For human renal biopsy specimens, D2-40–stained sections were scanned at low power to identify three LV “hotspots,” which were then examined at 40×. D2-40–positive vessels with a clearly defined lumen or well-defined linear vessel shape, but not single endothelial cells, were used for counting. Inflammatory cell density was also determined in the three areas of high-density infiltration using a method analogous to that for LV enumeration (66). In mouse models, LYVE-1 staining in renal sections was imaged at 40×. LYVE-1–positive vessels with a clearly defined lumen or well-defined linear vessel shape, but not single endothelial cells, were used for counting. The objectivity of LV counting was increased by imaging the LV in the longitudinal section of the murine kidney and subsequently calculating the numbers of LVs using Image-Pro Plus software. The number of LVs in each field was ranked from high to low. The top 20 fields were regarded as hotspots. The average value represented the density of the entire kidney. When assessing LVs in the RDLN, all LYVE-1 staining was imaged at 40×. The relative LYVE-1 area was calculated by Image-Pro Plus software. The LYVE-1 area in sham-operated mice was defined as the baseline value. For TUNEL and inflammatory cell counting, staining was carefully quantified in each slide by evaluating eight randomly chosen fields. All counting assessments were performed by two independent pathologists blinded to treatment conditions.

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