKB pathway leads to a reduction in cell death (1). These effects require interaction with podocin, which targets nephrin to lipid rafts at the plasma membrane and facilitates nephrin signaling (2,3). Ang II is used to induce a well established cell model for podocytes injury (4). Losartan is widely used for the treatment of a variety of glomerular diseases. However, the signaling mechanisms underlying the anti-proteinuria effects of losartan have not been well defined. In this study, we further investigated the role of nephrin in podocytes and its relation to proteinuria in kidney diseases. To study more clearly theoretical basis for the molecular mechanism of losartan anti-proteinuria and the special beneficial effects of losartan on podocyte injury.
Materials and methods Cells in culture Podocytes were cultured at 33 C in RPMI-1640 containing 10% fetal bovine serum (Gibco, Gaithersburg, MD, USA), Address for correspondence: Shengyou Yu, Guangzhou Medical University, Guangzhou First People’s Hospital, Guangdong Province, China. E-mail: [email protected]
History Received 12 August 2014 Revised 31 August 2014 Accepted 07 September 2014 Published online 29 September 2014
and then shifted to 37 C for differentiation. In the studies described below, all experiments were performed in growthrestricted podocytes, experiment set up control, Ang II and losartan group. Cell morphology was observed perturbation, and using image processing software to analyze the cell body of cell morphology and size of the difference after 8 h, 24 h and 48 h. The experiments were all repeated three times. RT-PCR analysis We used RT-PCR assay to confirm the expression of nephrin, total RNA was extracted from podocyte using trizol reagent according to the manufacturer’s instruction, and the RNA concentration was determined after the sample was dissolved in diethylpyrocarbonate-treated water. The assay was also run on an Applied Biosystems Model 7900 Genetic Analyzer (Lingley Ho 120, Warrington, UK) and the resulting data again were analyzed using the Applied Biosystems StatMiner software (GuangZhou, GuangDong, China). Independent experiment was repeated three times. Western blot analysis The podocytes were prepared for western blot analysis according to the method described (5,6). An analytical 10% SDS-PAGE was performed, and 30 mg of protein of each was analyzed, proteins in the SDS gels were transferred
2
S. Y. Yu
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
to a polyvinylidene difluoride (PVDF) membrane by an electroblot apparatus. The membranes were incubated with rabbit anti-nephrin antibody (Sigma Chemical Co, St Louis, MO); mouse anti-GAPDH antibody (Sigma Chemical Co). After rinsing three times with TTBS, the membranes were incubated with HRP-conjugated goat anti-rabbit or mouse IgG (Sigma Chemical Co) for 45 min at room temperature and then developed using ECL chemiluminescence reagent (Sigma Chemical Co). The specific protein bands were scanned and quantitated using densitometry in relation to the GAPDH, we repeated each western blot analysis using protein from three different and separate experiments. Assessment of apoptosis Podocytes were plated at a density of 7.5 104/cm2 and were allowed to attach to tissue culture plates for 24 h. The cells were then incubated with medium that contained 10% FBS in the presence or absence of losartan (Sigma Chemical Co). Ang II was added to the medium after 1 h. The percentage of apoptotic cells was assessed. Apoptosis was measured by staining with FITC-annexin V and PI (Sigma Chemical Co). Five hundred microliters of cell suspension liquid was taken and mixed with 5 ll FITC-annexin V and 5 ll PI (concentration 250 mg/l), avoiding light at 4 C for 10 min, excitation wavelength for 488 nm, each specimen collected 10 000 cells, and then relevant software analyzed and calculated the cell apoptosis rate. Statistical analysis Data were reported as mean ± SD with an equal to the number of experiments. Statistical evaluation was performed using a one-way ANOVA (two sided test), followed by LSD (equal variances assumed) for post hoc test between two groups, and also using the nonparametric tests (Mann–Whitney U-test) as a posttest. Values of p50.05 were considered as statistically significance.
Results Effect of losartan on Ang II-induced podocytes changes Podocytes were observed and photographed under inverted microscope. The cell bodies and nucleus of Ang II-induced podocytes were significantly decreased. Foot processes appeared retraction and the area of Ang II-inducted podocytes was significantly reduced (p50.05); foot processes was obviously retracted. However, after losartan treated, the area of podocytes was significantly greater at 8 h, 24 h and 48 h, the difference was significant (p50.05) (Figure 1). Effect of losartan on Ang II-induced nephrin mRNA expression We performed RT-PCR to measure the nephrin mRNA expression. Nephrin plays a central role in Ang II-induced apoptosis, but the role of nephrin is not well understood in podocyte apoptosis. RT-PCR showed that in the control, Ang II and losartan group, nephrin mRNA expression had no changes at 8 h (p40.05), incubation of the podocytes with Ang II markedly decreased nephrin mRNA levels at 24 and
J Recept Signal Transduct Res, Early Online: 1–5
48 h. In contrast, losartan completely prevented the mRNA decrease in nephrin induced by Ang II. These results showed that losartan significantly increased the nephrin mRNA levels (p50.05 versus Ang II). This suggests that losartan may inhibit podocyte apoptosis induced by Ang II by preventing the down-regulation of nephrin mRNA levels (Table 1). Effect of losartan on Ang II-induced nephrin protein expression We performed western blot analysis to measure the protein expression of nephrin. Nephrin plays a central role in Ang IIinduced apoptosis, but the role of nephrin is not well understood in podocyte apoptosis. Western blot analysis showed that in the control, Ang II and losartan group, nephrin protein expression had no changes at 8 h (p40.05), incubation of the podocytes with Ang II markedly decreased nephrin protein levels at 24 and 48 h. In contrast, losartan completely prevented the decrease in nephrin induced by Ang II. These results demonstrate that incubation of cultured podocytes with Ang II decreased the protein levels of nephrin and that addition of losartan prevented podocytes from decreasing nephrin levels in response to Ang II. Our results showed that losartan significantly increased the nephrin (p50.05 versus Ang II). This suggests that losartan may inhibit podocyte apoptosis induced by Ang II by preventing the downregulation of nephrin (Figure 2 and Table 2). Assessment of apoptosis We quantified podocyte apoptosis and measured viable cell number in cultured immortalized podocytes that were grown under restrictive conditions for 14 d and then were injured by exposure to Ang II. We first measured the percentage of apoptotic cells. Our results showed that losartan significantly decreased the number of apoptotic podocytes induced by Ang II (p50.05 versus control). The results confirmed that podocyte apoptosis was a dose-dependent manner. There was an increase in apoptotic cell number after 48 h of exposure to Ang II. In contrast, losartan significantly reduced Ang IIinduced apoptosis at 48 h. These results show that Ang IIinduced podocyte apoptosis is Ang II dependent and supports the notion that losartan exerts an antiapoptotic effect on cells that are exposed to Ang II through the regulation of losartan.
Discussion Nephrin is expressed in the glomerular podocytes, which regulates the structure, function of podocytes and maintain normal glomerular ultrafiltration. Its role in cell biology to date is limited to the podocyte, it appears likely that many glomerular disease pathways are directed via the slit diaphragm–protein complex, and the loss or dysregulation of nephrin will initiate potentially catastrophic downstream events within the podocyte (7). Podocyte injury is closely associated with the development of glomerulosclerosis in glomerular disease. Podocyte depletion as a result of apoptosis and the inability of podocytes to replicate causes glomerulosclerosis. Podocyte apoptosis is a major factor causing a decrease in podocyte number (8–11). In diabetic nephropathy, nephrin expression decreases as albuminuria
Role of nephrin
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
DOI: 10.3109/10799893.2014.963872
3
Figure 1. Ang II -induced podocytes changes at different time points (inverted-phase contrast microscope 200). Note: (a, d and g) the control group, 8 h, 24 h and 48 h, respectively (foot processes and the connection between podocytes are intact); (b): Ang II -induced 8 h; (c) losartan -treated 8 h; (e) Ang II -induced 24 h; (f) losartan-treated 24 h; (h) Ang II -induced 48 h (foot process retracted and lost, cell interconnected disappeared) and (i) losartan-treated 48 h.
Table 1. Nephrin mRNA levels at different time.
8h 24 h 48 h
The control
Ang II
Losartan
1.78 ± 0.11 1.82 ± 0.17 1.85 ± 0.20
1.57 ± 0.10 0.93 ± 0.06* 0.62 ± 0.03*
1.51 ± 0.10 1.62 ± 0.12 1.74 ± 0.14
Losartan prevents podocyte apoptosis induced by Ang II by preventing the down-regulation of nephrin mRNA levels. The nephrin mRNA levels increased at 8 h in the cells that were exposed to Ang II without losartan, whereas cells that were treated with losartan were resistant to Ang II-induced apoptosis.
progresses (12–14), and that this occurs independent of expression of other molecules located at the slit diaphragm of the podocyte, such as podocin and CD2AP, suggesting that decreased nephrin expression is not a non-specific marker of generalized podocyte dysfunction (15).
In this study, we found that nephrin plays an important role in Ang II-induced apoptosis, but the role of nephrin is not well understood in podocyte apoptosis. Western blot analysis showed that in the control, Ang II and losartan group, nephrin protein expression had no changes at 8 h (p40.05), incubation of the podocytes with Ang II markedly decreased nephrin protein levels at 24 and 48 h. Our results showed that losartan significantly decreased the nephrin (p50.05 versus Ang II). This suggests that losartan may inhibit podocyte apoptosis induced by Ang II by preventing the down-regulation of nephrin. We first tested the hypothesis that losartan has an inhibitory effect on Ang II-induced apoptosis in podocytes. To test this hypothesis, we quantified podocyte apoptosis and measured viable cell number in cultured immortalized podocytes that were grown under restrictive conditions for 14 d and then were injured by exposure to Ang II. We first measured the percentage of apoptotic cells. Our results
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
4
S. Y. Yu
J Recept Signal Transduct Res, Early Online: 1–5
Figure 2. The percentage of apoptosis measured by FITC-annexin V and PI staining. (a) The control; (b) Ang II and (c) losartan. Losartan prevents Ang II -induced apoptosis. The percentage of apoptotic cells, measured by FITC-annexin V and PI staining, increased at 24 h in the cells that were exposed to Ang II without losartan, whereas cells that were treated with losartan were resistant to Ang II -induced apoptosis. *p50.05 versus losartan ( ) Ang II (+).
Table 2. Nephrin protein levels at different time.
8h 24 h 48 h
The control
Ang II
0.164 ± 0.010 0.176 ± 0.011 0.179 ± 0.012
0.134 ± 0.005 0.087 ± 0.003* 0.053 ± 0.001*
Losartan
0.134 ± 0.003 0.146 ± 0.018 0.173 ± 0.021
Losartan may inhibit podocyte apoptosis induced by Ang II by preventing the downregulation of nephrin protein levels. Nephrin protein levels decreased at 24 h in the cells that were exposed to Ang II without losartan, whereas cells that were treated with losartan were resistant to Ang II -induced apoptosis. *p50.05 versus losartan ( )Ang II (+).
Journal of Receptors and Signal Transduction Downloaded from informahealthcare.com by McMaster University on 10/17/14 For personal use only.
DOI: 10.3109/10799893.2014.963872
showed that losartan significantly decreased the number of apoptotic podocytes induced by Ang II (p50.05 versus control). The results confirmed that podocyte apoptosis was a dose-dependent manner. There was a increase in apoptotic cell number after 48 h of exposure to Ang II. In contrast, losartan significantly reduced Ang II-induced apoptosis at 48 h. These results show that Ang II-induced podocyte apoptosis is Ang II dependent and supports the notion that losartan exerts an antiapoptotic effect on cells that are exposed to Ang II through the regulation of losartan. In summary, the major goal of this study was to explore the role of nephrin in Ang II-induced podocyte injury and proteinuria occurrence. Our present study revealed a novel molecular mechanism of Ang II-induced podocyte injury and proteinuria, which was associated with nephrin downregulation. We found that Ang II caused nephrin lower expression, and increased apoptosis. These results demonstrated that the direct harmful effect of Ang II on podocytes was mainly mediated by a mechanism that involved downregulation of nephrin. These novel findings provide new insights into the podocyte injury and proteinuria occurrence.
Declaration of interests The authors declare that they have no conflict of interests.
References 1. Foster RR, Saleem MA, Mathieson PW, et al. Vascular endothelial growth factor and nephrin interact and reduce apoptosis in human podocytes. Am J Physiol Renal Physiol 2005;288:48–57. 2. Huber TB, Kottgen M, Schilling B, et al. Interaction with podocin facilitates nephrin signaling. J Biol Chem 2001;276:41543–6.
Role of nephrin
5
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