Fig. 1: Glomerular podocytes exhibit BCAA catabolic defects in patients with DKD and DKD db/db mice. | Nature Communications

Fig. 1: Glomerular podocytes exhibit BCAA catabolic defects in patients with DKD and DKD db/db mice.

From: Branched-chain amino acids contribute to diabetic kidney disease progression via PKM2-mediated podocyte metabolic reprogramming and apoptosis

Fig. 1

a The levels of 4-Methyl-2-oxopentanoate and Methylmanic acid in the 24 hrs urine samples were tested by non-targeted metabolomics from male or female T2DM patients without DKD (non-DKD, n = 5) or patients with early DKD (n = 5). b Representative images of immunohistochemistry (IHC) staining of P-BCKDE1α (Ser 293) in glomeruli. The P-BCKDE1α (Ser 293) score was evaluated by IHC in glomerular sections. A total of 70-110 glomeruli from n = 6 patients per group were analyzed. c, d Upper, representative images of double staining of P-BCKDE1α (Ser 293) and PP2Cm with Wilms tumor-1 (WT-1, a podocyte-specific marker) in kidney sections. Bottom, quantification of P-BCKDE1α (Ser 293) and PP2Cm intensities in WT1 positive podocytes, normalized to DAPI. Male: p-BCKD/DAPI intensities (median (Q1–Q3)) 0.98 (0.86 − 1.15) versus 1.65 (1.32 − 2.35), n = 107 cells per group; PP2Cm/DAPI intensities 1.03 (0.87 − 1.16) versus 0.50 (0.35 − 0.69), n = 108 cells per group. Female: p-BCKD/DAPI intensities 0.96 (0.77 −1.24) versus 1.67 (1.27 −2.10), n = 107 cells per group; PP2Cm/DAPI intensities 0.94 (0.69 −1.22) versus 0.49 (0.38 −0.67), n = 107 cells per group. White boxes indicate the localization of WT1 with P-BCKDE1α or PP2Cm. e, f Representative Western blots of PP2Cm, P-BCKDE1α and BDK were detected in female and male C57BL/6 mice or db/db mice. n = 4 mice per group. g, h Representative double-staining images of PP2Cm, P-BCKDE1α (Ser 293) with WT1. n = 4 mice per group. i The concentration of BCAAs (including leucine, isoleucine and valine) or BCKAs (including KIC, KIV, KMV) were measured in isolated glomeruli, n = 4 per group. j Representative Western blots of BDK, PP2Cm, P-BCKDE1α (Ser 293) and total BCKDE1α in primary podocytes under low-glucose (LG, 5.5 mM glucose) or high-glucose (HG, 25 mM glucose) conditions for 24 hrs. 20 mM mannitol (Man) plus 5 mM glucose were treated as the osmotic control group, n = 4 total samples per group. The data were presented as the mean ± SD and were analyzed by unpaired two-tailed Student’s t test.

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