*2.6. Proteome Profiler Rat Cytokine Array*

Assays were performed essentially as described in the product protocol. Briefly, plasma was incubated with nitrocellulose membranes spotted with capture and control antibodies. After incubation, the membranes were washed and incubated with streptavidin HRP. In a deviation from protocol, we utilized SuperSignal West Femto (ThermoFisher, Madison, WI, USA; Cat# 34094) for chemiluminescent detection, as it gave a stronger signal. Blots were visualized on a FotoDyne gel doc system.

#### *2.7. Histology*

Formalin-fixed (10% formalin), paraffin-embedded, kidneys were cut into 5 um sections. Slides were deparaffinized, rehydrated from xylene through a graded ethanol series to water and subsequently treated as described below. Slides were scanned using a 20× objective in an Aperio Digital Pathology Slide Scanner. All H&E slides were reviewed by Dr. Weixiong Zhong, MD, PhD, transplant pathologist, and scored for ptc, glomerulitis (g), vasculitis (v)/intimal arteritis, interstitial inflammation (i) and C4d staining, according to Banff 2009 [21].

Separately, slides were assigned a blinded number, and non-overlapping digital images of renal tubules were taken at the interface between the medulla and the cortex from each H/E slide. Care was taken to not include large vessel lumens and glomeruli. Automated quantification of red and blue pixels in each 10× kidney image was performed using a custom macro written in ImageJ software (https://imagej.nih.gov/ij/index.html

accessed on 13 April 2021). Red pixels reflected proximal tubular thickness including brush border. Nuclei were quantified in the blue channel. The ratio of blue nuclear pixels to red tubules provided an Inflammatory Infiltration Score for the white blood cell infiltration in the post-transplant kidneys. Scores were averaged and plotted using Graphpad Prism.
