Detection and localization of HIV-1 DNA in renal tissues by in situ polymerase chain reaction.
The localization of HIV-1 DNA in renal tissues is critically important for understanding pathogenesis of HIV-associated nephropathy (HIVAN), but the clarification has been technically challenging. We applied in situ polymerase chain reaction (IS-PCR) to human renal tissues to demonstrate viral entry into the renal epithelial cells in vivo. To test the specificity of this method and to determine the cell types infected, we used IS-PCR followed by in situ hybridization (ISH) and IS-PCR followed by immunohistochemistry and histochemical counterstains. Brief 2 hour fixation in 4% paraformaldehyde had 92.9% sensitivity and 100% specificity for detection of viral DNA in renal biopsies of HIVAN patients, compared to 70.8% sensitivity and 66.7% specificity in renal biopsies fixed overnight in 10% formalin. Under optimized conditions, the only signals detectable in HIV-1 seronegative cases were false positives attributable to renal tubular apoptosis. In HIVAN cases, positive signal was observed in podocytes, parietal cells, renal tubular cells, and interstitial leukocytes. Immunohistochemical co-labeling for pan-T cell and macrophage markers revealed that the interstitial leukocytes with positivity for HIV-1 DNA included both T cells and macrophages. Application of ISH after IS-PCR showed the same distribution of signal as observed using IS-PCR alone, confirming the specificity of the technique. IS-PCR is a powerful technique to detect viral DNA in human tissue sections, but requires proper use of negative controls to set optimal fixation, protein digestion, and amplification conditions.
Tanji
N
N
Department of Pathology, Columbia University, College of Physicians and Surgeons, New York, USA. tanji@m.ehime-u.ac.jp
Ross
M D
MD
Tanji
K
K
Bruggeman
L A
LA
Markowitz
G S
GS
Klotman
P E
PE
D'Agati
V D
VD
D016263:AIDS-Associated Nephropathy / Q000473:pathology / Q000821:virology*; D000328:Adult; D000368:Aged; D015331:Cohort Studies; D004279:DNA, Viral / Q000032:analysis*; D005189:False Positive Reactions; D005260:Female; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007150:Immunohistochemistry; D017403:In Situ Hybridization / Q000379:methods*; D007668:Kidney / Q000473:pathology / Q000821:virology*; D008264:Macrophages / Q000473:pathology / Q000821:virology; D008297:Male; D008875:Middle Aged; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013601:T-Lymphocytes / Q000473:pathology / Q000821:virology
D004279:DNA, Viral