Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: Characteristics of the PIC nanocarriers. (A) Chemical structures of PEG-PLL block copolymers and P(Asp) homopolymers. (B) Size distribution of the PIC nanocarriers was measured by DLS.

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: Blocking Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: In vitro transfection of siRNAs complexed with PIC nanocarriers into mesangial cells. (A) Fluorescence photomicrograph of the cultured mesangial cells treated with the FITC-labeled nonsilencing control siRNAs (naked), FITC-labeled siRNAs complexed with the nanocarriers (PIC), FITC-labeled siRNAs encapsulated in HVJ-E (HVJ), or nontreated (NT) cells. The PIC nanocarriers, in contrast to HVJ-E, were shown to pass through the 0.2-μm-sized filter. Figures are representative of three independent experiments. Original magnification ×200. (B and C) Q-RT-PCR analysis of the MAPK1 expression in the cultured mesangial cells. The transfection of the MAPK1 siRNAs complexed with the nanocarriers significantly suppressed the MAPK1 mRNA expression at a concentration of more than 50 nM MAPK1 siRNAs in a dose-dependent manner. P values were calculated by ANOVA, mean ± SEM, n = 5. NSC/PIC, nonsilencing control siRNAs complexed with PIC nanocarriers; MPK/naked, naked MAPK1 siRNAs; MPK/PIC, MAPK1 siRNAs complexed with PIC nanocarriers.

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: In Vitro, Transfection, Fluorescence, Cell Culture, Labeling, Control, Reverse Transcription Polymerase Chain Reaction, Expressing, Concentration Assay

Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: In vivo and ex vivo optical imaging analysis. (A) Cy5-labeled control siRNAs complexed with the PIC nanocarriers accumulated in kidneys immediately after intraperitoneal injection and retained high fluorescence signals in kidneys for more than 3 hours. (B) Ex vivo imaging for kidneys 3.5 hours postinjection. Prolonged signal was detected in the whole kidneys, including cortical area of the mouse treated with siRNA/nanocarriers complex, compared with naked siRNAs. Figures are representative of three independent experiments. NT, nontreatment; naked, naked siRNAs; PIC, siRNAs complexed with the PIC nanocarriers.

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: In Vivo, Ex Vivo, Optical Imaging, Labeling, Control, Injection, Fluorescence, Imaging

Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: The PIC nanocarrier complex accumulates in glomeruli. (A) Confocal microscopy analysis and cross-sectional intensity measurements showing that Cy5-labeled siRNAs complexed with the PIC nanocarriers were taken up in the glomeruli at 3.5 or 5.5 hours after intraperitoneal injection. naked, naked siRNAs; PIC, siRNAs complexed with the PIC nanocarriers; HVJ, siRNAs encapsulated in HVJ-E. (B) Fluorescence photomicrograph of the kidney section at 6 hours after intraperitoneal injection of FITC-labeled P(Asp) complexed with the PIC nanocarriers (P(Asp)/PIC), compared with nontreatment (NT), naked P(Asp), and P(Asp) encapsulated in HVJ-E (P(Asp)/HVJ). The high-fluorescence intensity was detected in almost all glomeruli (yellow arrows and inset) of the mice treated with P(Asp)/PIC. Autofluorescence was found in tubular region. Figures are representative of three independent experiments. Original magnification ×200.

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: Confocal Microscopy, Labeling, Injection, Fluorescence

Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: Silencing of intraglomerular MAPK1 by MAPK1 siRNAs complexed with the PIC nanocarriers. (A) Q-RT-PCR analysis of MAPK1 mRNA expression in isolated glomeruli of MRL/lpr mice. NT, nontreatment; NSC/PIC, nonsilencing control siRNAs complexed with PIC nanocarriers; MPK/PIC, MAPK1 siRNAs complexed with PIC nanocarriers; MPK/HVJ, MAPK1 siRNAs encapsulated in HVJ-E. Nontreated BALB-c mice were used as control. P values were calculated by ANOVA, mean ± SEM, n = 5. (B) In situ hybridization for MAPK1 mRNA in the kidney sections using antisense (AS) and sense (S) probes. Original magnification ×200. (C) Western blots and densitometry analysis of the results. MAPK1 siRNAs complexed with the PIC nanocarriers suppressed the expression of both MAPK1 protein (left) and P-MAPK1 protein (right). P values were calculated by ANOVA, mean ± SEM, n = 4. (D) MAPK1 and P-MAPK1 immunostaining of the kidney sections. Original magnification ×200. (E) Densitometry analysis in panel D. Data were expressed as the positive-stained area of MAPK1 (left) or P-MAPK1 (right) in a glomerulus (%).

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Isolation, Control, In Situ Hybridization, Western Blot, Immunostaining, Staining

Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: Amelioration of the glomerular lesions by MAPK1 siRNAs complexed with the PIC nanocarriers. (A) Representative glomeruli (upper) and tubulointerstitial regions (lower) are shown. NT, nontreatment; NSC/PIC, nonsilencing control siRNAs complexed with PIC nanocarriers; MPK/PIC, MAPK1 siRNAs complexed with PIC nanocarriers; MPK/HVJ, MAPK1 siRNAs encapsulated in HVJ-E. Original magnification ×200. (B–E) Histologic analysis of the kidney sections. The glomerular sclerosis score (B), the number of glomeruli with global sclerosis (D), and the PAS-positive glomerular lesions (%) (E) were significantly decreased in the group treated with MAPK1 sRNAs complexed with the nanocarriers. Semiquantitative scoring of tubulointerstitial injury (C) showed no significant differences between the four groups. P values were calculated by ANOVA, mean ± SEM, n = 6.

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: Control

Journal: Journal of the American Society of Nephrology : JASN

Article Title: siRNA-Based Therapy Ameliorates Glomerulonephritis

doi: 10.1681/ASN.2009030295

Figure Lengend Snippet: TGF-β1, PAI-1, and fibronectin (FN) expression are suppressed by intraglomerular MAPK1 silencing. (A) Representative photomicrographs. Upper: In situ hybridization for TGF-β1 mRNA in the kidney sections. Lower: Immunohistochemistry for PAI-1 and FN expression in the glomeruli. NT, nontreatment; NSC/PIC, nonsilencing control siRNAs complexed with PIC nanocarriers; MPK/PIC, MAPK1 siRNAs complexed with PIC nanocarriers; MPK/HVJ, MAPK1 siRNAs encapsulated in HVJ-E; AS, antisense probe; S, sense probe. Original magnification ×200. (B) Q-RT-PCR analysis (upper) revealed that the treatment with MAPK1 siRNAs complexed with the PIC nanocarriers suppressed the expression of the TGF-β1 mRNA in the glomeruli of MRL/lpr mice. Nontreated BALB-c mice were used as controls. P values were calculated by ANOVA, mean ± SEM, n = 5. Densitometry analysis of PAI-1 and FN staining in glomeruli is shown in lower panel. Data were expressed as the positive staining area of PAI-1 or FN in each glomerulus (%). P values were calculated by ANOVA, mean ± SEM, n = 3.

Article Snippet: Intraglomerular Accumulation and Localization of the Nanocarriers Cross-sectional analysis by fluorescence intensity profiling revealed higher fluorescence signals at 3.5 hours in the glomeruli of the mice treated with the siRNA/nanocarrier complexes than in those treated with naked siRNAs or with siRNA/HVJ-E ( A).

Techniques: Expressing, In Situ Hybridization, Immunohistochemistry, Control, Reverse Transcription Polymerase Chain Reaction, Staining