The relationship among nephrin, podocin, CD2AP, and alpha-actinin might not be a true 'interaction' in podocyte

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

Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy

BACKGROUND: Recent disclosure of podocyte proteins has unraveled previously rather mysterious mechanisms that govern glomerular perm-selectivity in health and disease. Here we addressed the role of nephrin, CD2-associated protein (CD2AP), and podocin together with the integrity of the slit diaphragm in the pathogenesis of proteinuria of patients with diabetes and nephropathy. METHODS: Nephrin mRNA and protein expression were evaluated in parallel in adult diabetic patients by in situ hybridization and immunohistochemistry. For comparison, nondiabetic patients with minimal change nephrosis and normal control patients were evaluated. CD2AP and podocin expression by immunohistochemistry was also assessed. The filtration slit was analyzed by morphometry and transmission electron microscopy. RESULTS: Extracellular nephrin mRNA and protein were markedly reduced in diabetic patients. No changes were found in patients with minimal change versus controls. CD2AP and podocin were comparable in all subjects. Ultrastructural analysis showed in diabetic patients a remarkable reduction in the percentage of electron dense slit diaphragms, despite a frequency of the filtration slits comparable to control patients. CONCLUSION: Down-regulation of nephrin and loss of the electron dense structure of slit diaphragm indicate a novel mechanism accounting for proteinuria in diabetic nephropathy. To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical relevance. Reduction of nephrin in the context of normal expression of CD2AP and podocin can be taken reasonably as a specific marker of renal disease in diabetes. Therapies targeted at correcting podocyte nephrin might be of value for diabetic medicine.     

三种不同药物作用下足细胞分子的变化

目的 从足细胞分子的角度探讨抗蛋白尿药物作用的细胞分子机制。方法 建立阿霉素肾病大鼠模型。注射阿霉素后次日分别给予利生普利、泼尼松以及全反式维甲酸(ATRA)干预蛋白尿。注射阿霉素后第3、7、14、28 天每组处死6 只大鼠，留取肾脏标本。应用间接免疫荧光染色、实时PCR、Western印迹分别检测各个时间点nephrin、podocin、CD2相关蛋白(AP)、α辅肌动蛋白(actinin)-4 的分布、mRNA 和蛋白表达量的变化。应用免疫沉淀检测nephrin 与podocin、nephrin与CD2AP 分子间作用以及nephrin 磷酸化水平。结果 与对照组相比，第14天时肾病组尿蛋白显著增加(P < 0.01)。与肾病组相比，利生普利、泼尼松和ATRA干预后均显著降低了蛋白尿( P < 0.05)， 减轻了足突融合。通过分析不同时间点足细胞分子的表达，显示3种干预药物均引起了nephrin、podocin、CD2AP 表达的变化，维持了正常的nephrin 磷酸化水平，而且利生普利和泼尼松首先抑制了podocin 分子，而ATRA首先抑制了CD2AP 分子的异常变化。与此同时，nephrin、podocin、CD2AP 和α-actinin-4 分子的分布在干预后也趋于正常。此外，无论在肾病组还是干预组大鼠，nephrin 与podocin、nephrin 与CD2AP 分子间一直保持着共沉淀关系。 结论 利生普利、泼尼松和ATRA 都通过稳定重要的足细胞分子nephrin、podocin、CD2AP 来发挥它们的抗蛋白尿作用。     

EP1 receptor mediates Adriamycin-induced podocyte injury through activating p38 MAPK

Objective To investigate the effect of prostaglandin E2 receptor 1 (EP1) on Adriamycin (ADR)-induced glomerular podocytes injury and its possible mechanism. Methods (1) In vivo experiments: 6-8 weeks old male Balb/c mice were randomly divided into four groups: Control group; ADR group; EP1 agonist 17-phenyl PGE2+ADR group; EP1 antagonist SC-19220+ADR group. The mouse model of nephrotic syndrome was induced by injection of ADR (10 mg/kg) into tail vein, and then EP1 agonist (1 μg/g) and antagonist (25 μg/g) were administered respectively. Six weeks later, all mice were sacrificed and urine, blood and kidney tissues were collected. Detecting urine protein, blood chemistry, changes of renal pathology and podocyte-related proteins, electron microscopy changes of podocytes. (2) In vitro experiments: Podocytes were cultured in vitro and divided into different groups: Control group; ADR group (0.2 μmol/L); EP1 agonist (0.1, 1, 10 μmol/L)+ADR (0.2 μmol/L) group; antagonist (0.1, 0.5, 1 μmol/L)+ADR (0.2 μmol/L) group. The proliferation of podocytes was measured by CCK-8. Expression of PGE2 in podocytes was detected by ELISA. Indirect immunofluorescence was used to determine the localization of podocyte-related proteins nephrin, podocin and CD2AP. Expression of nephrin, podocin, CD2AP, COX2 in podocytes was detected by Western blotting and Real-time quantitative PCR. p38 MAPK or phospho-p38 MAPK was measured by Western blotting as well. Flow cytometry was used to detect cell apoptosis. Results (1) In vivo experiments: Compared with control group, obvious proteinuria, blood biochemical changes and renal pathological changes were observed in ADR group, proteinuria, blood biochemical and renal pathological changes were more serious in mice dealt with agonist, while antagonist could reduce ADR-induced injury (all P＜0.05). Results of immunohistochemistry showed that the expression of podocyte-related proteins nephrin, podocin and CD2AP in ADR group were significantly lower than those in control group, and EP1 agonist could further inhibit expression of these proteins, while antagonist could reverse this inhibitory effects (P＜0.05). Electron microscopic results showed that mice in ADR group appeared foot enlargement and fusion, and the agonist group further aggravated the injury, while antagonist intervention could inhibit the injury of podocytes. (2) In vitro experiments: Compared with control group, expression of PGE2 and COX2 were increased; mRNA and protein expression of nephrin, podocin, CD2AP were decreased, p38 MAPK activity and podocytes apoptosis were increased in ADR group (P＜0.05); Agonist could aggravate podocytes damage (P＜0.05), while Antagonist could down-regulate the expression of PGE2 and COX2, promote the expression of nephrin, podocin and CD2AP, and inhibit the activity of p38 MAPK and podocytes apoptosis (P＜0.05). The addition of p38 MAPK inhibitor(10 μmol/L) could reduce the inhibitory effect of EP1 agonist on the expression of podocyte-related proteins nephrin, podocin and CD2AP (P＜0.05). Conclusions EP1 receptor may activate the p38 MAPK signaling pathway to inhibit podocytes-related proteins nephrin, podocin and CD2AP, as well as mediate the ADR induced podocyte injury. Inhibition of EP1 receptor however have a protective effect.     

Nephrotic plasma alters slit diaphragm-dependent signaling and translocates nephrin, Podocin, and CD2 associated protein in cultured human podocytes

Podocytes are critical in maintaining the filtration barrier of the glomerulus and are dependent on the slit diaphragm (SD) proteins nephrin, podocin, and CD2-associated protein (CD2AP) to function optimally. The effects of normal human plasma and nephrotic plasma on podocytes were tested, focusing particularly on the SD complex. With the use of a conditionally immortalized human podocyte cell line, it first was shown that exposure to normal and non-nephrotic human plasma leads to a concentration of nephrin, podocin, CD2AP, and actin at the cell surface. Next, the effects of plasma from patients with nephrotic conditions to non-nephrotic conditions were compared. When exposed to all nephrotic plasma samples (and a non-human serum control), nephrin podocin and CD2AP assumed a cytoplasmic distribution; nephrin and synaptopodin were selectively downregulated, and the relocation of nephrin induced by nephrotic plasma could be rescued back to the plasma membrane by co-incubation with non-nephrotic plasma. Furthermore, intracellular calcium signaling was altered by nephrotic plasma, which was mediated by tyrosine kinase phosphorylation. With the use of nephrin mutant human cell lines, it was shown that this signaling and translocation response to normal plasma is nephrin dependent. This work demonstrates that nephrotic plasma seems to be deficient in factors that act via the podocyte SD complex, which are essential in maintaining its physiologic function.     

通过基因敲低探讨多个足细胞分子作用及分子间反应

目的 研究足细胞裂孔隔膜(SD)复合体分子nephrin&#65380;podocin和CD2AP,以及足细胞骨架蛋白α-辅肌动蛋白(actinin)-4的作用及分子间反应&#65377;方法 针对nephrin&#65380; podocin&#65380;CD2AP和α-actinin-4 mRNA序列分别设计并构建2个特异RNA干扰质粒-psiRNA-hH1GFPzeo,分别导入小鼠足细胞系MPC5以 “敲低”其表达&#65377;免疫荧光染色观察其分布方式&#65377;半定量RT-PCR和免疫蛋白印迹检测其mRNA和蛋白表达&#65377; 结果 (1) podocin敲低组(siPod966和siPod54):未检测到podocin及nephrin mRNA,其蛋白分别下降了92%&#65380;79%及82%&#65380;67%&#65377;而CD2AP mRNA和蛋白分别增加了62%&#65380;42%及71%&#65380;46%&#65377;α-actinin-4无变化&#65377;(2)nephrin敲低组(siNep492):未检测到nephrin mRNA和蛋白&#65377;而CD2AP mRNA和蛋白分别增加了35%&#65380;48%&#65377;Podocin和α-actinin-4无变化&#65377;(3)CD2AP敲低组(siCda744和siCda21):未检测到CD2AP mRNA,其蛋白分别下降了92%和83%&#65377;Nephrin mRNA和蛋白分别下降了60%&#65380;48%及76%&#65380;72%;而podocin mRNA和蛋白分别增加了38%&#65380;22%及56%&#65380;44%&#65377;Α-actinin-4无变化&#65377;(4)α-actinin-4敲低组(siAct1790和siAct319):α-actinin-4和nephrin 的mRNA分别下降了69%&#65380;58%及64%&#65380;49%;蛋白分别下降了81%和55%以及71%&#65380;64%&#65377;而podocin以及CD2AP mRNA分别增加了50%&#65380;34%及45%&#65380;28%;蛋白分别增加了64%&#65380;46%及65%&#65380;42%&#65377;(5)敲低nephrin&#65380;podocin和CD2AP后,这些表达量降低的分子的分布发生了明显改变,即以核周为主;而相应分子敲低后引起的podocin和CD2AP表达增加,其分布亦主要以核周染色增强为主&#65377;α-actinin-4即使表达降低,分布亦无变化,仍呈细丝状分布于胞质及足细胞伸出的突起中&#65377;结论 (1)在SD复合体分子中,nephrin可能具有相对独立的作用&#65377;(2) α-actinin-4对nephrin&#65380;podocin和CD2AP有直接或间接的作用&#65377;(3)足细胞分子间的作用和联系不总是“一致的”,可能是“单向的”&#65380;也可能是“双向的”&#65377;(4)nephrin&#65380;podocin&#65380;CD2AP和α-actinin-4在足细胞的分布有赖于其表达量的正常及正常的分子间反应&#65377;     

Clinical features and outcome of childhood minimal change nephrotic syndrome: is genetics involved?

The pathogenesis of minimal change nephrotic syndrome (MCNS) is still unknown. We performed a clinical and genetic evaluation of 104 adults (mean age 35 years) who presented with MCNS in childhood (mean follow-up 30 years). Clinical data and the present health status were evaluated. Also, the genes encoding the four major slit diaphragm proteins, nephrin, podocin, Neph1 and CD2-associated protein were sequenced in 38 patients with MCNS of varying severity. MCNS presented at the mean age of 5 years, and 80% of the patients relapsed 1–28 (median 3) times during childhood. The 14 subjects (14%) who had proteinuric episodes still in adulthood had a refractory disease already as children. The participants did not show a strong tendency for allergy or immune diseases, and no familial clustering of MCNS was observed. The genetic analyses revealed heterozygous amino acid changes in nephrin and podocin in 10 of the 38 patients studied. On the other hand, the genes coding for Neph1 and CD2AP were highly conserved and no amino acid substitutions were detected. In conclusion, MCNS is a multifactorial disease, in which genetics play a minor role. Allelic variants of the podocyte proteins may, however, modify the phenotype in occasional individuals.Anne-Tiina Lahdenkari and Maija Suvanto contributed equally     

Reduced podocin expression in minimal change disease and focal segmental glomerulosclerosis is related to the level of proteinuria

Background

Glomerular podocyte molecules are involved in the pathogenesis of congenital nephrotic syndrome. However, their role in primary nephrotic syndrome is not clear. This study investigated the expression of nephrin, podocin and synaptopodin in primary nephrotic syndrome.

Methods

Eighty-seven patients with primary nephrotic syndrome including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and membranoproliferative glomerulonephritis Type I (MPGN) were included in the study. Glomerular expression of nephrin, podocin and synaptopodin was studied in renal biopsies by immunofluorescence and immunohistochemistry. Correlation of expression with clinical and biochemical parameters was performed.

Results

The pattern of expression for all podocyte proteins in controls was uniform fine granular along the capillary walls towards the visceral epithelial cell aspect. Glomerular expression of nephrin was present in all renal biopsies and was similar to that in controls. Glomerular synaptopodin expression was seen in all MN and MPGN patients, while it was seen in 74 % (17/23) MCD and 93.5 % (29/31) FSGS. Reduced synaptopodin expression showed no correlation with clinical and biochemical factors. Podocin expression was present in 5/23 MCD (22 %), 3/31 FSGS (9.6 %), 13/17 MN (76.4 %) and 13/16 MPGN (81 %) patients. The reduced expression of podocin significantly correlated with the degree of proteinuria (p = 0.032). No correlation with age, gender and serum creatinine level was observed.

Conclusion

Reduction of glomerular podocin expression found in MCD and FSGS is related to the amount of proteinuria. Our findings suggest that alteration in podocyte phenotype may not be a primary event and may reflect the degree of podocyte injury in primary nephrotic syndrome.     

足细胞分子高表达致阿霉素肾病大鼠发生蛋白尿

目的 动态观察足细胞裂孔隔膜复合体分子nephrin、podocin、CD2AP及α-actinin-4在阿霉素肾病大鼠蛋白尿发生发展中的表达变化，探讨其在蛋白尿发生发展中的分子行为及其机制。方法 尾静脉注射阿霉素建立阿霉素肾病大鼠模型，3、7、14、28 d每组处死6只大鼠留取肾脏标本。应用间接免疫荧光染色、实时定量PCR、Western 印迹分别检测各个时间点nephrin、podocin、CD2AP、α-actinin-4的分布、mRNA和蛋白表达量的变化。结果 (1)14 d肾病组大鼠24 h尿蛋白显著高于对照组(P < 0.01)，增高持续到28 d。(2)透射电镜显示14 d肾病组足突不同程度变宽，28 d足突弥漫性融合。(3)与对照组相比，从第7天开始肾病组nephrin和podocin染色从沿肾小球毛细血管袢线样分布向不连续粗颗粒样的分布模式转变；CD2AP节段染色增强区逐渐扩大，有的区域呈斑片状和连续线状增强；α-actinin-4从沿肾小球毛细血管袢均匀的点线样分布向不均匀粗颗粒的分布转变，而且随时间进展这种转变逐渐加重。(4)与对照组相比，肾病组于7 d时nephrin mRNA表达显著增高(P < 0.01)；podocin mRNA表达14 d时显著增高(P < 0.05)，直至28 d(P < 0.05)；CD2AP mRNA表达28 d时显著增高(P < 0.05)。(5)与对照组相比，肾病组nephrin蛋白表达28 d时显著增高(P < 0.05)；podocin蛋白表达于7 d时显著增高(P < 0.05)，而28 d时又显著降低(P < 0.05)；CD2AP蛋白表达于14 d时显著增高(P < 0.05)，直至28 d (P < 0.05)。(6)α-actinin-4 mRNA与蛋白表达在实验过程中未出现明显变化。结论 nephrin、podocin和CD2AP的表达增加及分布异常是导致阿霉素肾病大鼠蛋白尿发生发展的分子机制，而分子表达的增加是足细胞抵抗损伤的一种代偿反应。     

Role of podocyte slit diaphragm as a filtration barrier

Although the role of glomerular basement membrane has been emphasised as the barrier for retaining plasma proteins in the past three decades, some recent studies have demonstrated that the slit diaphragm of the glomerular epithelial cell (podocyte) is the structure likely to be the barrier in the glomerular capillary wall. Nephrin and podocin were identified as gene products mutated in Finnish-type congenital nephrotic syndrome and autosomal recessive steroid-resistant nephrotic syndrome, respectively. Nephrin s located at the outer leaflet of plasma membranes of the slit diaphragm. Podocin is reported to have an interaction with nephrin. The anti-nephrin antibody is capable of inducing massive proteinuria, which indicates that nephrin is a key functional molecule in the slit diaphragm. The expression of nephrin and podocin was reduced in glomeruli of minimal change nephrotic syndrome, which suggested that the altered expression of these molecules contributes to the development of proteinuria also in acquired diseases. Some recent studies demonstrated that CD2-associated protein (CD2AP) is also a functional molecule in the slit diaphragm, and its expression is altered in membranous nephropathy. These observations suggested that alteration of the molecular arrangement in the slit diaphragm is involved in the development of proteinuria in several kinds of glomerular diseases.     

Expression of Fc epsilon receptor 2 in minimal change nephrotic syndrome]

In order to clarify the mechanism for elevation of serum IgE level in minimal change nephrotic syndrome (MCNS), we investigated Fc epsilon receptor 2 (Fc epsilon R2/CD23) expression, using fluorescein isothiocyanate (FITC) labeled anti-CD20 monoclonal antibody, phycoerythrin (PE) labeled anti-CD23 monoclonal antibody and two-color flow cytometry. Moreover, serum IgE level was examined by radio-immunosorbent test. The subjects included 25 cases of MCNS, 17 cases of focal glomerular sclerosis (FGS), and 25 healthy volunteers as controls. The patients in the nephrotic stage of MCNS demonstrated significantly elevated levels of serum IgE, while those in the remission stage of MCNS showed no change in their serum IgE levels. In patients with nephrotics of MCNS, CD23+ cells, CD20+ CD23+ cells and CD23/CD20 ratio were significantly increased compared to normal controls. Furthermore, CD23/CD20 ratio in MCNS was significantly correlated with serum IgE level. Concerning FGS, there were no differences in serum IgE level and Fc epsilon R2 expression compared to those of normal controls. These results suggest that Fc epsilon R2 expression may be important in the mechanism of elevated serum IgE level in MCNS.     

Investigation of CD68 positive monocytes/macrophage(CD68+ Mo/M phi) in urine and infiltrated tissue of various kidney diseases in children

In order to investigate the participation of monocytes/macrophages(Mo/M phi) in the progression of various kidney diseases of children, Mo/M phi in urine and that infiltrating renal tissue were both measured as the number of CD68 positive Mo/M phi (CD68+ Mo/M phi), using anti-CD68 antibody. The number of CD68+ Mo/M phi infiltrating in one glomerulus was significantly higher in Henoch-Sch?nlein purpura nephritis(HSPN) (p < 0.01) in comparison with that in minimal change nephrotic syndrome(MCNS) (p < 0.01), and a high tendency was found in IgA nephropathy (IgAN), proliferative glomerulonephritis (non-IgAN), focal segmental glomerulosclerosis(FSGS) and membranoproliferative glomerulonephritis (MPGN), respectively. The number of CD68+ Mo/M phi infiltrating one mm2 of tubulo-interstitium area was significantly higher in HSPN(p < 0.05), FSGS(p < 0.01), Alport's syndrome(p < 0.01), respectively, than that in MCNS. The number of CD68+ Mo/M phi in one milliliter of urine correlated significantly with both that infiltrating the glomerulus and the tubulo-interstitium(both p < 0.01). Moreover the number of urine CD68+ Mo/M phi in a clinically active stage was significantly higher than that in an inactive stage in the AGN(p < 0.05), IgAN(p < 0.05), HSPN(p < 0.05), non-IgAN(p < 0.01) and MPGN groups(p < 0.05), respectively. From these results, 1) It was suggested that the Mo/M phi infiltrating renal tissue participated in the development of various kidney diseases. 2) It was predicted that CD68+ Mo/M phi in urine reflected both the number of Mo/M phi infiltrating the glomerulus and that in the tubulo-interstitium. 3) It was suggested that the number of CD68+ Mo/M phi in urine indicated clinical activity in proliferative glomerulonephritis groups of children.     

Reduction of VEGF-A and CTGF expression in diabetic nephropathy is associated with podocyte loss

Micro-vascular and renal complications in diabetic patients are a considerable clinical challenge. In a previous study, we found a significant decrease in vascular endothelial growth factor A (VEGF-A) mRNA levels in glomeruli from patients with diabetic nephropathy (DN). We now set out to investigate the relationship between reduced VEGF-A and connective tissue growth factor (CTGF) expression levels, the number of podocytes, and the extent of interstitial fibrosis. Laser capture microdissection was applied to obtain glomerular RNA from 28 patients with DN and 22 controls. mRNA levels of VEGF-A, CTGF, nephrin, podocin, and Wilms tumor1 (WT1) were measured using real-time polymerase chain reaction. Protein expression was evaluated using immuno-stainings for VEGF-A and CTGF, as well as markers for podocytes (WT1) and endothelial cells (CD31). We found a significant decrease in glomerular mRNA levels for VEGF-A (2.5 times), CTGF (1.6), nephrin (2.8), podocin (3.3), and WT1 (1.7) in patients with DN. There was a significant correlation between expression of podocyte markers and VEGF-A mRNA levels, and an inverse correlation between podocin message and the extent of interstitial fibrosis. CD31-positive area was significantly decreased (3.2 times) in patients with DN. Reduction of angiogenic factors correlated with the extent of interstitial fibrosis. This downregulation was related to a reduction of podocytes in DN. The results may suggest that downregulation of VEGF-A and CTGF in DN is a result of podocyte loss.     

Expression of nephrin in acquired forms of nephrotic syndrome in childhood

Nephrin is a podocyte adhesion molecule located at the slit diaphragm between adjacent glomerular epithelial cells. Mutations in the gene encoding nephrin result in the absence of nephrin or alterations in nephrin causing massive proteinuria in patients with congenital nephrotic syndrome. Given the importance of nephrin to the structural integrity of the glomerular filtration barrier, we postulated that it might also be altered in acquired forms of nephrotic syndrome (NS). To test this hypothesis, frozen kidney biopsy sections from 29 pediatric patients with acquired NS and 5 controls were examined for expression of nephrin. The pathological diagnoses were minimal change disease (MCNS) (19) and focal segmental glomerulosclerosis (FSGS) (10). To determine if nephrin expression differed between children and adults with NS, 10 adult patients and 3 controls were also examined. Nephrin expression was evaluated by immunoperoxidase staining with a monoclonal antibody against the extracellular FnIII portion of human nephrin. In all cases, nephrin expression was seen along the glomerular basement membrane in a finely granular/linear pattern. Expression of nephrin was similar to controls in all 19 patients with MCNS and all 10 patients with FSGS. Areas of sclerosis in patients with FSGS did not demonstrate nephrin expression. A distinctly granular pattern to nephrin expression was seen in adult patients with NS as well as controls. These findings suggest that an alteration in nephrin expression is not a feature of acquired forms of NS in childhood.     

Expression of human nephrin mRNA in diabetic nephropathy.

BACKGROUND: Diabetic nephropathy (DN) is associated with functional changes in the filtration barrier, and microalbuminuria is a strong predictor of the development of overt DN. Nephrin is a novel podocyte-specific protein which localizes at the slit diaphragm. This study examines the expression of nephrin mRNA in the kidneys of type 2 diabetics with DN. METHODS: Renal tissues were obtained from 13 type 2 diabetics with DN. We also examined samples from five patients with minimal change nephrotic syndrome (MCNS) and five normal kidneys (normals) as control. The severity of DN was classified into two grades based on histopathological findings. DN grade 1 (DN1 = seven patients) presented mild mesangial expansion, and DN grade 2 (DN2 = six patients) moderate mesangial expansion. Nephrin mRNA was quantitated and localized by in situ hybridization. RESULTS: Cells positive for nephrin mRNA were detected exclusively in glomerular epithelial cells. The percentage of cells positive for nephrin mRNA in DN2 was significantly lower than in MCNS and normal kidneys. Furthermore, there was an inverse correlation between the percentage of cells positive for nephrin mRNA and extent of proteinuria. CONCLUSION: The low expression of nephrin mRNA may be closely linked to development and/or progression of proteinuria in human diabetic nephropathy.     

IgA肾病中足细胞损伤及其与蛋白尿的关系

目的 观察 IgA肾病（IgAN）患者足细胞损伤的各种表现，探讨其与蛋白尿的关系。 方法 收集35例伴有明显蛋白尿[尿蛋白量（24 h）>1.0 g]的IgAN患者肾活检组织作研究；以8例肾错构瘤患者术后切除肾和肾癌患者术后远离癌旁肾组织为正常对照。免疫组化方法观察肾组织细胞周期调节蛋白（p21、p27）、足细胞结构蛋白（nestin）、足细胞数目 （WT1）。用显微切割方法取出肾小球，通过实时定量PCR方法检测整合素（integrin）β1、nephrin和α辅肌动蛋白4（α-actinin 4）水平。电镜观察足细胞超微结构的改变。根据足细胞数目密度(Nv, n×106/μm3)将35例IgAN患者分为足细胞数目减少组（ Nv<52.49×106/μm3，n = 15）和足细胞数目正常组（Nv≥52.49×106/μm3，n = 20）。随访蛋白尿的转归情况，共18个月。 结果 （1）与正常对照组比较，IgAN患者肾小球内个别足细胞重新表达p21，而足细胞p27的表达明显降低（0.71±0.12比0.91±0.07，P < 0.05）。（2）IgAN患者足细胞nestin 蛋白表达比正常对照显著降低（13.40%±0.04%比 17.60%±0.04%，P < 0.05）；肾小球内integrin-β1 mRNA表达显著升高（12.54±5.20比1.02±0.30，P < 0.05），而nephrin及α-actinin4 mRNA无明显改变。（3）电镜下观察到明显的足突融合和足细胞从基底膜脱落。（4）IgAN患者足细胞数目密度比正常对照组显著减少(161.27±225.92比323.22±138.12，P < 0.05)，且与Lee氏分级相关。（5）足细胞数目密度、integrin-β1 mRNA与肾穿刺当时的尿蛋白量（24 h）呈负相关(r = -0.4483、-0.840, 均P < 0.05)。足细胞数目减少组较足细胞数目正常组的蛋白尿下降程度明显减少（P < 0.05）。 结论 伴蛋白尿的IgAN中存在足细胞的损伤，表现为足细胞周期调节蛋白、结构蛋白的改变，足突的融合及足细胞数目的减少，而足细胞损伤及足细胞数目减少会影响蛋白尿的发生和发展。     

Overexpression of interleukin-13 induces minimal-change-like nephropathy in rats

IL-13 has been implicated in the pathogenesis of minimal-change nephrotic syndrome. This study aimed to investigate the role of IL-13 on the development of proteinuria and expression of podocyte-related genes that are associated with nephrotic syndrome. IL-13 was overexpressed in Wistar rats through transfection of a mammalian expression vector cloned with the rat IL-13 gene, into the quadriceps by in vivo electroporation. Serum IL-13, albumin, cholesterol, and creatinine and urine albumin were measured serially. Kidneys were harvested after day 70 for histology and electron microscopy. Glomerular gene expression of nephrin, podocin, dystroglycan, B7-1, and IL-13 receptor subunits were examined using real-time PCR with hybridization probes and expressed as an index against beta-actin. Protein expression of these molecules was determined by immunofluorescence staining. The IL-13-transfected rats (n = 41) showed significant albuminuria, hypoalbuminemia, and hypercholesterolemia when compared with control rats (n = 17). No significant histologic changes were seen in glomeruli of IL-13-transfected rats. However, electron microscopy showed up to 80% of podocyte foot process fusion. Glomerular gene expression was significantly upregulated for B7-1, IL-4Ralpha, and IL-13Ralpha2 but downregulated for nephrin, podocin, and dystroglycan. Immunofluorescence staining intensity was reduced for nephrin, podocin, and dystroglycan but increased for B7-1 and IL-4Ralpha in IL-13-transfected rats compared with controls. In conclusion, these results suggest that IL-13 overexpression in the rat could lead to podocyte injury with downregulation of nephrin, podocin, and dystroglycan and a concurrent upregulation of B7-1 in the glomeruli, inducing a minimal change-like nephropathy that is characterized by increased proteinuria, hypoalbuminemia, hypercholesterolemia, and fusion of podocyte foot processes.