Figure 5

Dietary branched-chain amino acids suppressed phosphate-induced renal tubulointerstitial injury. Six-week-old male Wistar rats were randomly divided into the HPi + BCAA_0 (high phosphate [2.2%] without BCAA-supplemented diet) and HPi + BCAA_10 (high phosphate with BCAA-supplemented [10%] diet) groups. All results were obtained from the rats at 12 weeks of age, except for those of SDH enzymatic staining. The kidney samples for SDH staining were obtained at 7 weeks of age. (a) qRT-PCR analyses for Havcr1, Tnf, Icam1, Tgfb1, and Col1a1 in the kidney (N = 4 in each group: *P < 0.05, unpaired t-test). (b) Representative micrographs of Masson’s trichrome-stained kidney sections (bars = 100 µm). Interstitial fibrotic area was quantified (N = 4 in each group: *P < 0.05, unpaired t-test). (c) Nitrotyrosine-containing protein levels in the kidney were analyzed using western blotting (N = 9–11 in each group: ***P < 0.001, unpaired t-test). Full-length blots are presented in Supplementary Fig. S3. For the quantitative analysis, several blots were processed in parallel because the number of gel lanes was not sufficient to simultaneously analyze all samples in one electrophoresis gel. To enable comparison among parallelly processed blots, several lanes in the parallelly processed blots were applied with the same sample. (d) Representative electron micrographs of proximal tubules (bars = 5 µm) and light microscope images of enzymatically stained kidney cortices for SDH activity (bars = 100 µm) (N = 6–9 in each group: ***P < 0.001, unpaired t-test). All quantitative results are presented as a scatter plot with means ± standard deviations. qRT-PCR, quantitative reverse transcription-polymerase chain reaction; SDH, succinate dehydrogenase.