Nephrotic Plasma Alters Slit Diaphragm–Dependent Signaling and Translocates Nephrin, Podocin, and CD2 Associated Protein in Cultured Human Podocytes
Richard J.M. Coward*,
Rebecca R. Foster,
David Patton,
Lan Ni*,
Rachel Lennon*,
David O. Bates,
Steven J. Harper*,,
Peter W. Mathieson* and
Moin A. Saleem*
* Academic and Children’s Renal Unit, University of Bristol; Microvascular Research Laboratories, Department of Physiology, University of Bristol, Preclinical Veterinary School; and Faculty of Applied Sciences, University of the West of England, Bristol, United Kingdom
Address correspondence to: Dr. Moin A. Saleem, Academic Renal Unit, Southmead Hospital, Bristol, UK. Phone: +44-117-9596048; Fax: +44-117-9595438; E-mail: m.saleem{at}bristol.ac.uk
Podocytes are critical in maintaining the filtration barrierof 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 andnephrotic plasma on podocytes were tested, focusing particularlyon the SD complex. With the use of a conditionally immortalizedhuman podocyte cell line, it first was shown that exposure tonormal and non-nephrotic human plasma leads to a concentrationof nephrin, podocin, CD2AP, and actin at the cell surface. Next,the effects of plasma from patients with nephrotic conditionsto non-nephrotic conditions were compared. When exposed to allnephrotic plasma samples (and a non-human serum control), nephrinpodocin and CD2AP assumed a cytoplasmic distribution; nephrinand synaptopodin were selectively downregulated, and the relocationof nephrin induced by nephrotic plasma could be rescued backto the plasma membrane by co-incubation with non-nephrotic plasma.Furthermore, intracellular calcium signaling was altered bynephrotic plasma, which was mediated by tyrosine kinase phosphorylation.With the use of nephrin mutant human cell lines, it was shownthat this signaling and translocation response to normal plasmais nephrin dependent. This work demonstrates that nephroticplasma seems to be deficient in factors that act via the podocyteSD complex, which are essential in maintaining its physiologicfunction.
In recent years, the podocyte slit diaphragm (SD) proteins havebeen shown to be central to the development of congenital andearly childhood-onset human nephrotic syndromes through theseminal work of Kestila and Boute (1,2). Furthermore, the importanceof CD2-associated protein (CD2AP) is critical in maintainingthe integrity of the slit diaphragm (3) and has been demonstratedto be related closely to nephrin, podocin, and actin in humanpodocytes in culture (4). Of great interest is the role of theseproteins in the much more common acquired nephrotic conditions,but in vitro work has been hampered by the lack of a representativehuman podocyte cell line. The podocyte displays a highly developedarchitectural phenotype, and in particular the SD is a uniquetype of cell junction (5), the permeability characteristicsof which are determined by the unique proteins located there.Of these proteins, nephrin has been shown to be capable of signalingin combination with podocin (6), and these molecules are knownto be intimately related to the actin cytoskeleton and to CD2APin the podocyte (4,7). We hypothesized that these critical SDproteins are maintained in their structural conformation byfactors in normal plasma that signal through this molecularcomplex. We studied the properties of the SD in a conditionallyimmortalized human cell line (8) when cultured in plasma frompatients with no renal disease and compared these propertieswith plasma from patients with nephrotic conditions (three patientswith focal segmental glomerulosclerosis [FSGS] in relapse andthe same patients in remission) and a patient with Lupus nephritis.These were compared and contrasted with standard fetal calfserum (FCS) culture conditions.
We further examined how nephrin-deficient podocyte cell linesbehave in conditions that mimic the normal and nephrotic podocytemilieu to ascertain the importance of nephrin for the functionof the SD complex. To determine whether these nephrin-mediatedeffects alter cellular physiology, we also measured the intracellularcalcium handling of wild-type (WT) and nephrin mutant cellswhen exposed to plasma. We present evidence for the presenceof factors in normal human plasma that are essential for maintenanceof the mature SD complex.
Human Podocyte Cell Culture
Conditionally immortalized WT human podocyte cells were developedby the use of the temperature-sensitive large T antigen-SV40transgene as described previously (8). These cells have beenshown to differentiate fully by 14 d after switching from 33to 37°C. Experiments to compare normal plasma with diseaseplasma were performed after day 14. Cell passages between 5and 20 were used in all experiments. Podocytes were routinelycultured in RPMI-1640 medium with glutamine (R-8758; Sigma,St. Louis, MO) supplemented with 10% FCS (Life Technologies,Grand Island, NY) and insulin transferrin sodium selenite (SigmaI-1184; 1 ml/100 ml). FCS was substituted with human plasmaat the same concentration (10%) for the experiments. A secondhuman podocyte cell line, developed in the same way in our laboratory,derived from the normal pole of a kidney with Wilm’s tumor,was also used in some experiments to confirm further immunofluorescence(IF) findings. This was to exclude clonal variation as a potentialexplanation for differences seen.
Patient Samples
Plasma was obtained from patients who had undergone therapeuticplasma exchange for biopsy-proven FSGS (two boys aged 14 and12 yr; one girl aged 11 yr) in relapse and the same patientsin remission at the end of their plasma exchange course, togetherwith a 14-yr-old girl who required plasma exchange for systemiclupus erythematosus (SLE) and a control patient with no renaldisease but on prophylactic plasma exchange for cryoglobulinemia.Normal human plasma (and serum) was examined from an AB Rhesus-negativedonor (Sigma) and from healthy adult volunteers. The profilesof the plasma samples are shown in Table 1.
Rescue Experiments
With rescue experiments, day 14 podocytes were initially exposedto 48 h of nephrotic or non-nephrotic plasma, and then the mediumwas changed with the initial plasma replaced at the same concentration(10%) and 10% of nephrotic or non-nephrotic plasma added foran additional 48 h.
Nephrin Mutant Podocyte Development
A kidney was removed for therapeutic reasons from a Finnishinfant (male, 0.9 yr) who had congenital nephrotic syndromesecondary to the homozygous Finmajor mutation (nt121 del 2),which results in a two-nucleotide deletion in exon 2 of chromosome19 and subsequently a truncation of the protein (1). Anothernephrin mutant kidney (female, 1.8 yr) that was removed becauseof severe early-onset nephrotic syndrome as a result of a homozygousmissense exon 11 mutation was also studied. Full informed consentfrom the family and ethical committee approval were obtainedfor use of the discarded kidneys. Primary culture, subcloning,and propagation were carried out as described previously (8).These cell lines were transfected with both SV40 large T antigengene as described previously and a telomerase construct (9),to prevent senescence. This allows the cell line to replicatead infinitum at 33°C, while the SV40 large T antigen wasswitched on, but at 37°C, the large T antigen is silencedand the cell line can differentiate. The cell lines when transformedwere characterized and expressed WT1, synaptopodin, podocin,and CD2AP on IF and/or Western blotting. No nephrin was detectableon IF of the kidney sections and on IF or Western blotting onthe podocyte cell line of the Fin major kidney (Figure 1). Cellpassage between 7 and 15 were used for all experiments. Cultureconditions were identical to the WT podocytes described above.All experiments on mutant cells were carried out in the Finmajor cell line, and the second mutant cell line was used foradditional data in the calcium flux experiments.
Figure 1. Characterization of the Fin major human podocyte cell line. Immunofluorescence (IF) staining and Western blotting of differentiated cells, either Fin major nephrin–deficient (–/–) or wild-type (WT) podocytes. (A) Nephrin IF using a polyclonal rabbit antibody K2966. Expression in WT podocyte but not nephrin knockouts. WT podocyte cultured exclusively in FCS giving cytoplasmic distribution of nephrin. (B) Nephrin Western blot. No expression in Fin major cells. Actin loading shown. (C) WT1 expressed in the nuclei of Fin major and WT podocytes. (D) Non-immunized rabbit Ig control. All above antibodies of rabbit origin.
Antibodies
Primary nephrin antibodies used included a rabbit polyclonalantibody (10) that recognizes the intracellular fragment ofthe molecule and a panel of both rabbit polyclonal and mousemonoclonal antibodies against the extracellular domain of nephrinas described previously (4). These all were gifts from KarlTryggvason (Karolinska Institute, Stockholm, Sweden). A rabbitpolyclonal podocin c-terminal antibody was used for both Westernblotting and IF (gift from Corinne Antignac, Paris, France)(8). Other antibodies used included synaptopodin (Progen, Heidelberg,Germany) and polyclonal rabbit antibodies raised against CD2APand WT1 (Santa Cruz Biotechnology, Santa Cruz, CA). Secondaryspecies-specific horseradish peroxidase–associated antibodies(Sigma) and FITC- and TRITC-labeled antibodies (Jackson Immunoresearch,Philadelphia, PA) were also used. The F-actin cytoskeleton wasimaged using a directly conjugated Texas red–Phalloidinstain (Molecular Probes, Eugene OR) and Western blotted witha monoclonal antibody (Sigma).
Immunofluorescence
Immunolabeling was done as described previously (4). Imageswere obtained by using a Leica photomicroscope attached to aSpot 2 slider digital camera (Diagnostic Instruments, SterlingHeights, MI) and processed with Adobe Photoshop 5.0 software.All images were analyzed by an investigator who was blindedto the identity of the samples and ranked from cortical to cytoplasmicdistribution of the molecules on a scale of 0 to 3. At least20 cells per coverslip were ranked. Each blinded experimentwas carried out on three independent occasions. Images weretaken at x400 magnification.
Environmental Scanning Electron Microscopy
Environmental scanning electron microscopy was performed asdescribed previously on these cells (8). The images were categorizedby two blinded investigators and ranked according to surfaceblebbing and fine process formation on a scale from 0 to 4.For each condition, at least five images were obtained and ranked,with four to 15 cells seen per image. Representative imagesare shown.
Western Blotting and Protein Extraction
The exact techniques as described previously were used for proteinextraction and Western blotting (8). Protein was quantifiedusing a modified Bradford assay (11) to ensure equal loadingof lanes in comparative studies and confirmed by actin loading.
Lipid Raft Fraction Preparation
For preparation of low-density Triton X-100–insolublemembrane domains, cells were treated for 48 h as before witheither normal plasma or FSGS relapse plasma. The protocol wasfollowed exactly as described previously (12). Fractions thenwere probed with anti-nephrin (48E11) or anti-actin antibodyas above.
Intracellular Calcium Studies
Cells were grown on coverslips and then incubated with Fura2-AM, excited, and assayed using ratiometric fluorescence measurementas described before (13). Undiluted samples of relapse and remissionplasma from one patient with FSGS were perfused in random orderon the same coverslip with a 20-min wash period of HBSS media(Life Technologies BRL) in between. HBSS was also used as abaseline control. A 10-min preincubation of 70 µM genistein(Sigma) was used to block tyrosine kinase. Control cells ofimmortalized proximal tubular cells (HK2) (14) and human veinembryonic endothelial cells (HUVEC) (ATCC, Manassas, VA) weretreated in exactly the same manner as the podocytes, and theirresponse to plasma was recorded. There was a replicate of atleast four in all sets of experiments.
Nephrin, CD2AP, Podocin, and Actin
After 48 h of exposure, there were differences in the distributionof nephrin, podocin, and CD2AP in differentiated podocytes thatwere exposed to nephrotic compared with non-nephrotic patientplasma. Nephrotic plasma–exposed podocytes demonstrateda cytoplasmic distribution of all three molecules. Nephrin wasobserved in a diffuse cytoplasmic and filamentous pattern (averagescore, 0.4; Figure 2), podocin distribution was predominantlydiffusely cytoplasmic (average score, 1.0), and CD2AP was seenin unevenly distributed "spots" in a submembranous and cytoplasmiclocation (average score, 0.2; Figure 3), as described previously(15). Plasma from all patients with nephrotic syndromes resultedin the same protein distribution, regardless of the underlyingnephrotic cause. In contrast, samples from non-nephrotic individualsand healthy volunteers (data not shown) resulted in a highlyenriched cortical, plasma membrane, location of nephrin (averagescore, 2.3), podocin (2.0), and CD2AP (2.6) in the differentiatedpodocyte (Figures 2 and 3). These enriched areas were not presentcontinuously around the plasma membrane or indeed specificallyat cell–cell junctions but seemed prominent at the leadingedge of softly rounded cell protrusions, suggestive of a subtlealteration of cell shape to coincide with this distribution.A similar distribution was seen using samples from normal volunteers,patients with no previous renal disease, and patients who werein remission from established FSGS. Intriguing, human plasma(Figure 2) and serum (data not shown) also affected the locationof nephrin when compared with FCS in podocytes of the same passageand age, with increased cortically located nephrin in the humancompared with bovine samples. Prolonged culture in serum-freemedium resulted in podocyte detachment, granulation, and necrosis,so these conditions were not included in the control experiments.Cellular morphology was disrupted in the presence of plasmafrom nephrotic patients, which occurred as early as 6 h, withretraction of fine process formation (average score, 1.2) andblebbing (average score, 2.9) on the cell surface noted (Figure 4)when viewed by environmental scanning electron microscopy.This appearance was also seen in the FCS-treated cells, althoughnotably process retraction was not as widespread (average score,2.3). In contrast, in remission and normal plasma–treatedsamples, there were many long processes (average score, 3.3and 3.8, respectively) and very few surface blebs (1.9 and 1.4,respectively). Paired t test for either observation, betweenrelapse/FCS and normal/remission, was significant at <0.01.In conjunction with this morphologic change, actin patterningat 48 h was also altered between nephrotic/FCS and non-nephroticplasma. In cells that were treated with normal or remissionplasma, actin filaments in the cytoplasm were weak or absentand cortical actin distribution was strong (average score, 2.1),whereas in cells that were treated with nephrotic plasma orFCS, cytoplasmic actin filaments were strongly expressed throughout,and cortical distribution was relatively weaker (average score,1.0; paired t test P < 0.05; Figure 4).
Figure 2. Nephrin immunolocalization in WT human podocytes incubated with various human plasma samples. Differentiated cells incubated with various plasma samples. Ten percent plasma applied to the cells for 48 h. IF with monoclonal 48E11 antibody. Peripheral nephrin shown by arrows. Representative of three independent, blinded experiments. (A) Normal human plasma. (B) Plasma from patient with focal segmental glomerulosclerosis (FSGS) in remission. (C) Plasma from FSGS patient in relapse. (D) Plasma from patient with systemic lupus erythematosus. (E) FCS alone. (F) Control of nephrin 48E11 antibody applied to proximal tubular HK2 cells. Negative staining.
Figure 3. Effects of nephrotic and non-nephrotic plasma on CD2 associated protein (CD2AP) and podocin in WT human podocytes. Differentiated cells incubated with different plasma samples. Ten percent plasma applied to the cells for 48 h. Representative panels from three independent experiments. Peripheral location of the molecules arrowed. (A) CD2AP from patient in remission from FSGS. (B) CD2AP from normal patient plasma. (C) CD2AP from nephrotic patient (FSGS relapse). (D) Podocin from patient in remission from FSGS. (E) Podocin from normal patient plasma. (F) Podocin from nephrotic patient (FSGS relapse).
Figure 4. Effects on the actin cytoskeleton by human plasma samples and FCS on WT human podocytes. Differentiated cells incubated with different plasma samples. Ten percent plasma applied to the cells for 6 h. (A through D) Environmental scanning electron micrographs of the podocytes, of same passage and age, exposed to the following. (A) Normal human plasma. Process formation illustrated. (B) FSGS patient in remission. Again, process formation demonstrated by arrow. (C) Nephrotic (FSGS relapse). Lack of foot processes and blebs noted. Insets show expanded images of affected areas. (D) FCS. Shows similar blebs to C. (E through H) The actin cytoskeleton by phalloidin staining at 48 h after exposure to the following plasma samples. (E) Normal human plasma. Predominantly cortical distribution. (F) FSGS patient in remission, similar to E. (G) FSGS patient in relapse. Cytoplasmic actin stress fibers more prominent. (H) FCS. Cytoplasmic actin stress fibers more prominent.
Quantitatively, Western blotting revealed that nephrin was downregulatedby 48 h when podocytes were exposed to nephrotic plasma, butthe other SD proteins (podocin and CD2AP) were not affected(Figure 5, A and B). In addition, we used a protocol that isolateslipid rafts on the basis of their insolubility in TX-100 andlow buoyant density in sucrose density gradients (16). Nephrinwas seen in both the heavy (cytoskeletal bound) fraction andraft fractions in normal plasma–treated cells, which correlateswith its distribution in glomerular preparations (16), comparedwith just being present in the heavy fraction in FSGS relapseplasma–treated cells (Figure 5C). It is interesting thatof the molecules studied, the only one that was downregulatedearly, at 24 h, in response to nephrotic plasma was synaptopodin,an actin associated podocyte protein, suggesting that the podocyteswere becoming dedifferentiated (Figure 5A). This observationis consistent with studies of biopsy specimens showing downregulationof synaptopodin in affected podocytes in nephrotic diseases(17–19).
Figure 5. Western blot of podocyte proteins and their modulation by nephrotic plasma. Differentiated cells incubated with various plasma samples. Ten percent plasma applied to the cells for 24 or 48 h. Cells were lysed, and the lysates were probed with the following antibodies. (A) This shows representative blots of synaptopodin (at 24 h), and nephrin, podocin, CD2AP, and actin at 48 h (n = 4). (B) Densitometry on nephrin to actin ratio after 48 h of exposure. Nephrotic groups compared with non-nephrotic groups; four independent experiments. SEM shown. Data analyzed by a two-tailed nonparametric Wilcoxon test. (C) Sucrose density centrifugation of cells that were treated with either FSGS relapse plasma or normal human plasma. Fractions were probed with nephrin monoclonal Ab 48E11. Lighter fractions (1–4) correspond to those that normally contain lipid rafts (16). Control fractions were probed with actin to demonstrate efficiency of the gradient.
By analyzing podocytes from a patient with Fin major congenitalnephrotic syndrome, we showed that under control (FCS) conditions,CD2AP was present in its subplasma membrane and cytoplasmiclocation as before (average score, 0.3), but after 48 h of incubationin normal plasma, there was no translocation to the cell surface(average score, 0.6; P < 0.01; Figure 6). In addition, thecortical concentration of actin seen in response to normal plasmawith WT cells was less marked in the mutant cells, with no changeto the cytoplasmic filamentous stress fibers (average score,1.5 versus 1.9; P < 0.1). This demonstrated that CD2AP ispresent in nephrin-deficient cells but that nephrin needs tobe present to allow the plasma membrane translocation of CD2APand actin reorganization at the cell surface to occur.
Figure 6. Peripheral translocation of CD2AP is dependent on nephrin. WT podocytes and Fin major podocytes were initially cultured in FCS and then exposed for 48 h to normal human plasma. Cells then were immunostained as follows. For wild types: (A) The actin cytoskeleton shows cytoplasmic stress fibers when cultured in FCS, with weak cortical distribution. (B) CD2AP is diffusely distributed with intracytoplasmic spots in FCS culture. (C) On exposure to normal human plasma, the actin cytoskeleton assumes a thick cortical distribution, with almost complete loss of cytoplasmic filaments. (D) CD2AP translocates to the plasma membrane (high magnification view on inset panel). For the Fin major (–/–) podocytes: (E) The actin cytoskeleton is similar to WT podocytes with FCS. (F) CD2AP is diffuse and intracellular in distribution with FCS. (G) With normal human plasma, the actin cytoskeleton does not become cortically distributed but preserves the cytoplasmic stress fibers. (H) CD2AP remains intracellular, diffuse, and in spots. In areas where CD2AP seems to have reached the plasma membrane, the pattern is disrupted and remains submembranous (inset). Magnification, x400.
Rescue of Nephrin Translocation
We examined whether the nephrotic plasma was responsible forthe cytoplasmic relocation of nephrin directly via putativecirculating factor(s) or was due to factors that were missingfrom nephrotic plasma and normally found in human plasma thatwere important. We found that nephrotic plasma induced a cytoplasmicdistribution of nephrin, but this could be rescued and directedto the plasma membrane by subsequently adding an equal amountof non-nephrotic to nephrotic plasma (Figure 7). Importantly,this was not disease-specific, i.e., non-nephrotic plasma co-incubationcould rescue FSGS and SLE nephropathic states, which would argueagainst a specific inhibitory factor present in normal plasmathat may be antagonizing a "circulating factor" in FSGS (20).
Figure 7. Peripheral nephrin and synaptopodin rescue of nephrotic plasma exposed cells by non-nephrotic plasma in WT human podocytes. IF performed using the mouse monoclonal antibody 48E11 (top) or synaptopodin (bottom). Each experiment shows day 14 thermoswitched podocytes exposed to 48 h of initial plasma then fresh co-incubation of original plasma sample with another plasma sample. All samples applied at 10% concentration. The same passage number and culture time of podocyte were used in each experiment. Representative experiment shown. (A) Normal plasma (10%) followed by normal plasma–normal plasma (20%). Peripheral nephrin localization arrowed. (B) Nephrotic plasma for 48 h (10%) followed by nephrotic–nephrotic plasma (20%). Cytoplasmic nephrin distribution. (C) FCS 10% followed by FCS (20%). Cytoplasmic nephrin distribution. (D) Nephrotic plasma (10%) for 48 h followed by nephrotic plasma (10%) and normal plasma (10%) for 48 h. Nephrin again peripherally located (synaptodin up-regulated with normal plasma, Panel A and D). Magnification, x400.
Intracellular Calcium Signaling
We studied the effect of plasma from a nephrotic patient inrelapse and in remission on both WT and on two different nephrinmutant podocyte cell lines. We found in WT cells, with remissionplasma, that there was no [Ca2+] response (peak/baseline ratio= 1), whereas relapse plasma caused a statistically significantincrease in peak [Ca2+], which was inhibited by the tyrosinekinase inhibitor genistein. The effect on nephrin mutant cellswas revealing: With remission plasma, there was a considerableincrease in peak [Ca2+] in both mutant cell lines, comparedwith WT cells, suggesting that the presence of nephrin in thecorrect conformation and cellular location prevents an increasein [Ca2+] brought about by this plasma. With relapse plasma,there was a constant peak [Ca2+] response that was consistentbetween podocyte cell types (Figure 8, A and B). It is interestingthat genistein had no effect on the nephrin mutant cells’response to remission plasma (Figure 8C). This shows that theeffect of remission plasma on intracellular calcium is independentof tyrosine kinase activation in the absence of nephrin expression.This therefore supports the suggestion that nephrin plays arole in the calcium response in WT cells and that the responseseen in nephrin knockout cells is a result of perturbation ofthis normal pathway. The effect of plasma seemed to be podocytespecific in that neither relapse nor remission plasma causedany increase in [Ca2+] in either proximal tubular or human endothelialcells (Figure 8D).
Figure 8. Podocyte calcium flux data. Cells were grown on coverslips and then incubated with Fura 2-AM, excited, and assayed using ratiometric fluorescence measurement. Undiluted samples of relapse and remission plasma from one patient with FSGS were perfused in random order on the same coverslip with a 20-min wash period of HBSS medium in between. HBSS was also used as a baseline control. Data expressed as ratio of peak/baseline of [Ca2+]Intensity. (A) Relapse plasma significantly increases [Ca2+]i levels in differentiated WT podocytes (1.67 ± 0.19; paired t test P < 0.001; n = 9), exon 11 nephrin mutant podocytes (relapse, 1.5 ± 0.15; paired t test P < 0.001; n = 7) and Fin major podocytes (relapse, 1.6 ± 0.12; paired t test P < 0.001; n = 7) compared with baseline. Remission plasma does not significantly increase [Ca2+]I levels in WT podocytes (0.9 ± 0.146; t test P < 0.001; n = 9); however, it does in both exon 11 mutant podocytes (remission, 2.6 ± 0.3; paired t test P < 0.001; n = 7) and Fin major podocytes (remission, 2.2 ± 0.37; paired t test P < 0.005; n = 6) compared with baseline. (B) Preincubation of WT podocytes with 70 mM genistein attenuates the [Ca2+]i response stimulated by relapse plasma (relapse, 121.5 ± 34.7 nM [Ca2+] versus 54.8 ± 4.9 [Ca2+]; ANOVA Bonferroni multiple comparison test, P < 0.05, n = 5; genistein/relapse, 59.8 ± 12.9 nM [Ca2+] versus 44.3 ± 7.9 nM [Ca2+], NS). *P < 0.05. (C) Preincubation of Fin major podocytes with 70 mM genistein shows no significant abrogation of the increased [Ca2+] response to remission plasma (seen in A; n = 4; paired t test P < 0.004, remission versus remission/genistein). (D) Effect of the plasma is podocyte specific. The data are expressed as a ratio of peak/baseline [Ca2+]. In HK2 cells (a proximal tubular epithelial cell line) and human vein embryonic endothelial cells (HUVEC), neither remission nor relapse plasma has a significant effect on [Ca2+] flux (HK2 cell: remission 0.77 ± 0.14, relapse: 0.96 ± 0.11; HUVEC: remission 1 ± 0.11, relapse 0.8 ± 0.1 versus baseline; ANOVA Bonferroni multiple comparison test; n = 7).
Key podocyte SD molecules are critical in maintaining the filtrationbarrier of the kidney and preventing protein loss into the urine.This study shows that human plasma seems to contain factorsthat are crucial to the mature distribution of the SD proteinsnephrin, podocin, and CD2AP and that are absent from nephroticplasma. In conjunction with this mature distribution of SD proteins,we showed that actin is cortically reorganized. Furthermore,we have demonstrated using a human nephrin "knockout" cell linethat nephrin is crucial for mature distribution of the wholeSD complex, with or without plasma factors. Finally, our calciumflux data strongly suggest that this mature SD complex is involvedin mediating intracellular signaling, which is influenced bythese plasma factors.
Previous authors have studied the distribution of nephrin andpodocin in health and nephrosis, and some have found that theyare localized away from the SD in nephrotic syndromes (21,22).However, others have not demonstrated this (23,24). It has beennoted in several human biopsy studies (25,26) and animal models(27,28) that nephrin, podocin, and CD2AP distribution in nephroticdisease changes from a linear capillary loop pattern to a granularpattern, supportive of our observation of intracellular translocation.Previously, it has been impossible to study their location invitro, as cell lines have not convincingly expressed these proteins.We have shown that, in vitro, these molecules are located ina characteristic peripheral location in the presence of normalhuman plasma, which intuitively would seem to be the correcttargeting of these proteins and be analogous with the knownhuman SD location in health in vivo (10). Furthermore, our findingthat nephrin is vital to allow CD2AP to locate at the plasmamembrane of the cell and does not occur when nephrin mutantsare subjected to normal plasma suggests that the molecules areinterdependent on each other to find their normal location.Nephrin, via CD2AP, interacts directly with the actin cytoskeleton(which is the structural backbone of the foot process), andwe showed previously that disruption of nephrin results in concomitantdisruption of actin (4). Thus, the cytoplasmic relocalizationof the SD complex under nephrotic conditions could be relateddirectly to foot process effacement.
The identification of a circulating factor as the pathogenicmolecule in nephrotic syndromes, particularly minimal-changenephrosis and FSGS, has long been a holy grail for researchersin this field (29). A number of in vitro studies have been basedon studying albumin permeability of isolated glomeruli, whichfollow the trail of a putative circulating factor, althoughto date with inconsistent results. Our study for the first timeaddresses the question by the direct examination of the in vitropodocyte and, more specifically, the effect on SD proteins.The role of additional putative circulating factors in the pathogenesisof conditions such as FSGS (29) have been challenged recently(30,31). It has been suggested tentatively that nephrosis couldresult from the lack of a factor that is normally present inhuman plasma and not the addition of a circulating factor (30,31).In fact, there is tantalizing evidence to support the loss ofvital factor(s) in nephrosis from a study by Carraro et al.(30), in which plasma derived from proteinuric patients withNPHS2 gene mutations (congenital nephrotic patients) inducedalbumin permeability of isolated glomeruli in vitro, whereasurine from these patients (and not normal urine controls) blocksthis increased permeability, hence suggesting the loss of crucialfactors into the urine of nephrotic patients. Our findings providedirect evidence for this hypothesis. The observation that FCSas well as nephrotic plasma results in loss of peripheral nephrin-podocin-CD2APlocalization suggested to us that there is something missingin the medium that promotes mature SD complex distribution andcell signaling, and accordingly we found that normal plasmais able to rescue the cytoplasmic nephrin and move it to theperiphery of the cell. This was not specific to FSGS and seems,in relation to nephrin, to be associated with the nephroticsyndrome per se. Clearly, nephrotic syndromes are a heterogeneousgroup of diseases, and it may be that in disease such as FSGS,there is a primary loss or imbalance of important plasma factorsthat disrupt SD integrity and subsequently foot process morphology,whereas in other nephroses, such as those caused by SLE nephritis,the disruption is secondary to a generalized loss of proteins.There is a broad literature documenting that a proportion ofcases of FSGS is treatable by plasma exchange, suggesting removalof a pathogenic factor, and also by protein A immunoadsorption.However, there are many potential anomalies/variables in interpretingthese data, beyond the scope of this brief discussion. For example,in our schema, it is possible that plasma exchange (often beingreplaced by fresh plasma) is in fact reestablishing a naturalbalance of circulating cytokines, lipoproteins, etc. that incertain (although perhaps not all) circumstances allows restorationof the mature SD. Also, immunoadsorption has been shown to beeffective in non-FSGS nephrotic syndromes (32), favoring a nonspecificbeneficial effect. The details of which unique factors are presentin human plasma are clearly areas for future investigation,and this in vitro system would be a valuable resource to studythese questions.
Nephrin, podocin, and CD2AP are known to be associated, togetherwith actin in the podocyte in a lipid raft complex at the SD(4,7). One of the key functions postulated for lipid rafts isto facilitate rapid signaling events (33), and nephrin and podocinhave been shown to be interdependent in initiating an intracellularsignaling cascade (6), although the downstream consequencesof this signaling pathway remain to be determined. It is nowknown that membrane-associated nephrin is tyrosine phosphorylated,with the Src family kinases Fin and Yes present within the membranecomplex (34). In vivo deletion of Yes resulted in dramaticallyincreased nephrin phosphorylation, whereas deletion of Fyn resultsin reduced phosphorylation and coarsening of foot process morphology,suggesting complex regulation of nephrin signaling. We haveshown within minutes of exposure to nephrotic plasma that thereis enhanced calcium signaling relative to remission plasma.Moreover, there is a dramatic increase in calcium signalingby remission plasma in nephrin-deficient cells. This stronglysuggests that the intact SD complex at the plasma membrane,in response to steady-state signals from normal plasma, suppressesa calcium-mediated signaling cascade. Whether activation ofthis cascade is involved in the subsequent SD mislocalizationis yet to be determined. Together, these data lead us to postulatean updated concept for the pathogenesis of nephrotic syndromes.In this model, the podocyte in its mature form (i.e., intactnephrin-podocin-CD2AP complex at the SD, interdigitating footprocesses) is constantly maintained by a milieu of factors,some from the circulation and probably some from local autocrine/paracrineproduction (our own unpublished data). Disruption or loss ofthis balance, be it primary or secondary, results in disruptionof nephrin-mediated signaling, leading to loss of SD stabilityand hence reorganization of associated actin filaments, causingintracellular relocalization of the SD complex and foot processeffacement.
Nephrotic syndromes are a heterogeneous group of conditions,and this study addresses the cell biology of the podocyte inrelation to a subset of patients. Our results were consistentacross the samples, although it is certainly possible that inother nephrotic patients there may be alternative mechanismsat play. The observations that we describe, however, do fitcomfortably with the emerging knowledge of podocyte biology,in particular a role for the SD complex in signaling and actinregulation.
In conclusion, we have demonstrated that podocytes are affecteddirectly by nephrotic plasma with early nephrin-dependent signalingevents, followed by actin reorganization and interdependenttranslocation of the proteins nephrin, podocin, and CD2AP awayfrom the plasma membrane into the cytoplasm of the cell. Thisprocess can be rescued by normal plasma, suggesting the lossof a factor in nephrosis. At this stage, our understanding ofthe factors that are responsible for maintaining podocyte differentiationin vivo is simplistic, and we hope that this study will be astep toward a more detailed study of these in health and disease.
Acknowledgments
This study was supported by Wellchild UK, British Medical Association,Elizabeth Wherry and Charlotte Eyck Award, Southmead HospitalResearch Foundation, and The Wellcome Trust.
We thank Jussi Merenmies for arrangements to obtain the FM kidneyspecimen; Maria Bitsori and Felicity Woodward for help withimmunofluorescence experiments; and David Yearsley for additionalfundraising.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Kestila M, Lenkkeri U, Mannikko M, Lamerdin J, McCready P, Putaala H, Ruotsalainen V, Morita T, Nissinen M, Herva R, Kashtan CE, Peltonen L, Holmberg C, Olsen A, Tryggvason K: Positionally cloned gene for a novel glomerular protein—nephrin—is mutated in congenital nephrotic syndrome.
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Received for publication March 8, 2004.
Revision received November 22, 2004.
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Abstract
Introduction
