Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 1. The proteomic analysis of plasma was conducted across three groups: normal adults, diabetic patients, and patients with diabetic kidney disease. (A) A Venn diagram was utilized to analyze the sequenced proteins, where group D represents the plasma samples from diabetic patients, group DK denotes the plasma from DKD patients, and group nor corresponds to the plasma of normal adults. The numbers indicate the count of sequenced and annotated proteins. (B) Heat maps were generated to analyze the differential expression of plasma proteins in patients with diabetes and those with diabetic kidney disease. (C) A volcano plot illustrated the significant differences in plasma proteins between diabetic patients and those with DKD. (D) An analysis of protein–protein interactions highlighted significant differences in plasma proteins among patients with diabetes and diabetic kidney disease. (E) The analysis focused on plasma-specific proteins and their interactions in patients with DKD. (F, G) Investigated the enrichment of plasma-specific protein Gene Ontology (GO) and KEGG database pathways in patients with diabetic kidney disease. (H) The plasma levels of IGFBP2 and IGFBP4 in normal adults, diabetic patients, and diabetic kidney patients were quantified using Western blotting (WB). Data are presented as mean ± SD (n = 3). **p < .01.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Clinical Proteomics, Generated, Quantitative Proteomics, Protein-Protein interactions, Western Blot

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 2. IGFBP2 and IGFBP4 contribute to the progression of DKD in mice. (A) Enzyme-linked immunosorbent assay (ELISA) was employed to assess the levels of serum albumin, fasting glucose, and serum creatinine in both normal mice and diabetic mice with nephropathy. The CK group consisted of normal mice, while the DK group comprised diabetic mice with nephropathy. (B) Insulin tolerance tests were conducted on normal mice and mice with DKD, and the area under the curve for the test data was calculated. (C) ELISA was utilized to evaluate the changes in urinary albumin, urinary creatinine, and urinary glucose between normal mice and those with DKD. (D) Hematoxylin and eosin (HE) staining was performed to observe the pathological alterations in kidney tissue from normal mice and those with DKD, while periodic acid-Schiff (PAS) staining was used to assess glycogen deposition, and Masson’s tri chrome staining was applied to examine collagen deposition in the kidney tissue. Scale bar: 50 µm. (E) The expression levels of IGFBP2 and IGFBP4 proteins in the plasma of normal mice and those with DKD were analyzed via Western blotting (WB) from the second to the sixth week. (F) Correlation analyses were conducted to evaluate the relationship between plasma IGFBP2 and IGFBP4 expression levels and serum albumin, fasting glucose, and serum creat inine in DKD mice. Data are presented as mean ± SD. n = 3. *p < .05, **p < .01, ***p < .001, compared to CK.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, Expressing, Clinical Proteomics, Western Blot

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 3. The effects of IGFBP2 and IGFBP4 on the complement pathway in DKD mice were examined. (A) The alterations in IGFBP2, IGFBP4, and comple ment proteins C3, C4B, C5, and C9 in the renal tissues of both normal and DKD mice were assessed using immunohistochemistry, with a scale bar of 50 μm. (B) The levels of plasma IGFBP2, IGFBP4, and complement proteins C3, C4B, C5, and C9 in normal and DKD mice were analyzed via Western blotting (WB). (C) The changes in IGFBP2, IGFBP4, and complement proteins C3, C4B, C5, and C9 in the renal tissues of normal and DKD mice were also evaluated using WB. (D) Plasma levels of MAC and MBL were measured using ELISA in both normal and DKD mice. Data are presented as mean ± SD, with n = 3. Statistical significance is indicated as **p < .01 and ***p < .001.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Immunohistochemistry, Clinical Proteomics, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 4. There is a protein interaction between IGFBP2 and IGFBP4. (A) IGFBP2 was overexpressed in 293T cells, and the expression level of IGFBP2 was assessed using qPCR to confirm the successful construction of the model. The group with overexpressed IGFBP2 is referred to as oe-IGFBP2. (B) Western blotting (WB) was employed to measure the levels of IGFBP2 and IGFBP4 following the overexpression of IGFBP2 in 293T cells. (C) Co-immunoprecipitation (Co-IP) was utilized to detect the formation of the IGFBP2 and IGFBP4 complex. Data are presented as mean ± SD, with n = 3 and **p < .01.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Expressing, Western Blot, Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 5. The crosstalk effect among HK-2 cells, THP-1 cells, and primary human renal podocytes was investigated as follows: (A) the serum from DKD mice (10%) was added to HK-2 cells for culture for 24 h, after which relevant indicators were detected. The supernatant from HK-2 cells was then transferred to THP-1 cells to assess the functional changes in THP-1 cells, and subsequently, the THP-1 cell supernatant was added to primary human renal podocytes to observe their functional alterations. (B) The levels of IGFBP2 and IGFBP4 following HK-2 cell stimulation were measured. (C) The polarization of THP-1 cells was assessed using flow cytometry, where CD86% served as the marker for M1 polarization and CD206% indicated M2 polarization. (D) ELISA was employed to detect changes in complement proteins C3, C4B, C5, and C9 in THP-1 cells. (E) The levels of MAC and MBL in THP-1 cells were also measured using ELISA. (F) Podocyte proliferation was evaluated using the CCK-8 assay. (G) Apoptosis of primary human renal podocytes was analyzed through flow cytometry. (H) The expression of reactive oxygen species (ROS) in primary human renal podocytes was visualized by immunofluorescence, with green fluorescence indicating the intensity of ROS expression. Data are presented as mean ± SD (n = 3; *p < .05, **p < .01, ***p < .001).

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Functional Assay, Cell Stimulation, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Expressing, Immunofluorescence, Fluorescence

Journal: Renal Failure

Article Title: IGFBP2 and IGFBP4 interact to activate complement pathway in diabetic kidney disease

doi: 10.1080/0886022x.2024.2440528

Figure Lengend Snippet: Figure 6. Effects of exogenous addition of IGFBP2 and IGFBP4 on THP-1 cells and primary human renal podocytes. (A) Following the addition of recombi nant IGFBP2 and IGFBP4 proteins to THP-1 cells, the supernatant was collected for the assessment of related indices, which were subsequently applied to primary human renal podocytes to observe functional changes. (B) Different concentrations of recombinant IGFBP2 and IGFBP4 (50 ng/mL, 100 ng/mL, 200 ng/mL, and 400 ng/mL) were found to stimulate the levels of complement proteins C3, C4B, C5, C9, as well as MAC and MBL in THP-1 cells. (C) The effects of individually adding IGFBP2h or IGFBP4 recombinant protein, or their combined addition, on the levels of complement proteins C3, C4B, C5, C9, MAC, and MBL in THP-1 cells were assessed. (D) Flow cytometry analysis was conducted to determine the impact of IGFBP2h or IGFBP4 recombinant protein, either alone or in combination, on the polarization of THP-1 cells. (E) The influence of THP-1 cell supernatant on podocyte proliferation was eval uated using the CCK-8 assay. (F) The effect of THP-1 cell supernatant on podocyte apoptosis was measured through flow cytometry. (G) Immunofluorescence was utilized to observe the effect of THP-1 cell supernatant on ROS expression in primary human renal podocytes. (H) The impact of THP-1 cell supernatant on podocyte morphology was also assessed. Data are presented as mean ± SD. n = 3; *p < .05, **p < .01, ***p < .001.

Article Snippet: Primary antibodies were then added, including IGFbP4 (Pa5-25925, thermo Fisher, Waltham, Ma), IGFbP2 (Pa5-79450, thermo Fisher, Waltham, Ma), C3 (Pa5-21349, thermo Fisher, Waltham, Ma), C4b (22233-1-aP, Proteintech, Wuhan, China), C5 (ab275931, abcam, Cambridge, uK), C9 (ab168345, abcam, Cambridge, uK), GaPdH (ab8245, abcam, Cambridge, uK), and transferrin (Pa5-27306, thermo Fisher, Waltham, Ma). these were incubated overnight at 4 °C, followed by washing with tbSt buffer. the samples were then combined with Goat anti-Mouse IgG/HrP (SE131, Solarbio, beijing, China) and Goat anti-rabbit IgG H&L (HrP) (ab6721, abcam, Cambridge, uK), and incubated at room temperature for 2 h. ECL color development was performed, and gray values were measured using an automatic gel imaging analyzer.

Techniques: Functional Assay, Recombinant, Flow Cytometry, CCK-8 Assay, Immunofluorescence, Expressing