Early glomerular filtration defect and severe renal disease in podocin-deficient mice

Podocytes are specialized epithelial cells covering the basement membrane of the glomerulus in the kidney. The molecular mechanisms underlying the role of podocytes in glomerular filtration are still largely unknown. We generated podocin-deficient (Nphs2-/-) mice to investigate the function of podocin, a protein expressed at the insertion of the slit diaphragm in podocytes and defective in a subset of patients with steroid-resistant nephrotic syndrome and focal and segmental glomerulosclerosis. Nphs2-/- mice developed proteinuria during the antenatal period and died a few days after birth from renal failure caused by massive mesangial sclerosis. Electron microscopy revealed the extensive fusion of podocyte foot processes and the lack of a slit diaphragm in the remaining foot process junctions. Using real-time PCR and immunolabeling, we showed that the expression of other slit diaphragm components was modified in Nphs2-/- kidneys: the expression of the nephrin gene was downregulated, whereas that of the ZO1 and CD2AP genes appeared to be upregulated. Interestingly, the progression of the renal disease, as well as the presence or absence of renal vascular lesions, depends on the genetic background. Our data demonstrate the crucial role of podocin in the establishment of the glomerular filtration barrier and provide a suitable model for mapping and identifying modifier genes involved in glomerular diseases caused by podocyte injuries.     

An update: the role of Nephrin inside and outside the kidney

Nephrin is a key molecule in podocytes to maintain normal slit diaphragm structure. Nephin interacts with many other podocyte and slit diaphragm protein and also mediates important cell signaling pathways in podocytes. Loss of nephrin during the development leads to the congenital nephrotic syndrome in children. Reduction of nephrin expression is often observed in adult kidney diseases including diabetic nephropathy and HIV-associated nephropathy. The critical role of nephrin has been confirmed by different animal models with nephrin knockout and knockdown. Recent studies demonstrate that knockdown of nephrin expression in adult mice aggravates the progression of unilateral nephrectomy and Adriamycin-induced kidney disease. In addition to its critical role in maintaining normal glomerular filtration unit in the kidney, nephrin is also expressed in other organs. However, the exact role of nephrin in kidney and extra-renal organs has not been well characterized. Future studies are required to determine whether nephrin could be developed as a drug target to treat patients with kidney disease.     

Planar Cell Polarity Pathway Regulates Nephrin Endocytosis in Developing Podocytes

The noncanonical Wnt/planar cell polarity (PCP) pathway controls a variety of cell behaviors such as polarized protrusive cell activity, directional cell movement, and oriented cell division and is crucial for the normal development of many tissues. Mutations in the PCP genes cause malformation in multiple organs. Recently, the PCP pathway was shown to control endocytosis of PCP and non-PCP proteins necessary for cell shape remodeling and formation of specific junctional protein complexes. During formation of the renal glomerulus, the glomerular capillary becomes enveloped by highly specialized epithelial cells, podocytes, that display unique architecture and are connected via specialized cell-cell junctions (slit diaphragms) that restrict passage of protein into the urine; podocyte differentiation requires active remodeling of cytoskeleton and junctional protein complexes. We report here that in cultured human podocytes, activation of the PCP pathway significantly stimulates endocytosis of the core slit diaphragm protein, nephrin, via a clathrin/β-arrestin-dependent endocytic route. In contrast, depletion of the PCP protein Vangl2 leads to an increase of nephrin at the cell surface; loss of Vangl2 functions in Looptail mice results in disturbed glomerular maturation. We propose that the PCP pathway contributes to podocyte development by regulating nephrin turnover during junctional remodeling as the cells differentiate.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

MAGI-1 Interacts with Nephrin to Maintain Slit Diaphragm Structure through Enhanced Rap1 Activation in Podocytes

MAGI-1 is a multidomain cytosolic scaffolding protein that in the kidney is specifically located at the podocyte slit diaphragm, a specialized junction that is universally injured in proteinuric diseases. There it interacts with several essential molecules, including nephrin and neph1, which are required for slit diaphragm formation and as an intracellular signaling hub. Here, we show that diminished MAGI-1 expression in cultured podocytes reduced nephrin and neph1 membrane localization and weakened tight junction integrity. Global magi1 knock-out mice, however, demonstrated normal glomerular histology and function into adulthood. We hypothesized that a second mild but complementary genetic insult might induce glomerular disease susceptibility in these mice. To identify such a gene, we utilized the developing fly eye to test for functional complementation between MAGI and its binding partners. In this way, we identified diminished expression of fly Hibris (nephrin) or Roughest (neph1) as dramatically exacerbating the effects of MAGI depletion. Indeed, when these combinations were studied in mice, the addition of nephrin, but not neph1, heterozygosity to homozygous deletion of MAGI-1 resulted in spontaneous glomerulosclerosis. In cultured podocytes, MAGI-1 depletion reduced intercellular contact-induced Rap1 activation, a pathway critical for proper podocyte function. Similarly, magi1 knock-out mice showed diminished glomerular Rap1 activation, an effect dramatically enhanced by concomitant nephrin haploinsufficiency. Finally, combined overexpression of MAGI-1 and nephrin increased Rap1 activation, but not when substituting a mutant MAGI-1 that cannot bind nephrin. We conclude that the interaction between nephrin and MAGI-1 regulates Rap1 activation in podocytes to maintain long term slit diaphragm structure.     

Nephrin--a unique structural and signaling protein of the kidney filter

Since the discovery of nephrin, the first integral component of the slit diaphragm to be identified, the podocyte slit pore has become a major focus in research concerning the glomerular filtration barrier. Nephrin is a central component of the glomerular ultrafilter, with both structural and signaling functions. The extracellular domain of nephrin and other components of the slit diaphragm seem to form a porous molecular sieve. The intracellular domain of nephrin is associated with linker proteins, such as CD2-associated protein and Nck proteins that can connect nephrin to the actin cytoskeleton. Alterations in nephrin interactions with other proteins during development or injury can lead to complex signaling reactions aimed at establishing or restoring the filter function.     

Advanced oxidation protein products decrease expression of nephrin and podocin in podocytes via ROS-dependent activation of p38 MAPK

Accumulation of plasma advanced oxidation protein products(AOPPs) promotes progression of proteinuria and glomerulo-sclerosis.To investigate the molecular basis of AOPPs-induced proteinuria,normal Sprague-Dawley rats were treated with AOPPs-modified rat serum albumin.The expression of glomerular podocyte slit diaphragm(PSD)-associated proteins,nephrin and podocin,was significantly decreased coincident with the onset of albuminuria in rats treated with AOPPs.Chronic inhibi-tion of NADPH oxidase by apocynin p...     

Nephrin expression is increased in anti-Thy1.1-induced glomerulonephritis in rats

Nephrin is an important constituent of the glomerular filtration barrier and alteration of its expression is associated with severe proteinuria. In this study we show that injection of an anti-Thy1.1 antibody in rats not only induces a mesangioproliferative glomerulonephritis associated with increased proteinuria, but also leads to a sustained increase of nephrin mRNA and protein expression in renal glomeruli over a time period of 29 days. In contrast, podocin and CD2AP, two proteins shown to interact with nephrin in the slit diaphragm, are acutely downregulated at days 3-7 and, thereafter, recovered again to normal levels after 29 days. Interestingly, immunofluorescence staining of kidney sections at day 10 of the disease shows a highly heterogeneous pattern, in that some podocytes show complete absence of nephrin, whereas others show highly accumulated staining for nephrin compared to control sections, which in total results in an increased level of nephrin per glomerulus. In summary, our data show that in the course of mesangioproliferative glomerulonephritis in rats, an upregulation of nephrin expression occurs with a concomitant transient downregulation of podocin and CD2AP which may account for a highly dysregulated filtration barrier and increased proteinuria.     

Renal Effects and Underlying Molecular Mechanisms of Long-Term Salt Content Diets in Spontaneously Hypertensive Rats

Several evidences have shown that salt excess is an important determinant of cardiovascular and renal derangement in hypertension. The present study aimed to investigate the renal effects of chronic high or low salt intake in the context of hypertension and to elucidate the molecular mechanisms underlying such effects. To this end, newly weaned male SHR were fed with diets only differing in NaCl content: normal salt (NS: 0.3%), low salt (LS: 0.03%), and high salt diet (HS: 3%) until 7 months of age. Analysis of renal function, morphology, and evaluation of the expression of the main molecular components involved in the renal handling of albumin, including podocyte slit-diaphragm proteins and proximal tubule endocytic receptors were performed. The relationship between diets and the balance of the renal angiotensin-converting enzyme (ACE) and ACE2 enzymes was also examined. HS produced glomerular hypertrophy and decreased ACE2 and nephrin expressions, loss of morphological integrity of the podocyte processes, and increased proteinuria, characterized by loss of albumin and high molecular weight proteins. Conversely, severe hypertension was attenuated and renal dysfunction was prevented by LS since proteinuria was much lower than in the NS SHRs. This was associated with a decrease in kidney ACE/ACE2 protein and activity ratio and increased cubilin renal expression. Taken together, these results suggest that LS attenuates hypertension progression in SHRs and preserves renal function. The mechanisms partially explaining these findings include modulation of the intrarenal ACE/ACE2 balance and the increased cubilin expression. Importantly, HS worsens hypertensive kidney injury and decreases the expression nephrin, a key component of the slit diaphragm.     

Rosuvastatin beneficially alters the glomerular structure of kidneys from spontaneously hypertensive rats (SHRs)

The incidence of chronic renal diseases is increasing worldwide, and there is a great need to identify therapies capable of arresting or reducing disease progression. The current treatment of chronic nephropathies is limited to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers, but increasing clinical and experimental evidence suggests that statins could play a therapeutic role. Ultrastructural studies have shown the presence of gap junctions within all the cells of the glomerulus and podocytes have been found to contain primarily connexin-43. The present study aims to observe the beneficial effects of rosuvastatin on structural and ultrastructural renal morphology and on glomerular connexin-43 expression in normotensive rats and spontaneously hypertensive rats (SHR). Rats were randomly allocated into four groups: WKY-C: normotensive animals no receiving rosuvastatin; WKY-ROS: normotensive animals receiving rosuvastatin; SHR-C: hypertensive animals no receiving rosuvastatin; SHR-ROS: hypertensive animals receiving rosuvastatin. Our results show no differences in blood urea, creatinine, uric acid and creatine phosphokinase levels between the groups, however, there was an decreasing of 24-h protein excretion in SHR-ROS. Capsular area in SHR-ROS was decreased, however, there was no alteration in urinary space. By transmission electron microscopy the slit diaphragm and podocyte foot processes were more preserved in SHR-ROS. By scanning electron microscopy the podocyte foot processes were more preserved in SHR-ROS. Increased connexin-43 immunofluorescence was observed in glomeruli of WKY-ROS and SHR-ROS. In conclusion, we hypothesize that renal pleiotropic effect of rosuvastatin can be a therapeutic tool for improving kidney ultrastructure and, consequently, renal function in hypertensive individuals.     

The activation of extracellular signal-regulated kinase is responsible for podocyte injury

Podocyte and its slit diaphragm play an important role in maintaining normal glomerular filtration barrier function and structure. Podocyte apoptosis and slit diaphragm injury leads to proteinuria and glomerulosclerosis. However, the molecular mechanism of podocyte injury remains poorly understood. The family of mitogen-activated protein kinases including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase, and p38 signal pathways, are implicated in the progression of various glomerulopathies. However, the role of the activated signal pathway(s) in podocyte injury is elusive. This study examined phosphorylation of ERK in rat puromycin aminonucleoside (PAN) nephropathy as well as conditionally immortalized mouse podocyte treated with PAN in vitro. The effect of treatment with U0126, an inhibitor of ERK, was also investigated. In PAN nephropathy, the phosphorylation of ERK was marked. In podocyte injury, the marked and sustained activation of ERK pathway was also observed before the appearance of significant podocyte apoptosis. Pretreatment with U0126 to podocyte completely inhibited ERK activation, with complete suppression podocyte apoptosis and ameliorated nephrin protein expression along with the phosphorylation of nephrin in podocyte injury. In cultured podocyte, PAN induced actin recorganition, and U0126 inhibited such change. However, U0126 did not recovery the phosphorylation change of neph1 in podocyte injury. We concluded that the sustained activation of ERK along with the phosphorylation of neph1 might be necessary for podocyte injury. The study here suggested that ERK might become a potential target for therapeutic intervention to prevent podocytes from injury which will result in proteinuria.     

Increased urinary excretion of nephrin, podocalyxin, and βig-h3 in women with preeclampsia

Emerging evidence has shown that podocyte injury and reduced specific podocyte protein expressions contribute to proteinuria in preeclampsia. We collected urine specimens from women with preeclampsia to study whether podocyte-specific protein shedding is associated with renal barrier dysfunction. Urine specimens from women with normal pregnancies and from pregnant women complicated by chronic hypertension were used for comparison. We determined soluble podocyte slit protein nephrin levels in the urine specimens. Podocalyxin, βig-h3, and VEGF concentrations were also measured. We found that nephrin and podocalyxin were barely detectable in the urine specimens from normal pregnant women and from women with chronic hypertension. In preeclampsia, urinary nephrin and podocalyxin concentrations were significantly increased and highly correlated to each other, r(2) = 0.595. Nephrin and podocalyxin were also correlated with urine protein concentrations. βig-h3 was detected in the urine specimens from women with preeclampsia, and it is highly correlated with nephrin and podocalyxin concentrations in preeclampsia. βig-h3 was undetectable in normal pregnancy and pregnancy complicated by chronic hypertension. Elevated VEGF levels were also found in women with preeclampsia compared with those of normal pregnancy and pregnancy complicated by chronic hypertension. These results provide strong evidence that podocyte protein shedding occurs in preeclampsia, and their levels are associated with proteinuria. The finding of urinary βig-h3 excretion in preeclampsia suggests that increased transforming growth factor activity might also be involved in the kidney lesion in this pregnancy disorder.     

Ischemic injury to kidney induces glomerular podocyte effacement and dissociation of slit diaphragm proteins Neph1 and ZO-1

Glomerular injury is often characterized by the effacement of podocytes, loss of slit diaphragms, and proteinuria. Renal ischemia or the loss of blood flow to the kidneys has been widely associated with tubular and endothelial injury but rarely has been shown to induce podocyte damage and disruption of the slit diaphragm. In this study, we have used an in vivo rat ischemic model to demonstrate that renal ischemia induces podocyte effacement with loss of slit diaphragm and proteinuria. Biochemical analysis of the ischemic glomerulus shows that ischemia induces rapid loss of interaction between slit diaphragm junctional proteins Neph1 and ZO-1. To further understand the effect of ischemia on molecular interactions between slit diaphragm proteins, a cell culture model was employed to study the binding between Neph1 and ZO-1. Under physiologic conditions, Neph1 co-localized with ZO-1 at cell-cell contacts in cultured human podocytes. Induction of injury by ATP depletion resulted in rapid loss of Neph1 and ZO-1 binding and redistribution of Neph1 and ZO-1 proteins from cell membrane to the cytoplasm. Recovery resulted in increased Neph1 tyrosine phosphorylation, restoring Neph1 and ZO-1 binding and their localization at the cell membrane. We further demonstrate that tyrosine phosphorylation of Neph1 mediated by Fyn results in significantly increased Neph1 and ZO-1 binding, suggesting a critical role for Neph1 tyrosine phosphorylation in reorganizing the Neph1-ZO-1 complex. This study documents that renal ischemia induces dynamic changes in the molecular interactions between slit diaphragm proteins, leading to podocyte damage and proteinuria.     

Super-resolved local recruitment of CLDN5 to filtration slits implicates a direct relationship with podocyte foot process effacement

Under healthy conditions, foot processes of neighbouring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Besides slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D-structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin-5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement.     

Vitamin D3 ameliorates podocyte injury through the nephrin signalling pathway

Renal podocytes form the main filtration barrier possessing unique phenotype maintained by proteins including podocalyxin and nephrin, which are modulated in pathological conditions. In diabetic nephropathy (DN), podocytes become structurally and functionally compromised. Nephrin, a structural backbone protein of the slit diaphragm, acts as regulator of podocyte intracellular signalling with renoprotective role. Vitamin D₃ through its receptor, VDR, provides renal protection in DN but limited data exist about its effect on podocytes. In this study, we used isolated rat glomeruli to assess podocalyxin and nephrin expression after treatment with the 1,25‐dihydroxyvitamin D₃ analogue paricalcitol in the presence of normal and diabetic glucose levels. The role of 1,25‐dihydroxyvitamin D₃ (calcitriol) and its analogue, paricalcitol, on podocyte morphology and survival was also investigated in the streptozotocin (STZ)‐diabetic animal model. In our ex vivo model, glomeruli exhibited high glucose‐mediated down‐regulation of podocalyxin, and nephrin, while paricalcitol reversed the high glucose‐induced decrease of nephrin and podocalyxin expression. Paricalcitol treatment enhanced VDR expression and promoted VDR and RXR co‐localization in the nucleus. Our data also indicated that hyperglycaemia impaired survival of cultured glomeruli and suggested that the implemented nephrin down‐regulation was reversed by paricalcitol treatment, initiating Akt signal transduction which may be involved in glomerular survival. Our findings were further verified in vivo, as in the STZ‐diabetic animal model, calcitriol and paricalcitol treatment resulted in significant amelioration of hyperglycaemia and restoration of nephrin signalling, suggesting that calcitriol and paricalcitol may provide molecular bases for protection against loss of the permselective renal barrier in DN.     

TM4SF10 and ADAP interaction in podocytes: role in Fyn activity and nephrin phosphorylation

TM4SF10 [transmembrane tetra(4)-span family 10] is a claudin-like cell junction protein that is transiently expressed during podocyte development where its expression is downregulated in differentiating podocytes coincident with the appearance of nephrin at the slit diaphragm. In a yeast two-hybrid screen, we identified adhesion and degranulation-promoting adaptor protein (ADAP), a well-known Fyn substrate and Fyn binding partner, as a TM4SF10 interacting protein in mouse kidney. Using coimmunoprecipitation and immunohistochemistry experiments in cultured human podocytes, we show that TM4SF10 colocalizes with Fyn and ADAP but does not form a stable complex with Fyn. Cytoskeletal changes and phosphorylation events mediated by Fyn activity were reversed by TM4SF10 overexpression, including a decrease in the activating tyrosine phosphorylation of Fyn (Y(421)), suggesting TM4SF10 may have a regulatory role in suppressing Fyn activity. In addition, TM4SF10 was reexpressed following podocyte injury by puromycin aminonucleoside treatment, and its expression enhanced the abundance of high-molecular-weight forms of nephrin indicating it may participate in a mechanism controlling nephrin's appearance at the plasma membrane. Therefore, these studies have identified ADAP as another Fyn adapter protein expressed in podocytes, and that TM4SF10, possibly through ADAP, may regulate Fyn activity. Since TM4SF10 expression is temporally regulated during kidney development, these studies may help define a mechanism by which the slit diaphragm matures as a highly specialized cell junction during podocyte differentiation.     

Diversities of podocyte molecular changes induced by different antiproteinuria drugs

Nephrin, podocin, CD2AP, and alpha-actinin-4 are important podocyte proteins that help maintain the integrity of the slit diaphragm and prevent proteinuria. Studies have shown that angiotensin-converting enzyme inhibitors, glucocorticoids, and all-trans retinoic acid (ATRA) have antiproteinuric effects. However, it is still unclear whether these drugs, with different pharmacological mechanisms, lead to a reduction in proteinuria by changing the expression and distribution of these important podocyte proteins. In this study, changes in the expression and distribution of nephrin, podocin, CD2AP, and alpha-actinin-4 were dynamically detected in Adriamycin-induced nephrotic (ADR) rats treated with three different drugs: lisinopril, prednisone, and ATRA. Nephropathy was induced by an intravenous injection of Adriamycin. After Adriamycin injection, rats received lisinopril, prednisone, and ATRA treatment, respectively. Renal tissues were collected at Days 3, 7, 14, and 28. The distribution and the expression of messenger RNA and protein of nephrin, podocin, CD2AP, and alpha-actinin-4 were detected by indirect immunofluorescence, real-time polymerase chain reaction, and Western blotting, respectively. With the intervention of lisinopril, prednisone, and ATRA, changes in the expression of nephrin, podocin, and CD2AP were diverse, which was different from that detected in ADR rats. After lisinopril and prednisone intervention, podocin exhibited prominent earlier changes compared with those of nephrin and CD2AP, whereas CD2AP showed more prominent changes after ATRA intervention. There was no change in the expression of alpha-actinin-4 molecule. In summary, we conclude that the antiproteinuric effects of lisinopril, prednisone, and ATRA were achieved by changes in the expression and distribution of the important podocyte molecules nephrin, podocin, CD2AP, and alpha-actinin-4. The pattern in the change of podocyte molecules after lisinopril and prednisone intervention was similar, but the pattern in the change of podocyte molecules after ATRA intervention was different from that of lisinopril or prednisone intervention.