Intravenous clusterin administration reduces myocardial infarct size in rats.

BACKGROUND: Clusterin (Apolipoprotein J), a plasma protein with cytoprotective and complement-inhibiting activities, localizes in the infarcted heart during myocardial infarction (MI). Recently, we have shown a protective effect of exogenous clusterin in vitro on ischaemically challenged cardiomyocytes independent of complement. We therefore hypothesized that intravenous clusterin administration would reduce myocardial infarction damage. METHODS: Wistar rats undergoing experimental MI, induced by 40 min ligation of a coronary vessel, were treated with either clusterin (n=15) or vehicle (n=13) intravenously, for 3 days post-MI. After 4 weeks, hearts were analysed. The putative role of megalin, a clusterin receptor, was also studied. RESULTS: Administration of human clusterin significantly reduced both infarct size (with 75 ± 5%) and death of animals (23% vehicle group vs. 0% clusterin group). Importantly, histochemical analysis showed no signs of impaired wound healing in the clusterin group. In addition, significantly increased numbers of macrophages were found in the clusterin group. We also found that the clusterin receptor megalin was present on cardiomyocytes in vitro which, however, was not influenced by ischaemia. Human clusterin co-localized with this receptor in vitro, but not in the human heart. In addition, using a megalin inhibitor, we found that clusterin did not exert its protective effect on cardiomyocytes through megalin. CONCLUSIONS: Our results thus show that clusterin has a protective effect on cardiomyocytes after acute myocardial infarction in vivo, independent of its receptor megalin. This indicates that clusterin, or a clusterin derivate, is a potential therapeutic agent in the treatment of MI.

Intramolecular epitope spreading in Heymann nephritis.

Immunization with megalin induces active Heymann nephritis, which reproduces features of human idiopathic membranous glomerulonephritis. Megalin is a complex immunological target with four discrete ligand-binding domains (LBDs) that may contain epitopes to which pathogenic autoantibodies are directed. Recently, a 236-residue N-terminal fragment, termed "L6," that spans the first LBD was shown to induce autoantibodies and severe disease. We used this model to examine epitope-specific contributions to pathogenesis. Sera obtained from rats 4 weeks after immunization with L6 demonstrated reactivity only with the L6 fragment on Western blot, whereas sera obtained after 8 weeks demonstrated reactivity with all four recombinant fragments of interest (L6 and LBDs II, III, and IV). We demonstrated that the L6 immunogen does not contain the epitopes responsible for the reactivity to the LBD fragments. Therefore, the appearance of antibodies directed at LBD fragments several weeks after the primary immune response suggests intramolecular epitope spreading. In vivo, we observed a temporal association between increased proteinuria and the appearance of antibodies to LBD fragments. These data implicate B cell epitope spreading in antibody-mediated pathogenesis of active Heymann nephritis, a model that should prove valuable for further study of autoimmune dysregulation.

Routes to covalent catalysis by reactive selection for nascent protein nucleophiles.

Reactivity-based selection strategies have been used to enrich combinatorial libraries for encoded biocatalysts having revised substrate specificity or altered catalytic activity. This approach can also assist in artificial evolution of enzyme catalysis from protein templates without bias for predefined catalytic sites. The prevalence of covalent intermediates in enzymatic mechanisms suggests the universal utility of the covalent complex as the basis for selection. Covalent selection by phosphonate ester exchange was applied to a phage display library of antibody variable fragments (scFv) to sample the scope and mechanism of chemical reactivity in a naive molecular library. Selected scFv segregated into structurally related covalent and noncovalent binders. Clones that reacted covalently utilized tyrosine residues exclusively as the nucleophile. Two motifs were identified by structural analysis, recruiting distinct Tyr residues of the light chain. Most clones employed Tyr32 in CDR-L1, whereas a unique clone (A.17) reacted at Tyr36 in FR-L2. Enhanced phosphonylation kinetics and modest amidase activity of A.17 suggested a primitive catalytic site. Covalent selection may thus provide access to protein molecules that approximate an early apparatus for covalent catalysis.

Mitochondrial encephalopathy with lactic acidosis and stroke-like episodes syndrome with hypothyroidism and focal segmental glomerulosclerosis in a paediatric patient.

Herein, we report on a paediatric patient with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) who was hospitalized for acute on chronic renal insufficiency, seizures and deterioration of the level of consciousness. She also had hypertension, hypothyroidism and nephrotic range proteinuria. Kidney biopsy revealed many sclerotic glomeruli and focal segmental glomerulosclerosis (FSGS). Glomerulopathy is rare in patients with MELAS, and FSGS has been reported only in a few patients. The histopathological features of the renal biopsy suggested that the aetiology of the FSGS may have been secondary to chronic renal injury rather than from a primary immunologic cause. Moreover, our case is unique in that, the coexistence of MELAS, hypothalamic hypothyroidism and FSGS has not been reported in the past. The purpose of this report is to increase the awareness of health-care professionals, especially in the fields of paediatrics, neurology, endocrinology and nephrology, regarding the manifestations and complications of MELAS.

Treatment of membranous nephropathy in children.

Membranous nephropathy (MN) is not a common pediatric glomerular disease and not a common cause of idiopathic nephrotic syndrome (NS) in children. Because of the rarity of the disease, there is only a limited amount of uncontrolled data and no controlled data available in children regarding the treatment of MN. Older uncontrolled data indicate that nearly a quarter of children with NS, whether untreated or treated with various immunosuppressive agents, develop chronic renal failure. Current recommendations for treatment both for children presenting with or without NS therefore are based on controlled data obtained in adults with MN. All children should receive angiotensin-converting enzyme (ACE) inhibitors or angiotensin-receptor blockers (ARBs). Children with NS may be treated initially with corticosteroids. If a satisfactory response is not obtained with corticosteroids, then treatment with cyclosporine or chlorambucil can be tried. The protocols of treatment with these drugs are described in this article.

Early and late recipient graft function and donor outcome after laparoscopic vs open adult live donor nephrectomy for pediatric renal transplantation.

BACKGROUND: Laparoscopically procured live donor kidney grafts are increasingly transplanted into pediatric recipients. The safety and efficacy of this changed surgical practice are unknown. HYPOTHESIS: Outcomes of laparoscopic vs open donor grafts in recipients 18 years and younger are equivalent. DESIGN AND SETTING: Retrospective review at an academic tertiary care referral center. PATIENTS: Eleven consecutive pediatric recipients of laparoscopically procured kidneys between April 1, 1997, and December 31, 2001, were pair matched for age with 11 recipients of openly procured kidneys between December 1, 1991, and March 31, 1997; the 22 adult donors were also studied. MAIN OUTCOME MEASURES: Recipients: surgical complications, graft function and survival. Donors: perioperative morbidity and length of hospital stay. RESULTS: Twenty (91%) of 22 kidneys were donated by a parent of the recipient. In recipients of laparoscopically procured grafts, we observed significantly lower creatinine clearances and higher creatinine levels on days 1, 4, and 6, but by 1 month, graft function was similar in both groups. No significant differences in surgical complications, delayed function, acute and chronic rejection, and graft survival rates were found. No laparoscopic or open donor required blood transfusion, reoperation, or hospital readmission. One laparoscopic donor (9%) was converted to open nephrectomy. For laparoscopic vs open donors, median operative time was longer (difference, 67 min; P =.08), but median postoperative length of stay was significantly shorter (3 vs 5 days; P =.02). CONCLUSIONS: Laparoscopic live donor nephrectomy has no adverse impact on pediatric recipient outcomes. For donors, the laparoscopic operation is safe and the hospital stay is shortened. These results support the continued use of laparoscopically procured live donor kidneys in pediatric renal transplantation.

Idiopathic membranous nephropathy: an autoimmune disease.

For more than 50 years researchers have debated the evidence for an autoimmune basis of human idiopathic membranous nephritis (MN). Work published in the past 2 years has substantially strengthened the belief that MN is indeed an autoimmune disease of the kidney. Autoantibodies of the IgG4 subclass to at least three podocyte membrane proteins including phospholipase A(2)-receptor, aldose reductase, and manganese superoxide dismutase have been detected by immunoblotting in sera as well as in acid eluates prepared from renal biopsy tissue of patients with this disease, using either whole tissue or microdissected glomeruli from frozen sections. In each case the podocyte antigen has been shown to co-localize with the subepithelial glomerular immune deposits in renal tissue of the same patients. It is not certain if any of these podocyte proteins is an inciting/primary autoantigen or whether they are secondary antigens recruited by intermolecular epitope-spreading, initiating from a yet-to-be-discovered autoantigen. Although it is clear that autoantibodies to podocyte membrane proteins are elicited in idiopathic MN and contribute to the formation of the subepithelial deposits, many questions remain concerning the triggers for their development and their contribution toward proteinuria and progression of the disease.

Nested N-terminal megalin fragments induce high-titer autoantibody and attenuated Heymann nephritis.

It was shown previously that an N-terminal fragment (nM60) that encompasses amino acid residues 1 to 563 of megalin could induce active Heymann nephritis (AHN) as efficiently as the native protein. For delineation of a minimal structure within this fragment that is sufficient to induce AHN, smaller protein fragments that encompass residues 1 to 236 (L6), 1 to 195 (L5), 1 to 156 (L4), and 1 to 120 (L3), representing successive C-terminal truncations within ligand-binding repeats of nM60, were cloned and produced in a baculovirus insect cell expression system. Protein fragments L4, L5, and L6 clearly were glycosylated. All four fragments stimulated proliferation of megalin-sensitized lymph node cells and induced high-titer anti-megalin autoantibodies in Lewis rats. A full-blown disease, as assessed by severity of proteinuria, was observed in rats that were immunized with L6 and L5, whereas animals that were immunized with L4 and L3 developed only mild disease. The proteinuria levels correlated with staining for complement (C3, C5b-9) and IgG1 isotype in glomerular immune deposits. The results suggest that one or more molecular determinants on the region that comprises amino acid residues 157 to 236 contribute to the induction of a full-blown form of AHN. Study of the structure, conformation, and posttranslational modifications of these determinants could provide greater insight into the molecular correlates of immunopathogenesis in this disease model.

Conformation and glycosylation of a megalin fragment correlate with nephritogenicity in Heymann nephritis.

Active Heymann nephritis (AHN), a rat model of autoimmune glomerulonephritis, is induced by immunization with autologous megalin, a 600-kDa cell surface glycoprotein isolated from crude renal extracts. Recombinant proteins containing a 563-residue N-terminal sequence of megalin were obtained from Escherichia coli and baculovirus-insect cell expression systems. Rats immunized with the soluble, secreted protein encoded by a baculovirus construct elicited high titer anti-megalin autoantibodies and developed glomerular immune deposits and elevated proteinuria consistent with AHN. Rats treated with the bacterial or nonsecreted insect cell proteins produced a milder anti-megalin response and did not develop the disease. Nephritogenicity appeared to correlate with conformational or other structural features of native megalin. All three recombinant proteins were reactive in Western blots with rabbit anti-megalin antiserum, whereas the insect cell-derived proteins reacted preferentially in Western blot and ELISA with anti-megalin autoantibodies from rats with AHN induced by native megalin. Only the secreted insect cell product was stained in a lectin blot, suggesting its specific glycosylation. These observations provide evidence that a megalin N-terminal domain includes B and T cell epitopes sufficient for a pathogenic autoimmune response and that a native-like conformation and glycosylation are essential for the induction of disease. The importance of conformational B cell epitopes for pathogenic autoantibodies recapitulates observations made in other models of organ-specific autoimmune disease. Glycosidic modifications could influence the presentation of either B or T cell epitopes in AHN, consistent with emerging evidence of the role of post-translational modifications in pathogenic autoimmune responses.

Life-threatening extrarenal lupus in children despite improvement in serologic findings.

Systemic lupus erythematosus (SLE) is an autoimmune disorder with the potential for multiorgan involvement. Serologic tests are helpful in establishing the diagnosis of SLE and predicting disease flares. However, there are few data on the relationship between the onset of new organ involvement and lupus serologies, especially in children. This report details our experience in managing two children with lupus nephritis. Both developed life-threatening extrarenal complications (cerebritis and carditis) soon after receiving high-dose immunosuppressive therapy and despite normalizing serologies. This lack of concordance between serologies and the development of carditis and cerebritis needs to be recognized so that health care professionals treating children with SLE can promptly intensify immunosuppressive medications and avoid life-threatening delays from seeking alternative explanations for symptomatology.

QTc interval in children with chronic renal failure and with renal transplants.

Prolongation of the QTc interval, a risk factor for cardiac arrhythmias, has been observed in adult hemodialysis patients; there are few data on the QTc interval in children with chronic renal failure (CRF) and following renal transplantation (Tx). The purpose of our study was to determine the QTc interval in children with CRF and post renal Tx. Twenty children with CRF and 16 children with renal Tx who were followed at the University of California, Davis, underwent prospective EKG monitoring. The mean QTc interval in the CRF and post-Tx cohorts was normal at 407.9 ms and 408.2 ms, respectively. None of the children with CRF had QTc prolongation. Two Tx recipients had QTc prolongation; both had cardiac dilatation on echocardiography (ECHO). There was no correlation between the QTc interval and the creatinine clearance in either group. However, a significant correlation was noted between QTc prolongation and cardiac dilatation on ECHO in the Tx group (P=0.02, Fisher's exact test). In conclusion, QTc prolongation is uncommon in children with CRF and following Tx, in the absence of cardiac dilatation. However, caution is still needed before prescribing medications known to cause QTc prolongation.

Microhematuria after renal transplantation in children.

The renal transplant (Tx) recipient is at risk for developing various complications including urolithiasis, the only manifestation of which may be hematuria. However, there are no data on the prevalence of microscopic hematuria in renal Tx recipients. The objective of our study was to determine the prevalence of microhematuria in our pediatric Tx patients and to investigate the causes of microhematuria. Records of all pediatric renal Tx recipients followed at our center from September 1999 to September 2000 were retrospectively reviewed; of the 21 patients, seven (33%) had persistent microscopic hematuria that was first noted 2.9 years post-Tx. Patients with and without hematuria had similar baseline characteristics. Only one patient had pre-existing hematuria that continued post-Tx. The etiology of hematuria in the other six patients was: recurrent IgA nephropathy (one patient), CMV nephritis (one patient), and unexplained (four patients). None had renal calculi or hypercalciuria. Three of the four patients with unexplained hematuria have chronic allograft nephropathy, and the fourth (original disease dysplasia) has hypocomplementemia. At their last follow-up, 5.3 years after onset of hematuria, all patients are alive with stable allograft function. In conclusion, microscopic hematuria is not uncommon in pediatric renal Tx recipients. While causes of post-Tx hematuria are diverse, stones are not commonly seen. Whether chronic allograft nephropathy per se can be implicated as a cause of hematuria remains to be determined. Renal biopsies should be considered at the onset of hematuria if proteinuria and/or deterioration in renal function are seen concomitantly, to look for recurrent or de novo glomerulonephritis.

Pilot study describing the use of pravastatin in pediatric renal transplant recipients.

Renal transplant (Tx) recipients frequently develop hypercholesterolemia. Pravastatin (P) has been shown to be effective in adult renal Tx recipients, not only in reducing serum cholesterol, but possibly also in decreasing graft rejection. However, there are no data on the use of P in children following renal transplantation. We conducted a retrospective case-control study evaluating the safety and efficacy of P (10-20 mg/day) in reducing hypercholesterolemia, when used pre-emptively in the post-Tx period in seven children, compared with an historical control (C) group of nine children who had not received P. The two groups were comparable with respect to their demographics and in their pretransplant serum cholesterol. Compared with the C group, the mean serum cholesterol in the P group was lower at 3 months (159 mg/dL vs. 225 mg/dL), 6 months (134 mg/dL vs. 200 mg/dL), 9 months (134 mg/dL vs. 209 mg/dL), and 12 months (125 mg/dL vs. 195 mg/dL) (p < 0.005 for all, Student's two-tailed t-test). At 1 month only 43% of the P group had hypercholesterolemia compared with 67% of the controls; by 12 months this difference was even more significant (0% in the P group vs. 45% in the C group). None of the treated patients developed any adverse reactions. This study demonstrates that the pre-emptive use of P in pediatric renal Tx recipients appears to be effective in significantly reducing serum cholesterol. Whether this effect will translate into improved allograft and patient survival in the long term cannot be predicted at present and will require additional studies to evaluate.

A small N-terminal 60-kD fragment of gp600 (megalin), the major autoantigen of active Heymann nephritis, can induce a full-blown disease.

Active Heymann nephritis of rat, an autoimmune glomerular disease, is an immunohistological, ultrastructural, and clinical model of human membranous glomerulonephritis. Both diseases in their full-blown form are characterized by (1) the formation of large, subepithelial glomerular immune deposits, which stain for IgG, C3, and membrane attack (C5b-9) components of complement and (2) the excretion of large amounts of protein in the urine (proteinuria). The target autoantigen of active Heymann nephritis is a large transmembrane renal glycoprotein with a molecular weight of approximately 600 kD, variously named gp600, gp330, LRP-2, or "megalin." This study was performed to identify the region in this enormously large glycoprotein that would produce full-blown active Heymann nephritis. A stable, small (60-kD) proteolytic fragment of gp600 was isolated and localized to the N-terminal end of the molecule using Western blot, sequencing, and amino acid analyses. Based on its primary structure, this fragment contains approximately 60 cysteine residues, the cross-linking of which to each other probably explains its stability. Immunization of rats with this fragment induced a full-blown disease that was comparable to the disease induced by a preparation containing the whole protein. These results indicate that this small fragment, retaining the natural disulfide bonds and probably its overall structure, contains those B and T cell epitopes that are sufficient to produce this organ-specific autoimmune disease.

Bilateral infundibulopelvic stenosis without renal insufficiency: is surgery necessary?

The following case describes the clinical course of a patient with bilateral infundibulopelvic stenosis from her initial presentation at age 2 through the age of 14 years. This condition is associated with hypoplasia of segments of the upper collecting system and is characterized by dilated calyces that drain through stenotic infundibulae. Our patient is unique in that, although her renal function has slowly deteriorated with time, she has a persistently non-obstructive pattern on dynamic imaging studies. Only a minority of patients reported in the literature with this condition progress to renal insufficiency or failure. Although some patients have undergone surgery for presumed obstruction, surgical intervention has no proven benefit. Patients are at risk for hyperfiltration injury and should be followed for the development of hypertension, proteinuria and renal insufficiency. Without evidence of obstruction, medical management remains the cornerstone of treatment.