Laser capture microdissection and multiplex-tandem PCR analysis of proximal tubular epithelial cell signaling in human kidney disease.
Interstitial fibrosis, a histological process common to many kidney diseases, is the precursor state to end stage kidney disease, a devastating and costly outcome for the patient and the health system. Fibrosis is historically associated with chronic kidney disease (CKD) but emerging evidence is now linking many forms of acute kidney disease (AKD) with the development of CKD. Indeed, we and others have observed at least some degree of fibrosis in up to 50% of clinically defined cases of AKD. Epithelial cells of the proximal tubule (PTEC) are central in the development of kidney interstitial fibrosis. We combine the novel techniques of laser capture microdissection and multiplex-tandem PCR to identify and quantitate "real time" gene transcription profiles of purified PTEC isolated from human kidney biopsies that describe signaling pathways associated with this pathological fibrotic process. Our results: (i) confirm previous in-vitro and animal model studies; kidney injury molecule-1 is up-regulated in patients with acute tubular injury, inflammation, neutrophil infiltration and a range of chronic disease diagnoses, (ii) provide data to inform treatment; complement component 3 expression correlates with inflammation and acute tubular injury, (iii) identify potential new biomarkers; proline 4-hydroxylase transcription is down-regulated and vimentin is up-regulated across kidney diseases, (iv) describe previously unrecognized feedback mechanisms within PTEC; Smad-3 is down-regulated in many kidney diseases suggesting a possible negative feedback loop for TGF-β in the disease state, whilst tight junction protein-1 is up-regulated in many kidney diseases, suggesting feedback interactions with vimentin expression. These data demonstrate that the combined techniques of laser capture microdissection and multiplex-tandem PCR have the power to study molecular signaling within single cell populations derived from clinically sourced tissue.
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia ; Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Queensland, Australia ; Medical School, University of Queensland, Brisbane, Queensland, Australia.
Ray
R
Wilkinson
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Xiangju
X
Wang
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Andrew J
AJ
Kassianos
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Steven
S
Zuryn
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Kathrein E
KE
Roper
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Andrew
A
Osborne
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Sandeep
S
Sampangi
Pathology Queensland, Brisbane, Queensland, Australia.
Leo
L
Francis
Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Vishwas
V
Raghunath
Conjoint Kidney Research Laboratory, Pathology Queensland, Brisbane, Queensland, Australia ; Department of Renal Medicine, Royal Brisbane and Women's Hospital, Brisbane, Queensland, Australia.
Helen
H
Healy
D058186:Acute Kidney Injury / Q000503:physiopathology*; D000704:Analysis of Variance; D004847:Epithelial Cells / Q000502:physiology*; D005355:Fibrosis; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D007150:Immunohistochemistry; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000503:physiopathology; D061346:Laser Capture Microdissection / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*