Focal segmental glomerulosclerosis (FSGS) is characterized by steroid resistant nephrotic syndrome and progression to end-stage renal disease. Proteinuria in certain patients with FSGS may be caused by a circulating factor (FSGS permeability factor [FSPF]). The current report documents the biochemical characteristics and the biological and molecular effects of 70% ammonium sulfate supernatant of plasma from patients with recurrence of FSGS after transplantation (FSGS 70% supernatant). FS permeability activity, defined as the capacity of plasma from patients with FSGS to increase albumin permeability (Palb) of isolated glomeruli, was assessed in vitro. Permeability activity was not affected by lyophilization. FSPF bound strongly to matrices containing Mono-Q anion exchanger or protein A. It eluted from matrix-bound Cibacron blue F3GA over a wide range of salt concentrations, indicating a potential binding with other proteins, such as albumin. FSPF caused a maximal increase in Palb within 2 mins of incubation in vitro. Cellular proteins isolated from glomeruli with increased Palb showed decreased tyrosine phosphorylation of focal adhesion kinase, paxillin, and other proteins. Tyrosine phosphatase inhibition prevented the increase in Palb. Intravenous administration of as little as 3 mg protein in FSGS 70% supernatant increased Palb, while 9 mg or more were required to produce proteinuria. We conclude that FSPF is a low-molecular-weight protein, carries an anionic charge, and binds to protein A. Effects of FSPF on the glomerular permeability barrier are rapid and dose dependent and involve signaling through altered phosphorylation of cellular proteins. Identification of these biochemical and biological characteristics may be used to design strategies for removing FSPF from circulation and for purification and identification of this factor.
ShiikiH, NishinoT, UyamaH, KimuraT, NishimotoK, IwanoM, KanauchiM, FujiiY, DohiK. Clinical and morphological predictors of renal outcome in adult patients with focal and segmental glomerulosclerosis (FSGS). Clin Nephrol46: 362–368, 1996.
2.
D'agatiV. The many masks of focal segmental glomerulosclerosis. Kidney Int46: 1223–1241, 1994.
3.
HaasM, SpargoBH, CoventryS. Increasing incidence of focal segmental glomerulosclerosis among adult nephropathies. Am J Kidney Dis26: 740–750, 1995.
4.
HaasM, MeehanSM, KarrisonTG, SpargoBH. Changing etiologies of unexplained adult nephrotic syndrome: a comparison of renal biopsy findings from 1976–1979 and 1995–1997. Am J Kidney Dis30: 621–631, 1997.
5.
SorofJM, HawkinsEP, BrewerED, BoydstunII, KaleAS, PowellDR. Age and ethnicity affect the risk and outcome of focal segmental glomerulosclerosis. Pediatr Nephrol12: 764–768, 1998.
KorbetSM, GenchiRM, BorokRZ, SchwartzMM. The racial prevalence of glomerular lesions in nephrotic adults. Am J Kidney Dis27: 647–651, 1996.
8.
FaubertPF, PorushJG. Familial focal segmental glomerulosclerosis: nine cases in four families and review of the literature. Am J Kidney Dis30: 265–270, 1997.
9.
FelldinM, NordenG, SvalanderC, NybergG. Focal segmental glomerulosclerosis in a kidney transplant population: hereditary and sporadic forms. Transpl Int11: 16–21, 1998.
10.
KestilaM, LenkkeriU, MannikkoM, LamerdinJ, McCreadyP, PutaalaH, RuotsalainenV, MoritaT, NissinenM, HervaR, KashtanCE, PeltonenL, HolmbergC, OlsenA, TryggvasonK. Positionally cloned gene for a novel glomerular protein-nephrin is mutated in congenital nephrotic syndrome. Mol Cell1: 557–582, 1998.
TsukaguchiH, YagerH, DawbornJ, JostL, CohlmiaJ, AbreuPF, PereiraAB, PollakMR. A locus for adolescent and adult onset familial focal segmental glomerulosclerosis on chromosome 1q25-31. J Am Soc Nephrol11: 1674–1680, 2000.
WinnMP, ConlonPJ, LynnKL, HowellDN, SlotterbeckBD, SmithAH, GrahamFL, BembeM, QuarlesLD, Pericak-VanceMA, VanceJM. Linkage of a gene causing familial focal segmental glomerulosclerosis to chromosome 11 and further evidence of genetic heterogeneity. Genomics58: 113–120, 1999.
15.
BouteN, GribouvalO, RoselliS, BenessyF, LeeH, FuchshuberA, DahanK, GublerMC, NiaudetP, AntignacC. NPHS2, encoding the glomerular protein podocin, is mutated in autosomal recessive steroid-resistant nephrotic syndrome. Nat Genet25: 125, 2000.
16.
KambhamN, TanjiN, SeigleRL, MarkowitzGS, PulkkinenL, UittoJ, D'agatiVD. A mutation in beta4 integrin is associated with congenital FSGS and epidermolysis bullosa. Am J Kidney Dis36: 190–196, 2000.
Breiteneder-GeleffS, MatsuiK, SoleimanA, MeranerP, PoczewskiH, KaltR, SchaffnerG, KerjaschkiD. Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis. Am J Pathol151: 1141–1152, 1997.
19.
JohnsonRJ, GretchDR, YamabeH, HartJ, BacchiCE, HartwellP, CouserWG, CoreyL, WenerMH, AlpersCE, WillsonR. Membranoproliferative glomerulonephritis associated with hepatitis C virus infection. N Engl J Med18: 505–506, 1993.
20.
MoudgilA, NastCC, BaggaA, WeiL, NurmametA, CohenAH, JordanSC, ToyodaM. Association of parvovirus B19 infection with idiopathic collapsing glomerulopathy. Kidney Int59: 2126–2133, 2001.
21.
HariharanS, PeddiVR, SavinVJ, JohnsonCP, FirstMR, RozaAM, AdamsMB. Recurrent and de novo renal diseases after renal transplantation: a report from the renal allograft disease registry. Am J Kidney Dis31: 928–931, 1998.
22.
OdoricoJS, KnechtleSJ, RayhillSC, PirschJD, D'alessandroAM, BelzerFO, SollingerHW. The influence of native nephrectomy on the incidence of recurrent disease following renal transplantation for primary glomerulonephritis. Transplantation61: 228–234, 1996.
23.
MullerT, SikoraP, OffnerG, HoyerPF, BrodehlJ. Recurrence of renal disease after kidney transplantation in children: 24 years of experience in a single center. Clin Nephrol49: 82–90, 1998.
24.
ArteroM, SharmaR, SavinVJ, VincentiF. Plasmapheresis reduces proteinuria and serum capacity to injure glomeruli in patients with recurrent focal glomerulosclerosis. Am J Kidney Dis23: 574–581, 1994.
25.
CrenshawG, BiglerS, SalemM, CrookED. Focal segmental glomerulosclerosis in African Americans: effects of steroids and angiotensin converting enzyme inhibitors. Am J Med Sci319: 320–325, 2000.
26.
TuneBM, MendozaSA. Treatment of the idiopathic nephrotic syndrome: regimens and outcomes in children and adults. J Am Soc Nephrol8: 824–832, 1997.
27.
FeldSM, FigueroaP, SavinV, NastCC, SharmaR, SharmaM, HirschbergR, AdlerSG. Plasmapheresis in the treatment of steroid-resistant focal segmental glomerulosclerosis in native kidneys. Am J Kidney Dis32: 230–237, 1998.
28.
GinsburgDS, DauP. Plasmapheresis in the treatment of steroid-resistant focal segmental glomerulosclerosis. Clin Nephrol48: 282–287, 1997.
29.
GodfrinY, DantalJ, PerrettoS, HristeaD, LegendreC, KreisH, SoulillouJP. Study of the in vitro effect on glomerular albumin permselectivity of serum before and after renal transplantation in focal segmental glomerulosclerosis. Transplantation64: 1711–1715, 1997.
ZimmermanSW. Increased urinary protein excretion in the rat produced by serum from patients with recurrent focal glomerulosclerosis after transplantation. Clin Nephrol22: 32–38, 1984.
32.
SavinVJ, SharmaR, SharmaM, McCarthyET, SwanS, EllisE, LovellH, WaradyB, GunwarS, ChonkoAM, ArteroM, VincentiF. Circulating factor associated with increased glomerular permeability to albumin in recurrent focal glomerulosclerosis. N Engl J Med334: 878–883, 1996.
33.
SharmaM, SharmaR, McCarthyET, SavinVJ. “The FSGS factor”: enrichment and in vivo effect of activity from focal segmental glomerulosclerosis plasma. J Am Soc Nephrol10: 552–561, 1999.
34.
SharmaM, SharmaR, ReddySR, McCarthyET, SavinVJ. Proteinuria after injection of human focal segmental glomerulosclerosis factor. Transplantation73: 366–372, 2002.
35.
SharmaR, SharmaM, McCarthyET, GeXL, SavinVJ. Components of normal serum block the focal segmental glomerulosclerosis factor activity in vitro. Kidney Int58: 1973–1979, 2000.
36.
CandianoG, MusanteL, CarraroM, FacciniL, CampanacciL, ZennaroC, ArteroM, GinevriF, PerfumoF, GusmanoR, GhiggeriG. Apolipoproteins prevent glomerular albumin permeability induced in vitro by serum from patients with focal segmental glomerulosclerosis. J Am Soc Nephrol12: 143–150, 2001.
37.
BradfordMM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem72: 248–254, 1976.
38.
SavinVJ, SharmaR, LovellHB, WellingDJ. Measurement of albumin reflection coefficient with isolated rat glomeruli. J Am Soc Nephrol3: 1260–1269, 1992.
39.
GianazzaE, ArnaudP. Chromatography of plasma proteins on immobilized Cibacron Blue-F3-GA. Biochem J203: 637–641, 1982.
40.
GianazzaE, ArnaudP. A general method for fractionation of plasma proteins: dye ligand affinity chromatography on immobilized Cibacron Blue-F3-GA. Biochem J201: 129–136, 1982.
41.
CutatrecasasP, ParikhI. Insulin receptors. In: JakobyWB, WichekM, Eds. Methods in Enzymology. New York: Academic Press, Vol 34: pp 653–671, 1974.
42.
MolinariAM, MediciNM, MoncharmontB, PucaGA. Estradiol receptor of calf uterus: interaction with heparin-agarose and purification. Proc Natl Acad Sci U S A74: 4886–4890, 1977.
43.
ComptonBL, LewisMA, WhigmanF, GeraldSJ, CountrymanGE. Analytical potential of protein A for affinity chromatography of polyclonal and monoclonal antibodies. Anal Chem61: 1314–1317, 1989.
44.
KenneyAC. Ion exchange chromatography of proteins. In: KenneyA, FowellS, Eds. Practical Protein Chromatography—Methods in Molecular Biology. Totowa, NJ: The Humana Press, Vol 11: pp 249–258, 1992.
45.
OldbergA, KjellenL, HookM. Cell surface heparan sulfate: isolation and characterization of a proteoglycan from rat liver membranes. J Biol Chem254: 8505–8510, 1979.
46.
RossomandoEF. Ion exchange chromatography. In: DeutscherMP, Ed. Methods in Enzymology. New York: Academic Press, Vol 182: pp 309–316, 1990.
47.
LowryOH, RosenbroughNJ, FarrAL, RandallRJ. Protein measurement with the Folin phenol reagent. J Biol Chem193: 265–275, 1951.
48.
KoukouritakiSB, TamizuddinA, LianosEA. Enhanced expression of the cytoskeletal- associated protein, paxillin, in experimental nephrotic syndrome. J Investig Med46: 284–289, 1998.
49.
MowryJ, EttengerRB, SharmaR, SavinVJ. Increase albumin permeability (Palb) in rat glomeruli induced by sera of patients with recurrent focal segmental glomerulosclerosis (FSGS): implications in renal transplantation. J Am Soc Nephrol5: 1026, 1994.
50.
SharmaR, SharmaM, Ge XiuLi, McCarthyET, SavinVJ. Cyclosporine protects glomeruli from FSGS factor via an increase in glomerular cAMP. Transplantation27: 1916–1920, 1996.
SharmaM, LiJZ, SharmaR, ArteroM, GeXL, McCarthyET, WongHY, SavinVJ. Inhibitory effect of Tripterygium wilfordii multiglyco-side on increased glomerular albumin permeability in vitro. Nephrol Dial Transplant12: 2064–2068, 1997.
53.
Dall'AmicoR, GhiggeriG, CarraroM, ArteroM, GhioL, ZamoraniE, ZennaroC, BasileG, MontiniG, RivabellaL, CardilloM, ScalamognaM, GinevriF. Prediction and treatment of recurrent focal segmental glomerulosclerosis after renal transplantation in children. Am J Kidney Dis34: 1048–1055, 1999.
54.
CattranD, NeogiT, McCarthyET, SavinVJ, SharmaR. Serial estimates of serum permeability activity and clinical correlates in patients with native kidney focal segmental glomerulosclerosis. J Am Soc Nephrol14: 448–453, 2003.
Dall'AmicoR, GhiggeriG, CarraroM, ArteroM, GhioL, ZamoraniE, ZennaroC, BasileG, MontiniG, RivabellaL, CardilloM, ScalamognaM, GinevriF. Prediction and treatment of recurrent focal segmental glomerulosclerosis after renal transplantation in children. Am J Kidney Dis34: 1048–1055, 1999.
57.
LiB, YaoJ, MoriokaT, OiteT. Nitric oxide increases albumin permeability of isolated rat glomeruli via a phosphorylation-dependent mechanism. J Am Soc Nephrol12: 2616–2624, 2001.
58.
MusanteL, CandianoG, BruschiM, ZennaroC, CarraroM, ArteroM, GiuffridaMG, ContiA, SantucciA, GhiggeriGM. Characterization of plasma factors that alter the permeability to albumin within isolated glomeruli. Proteomics2: 197–205, 2002.
59.
MusanteL, CandianoG, ZennaroC, BruschiM, CarraroM, ArteroM, GhiggeriGM. Humoral permeability factors in the nephrotic syndrome: a compendium and prospectus. J Nephrol14(Suppl 4):S48–S50, 2001.
60.
GhiggeriGM, ArteroM, CarraroM, PerfumoF. Permeability plasma factors in nephrotic syndrome: more than one factor, more than one inhibitor. Nephrol Dial Transplant16: 882–885, 2001.
61.
CarraroM, CaridiG, BruschiM, ArteroM, BertelliR, ZennaroC, MusanteL, CandianoG, PerfumoF, GhiggeriGM. Serum glomerular permeability activity in patients with podocin mutations (NPHS2) and steroid-resistant nephrotic syndrome. J Am Soc Nephrol13: 1946–1952, 2002.
62.
CarraroM, ZennaroC, CandianoG, MusanteL, BruschiM, GhiggeriGM, ArteroM, FacciniL. Nephrotic urine prevents increased rat glomerular albumin permeability induced by serum from the same patient with idiopathic nephrotic syndrome. Nephrol Dial Transplant18: 689–693, 2003.
63.
AdamsGD, RamsayJr.. Optimizing the lyophilization cycle and the consequences of collapse on the pharmaceutical acceptability of Erwinia l-asparaginase. J Pharm Sci85: 1301–1305, 1996.
64.
AllisonSD, RandolphTW, ManningMC, MiddletonK, DavisA, CarpenterJF. Effects of drying methods and additives on structure and function of actin: mechanisms of dehydration-induced damage and its inhibition. Arch Biochem Biophys358: 171–181, 1998.
PoxonSW, HughesJA. The effect of lyophilization on plasmid DNA activity. Pharm Dev Tech5: 115–122, 2000.
67.
Pikal-ClelKA, CarpenterJF. Lyophilization-induced protein denaturation in phosphate buffer systems: monomeric and tetrameric beta-galactosidase. J Pharm Sci90: 1255–1268, 2001.
68.
BarbareeJM. Methods for determining favorable conditions for freeze-drying biological reference materials. Dev Biol Standard36: 335–341, 1976.
69.
DawsonPJ. Effect of formulation and freeze-drying on the long-term stability of rDNA-derived cytokines. Dev Biol Standard74: 273–282, 1992.
70.
PetersT, SjoholmI. Albumin—Structure, biosynthesis, function. Copenhagen: Federation of European Biochemical Societies, Vol 50, Colloquium B9, 1977.
71.
RosenoerVM, OratizM, RothschildMA. Albumin Structure, Function and Uses. New York: Pergamon Press, 1977.
72.
HennessenW. International Symposium on Standardization of Albumin, Plasma Substitutes and Plasmapheresis: The International Association of Biological Standardization. Basel: S. Karger, Vol 48, 1981.
73.
SharmaM, GeX, SharmaR, McCarthyET, SavinVJ. Partial purification of protein permeability factor from focal segmental glomerulosclerosis (FSGS) sera. J Am Soc Nephrol6: 884, 1995.
74.
Le BerreL, GodfrinY, Lafond-PuyetL, PerrettoS, Le CarrerD, BouhoursJF, SoulillouJP, DantalJ. Effect of plasma fractions from patients with focal and segmental glomerulosclerosis on rat proteinuria. Kidney Int58: 2502–2511, 2000.
75.
MusoE, MuneM, FujiiY, ImaiE, UedaN, HattaK, ImadaA, TakemuraT, MikiS, KuwaharaT, TakamitsuY, TsubakiharaY, KansaiFGS, LDL Apheresis Treatment (K-FLAT) Study Group:significantly rapid relief from steroid-resistant nephrotic syndrome by LDL apheresis compared with steroid monotherapy. Nephron89: 408–415, 2001.
76.
TojoK, SakaiS, MiyaharaT. Therapeutic trial of low-density lipoprotein apheresis (LDL-A) in conjunction with double filtration plasmapheresis (DFPP) in drug-resistant nephrotic syndrome due to focal glomerular sclerosis (FGS). Prog Clin Biol Res337: 193–194, 1990.
77.
EsnaultVL, BesnierD, TestaA, CovilleP, SimonP, SubraJF, AudrainMA. Effect of protein A immunoadsorption in nephrotic syndrome of various etiologies. J Am Soc Nephrol10: 2014–2017, 1999.
78.
SabatinoB, Van LiewJB. The renal handling of IgG in the aging rat and in experimental kidney disease. Proc Soc Exp Biol Med216: 414–443, 1997.
79.
ReiserJ, PixleyFJ, HugA, KrizW, SmoyerWE, StanleyER, MundelP. Regulation of mouse podocyte process dynamics by protein tyrosine phosphatases. Kidney Int57: 2035–2042, 2000.
80.
SharmaR, SharmaM, VamosS, McCarthyET, SavinVJ, WiegmannTB. Both subtype 1 and 2 receptors of angiotensin II participate in regulation of intracellular calcium in glomerular epithelial cells. J Lab Clin Med138: 40–49, 2001.
81.
AtkinsonKJ, RaoRK. Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions. Am J Physiol280:G1280–G1288, 2001.
82.
RaoRK, LiL, BakerRD, BakerSS, GuptaA. Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer. Am J Physiol279:G332–G340, 2000.
83.
Collares-BuzatoCB, JepsonMA, SimmonsNL, HirstBH. Increased tyrosine phosphorylation causes redistribution of adherens junction and tight junction proteins and perturbs paracellular barrier function in MDCK epithelia. Eur J Cell Biol76: 85–92, 1998.
84.
KarczewskiJ, GrootJ. Molecular physiology and pathophysiology of tight junctions III. Tight junction regulation by intracellular messengers: differences in response within and between epithelia. Am J Physiol279:G660–G665, 2000.
85.
YuanY, MengFY, HuangQ, HawkerJ, WuHM. Tyrosine phosphorylation of paxillin/pp15FAK and microvascular endothelial barrier function. Am J Physiol275(1 Pt 2):H84–H93, 1998.
86.
GarciaJG, SchaphorstKL, VerinAD, VepaS, PattersonCE, NatarajanV. Diperoxovanadate alters endothelial cell focal contacts and barrier function: role of tyrosine phosphorylation. J App Physiol89: 2333–2343, 2000.
87.
RomerLH, McLeanN, TurnerCE, BurridgeK. Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells. Mol Biol Cell5: 349–361, 1994.
88.
DefilippiP, BozzoC, VolpeG, RomanoG, VenturinoM, SilengoL, TaroneG. Integrin-mediated signal transduction in human endothelial cells: analysis of tyrosine phosphorylation events. Cell Adhes Commun2: 75–86, 1994.
