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hep26599-sup-0001-suppfig1.tif4959KSuppl. Fig. 1. 3d CBDL mice show altered AQP2 expression on collecting ducts but regular NKCC2 expression of the thick ascending limb of Henle . (A,B) Immunohistochemistry for AQP2 (left image) and NKCC2 (right image) in serial kidney sections of 3d sham-operated mice (A) and 3d CBDL mice (B). (A) Normal AQP2 expression on collecting duct cells and NKCC2-positive thick ascending limbs of Henle in 3d sham-operated mice. Dashed ellipse indicates the identical collecting duct in both sections. (B) Altered AQP2 expression on an injured collecting duct (dashed ellipse) in 3d CBDL mouse (3d CBDL), whereas the surrounding parts of the thick ascending limbs of Henle show regular NKCC2 expression.
hep26599-sup-0002-suppfig2.tif5289KSuppl. Fig. 2. Exfoliation of epithelium of collecting ducts in 3d CBDL mice. (A, B) Immunofluorescence microscopy for collecting duct-specific AQP2 and AE1 for type A intercalated cells demonstrating (A) continuous epithelial layer with regular AQP2 and AE1 expression on collecting ducts in a sham-operated control mouse (Control). (B) In contrast, collecting ducts of a 3d CBDL mouse (3d CBDL) show an exfoliated AQP2 positive epithelium (white arrows) coalescing to intraluminal casts (white asterisk). There is no increase in number of type A intercalated cells staining positive for AE1. (C, D) Immunofluorescence microscopy for AQP2 and pendrin for non type A intercalated cells showing (C) continuous epithelial layer with regular AQP2 and pendrin expression in cortical collecting ducts of a control mouse (Control). (D) No evidence for proliferation of non-type A intercalated cells but loss of epithelial membrane integrity (white arrows) in 3d CBDL mouse.
hep26599-sup-0003-suppfig3.tif1931KSuppl. Fig. 3. Portal venous injection of UDC-NBD-lysine demonstrates positive staining of bile ducts and bile infarcts in livers of 3d CBDL mice. (A,B) Injection of UDC-NBD-lysine (green) into the portal vein (pv) of 3d CBDL mice demonstrates strict luminal staining of bile duct (bd) and UDC-NBD-lysine leaking out into bile infarcts (indicated by asterisks); B shows bile infarct in higher magnification.
hep26599-sup-0004-suppfig4.tif5217KSuppl. Fig. 4 . Renal overexpression of VCAM in response to CBDL. (A) Immunohistochemistry for VCAM in sham-operated control, 3w, 6w, and 8w CBDL mouse. VCAM expression increases over time in CBDL mice and is primarily expressed in tubular epithelial cells. G, glomeruli. (B) Renal VCAM m-RNA expression in controls (8w sham-operated), 3w, 6w, and 8w CBDL mice. Expression levels of VCAM are normalized to the housekeeping gene 36b4. * P < 0.05 vs. control. (C) Induced renal VCAM protein expression in 8w CBDL mice.
hep26599-sup-0005-suppfig5.tif5036KSuppl. Fig. 5. norUDCA protects 3d CBDL mice from tubular epithelial injury. (A,B) PAS staining of kidneys of chow-fed 3d CBDL mice (A) and norUDCA-prefed 3d CBDL mice (B). In contrast to chow-fed CBDL mice, norUDCA-prefed CBDL mice show no evidence for collecting duct tubular epithelial injury. (C,D) Immunohistochemistry for AQP2 in chow-fed (C) and norUDCA-fed 3d CBDL mice (D). AQP2 positive collecting ducts of chow-fed 3d CBDL mice show discontinuous AQP2 staining (black arrows), whereas AQP2 positive collecting ducts in norUDCA-prefed mice appear normal.
hep26599-sup-0006-suppfig6.tif5192KSuppl. Fig. 6. Cholestatic patients with end-stage liver disease show cholemic nephropathy with biliary casts and tubulointerstitial nephritis. (A-B) Representative liver histology of a cholestatic patient with end-stage liver disease. Sirius red (A) and PAS staining (B) show enlarged and broadened portal tracts with ductular proliferation and portal fibrosis as characteristically observed in liver fibrosis of the biliary type. In addition, images show characteristic ductular cholestasis indicated by black arrowheads. pv, portal vein. Findings are representative of 4 patients. (C-F) PAS staining of kidney tissue of two different cholestatic patients with end-stage liver disease demonstrates cast formation at the level of distal tubuli and collecting ducts. (C, D) PAS-positive casts within distal tubuli and collecting ducts in patient A. (E, F) Intraluminal casts and peritubular inflammatory infiltrate in patient B. Findings are representative of 4 patients. Original magnification x20 for A and B, x40 for C and E, x60 for D and F.
hep26599-sup-0007-suppfig7.tif2053KSuppl. Fig. 7. Proposed pathobiology of cholemic nephropathy. Severe forms of cholestasis with excessive alternative urinary elimination of cholephiles leads to (1) damage of collecting duct tubular epithelial cells and basement membranes with leakage of urine into the kidney parenchyma, and at least partial collecting duct obstruction due to cell detritus and protein casts leading to (2) the induction of a reactive tubular epithelial cell phenotype with overexpression of proinflammatory and profibrogenetic cytokines, interstitial nephritis, and consequently (3) tubulointerstitial kidney fibrosis.
hep26599-sup-0008-suppinfo.doc60KSupporting Information tables

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