Ellen T. McCarthy

Circulating Permeability Factors in Idiopathic Nephrotic Syndrome and Focal Segmental Glomerulosclerosis

Circulating permeability factors may be important in idiopathic nephrotic syndrome (INS) including focal segmental glomerulosclerosis (FSGS) and in recurrence after renal transplantation. Evidence for plasma factors includes posttransplant recurrence of proteinuria and its response to plasmapheresis or immunoadsorption and induction of proteinuria in experimental animals by infusion of patient plasma or its fractions. The authors and other investigators have used proteomic techniques to seek pathogenic molecules. The authors have recently proposed cardiotrophin-like cytokine-1 (CLC-1) as an active factor in FSGS. Other potential permeability factors include hemopexin and vascular permeability factor in minimal change nephrotic syndrome (MCNS) and soluble urokinase receptor in FSGS. In the authors studies, in vitro plasma permeability activity is blocked by diverse substances that may decrease levels of active molecules or block the effects of circulating permeability factors. It has been shown that the simple sugar galactose blocks the effect of FSGS serum on albumin permeability in vitro and decreases permeability activity when administered to patients. Because identities of permeability factors and their mechanisms of action are not well defined, therapy of INS/FSGS is empiric. Corticosteroids are the mainstay of initial therapy whereas calcineurin inhibitors such as cyclosporine A (CsA) and immunosuppressive medications provide adjunctive therapy. Nonspecific therapies such as blocking the renin-angiotensin system and controlling blood pressure and plasma lipids may also diminish proteinuria and slow progression. Identification of molecules that initiate proteinuria and application of findings from in vitro studies may lead to development of new treatments to arrest progression and prevent recurrence after transplantation.

Galactose binds to focal segmental glomerulosclerosis permeability factor and inhibits its activity

Focal segmental glomerulosclerosis (FSGS) is associated with circulating permeability activity (Palb) and recurs after transplantation in about 30% of patients. The FS permeability factor (FSPF) consists of anionic low-molecular-weight protein(s) that might be excluded by the anionic filtration barrier. We postulated that FSPF may interact with sugars of the glycocalyx, and we tested its affinity for sugars using column chromatography. FSPF showed high affinity for galactose; Palb activity was absent from unbound material and present in eluate after dialysis to remove galactose. In parallel studies, Palb activity of serum was lost after adding galactose > or = 10(-12) M. To determine whether galactose also abolishes plasma Palb activity in vivo, a patient with posttransplant FSGS was given galactose and serum samples were collected. Intravenous infusion of galactose decreased Palb from 0.88 before infusion to undetectable levels postinfusion and at 48 hours. Oral galactose diminished Palb activity; Palb reached a nadir after 2 weeks and remained low for at least 4 weeks after galactose was discontinued. We conclude that FSPF has high affinity for galactose based on chromatography. Additionally, galactose inactivates FSPF and may lead to its clearance from plasma. The interaction between FSPF and glomeruli may depend on FSPF binding to galactose, and the FSPF-galactose complex may be susceptible to uptake by galactose-binding proteins and to catabolism. We propose testing galactose as a novel nontoxic therapy for nephrotic syndrome in FSGS to determine whether galactose slows progression and whether pretransplant therapy decreases rates of recurrence and graft loss.

Documentation of angiotensin II receptors in glomerular epithelial cells

Angiotensin II decreases glomerular filtration rate, renal plasma flow, and glomerular capillary hydraulic conductivity. Although angiotensin II receptors have been demonstrated in mesangial cells and proximal tubule cells, the presence of angiotensin II receptors in glomerular epithelial cells has not previously been shown. Previously, we have reported that angiotensin II caused an accumulation of cAMP and a reorganization of the actin cytoskeleton in cultured glomerular epithelial cells. Current studies were conducted to verify the presence of angiotensin II receptors by immunological and non-peptide receptor ligand binding techniques and to ascertain the activation of intracellular signal transduction in glomerular epithelial cells in response to angiotensin II. Confluent monolayer cultures of glomerular epithelial cells were incubated with angiotensin II, with or without losartan and/or PD-123,319 in the medium. Membrane vesicle preparations were obtained by homogenization of washed cells followed by centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane proteins followed by multiscreen immunoblotting was used to determine the presence of angiotensin II receptor type 1 (AT1) or type 2 (AT2). Angiotensin II-mediated signal transduction in glomerular epithelial cells was studied by measuring the levels of cAMP, using radioimmunoassay. Results obtained in these experiments showed the presence of both AT1 and AT2 receptor types in glomerular epithelial cells. Angiotensin II was found to cause an accumulation of cAMP in glomerular epithelial cells, which could be prevented only by simultaneous use of losartan and PD-123,319, antagonists for AT1 and AT2, respectively. The presence of both AT1 and AT2 receptors and an increase in cAMP indicate that glomerular epithelial cells respond to angiotensin II in a manner distinct from that of mesangial cells or proximal tubular epithelial cells. Our results suggest that glomerular epithelial cells participate in angiotensin II-mediated control of the glomerular filtration barrier.

Proteinuria after injection of human focal segmental glomerulosclerosis factor

BACKGROUNDnRecurrent focal segmental glomerulosclerosis (FSGS) is heralded by proteinuria that may remit after treatment with plasmapheresis or immunoadsorption. Study of recurrent FSGS has been hampered by lack of an animal model that exhibits a pattern of proteinuria that mimics human disease. We have obtained a component of FSGS patient plasma (FSGS factor) that increases glomerular albumin permeability (P(alb)) in vitro and causes transient proteinuria in vivo.nnnMETHODSnPlasma fractions containing FSGS factor and comparable plasma fractions from normal donors were injected into normal male Sprague-Dawley rats. Urinary protein, albumin, and creatinine were measured at various time points. Additionally, plasma samples from test animals were collected after injection and tested for FS activity defined by increased P(alb). Finally, glomeruli were isolated from animals after injection and P(alb) of these glomeruli tested.nnnRESULTSnProteinuria and albuminuria were increased by 24 hr after injection with FSGS factor, and returned to baseline by 48 hr after injection. Injection with the same fraction of normal plasma had no effect on urinary protein. FSGS factor increased urinary protein in a dose-dependent manner. Serum collected from rats 15 or 60 min after injection with FSGS factor increased P(alb) of glomeruli in vitro, whereas serum collected 3 or more hours after injection had no effect. Glomeruli isolated from rats receiving injections with FSGS factor had increased in vitro P(alb) compared with glomeruli from rats injected with a fraction from normal plasma.nnnCONCLUSIONSnWe have demonstrated that a single injection of FSGS factor increases P(alb) and, causes transient albuminuria and proteinuria in rats. FS activity in the plasma of recipient rats is also transient. This is the first detailed description of the time course and dose-dependence of proteinuria caused by FSGS factor in an animal model.

Bidirectional relationship between chronic kidney and periodontal disease: a study using structural equation modeling

Periodontal disease is associated with diabetes, heart disease, and chronic kidney disease (CKD), relationships postulated to be due in part to vascular inflammation. A bidirectional relationship between CKD and periodontal disease is plausible, though this relationship has not been previously reported. In this study, we assessed the potential for connections between CKD and periodontal disease, and mediators of these relationships using structural equation models of data from 11,211 adults ≥ 18 years of age who participated in the Third National Health and Nutrition Examination Survey. Multivariable logistic regression models were used to test the hypothesis that periodontal disease was independently associated with CKD. Given the potential that the periodontal disease and CKD relationship may be bidirectional, a two-step analytic approach was used that involved tests for mediation and structural equation models to examine more complex direct and indirect effects of periodontal disease on CKD, and vice versa. In two separate models, periodontal disease (adjusted odds ratio of 1.62), edentulism (adjusted odds ratio of 1.83), and the periodontal disease score were associated with CKD when simultaneously adjusting for 14 other factors. Altogether, three of four structural equation models support the hypothesized relationship. Thus, our analyses support a bidirectional relationship between CKD and periodontal disease, mediated by hypertension and the duration of diabetes.

Transforming Growth Factor-β, 20-HETE Interaction, and Glomerular Injury in Dahl Salt-Sensitive Rats

This study examined the role of transforming growth factor-&bgr; (TGF-&bgr;) in altering the glomerular permeability to albumin (Palb) during hypertension development in Dahl salt-sensitive (Dahl S) rats and whether TGF-&bgr; acts by inhibiting the glomerular production of 20-HETE. The results indicate that the renal expression of TGF-&bgr; doubles in Dahl S rats fed a high-salt diet for 7 days, and this is associated with a marked rise in Palb from 0.19±0.04 to 0.75±0.01 and changes in the ultrastructure of the glomerular filtration barrier. Chronic treatment of Dahl S rats with a TGF-&bgr; neutralizing antibody prevented the increase in Palb and preserved the structure of glomerular capillaries. It had no effect on the rise in blood pressure produced by the high-salt diet. In other studies, preincubation of glomeruli isolated from Sprague Dawley rats with TGF-&bgr;1 (10 ng/mL) for 15 minutes increased Palb from 0.01±0.01 to 0.60±0.02. This was associated with inhibition of the glomerular production of 20-HETE from 221±11 to 3.4±0.5 &mgr;g per 30 minutes per milligram of protein. Pretreatment of Sprague Dawley glomeruli with a stable analog of 20-HETE, 20-hydroxyeicosa-5(Z), 14(Z)-dienoic acid, reduced baseline Palb and opposed the effects of TGF-&bgr; to increase Palb. These studies indicate that upregulation of the glomerular formation of TGF-&bgr; may contribute to the development of proteinuria and glomerular injury early in hypertension development in Dahl S rats by increasing Palb through inhibition of the glomerular production of 20-HETE.

Cyclosporine protects glomeruli from FSGS factor via an increase in glomerular cAMP.

Cyclosporine (CsA) administration to patients with recurrent focal segmental glomerulosclerosis (FSGS) after transplantation results in remission of proteinuria. We have shown that sera from patients with recurrent FSGS can increase the glomerular albumin permeability (Palbumin) and that increase in glomerular cAMP levels can alter the permeability characteristics of glomeruli in vitro. The purpose of this study was to determine if the increased glomerular levels of cAMP were related to the protective effects of CsA on an increase in Palbumin by FSGS sera. Glomeruli from Sprague-Dawley rats following intraperitoneal administration of CsA (25 mg/kg/day), cremophore (25 mg/kg/day), or saline for 5 days were incubated with 1:50 dilution of serum from three FSGS patients or with pooled normal human serum prior to calculation of Palbumin. Glomerular cAMP was measured by radioimmunoassay. Glomerular ultrastructural changes were assessed by transmission electron microscopy (TEM). Serum from three FSGS patients markedly increased Palbumin of glomeruli from saline or cremophore treated rats (saline, 0.68+/-0.08; 0.72+/-0.07; 0.70+/-0.07; and cremophore, 0.79+/-0.05; 0.81+/-0.02; 0.79+/-0.01; n=25 glomeruli in each group). In contrast Palbumin of glomeruli from CsA treated rats was not increased by any of the three FSGS sera tested (0.03+/-0.02; 0.04+/-0.05; 0.02+/-0.07, n=25 glomeruli in each group). Glomerular cAMP (pmol/mg of protein) increased 5 fold in CsA treated rats (328+/-26; 5 rats) compared with cremophore or saline treated rats (87+/-24 and 65+/-23, P<0.01; 5 rats in each group). The glomerular basement membrane appeared to be thickened and the lamina densa had an irregular appearance after treatment with CsA. No ultrastructural changes of glomerular epithelial or endothelial cells were evident. We conclude that CsA may have a direct protective effect on the glomerular filtration barrier in FSGS. We postulate that increased levels of glomerular cAMP by CsA may play an important role in protecting the glomerular Palbumin effect of the FSGS factor and may contribute to remission of proteinuria in FSGS patients.

LPS and PAN-induced podocyte injury in an in vitro model of minimal change disease: changes in TLR profile.

Minimal change disease (MCD), the most common idiopathic nephrotic syndrome in children, is characterized by proteinuria and loss of glomerular visceral epithelial cell (podocyte) ultrastructure. Lipopolysaccharide (LPS) and puromycin aminonucleoside (PAN) are used to study podocyte injury in models of MCD in vivo and in vitro. We hypothesized that LPS and PAN influence components of the innate immune system in podocytes such as the Toll-Like Receptor (TLRs), TLR adapter molecules, and associated cytokines. Our results show that cultured human podocytes constitutively express TLRs 1–6 and TLR-10, but not TLRs 7–9. LPS (25xa0μg/ml) or PAN (60xa0μg/ml) caused comparable derangement of the actin cytoskeleton in podocytes. Quantitative RT-PCR analysis show that LPS differentially up-regulated the expression of genes for TLRs (1u2009>u20094u2009≥u20092u2009>u20093u2009>u20096u2009>u20095), the adapter molecule, MyD88, and transcription factor NF-κB within one hour. LPS also caused increased levels of IL-6, IL-8 and MCP1 without exerting any effect on TNF-α, IFN-α or TGF-β1 at 24xa0h. Immunofluorescence intensity analysis of confocal microscopy images showed that LPS induced a significant increase in nuclear translocation of NF-κB by 6xa0h. In contrast, PAN-induced only small changes in the expression of TLRs 2–6 that included a persistent increase in TLRs 2 and 5, a transient increase in TLR-4, and a gradual increase in TLRs 3 and 6 between 1 and 6xa0h. Correspondingly, it did not alter pro-inflammatory cytokine levels in podocytes. However, PAN induced a low but significant increase in NF-κB nuclear translocation within one hour that remained unchanged up to 6xa0h. In summary, these novel findings show that LPS, a known TLR-4 ligand, induced the gene expression of multiple TLRs with maximum effect on the expression of TLR-1 suggesting a loss of receptor selectivity and induction of receptor interactions in podocytes. A comparable derangement of the podocyte cytoskeleton and significant increase in the nuclear translocation of NF-κB by PAN suggest that disparate but complementary mechanisms may contribute to the development of podocytopathy in MCD.

ADMA injures the glomerular filtration barrier: role of nitric oxide and superoxide

Chronic kidney disease (CKD) is associated with decreased renal nitric oxide (NO) production and increased plasma levels of methylarginines. The naturally occurring guanidino-methylated arginines N-monomethyl-l-arginine (l-NMMA) and asymmetric dimethyl-l-arginine (ADMA) inhibit NO synthase activity. We hypothesized that ADMA and l-NMMA compromise the integrity of the glomerular filtration barrier via NO depletion. We studied the effect of ADMA on albumin permeability (P(alb)) in isolated glomeruli and examined whether this effect involves NO- and superoxide (O(2)(*-))-dependent mechanisms. ADMA at concentrations found in circulation of patients with CKD decreased cGMP and increased P(alb) in a dose-dependent manner. A similar increase in P(alb) was caused by l-NMMA but at a concentration two orders of magnitude higher than that of ADMA. NO donor DETA-NONOate or cGMP analog abrogated the effect of ADMA on P(alb). The SOD mimetic tempol or the NAD(P)H oxidase inhibitor apocynin also prevented the ADMA-induced increase in P(alb). The NO-independent soluble guanylyl cyclase (sGC) activator BAY 41-2272, at concentrations that increased glomerular cGMP production, attenuated the ADMA-induced increase in P(alb). Furthermore, sGC incapacitation by the heme site-selective inhibitor ODQ increased P(alb). We conclude that ADMA compromises the integrity of the filtration barrier by altering the bioavailability of NO and O(2)(*-) and that NO-independent activation of sGC preserves the integrity of this barrier under conditions of NO depletion. NO-independent activation of sGS may be a useful pharmacotherapeutic approach for preservation of glomerular function in CKD thereby reducing the risk for cardiovascular events.

Serial Estimates of Serum Permeability Activity and Clinical Correlates in Patients with Native Kidney Focal Segmental Glomerulosclerosis

A serum or plasma factor in certain patients with focal segmental glomerulosclerosis (FSGS) has been found to increase glomerular albumin permeability (P(alb)) and causes proteinuria in experimental animals. High P(alb) is associated with recurrence of FSGS after transplantation, but serial studies of P(alb) activity in patients with native kidney FSGS have not been performed, and the relationship between P(alb) and remission of proteinuria is not known. This study was designed to determine P(alb) activity before, during, and after 24 wk of treatment with cyclosporine or placebo given as part of a randomized controlled trial in steroid-resistant FSGS patients with nephrotic range proteinuria. Pretreatment P(alb) averaged 0.36 +/- 0.22 and was not significantly different between treatment groups and was not altered during or after the test medication. There was no association between P(alb) activity and remission or relapse in proteinuria. The average P(alb) activity in native kidney FSGS was lower than in previously reported patients with posttransplant recurrence of the disease, and its level did not vary during the course of the study. The antiproteinuric effect of cyclosporine appeared independent of changes in P(alb). This finding is consistent with a direct effect of cyclosporine on glomerular barrier function and/or that within this group of patients the variations in proteinuria are not reflected in changes in Palb because of its limits in terms of reproducibility and responsiveness.