M-type phospholipase A2 receptor as target antigen in idiopathic membranous nephropathy.

BACKGROUND: Idiopathic membranous nephropathy, a common form of the nephrotic syndrome, is an antibody-mediated autoimmune glomerular disease. Serologic diagnosis has been elusive because the target antigen is unknown. METHODS: We performed Western blotting of protein extracts from normal human glomeruli with serum samples from patients with idiopathic or secondary membranous nephropathy or other proteinuric or autoimmune diseases and from normal controls. We used mass spectrometry to analyze the reactive protein bands and confirmed the identity and location of the target antigen with a monospecific antibody. RESULTS: Serum samples from 26 of 37 patients (70%) with idiopathic but not secondary membranous nephropathy specifically identified a 185-kD glycoprotein in nonreduced glomerular extract. Mass spectrometry of the reactive protein band detected the M-type phospholipase A(2) receptor (PLA(2)R). Reactive serum specimens recognized recombinant PLA(2)R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA(2)R antibody. Anti-PLA(2)R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA(2)R was expressed in podocytes in normal human glomeruli and colocalized with IgG4 in immune deposits in glomeruli of patients with membranous nephropathy. IgG eluted from such deposits in patients with idiopathic membranous nephropathy, but not in those with lupus membranous or IgA nephropathy, recognized PLA(2)R. CONCLUSIONS: A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA(2)R. PLA(2)R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA(2)R is a major antigen in this disease.

Rituximab-induced depletion of anti-PLA2R autoantibodies predicts response in membranous nephropathy.

Autoantibodies to the M-type phospholipase A(2) receptor (PLA(2)R) are sensitive and specific for idiopathic membranous nephropathy. The anti-B cell agent rituximab is a promising therapy for this disease, but biomarkers of early response to treatment currently do not exist. Here, we investigated whether levels of anti-PLA(2)R correlate with the immunological activity of membranous nephropathy, potentially exhibiting a more rapid response to treatment than clinical parameters such as proteinuria. We measured the amount of anti-PLA(2)R using Western blot immunoassay in serial serum samples from a total of 35 patients treated with rituximab for membranous nephropathy in two distinct cohorts. Pretreatment samples from 25 of 35 (71%) patients contained anti-PLA(2)R, and these autoantibodies declined or disappeared in 17 (68%) of these patients within 12 months after rituximab. Those who demonstrated this immunologic response fared better clinically: 59% and 88% attained complete or partial remission by 12 and 24 months, respectively, compared with 0% and 33% among those with persistent anti-PLA(2)R levels. Changes in antibody levels preceded changes in proteinuria. One subject who relapsed during follow-up had a concomitant return of anti-PLA(2)R. In summary, measuring anti-PLA(2)R levels by immunoassay may be a method to follow and predict response to treatment with rituximab in membranous nephropathy.

The fate of Notch-deficient nephrogenic progenitor cells during metanephric kidney development.

To determine which nephron segments require Notch signals for development, we conditionally deleted Rbpj, a transcription factor required for canonical Notch signaling, in nephrogenic progenitors (NPs) of the metanephric mesenchyme. The retinoic acid receptor-ß2 (Rarb2) promoter efficiently directed Cre-recombinase (Cre) activity to these progenitors. Conditional knockout of Rbpj in mice (Rarb2Cre(+)/Rbpj (f/-)) caused severe renal hypoplasia, as indicated by a 70-95% reduction in nephron number and the development of tubular cysts. To track the fate of NPs following Rarb2Cre expression, we labeled them with membrane-associated enhanced green fluorescent protein (GFP). In TomatoGFP(+)/Rarb2Cre(+) control mice, NPs differentiated into epithelia of all nephron segments, except into collecting ducts. In TomatoGFP(+)/Rarb2Cre(+)/Rbpj (f/-) conditional knockout mice, NPs developed into podocytes or distal tubular epithelia, indicating that canonical Notch signals were not required for mesenchymal-to-epithelial transition or for the specification of these nephron segments. Conversely, the few proximal tubules and associated cysts that developed in these mice were derived from the 5-10% of NPs that had failed to express Cre and, therefore, had intact Notch signaling. Thus, our fate mapping studies establish that the profound effect of Notch signaling on nephrogenesis is due to the specification of proximal but not distal tubules or podocytes.

Induction of heat shock protein 70 inhibits ischemic renal injury.

Heat shock protein 70 (Hsp70) is a potent antiapoptotic agent. Here, we tested whether it directly regulates renal cell survival and organ function in a model of transient renal ischemia using Hsp70 knockout, heterozygous, and wild-type mice. The kidney cortical Hsp70 content inversely correlated with tubular injury, apoptosis, and organ dysfunction after injury. In knockout mice, ischemia caused changes in the activity of Akt and glycogen synthase kinase 3-ß (kinases that regulate the proapoptotic protein Bax), increased active Bax, and activated the proapoptotic protease caspase 3. As these changes were significantly reduced in the wild-type mice, we tested whether Hsp70 influences ischemia-induced apoptosis. An Hsp70 inducer, geranylgeranylacetone, increased Hsp70 expression in heterozygous and wild-type mice, and reduced both ischemic tubular injury and organ dysfunction. When administered after ischemia, this inducer also decreased tubular injury and organ failure in wild-type mice but did not protect the knockout mice. ATP depletion in vitro caused greater mitochondrial Bax accumulation and death in primary proximal tubule cells harvested from knockout compared with wild-type mice and altered serine phosphorylation of a Bax peptide at the Akt-specific target site. In contrast, lentiviral-mediated Hsp70 repletion decreased mitochondrial Bax accumulation and rescued Hsp70 knockout cells from death. Thus, increasing Hsp70 either before or after ischemic injury preserves renal function by attenuating acute kidney injury.

Hexokinase regulates Bax-mediated mitochondrial membrane injury following ischemic stress.

Hexokinase (HK), the rate-limiting enzyme in glycolysis, controls cell survival by promoting metabolism and/or inhibiting apoptosis. Since HK isoforms I and II have mitochondrial targeting sequences, we attempted to separate the protective effects of HK on cell metabolism from those on apoptosis. We exposed renal epithelial cells to metabolic stress causing ATP depletion in the absence of glucose and found that this activated glycogen synthase kinase 3ß (GSK3ß) and Bax caused mitochondrial membrane injury and apoptosis. ATP depletion led to a progressive HK II dissociation from mitochondria, released mitochondrial apoptosis inducing factor and cytochrome c into the cytosol, activated caspase-3, and reduced cell survival. Compared with control, adenoviral-mediated HK I or II overexpression improved cell survival following stress, but did not prevent GSK3ß or Bax activation, improve ATP content, or reduce mitochondrial fragmentation. HK I or HK II overexpression increased mitochondria-associated isoform-specific HK content, and decreased mitochondrial membrane injury and apoptosis after stress. In vivo, HK II localized exclusively to the proximal tubule. Ischemia reduced total renal HK II content and dissociated HK II from proximal tubule mitochondria. In cells overexpressing HK II, Bax and HK II did not interact before or after stress. While the mechanism by which HK antagonizes Bax-mediated apoptosis is unresolved by these studies, one possible scenario is that the two proteins compete for a common binding site on the outer mitochondrial membrane.

Plazomicin for the treatment of multidrug-resistant Klebsiella bacteraemia in a patient with underlying chronic kidney disease and acute renal failure requiring renal replacement therapy.

A 75-year-old man presented with shortness of breath and somnolence and was found to have urosepsis. Blood and urine cultures subsequently grew multidrug-resistant (MDR) Klebsiella pneumoniae (Kp) with the New Delhi metallo-ß-lactamase gene. The patient was treated successfully with plazomicin and meropenem/vaborbactam combination therapy. The course was complicated by acute kidney injury temporarily requiring haemodialysis, gastrointestinal bleed requiring multiple transfusions and hospital readmission with blood cultures again positive with MDR Kp. Plazomicin drug levels were persistently high during treatment, suggesting that therapeutic drug monitoring may be needed to safely use this drug in patients with severe renal dysfunction. This case marks the first use of plazomicin for bacteraemia in the literature outside of a clinical trial and demonstrates its safe and effective use in a patient with advanced renal disease, and provides important insights about dosing and therapeutic drug monitoring considerations in this patient population.

Evaluating the role of nucleic acid antigens in murine models of systemic lupus erythematosus.

Impaired apoptotic cell clearance is thought to contribute to the pathogenesis of systemic autoimmune disease, in particular systemic lupus erythematosus (SLE). Endogenous RNA- and DNA-containing autoantigens released from dying cells can engage Toll-like receptors (TLR) 7/8 and TLR9, respectively in a number of immune cell types, thereby promoting innate and adaptive immune responses. Mouse models of lupus reliably phenocopy many of the characteristic features of SLE in humans and these models have proved invaluable in defining disease mechanisms. TLR7 signaling is essential for the development of autoantibodies to RNA and RNA-associated proteins like Sm and RNP, while TLR9 signaling is important for the development of antibodies to DNA and chromatin. TLR7 deficiency ameliorates end-organ disease, but, surprisingly, TLR9 deficiency exacerbates disease, possibly as a result of TLR7 overactivity in TLR9-deficient mice. Deficiency of interferon regulatory factor 5 (IRF5) inhibits autoantibody production and ameliorates disease likely due to its role in both TLR7 and TLR9 signaling. In this report we describe methods to analyze two commonly used mouse models of SLE in which TLRs and/or IRF5 have been shown to play a role in disease pathogenesis.

Role of mitofusin 2 in the renal stress response.

The role of mitofusin 2 (MFN2), a key regulator of mitochondrial morphology and function in the renal stress response is unknown. To assess its role, the MFN2 floxed gene was conditionally deleted in the kidney of mice (MFN2 cKO) by Pax2 promoter driven Cre expression (Pax2Cre). MFN2 cKO caused severe mitochondrial fragmentation in renal epithelial cells that are critical for normal kidney tubular function. However, despite a small (20%) decrease in nephron number, newborn cKO pups had organ or tubular function that did not differ from littermate Cre-negative pups. MFN2 deficiency in proximal tubule epithelial cells in primary culture induced mitochondrial fragmentation but did not significantly alter ATP turnover, maximal mitochondrial oxidative reserve capacity, or the low level of oxygen consumption during cyanide exposure. MFN2 deficiency also did not increase apoptosis of tubule epithelial cells under non-stress conditions. In contrast, metabolic stress caused by ATP depletion exacerbated mitochondrial outer membrane injury and increased apoptosis by 80% in MFN2 deficient vs. control cells. Despite similar stress-induced Bax 6A7 epitope exposure in MFN2 deficient and control cells, MFN2 deficiency significantly increased mitochondrial Bax accumulation and was associated with greater release of both apoptosis inducing factor and cytochrome c. In conclusion, MFN2 deficiency in the kidney causes mitochondrial fragmentation but does not affect kidney or tubular function during development or under non-stress conditions. However, MFN2 deficiency exacerbates renal epithelial cell injury by promoting Bax-mediated mitochondrial outer membrane injury and apoptosis.

A comparison of the acute hemodynamic and delayed effects of 50% exchange transfusion with two different cross-linked hemoglobin based oxygen carrying solutions and Pentastarch.

BACKGROUND: Hemoglobin based oxygen carrying solutions (HBOC) have been designed to combine the beneficial effects of colloidal solutions with oxygen carrying capacity. Clinical trials in humans using HBOCs have had variable results. METHODS: We used a rodent 50% exchange model to compare Hemolink and Hemopure HBOC to autologous blood and Pentastarch solution. We monitored hemodynamic parameters, hemoglobin clearance, weight gain and hematocrit over a five-day period. RESULTS: Acute hemodynamic effects between the two HBOCs were similar with mild vasoconstriction. Cardiac output, systemic vascular resistance and renal function were similar to that seen with blood. HBOC's were associated with hemoglobinuria with a half-life in the circulation of 13.8 hrs for Hemolink and 19.2 hrs for Hemopure. Animals resuscitated with HBOCs exhibited delayed weight gain. CONCLUSION: Hemodynamic effects in rodents exchange-transfused with blood, Hemolink, or Hemopure were similar. The delayed weight gain observed with the HBOCs must be investigated.

Rapamycin ameliorates proteinuria-associated tubulointerstitial inflammation and fibrosis in experimental membranous nephropathy.

Proteinuria is a risk factor for progression of chronic renal failure. A model of proteinuria-associated tubulointerstitial injury was developed and was used to examine the therapeutic effect of rapamycin. Two studies were performed. In study A, proteinuric rats were given sheep anti-Fx1A to induce experimental membranous nephropathy; control rats received normal sheep serum. Four weeks later, groups were subdivided and underwent laparotomy alone (two kidneys), nephrectomy alone (one kidney), or nephrectomy with polectomy (0.6 kidney). Renal function and morphology were evaluated 4 wk later. Whereas control rats never developed proteinuria, anti-Fx1A induced severe proteinuria. Proteinuria was unaffected by renal mass reduction. Proteinuric rats developed tubulointerstitial disease that was most severe in rats with 0.6 kidneys. Renal function (GFR) was reduced by loss of renal mass and was reduced further in proteinuric rats with 0.6 kidneys. In study B, the effect of rapamycin on the expression of candidate proinflammatory and profibrotic genes and the progression of proteinuria-associated renal disease were examined. All rats received an injection of anti-Fx1A and were nephrectomized and then divided into groups to receive rapamycin or vehicle. Gene expression, renal morphology, and GFR were evaluated after 4, 8, and 12 wk. Rapamycin reduced expression of the proinflammatory and profibrotic genes (monocyte chemotactic protein-1, vascular endothelial growth factor, PDGF, TGF-beta(1), and type 1 collagen). Tubulointerstitial inflammation and progression of interstitial fibrosis that were present in vehicle-treated rats were ameliorated by rapamycin. Rapamycin also completely inhibited compensatory renal hypertrophy. In summary, rapamycin ameliorates the tubulointerstitial disease associated with chronic proteinuria and loss of renal mass.