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Proteomic analyses of mouse urinary exosomes. (A) Confirmation of urinary exosome morphology by electron microscopy. Urinary exosomes were extracted through differential centrifugation and verified using an electron microscope (JEOL JEM-1011 TEM, JEOL United States Inc., Peabody, MA). The image represents one of three independent experiments. (B) Venn Diagram analysis revealed that mice at 4 months old shared 16,079 peptides in their urinary exosomes with mice at 2 months old, while having 1,866 unique peptides. Conversely, mice at 2 months old also possessed 1,979 unique peptides. (C) A Volcano plot of 164 proteins increased and 176 protein decreased when SCD mice reached 4 month old compared with 2 month old. FC, fold change. (D) . Western blot analysis of protein abundance of heparanase (HPSE), cathepsin C (CATC), α2-macroglobulin (A2M), integrin αV (ITAV) and heat shock protein 27 (HSP27) in the urinary exosomes of non SCD (heterozygote) and SCD mice.

Journal: Frontiers in Physiology

Article Title: Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study

doi: 10.3389/fphys.2024.1300667

Figure Lengend Snippet: Proteomic analyses of mouse urinary exosomes. (A) Confirmation of urinary exosome morphology by electron microscopy. Urinary exosomes were extracted through differential centrifugation and verified using an electron microscope (JEOL JEM-1011 TEM, JEOL United States Inc., Peabody, MA). The image represents one of three independent experiments. (B) Venn Diagram analysis revealed that mice at 4 months old shared 16,079 peptides in their urinary exosomes with mice at 2 months old, while having 1,866 unique peptides. Conversely, mice at 2 months old also possessed 1,979 unique peptides. (C) A Volcano plot of 164 proteins increased and 176 protein decreased when SCD mice reached 4 month old compared with 2 month old. FC, fold change. (D) . Western blot analysis of protein abundance of heparanase (HPSE), cathepsin C (CATC), α2-macroglobulin (A2M), integrin αV (ITAV) and heat shock protein 27 (HSP27) in the urinary exosomes of non SCD (heterozygote) and SCD mice.

Article Snippet: The membrane was probed with a primary antibody against heparanase (1:1000 dilution, Proteintech 24529-1-AP), cathepsin c (1:1000 dilution, ThermoFisher PA5-37849), α2-macroglobulin (1:1000 dilution, Proteintech 66126-1-Ig), SERCA3 (1:500 dilution, Proteintech 13619-1-AP), integrin αV (1:500 dilution, Proteintech 27096-1-1AP), podocalyxin (1:500 dilution, Proteintech 18150-1-AP), HSP27 (1:1000 dilution, Cell Signaling 2402), β-actin (1:1000 dilution Cell Signaling 3700), SHP-1 (1:500 dilution, SC-287) or Tamm-Horsfall protein (1:2500 dilution, SC-271022) at 4°C overnight.

Techniques: Electron Microscopy, Centrifugation, Microscopy, Western Blot, Quantitative Proteomics

Proteins shortlisted for further evaluation.

Journal: Frontiers in Physiology

Article Title: Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study

doi: 10.3389/fphys.2024.1300667

Figure Lengend Snippet: Proteins shortlisted for further evaluation.

Article Snippet: The membrane was probed with a primary antibody against heparanase (1:1000 dilution, Proteintech 24529-1-AP), cathepsin c (1:1000 dilution, ThermoFisher PA5-37849), α2-macroglobulin (1:1000 dilution, Proteintech 66126-1-Ig), SERCA3 (1:500 dilution, Proteintech 13619-1-AP), integrin αV (1:500 dilution, Proteintech 27096-1-1AP), podocalyxin (1:500 dilution, Proteintech 18150-1-AP), HSP27 (1:1000 dilution, Cell Signaling 2402), β-actin (1:1000 dilution Cell Signaling 3700), SHP-1 (1:500 dilution, SC-287) or Tamm-Horsfall protein (1:2500 dilution, SC-271022) at 4°C overnight.

Techniques: Infection

Excretion of α2-macroglobulin protein in both urinary exosomes and urine is increased in parallel with albuminuria (Alb) in male and female SCD subjects. However, female subjects exhibit better correlations between urine exosomal α2-macroglobulin excretion and the urine albumin creatinine ratio (UACR), as well as in α2-macroglobulin excretion between urinary exosomes and urine, compared to their male counterparts. (A) Western blot analyses of α2-macroglobulin protein in the urinary exosomes (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (B,C) Only female subjects, but not male subjects, displayed a correlation in excretion of α2-macroglobulin in the urinary exosomes with UACR (Pearson test, Males: n = 9 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (D) Western blot analyses of α2-macroglobulin protein in both male and female subjects’ urine (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (E,F) Increased excretion of α2-macroglobulin protein in the urinary exosomes correlated better with its excretion in urine in female subjects than in male subjects (Pearson test, Males: n = 9 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (* p < 0.05, two-tailed t -test).

Journal: Frontiers in Physiology

Article Title: Proteomic analyses of urinary exosomes identify novel potential biomarkers for early diagnosis of sickle cell nephropathy, a sex-based study

doi: 10.3389/fphys.2024.1300667

Figure Lengend Snippet: Excretion of α2-macroglobulin protein in both urinary exosomes and urine is increased in parallel with albuminuria (Alb) in male and female SCD subjects. However, female subjects exhibit better correlations between urine exosomal α2-macroglobulin excretion and the urine albumin creatinine ratio (UACR), as well as in α2-macroglobulin excretion between urinary exosomes and urine, compared to their male counterparts. (A) Western blot analyses of α2-macroglobulin protein in the urinary exosomes (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (B,C) Only female subjects, but not male subjects, displayed a correlation in excretion of α2-macroglobulin in the urinary exosomes with UACR (Pearson test, Males: n = 9 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (D) Western blot analyses of α2-macroglobulin protein in both male and female subjects’ urine (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (E,F) Increased excretion of α2-macroglobulin protein in the urinary exosomes correlated better with its excretion in urine in female subjects than in male subjects (Pearson test, Males: n = 9 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (* p < 0.05, two-tailed t -test).

Article Snippet: The membrane was probed with a primary antibody against heparanase (1:1000 dilution, Proteintech 24529-1-AP), cathepsin c (1:1000 dilution, ThermoFisher PA5-37849), α2-macroglobulin (1:1000 dilution, Proteintech 66126-1-Ig), SERCA3 (1:500 dilution, Proteintech 13619-1-AP), integrin αV (1:500 dilution, Proteintech 27096-1-1AP), podocalyxin (1:500 dilution, Proteintech 18150-1-AP), HSP27 (1:1000 dilution, Cell Signaling 2402), β-actin (1:1000 dilution Cell Signaling 3700), SHP-1 (1:500 dilution, SC-287) or Tamm-Horsfall protein (1:2500 dilution, SC-271022) at 4°C overnight.

Techniques: Western Blot, Two Tailed Test