Identification of a New Complement Factor H Mutation in a Patient With Pregnancy-Related Acute Kidney Injury

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Exposure to Nerve Growth Factor Worsens Nephrotoxic Effect Induced by Cyclosporine A in HK-2 Cells

<div><p>Nerve growth factor is a neurotrophin that promotes cell growth, differentiation, survival and death through two different receptors: TrkA^NTR and p75^NTR. Nerve growth factor serum concentrations increase during many inflammatory and autoimmune diseases, glomerulonephritis, chronic kidney disease, end-stage renal disease and, particularly, in renal transplant. Considering that nerve growth factor exerts beneficial effects in the treatment of major central and peripheral neurodegenerative diseases, skin and corneal ulcers, we asked whether nerve growth factor could also exert a role in Cyclosporine A-induced graft nephrotoxicity. Our hypothesis was raised from basic evidence indicating that Cyclosporine A-inhibition of calcineurin-NFAT pathway increases nerve growth factor expression levels. Therefore, we investigated the involvement of nerve growth factor and its receptors in the damage exerted by Cyclosporine A in tubular renal cells, HK-2. Our results showed that in HK-2 cells combined treatment with Cyclosporine A + nerve growth factor induced a significant reduction in cell vitality concomitant with a down-regulation of Cyclin D1 and up-regulation of p21 levels respect to cells treated with Cyclosporine A alone. Moreover functional experiments showed that the co-treatment significantly up-regulated human p21promoter activity by involvement of the Sp1 transcription factor, whose nuclear content was negatively regulated by activated NFATc1. In addition we observed that the combined exposure to Cyclosporine A + nerve growth factor promoted an up-regulation of p75 ^NTR and its target genes, p53 and BAD leading to the activation of intrinsic apoptosis. Finally, the chemical inhibition of p75^NTR down-regulated the intrinsic apoptotic signal. We describe two new mechanisms by which nerve growth factor promotes growth arrest and apoptosis in tubular renal cells exposed to Cyclosporine A.</p> </div

CSA plus NGF treatment activates intrinsic apoptosis via p75^NTR.

<p>(A) caspase 8 and (B) caspase 9 activities in HK-2 cells transfected for 72 h with siRNA targeted human p75^NTR mRNA sequence or with a control siRNA and then treated with CsA 10nM and NGF 100ng/ml alone or in combination for 72h. *p<0.05 combined-treated versus untreated cells; § p<0.05 compared with cells transfected with a control siRNA and treated with CsA+NGF; NS: not significant. The results represent the means ± SD of 3 independent experiments, each performed in triplicate.</p

CSA up-regulates NGF protein levels via NFATc1.

<p>(A) WB of TrKA^NTR (Antibody dilution 1:300), p75^NTR (1:300) and NGF (1:300) basal protein expression in HK-2 cells. β-Actin (1:10000) was used as loading control. (B) HK-2 cells were untreated (c) or treated for 48h with CsA 10nM-100nM-1μM-4μM-8μM before lysis. Equal amounts of total cellular extract were analyzed for NGF levels by western blotting. β-Actin was used as loading control. Bars represent the means ± SD of 3 separate experiments between NGF/β-Actin levels in which band intensities were evaluated as density arbitrary units and expressed as percentages of the control (100%). *p<0.05 vs c. (C) HK-2 cells were treated with CsA 10nM for 3h. Nuclear and cytosolic fractions were analyzed by immunoblotting using anti-NFATc1 Ab (1:100). β-Actin and Lamin B (1:10000) were used respectively as cytosolic and nuclear loading controls. Immunoblots show a single representative of 3 separate experiments. The upper panels represent the means ± SD of 3 separate experiments between NFATc1/β-Actin and NFATc1/Lamin B levels in which band intensities were evaluated as optical density arbitrary units and expressed as percentages of the control which was assumed to be 100%. *p<0.05 vs c. (D) WB of NFATc1 and NGF in total extracts from HK-2 cells treated with CsA 10nM, PMA 100nM+Io 2.5μM and CsA+PMA+Io β-Actin was used as loading control. The upper panels represent the means ± SD of 3 separate experiments between NFATc1/β-Actin and NGF/β-Actin levels in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05 vs c; **p<0.05 vs CsA-treated cells. All immunoblots show a single representative of 3 separate experiments. Secondary antibodies dilutions used are: goat anti-mouse (1:2000) or anti-rabbit (1:7000) or donkey anti-goat (1:3000) IgG. </p

Activated NFATc1 negatively modulates Sp1 nuclear content.

<p>(A) HK-2 cells were untreated (c) or treated with CsA 10nM, NGF 100ng/ml alone or in combination for 15h before lysis. Nuclear and cytosolic expression of NFATc1 (Antibody dilution 1:100) and Sp1 (1:1000) were evaluated. Lamin B (1:10000) and β-Actin (1:10000) were used respectively as nuclear and cytosolic loading controls. The upper panels represent the means ± SD of 3 separate experiments between Sp1/Lamin B or Sp1/β-Actin and NFATc1/Lamin B or NFATc1/β-Actin levels in which band intensities were evaluated as optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05 compared with c; **p<0.05 compared with cells treated with CsA alone (B) NFATc1 and Sp1 nuclear and cytosolic protein content of HK-2 cells untreated (c) or treated for 15 h with CsA 10nM+NGF 100ng/ml, PMA 100nM+Io 2.5μM, PMA+Io+CsA+NGF. The upper panels represent the means ± SD of 3 separate experiments between Sp1/Lamin B or Sp1/β-Actin and NFATc1/Lamin B or NFATc1/β-Actin levels in which band intensities were evaluated as optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05 vs untreated cells. (C) HK-2 cells were untreated (c) or treated with CsA 10nM, NGF 100ng/ml alone or in combination for 24h. Total cell lysates were immunoprecipitated with anti-Sp1 antibody (IP:Sp1) and then subjected to immunoblot analyses with anti-NFATc1 and anti-Sp1 antibodies. The upper panels represent the means ± SD of 3 separate experiments between NFATc1/Sp1 levels in which band intensities were evaluated as optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05 vs untreated cells; **p<0.05 vs cells CsA+NGF-treated. All immunoblots show a single representative of 3 separate experiments. Secondary antibodies dilutions used are: goat anti-mouse (1:2000) or anti-rabbit (1:7000) or donkey anti-goat (1:3000) IgG.</p

Combined treatment with CsA plus NGF inhibits cell growth inducing p21 promoter activity.

<p>(A) MTT growth assays in HK-2 cells untreated (c) or treated with CsA (1–10nM) (A) or NGF (100–250–500ng/ml) (B). Cell proliferation is expressed as fold change respect to control. *p<0.05 vs c ; NS: not significant. (C) MTT growth assay in HK-2 cells untreated (c) or treated with CsA 10nM, NGF 100 ng/ml, alone or in combination for 48h. Results are expressed as fold change respect to c. *p<0.05 compared with c. **p<0.05 compared with CsA-treated cells. (D) CD1 and p21 mRNA content, evaluated by real time RT-PCR after 24 hours of exposure to CsA 10nM and NGF 100ng/ml alone or in combination. Each sample was normalized to its GAPDH mRNA content. *p<0.05 compared with c; **p<0.05 compared with CsA-treated cells. (E) HK-2 cells were untreated (c) or treated for 24h with CsA 10nM and NGF 100ng/ml alone or in combination. CD1 (Antibody dilution 1:500) and p21 (1:500) protein levels were determined by WB. β-Actin (1:10000) was used as loading control. Immunoblots show a single representative of 3 separate experiments. The upper panels represent the means ± SD of 3 separate experiments between CD1/β-Actin and p21/β-Actin levels in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05; **p<0.05 compared with CsA-treated cells. (F) HK-2 cells, transiently transfected with p21 promoter, were untreated (c) or treated for 15h with CsA 10nM, NGF 100ng/ml, Mithramycin (M) 100nM alone or in combination and then luciferase activity was measured. Luciferase activity of untreated cells was set as 1-fold induction, upon which treatments were calculated. *p<0.05 compared with c; **p<0.05 compared with cells CsA+NGF-treated. Bars represent the means ± SD of 3 different experiments each performed in triplicate. Secondary antibodies dilutions used are: goat anti-mouse (1:2000) or anti-rabbit (1:7000). </p

Combined treatment with CSA plus NGF activates intrinsic apoptosis via p75^NTR.

<p>(A) Real time RT-PCR analysis of TrKA^NTR and p75^NTR mRNA treated for 24h with CsA 10nM and NGF 100ng/ml alone or in combination. *p<0.05 compared with c; **p<0.05 compared with cells treated with CsA alone. (B) HK-2 cells were untreated (c) or treated with CsA 10nM and NGF 100ng/ml alone or in combination. Levels of phosphorylated (p) Akt (Ser473) (Antibody dilution 1:1000), mtor (1:500), MAPK (Thr202/Tyr204) (1:1000), JNK (Thr183/Tyr185) (1:200) and total non-phosphorylated proteins (1:1000) were measured in cellular extracts by WB. β-Actin (1:10000) was used as loading control. Numbers over the blots represent the average fold change versus control. (C) p53 and BAD mRNA expression in HK-2 cells untreated (c) or treated for 24h with CsA 10nM and NGF 100ng/ml alone or in combination. *p<0.05 compared with c; **p<0.05 compared with CsA-treated cells. (D) WB of p75^NTR in HK-2 cells transfected for 72h with siRNA targeted human p75^NTR mRNA sequence or with a control siRNA. β-Actin was used as loading control. The upper panels represent the means ± SD of 3 separate experiments between NGF/β-Actin levels in which band intensities were evaluated in terms of optical density arbitrary units and expressed as percentages of the control (100%). *p<0.05. (E) p53 and BAD mRNA levels in HK-2 cells transfected with p75^NTRsiRNA or control-siRNA for 72 hours followed by CsA 10nM and NGF 100ng/ml treatment alone or in combination for 24 hours. *p<0.05 compared to untreated cells (c), **p<0.05 compared with CsA-treated cells; § p<0.05 compared with cells CsA+NGF-treated and transfected with control-siRNA. All mRNA sample were normalized to its GAPDH mRNA content. Bars represent the means ± SD of 3 independent experiments, each performed in triplicate. All immunoblots show a single representative of 3 separate experiments. Secondary antibodies dilutions used are: goat anti-mouse (1:2000) or anti-rabbit (1:7000) IgG or donkey anti-goat (1:3000) .</p