Chronic ethanol ingestion is fibrogenic in rat kidney.

A) Fibrotic protein expression in kidney. Rats were fed the standard Lieber-deCarli liquid ethanol diet, or its isomaltose control, for 28 days before kidneys were excised, perfused with saline, fixed and sectioned as described in “Methods.” Sections were rehydrated and stained with anti-TGF-ß1, anti- α-smooth muscle actin, anti-collagen IV or anti-collagen 1 antibodies, and SP-streptavidin-conjugated secondary antibody for anti-TGF-ß1, or fluorescent secondary antibodies for the other sections as stated in “Methods.” TGF-ß sections were developed with horseradish peroxidase and DAB/metal chromogenic solution. B) mRNAs encoding fibrotic proteins were induced in kidneys of rats ingesting ethanol. Total RNA was isolated from ethanol and pair-fed kidneys from rats at the end of the feeding trial, and mRNA was quantified by SYBR Green one-step Reverse Transcription-PCR for the stated mRNAs and ribosomal 18S with the Bio-Rad MyiQ real-time PCR detection system. mRNA expression was normalized to 18S RNA content and 2-ΔΔCT was used to calculate the fold changes. Data are expressed as mean±SEM (n = 4), and p<0.05 (*) was considered as significant. C) Ethanol induced extracellular fibril deposition in kidneys of rats chronically metabolizing ethanol. Kidneys of control pair-fed or ethanol fed rats were isolated, fixed, sectioned and prepared for electron microscopy as described in “Methods.” The micrograph from the control animal shows erythrocytes within a capillary separating three epithelial cells, in contrast to the disorganized milieu with extracellular fibrils found in the kidney of ethanol-fed rats. The inset to the right is an expanded micrograph with extracellular fibrils highlighted by an arrow.