Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease: methodological pitfalls and their solution

Connie P. C. Ow, Md Mahbub Ullah, Jennifer P. Ngo, Adheeshee Sayakkarage, Roger G. Evans

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Pimonidazole adduct immunohistochemistry is one of the few available methods for assessing renal tissue hypoxia at the cellular level. It appears to be prone to artifactual false positive staining under some circumstances. Here, we assessed the nature of this false positive staining and, having determined how to avoid it, reexamined the nature of cellular hypoxia in rat models of kidney disease. When a mouse-derived anti-pimonidazole primary antibody was used, two types of staining were observed. First, there was diffuse staining of the cytoplasm of tubular epithelial cells, which was largely absent when the primary antibody was omitted from the incubation protocol or in tissues known not to contain pimonidazole adducts. Second, there was staining of the apical membranes of tubular epithelial cells, debris within the lumen of renal tubules, including tubular casts, and the interstitium; this latter staining was present even when the primary antibody was omitted from the incubation protocol. Such false positive staining was particularly prominent in acutely injured kidneys. It could not be avoided by preincubation of sections with a mouse IgG blocking reagent. Furthermore, preadsorption of the secondary antibody against rat Ig abolished all staining; however, when a rabbit-derived polyclonal anti-pimonidazole primary antibody was used, the false positive staining was largely avoided. Using this method, we confirmed the presence of hypoxia, localized mainly to the tubular epithelium, in the acute phase of severe renal ischemia-reperfusion injury, adenine-induced chronic kidney disease, and polycystic kidney disease. We conclude that this new method provides improved detection of renal cellular hypoxia.

Original languageEnglish
Pages (from-to)F322-F332
Number of pages11
JournalAmerican Journal of Physiology - Renal Physiology
Volume317
Issue number2
DOIs
Publication statusPublished - 1 Aug 2019

Keywords

  • acute kidney injury
  • chronic kidney disease
  • hypoxia
  • ischemia
  • kidney
  • pimonidazole

Cite this

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title = "Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease: methodological pitfalls and their solution",
abstract = "Pimonidazole adduct immunohistochemistry is one of the few available methods for assessing renal tissue hypoxia at the cellular level. It appears to be prone to artifactual false positive staining under some circumstances. Here, we assessed the nature of this false positive staining and, having determined how to avoid it, reexamined the nature of cellular hypoxia in rat models of kidney disease. When a mouse-derived anti-pimonidazole primary antibody was used, two types of staining were observed. First, there was diffuse staining of the cytoplasm of tubular epithelial cells, which was largely absent when the primary antibody was omitted from the incubation protocol or in tissues known not to contain pimonidazole adducts. Second, there was staining of the apical membranes of tubular epithelial cells, debris within the lumen of renal tubules, including tubular casts, and the interstitium; this latter staining was present even when the primary antibody was omitted from the incubation protocol. Such false positive staining was particularly prominent in acutely injured kidneys. It could not be avoided by preincubation of sections with a mouse IgG blocking reagent. Furthermore, preadsorption of the secondary antibody against rat Ig abolished all staining; however, when a rabbit-derived polyclonal anti-pimonidazole primary antibody was used, the false positive staining was largely avoided. Using this method, we confirmed the presence of hypoxia, localized mainly to the tubular epithelium, in the acute phase of severe renal ischemia-reperfusion injury, adenine-induced chronic kidney disease, and polycystic kidney disease. We conclude that this new method provides improved detection of renal cellular hypoxia.",
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Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease : methodological pitfalls and their solution. / Ow, Connie P. C.; Ullah, Md Mahbub; Ngo, Jennifer P.; Sayakkarage, Adheeshee; Evans, Roger G.

In: American Journal of Physiology - Renal Physiology, Vol. 317, No. 2, 01.08.2019, p. F322-F332.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Detection of cellular hypoxia by pimonidazole adduct immunohistochemistry in kidney disease

T2 - methodological pitfalls and their solution

AU - Ow, Connie P. C.

AU - Ullah, Md Mahbub

AU - Ngo, Jennifer P.

AU - Sayakkarage, Adheeshee

AU - Evans, Roger G.

PY - 2019/8/1

Y1 - 2019/8/1

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KW - acute kidney injury

KW - chronic kidney disease

KW - hypoxia

KW - ischemia

KW - kidney

KW - pimonidazole

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DO - 10.1152/ajprenal.00219.2019

M3 - Article

VL - 317

SP - F322-F332

JO - American Journal of Physiology - Renal Physiology

JF - American Journal of Physiology - Renal Physiology

SN - 1522-1466

IS - 2

ER -