Electronic Supplementary Material 1 : Kidney biopsy analysis

Initial biopsy processing

Specimens were fixed in alcoholic Bouin’s fixative with standard embedding. Both Periodic Acid-Schiff and Masson trichrome stains were performed on all specimens. All of the slides were reviewed by two experienced renal pathologists (DN, GH) blinded to the clinical and laboratory data.

Evaluation of lesions

A variety of morphologic lesions including glomerular, arterial, arteriolar, renal tubular and interstitial lesions was assessed (see Table below). Before beginning the analysis the two pathologists involved reviewed together a number of cases to agree on the scoring for the lesions. Then the cases were reviewed separately, and there was generally quite good agreement between the two observers on basic morphologic lesions. Where there were differences between them, the average of the two values was used, an approach usually considered adequate for this kind of study. Lesions not encountered in any of the 19 cases or considered non significant are not presented in the result section.

Apoptosis evaluation

Special attention was paid regarding apoptosis evaluation. Presence of apoptosis was assessed by three different techniques 1) presence of tubular apoptotic bodies on routine microscopy, 2) TUNEL (terminal-deoxynucleotidyl-transferase-mediated dUTP–digoxigenin nick end labelling) using an ApopTag Kit (Cat. No. S7100, Intergen, Norcross, GA, USA), 3) activated caspase 3 labelling with a polyclonal rabbit antibody against human cleaved caspase-3 (Asp175, Cell Signaling Technology, Danvers, MA), detected with streptavidin-biotin-peroxidase.

400X-magnification pictures of 10 successive cortical and medullary fields were taken in each case for counting tubular nuclei and apoptotic bodies on routine Masson trichrome stain by one of the authors (GH). TUNEL- and activated caspase-3-positive stains were evaluated similarly, with results expressed as number of postive cells/photograph.

Table. List of lesions assessed in biopsies. Unless stated otherwise, the parameters were graded from each biopsy overall.

Morphologic Parameter / Grading / Description of Grading
Glomeruli
Total glomeruli / Count / Total glomeruli in biopsy
Sclerotic glomeruli / Count / Total sclerotic glomeruli
Glomerular macrophages / Count/gl / All glomeruli counted, averaged
Glomerular PMNs / Count/gl / All glomeruli counted, averaged
Mesangial hyperplasia / 0 – 4+ / Semiquantiative estimate; global value for all glomeruli
Glomerular capillary dilatation / 0 – 4+ / Semiquantiative estimate; global value for all glomeruli
Glomerular capillary thrombi / 0 – 1 / Present or absent
Glomerular fibrin tactoids / 0 – 1 / Present or absent
Other glomerular lesions / ---- / Description of lesion
Tubular Lesions
Brush border / 0 – 4+ / Proportion of brush border that is intact: 100% = 4+
Proximal tubular “necrosis” / 0 – 4+ / Proportion of proximal tubular cells with necrotic, pyknotic, apoptotic, or absent nuclei1,2
Proximal tubule flattening / 0 – 4+ / Proportion of proximal tubules with flattened or absent cytoplasm2
Distal nephron “necrosis” / Proportion of distal nephron l tubular cells with necrotic, pyknotic, apoptotic, or absent nuclei1,2
Distal nephron flattening / 0 – 4+ / Proportion of distal nephron segments with flattened or absent cytoplasm2
Tubular vacuolization / 0 – 4+ / Overall amount and degree of tubular distorsion is basis of grading
Epithelial cytoplasmic degeneration / 0 – 4+ / Loss of continuity of cytoplasm, fragmentation, usually accompanied by pyknosis. Grading: % of cells showing these changes2
Tubular epithelial detachment / Detachment of from tubular basement membrane. Grading : % of cells detached2
Other Lesions
Interstitial inflammation / 0 – 4+ / Proportion of parenchyma occupied by inflammation3
Tubular atrophy / 0 – 4+ / Percentage of tubules that were atrophic3
Interstitial fibrosis / 0 – 4+ / Percentage of parenchyma occupied by interstitial fibrous tissue3
Interstitial edema / 0 – 4+ / Tends to involve parenchyma globally; grading based on degree of separation of
previously normal tubules (which should touch) from one another
Hyaline casts / 0 – 2+ / 0 = Absent; 1 = Occasional; 2 = Numerous
Granular casts / 0 – 2+ / 0 = Absent; 1 = Occasional; 2 = Numerous
Tubular luminal macrophages / Count
Tubular luminal cytoplasmic Debris / 0 – 4+ / Percentage of tubules with debris in lumen
Rbcs or rbc fragments – tubular lumen / 0 – 1 / Present or absent
Vascular Lesions
Arteriosclerosis / Severity of luminal occlusion
Arterial thrombi, fresh or organized / 0 – 1 / Present or absent
Arterial dilatation / 0 – 2+ / 0 = Absent; 1 = Definitely dilated; 2 = Severely dilated.
Arteriolosclerosis / 0 – 2+ / 0 = Absent; 1= Present, mild; 2 = Extensive and/or obstructive
Arteriolar thrombi, fresh or organized / 0 - 1 / Present/ absent.
Arterial/-olar smooth muscle swelling / 0 – 2+ / 0 = Absent; 1= Present, mild/focal; 2 = Extensive/marked
Interstitial capillary dilatation/congestion / 0 – 2+ / 0 = Absent; 1 = Definitely dilated; 2 = Severely dilated.
Interstitial hemorrhage / 0 – 1 / Present or absent (predominantly deep medulla)
Quantitative Interstitial Capillary Leukocyte Counts
Cortical macrophages4 / Count / 10 successive cortical fields counted; averaged
Cortical PMNs / Count / 10 successive cortical fields counted; averaged
Medullary macrophages4 / Count / 10 successive medullary fields counted; averaged
Medullary PMNs / Count / 10 successive medullary fields counted; averaged

1 – “Necrosis” (an old and largely incorrect term) is used here as shorthand for the global proportion of nuclei at that nephron level that were abnormal (necrotic, pyknotic, apoptotic, or were simply absent). Absence of nuclei was only diagnosed when more than half of the circumference of several tubules contained no nuclei.

2 – Grading based on the proportion of tubules involved at that nephron level: 0 – None; 0.5 + - <5% of tubules; 1+ - 5-25% of tubules; 2+ - 25-50% of tubules; 3+ - 50-75% of tubules; 4+ - > 75% of tubules. For cytoplasmic degeneration and epithelial cell detachment, grades refer to percentage of all tubules, using this basic schema.

3 – Graded according to percentage of parenchyma involved: 0 - No lesion; 0.5 +– Present, but in less than 5% of parenchyma; 1+ - Occupying up to 25% of parenchyma; 2+ - 25-50% of parenchyma involved, 3+ - Up to 75%; 4+ - > 75% of parenchyma involved.

4 –In a pre study on 10 biopsies, excellent correlations were found between counts after CD68 staining, a macrophage marker, and the counts on routine microscopy, e.g., for glomeruli, r = 0.72, p <0.001.