Fig 1: Elevated excretion levels of heparanase protein in urinary exosomes and urine are positively associated with albuminuria (Alb) in both male and female SCD subjects. However, only female subjects demonstrate stronger correlations between the excretion of urine exosomal heparanase and urine albumin creatinine ratio (UACR) and in excretion of heparanase between urinary exosomes and urine compared to male subjects. (A) Conformation of extraction of urinary exosomes by electron microscopy, a representative of three independent experiments. Urinary exosomes extraction and electron microscopic analysis were performed as described in Figure 3A. (B) Conformation of extraction of urinary exosomes by Western blot analysis. Each lane was loaded with 30 µg proteins. (C) Both male and female SCD subjects increased excretion of heparanase in their urine exosomes upon developing albuminuria as determined by Western blot analyses (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (D,E) Increased excretion of heparanase in the urinary exosomes correlated with UACR only in female subjects, but not 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). (F) Western blot analyses indicate that both male and female SCD subjects increased excretion of heparanase protein in their urine upon developing albuminuria (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (G,H) Only female subjects demonstrated a good correlation in excretion of heparinase protein between urinary exosomes and urine (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).
Fig 2: 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).
Fig 3: There is no association of excretion of Tamm-Horsfall protein (THP), β-actin, or SHP-1 protein in urinary exosomes with albuminuria in either male or female subjects. There is no association between excretion of THP and urine albumin creatinine ratio (UACR) in either male or female subjects. Only female subjects display a weak correlation in excretion of THP between urinary exosomes and urine. (A) Western blot analyses of THP in urinary exosomes (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (B,C) No correlation was observed between the excretion of THP in urinary exosomes and UACR in either sex (Pearson test, Males: n = 9 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (D) There is no association of urinary excretion of THP with albuminuria in either male or female subjects as examined by Western blot analysis (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb). (E,F) A weak correlation in the excretion of THP between urinary exosomes and urine was found only in female subjects (Pearson test, Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (G,H) No correlation was found in the excretion of β-actin or SHP-1 protein in urinary exosomes with albuminuria in either sex (Males: n = 10 for No Alb, n = 7 for Alb, Females: n = 8 for No Alb, n = 5 for Alb).
Fig 4: Both male and female SCD subjects with albuminuria increases excretion of SERCA3 protein in the urinary exosomes. However, only female subjects show a correlation of urine exosomal excretion of SERCA3 protein with the urine albumin creatinine ratio (UACR), whereas male subjects do not. (A) Western blot analyses of SERCA3 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) A strong correlation between the urine exosomal excretion of SERCA3 protein and UACR was observed only in female subjects, with no such correlation found 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).
Fig 5: Elevated levels of cathepsin C protein excretion in both urinary exosomes and urine demonstrate a positive association with albuminuria (Alb) in male and female SCD subjects. Importantly, the correlations between urine exosomal cathepsin C excretion and the urine albumin creatinine ratio (UACR), as well as cathepsin C excretion between urinary exosomes and urine, are particularly pronounced in female subjects compared to their male counterparts. (A) Western blot analyses of cathepsin C 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) The elevated excretion of cathepsin C in urinary exosomes exhibited a stronger correlation with urine UACR in female subjects compared to male subjects (Pearson test, Males: n = 9 for No Alb, n = 7 for Alb, Females: n = 7 for No Alb, n = 5 for Alb). (D) Both male and female subjects with albuminuria tended to increase urinary excretion of cathepsin C as shown by Western blot analyses (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 cathepsin C in the urinary exosomes correlated with its excretion in urine in female subjects, but not 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).
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