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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-β2 (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. [ABSTRACT FROM AUTHOR]

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 A2 receptor (PLA2R). Reactive serum specimens recognized recombinant PLA2R and bound the same 185-kD glomerular protein as did the monospecific anti-PLA2R antibody. Anti-PLA2R autoantibodies in serum samples from patients with membranous nephropathy were mainly IgG4, the predominant immunoglobulin subclass in glomerular deposits. PLA2R 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 PLA2R. Conclusions: A majority of patients with idiopathic membranous nephropathy have antibodies against a conformation-dependent epitope in PLA2R. PLA2R is present in normal podocytes and in immune deposits in patients with idiopathic membranous nephropathy, indicating that PLA2R is a major antigen in this disease. N Engl J Med 2009;361:11-21. [ABSTRACT FROM AUTHOR]

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 TM and Hemopure TM 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 TM , or Hemopure TM were similar. The delayed weight gain observed with the HBOCs must be investigated. [ABSTRACT FROM AUTHOR]

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-β (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. [ABSTRACT FROM AUTHOR]

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 3β (GSK3β) 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 GSK3β 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background Ischemic AKI lacks a urinary marker for early diagnosis and an effective therapy. Differential nucleophosmin (NPM) phosphorylation is a potential early marker of ischemic renal cell injury and a therapeutic target. Methods Differential NPM phosphorylation was assessed by mass spectrometry in NPM harvested from murine and human primary renal epithelial cells, fresh kidney tissue, and urine before and after ischemic injury. The biologic behavior and toxicity of NPM was assessed using phospho-NPM mutant proteins that either mimic stress-induced or normal NPM phosphorylation. Peptides designed to interfere with NPM function were used to explore NPM as a therapeutic target. Results Within hours of stress, virtually identical phosphorylation changes were detected at distinct serine/threonine sites in NPM harvested from primary renal cells, tissue, and urine. A phosphomimic NPM protein that replicated phosphorylation under stress localized to the cytosol, formed monomers that interacted with Bax, a cell death protein, coaccumulated with Bax in isolated mitochondria, and significantly increased cell death after stress; wild-type NPM or a phosphomimic NPM with a normal phosphorylation configuration did not. Three renal targeted peptides designed to interfere with NPM at distinct functional sites significantly protected against cell death, and a single dose of one peptide administered several hours after ischemia that would be lethal in untreated mice significantly reduced AKI severity and improved survival. Conclusions These findings establish phosphorylated NPM as a potential early marker of ischemic AKI that links early diagnosis with effective therapeutic interventions., (Copyright © 2019 by the American Society of Nephrology.)

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.

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.

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-β (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., (© 2011 International Society of Nephrology)

Polymorphisms in the transcription factor IFN regulatory factor 5 (IRF5) are strongly associated in human genetic studies with an increased risk of developing the autoimmune disease systemic lupus erythematosus. However, the biological role of IRF5 in lupus pathogenesis has not previously been tested in an animal model. In this study, we show that IRF5 is absolutely required for disease development in the FcgammaRIIB(-/-)Yaa and FcgammaRIIB(-/-) lupus models. In contrast to IRF5-sufficient FcgammaRIIB(-/-)Yaa mice, IRF5-deficient FcgammaRIIB(-/-)Yaa mice do not develop lupus manifestations and have a phenotype comparable to wild-type mice. Strikingly, full expression of IRF5 is required for the development of autoimmunity, as IRF5 heterozygotes had dramatically reduced disease. One effect of IRF5 is to induce the production of the type I IFN, IFN-alpha, a cytokine implicated in lupus pathogenesis. To address the mechanism by which IRF5 promotes disease, we evaluated FcgammaRIIB(-/-)Yaa mice lacking the type I IFN receptor subunit 1. Unlike the IRF5-deficient and IRF5-heterozygous FcgammaRIIB(-/-)Yaa mice, type I IFN receptor subunit 1-deficient FcgammaRIIB(-/-)Yaa mice maintained a substantial level of residual disease. Furthermore, in FcgammaRIIB(-/-) mice lacking Yaa, IRF5-deficiency also markedly reduced disease manifestations, indicating that the beneficial effects of IRF5 deficiency in FcgammaRIIB(-/-)Yaa mice are not due only to inhibition of the enhanced TLR7 signaling associated with the Yaa mutation. Overall, we demonstrate that IRF5 plays an essential role in lupus pathogenesis in murine models and that this is mediated through pathways beyond that of type I IFN production.

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease for which current therapy is suboptimal. SLE is characterized by autoantibody production, with renal disease and premature atherosclerosis being common and severe manifestations causing appreciable morbidity and mortality. Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists are widely used in the treatment of diabetes mellitus for their insulin-sensitizing properties, but also have immunomodulatory effects. In this report, we show that the PPARgamma agonist rosiglitazone reduces autoantibody production, renal disease, and atherosclerosis in mouse models of SLE. The beneficial effect of rosiglitazone on SLE manifestations depends on the induction of adiponectin, because rosiglitazone has no effect on autoantibody production or renal disease in lupus mice that lack adiponectin. In addition, lupus mice that lack adiponectin develop more severe disease than adiponectin-sufficient lupus mice, indicating that endogenous adiponectin is involved in regulating disease activity. Furthermore, administration of exogenous adiponectin ameliorates disease. These experiments suggest that PPARgamma agonists may be useful agents for the treatment of SLE. They also demonstrate that induction of adiponectin is a major mechanism underlying the immunomodulatory effects of PPARgamma agonists.

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.