Introduction: The medical uses of optical imaging are revolutionizing medicine; optical imaging is undergoing explosive growth fuelled by advances in high-sensitivity detectors. These light based systems utilise multiphoton microscopy (MPM) to provide high resolution and quantitative imaging of cellular metabolism in in situ and in vivo biological tissues and organs – in space (three dimensions), in time, in spectra, and in fluorescence anisotropy (i.e. a total of 6 dimensions). MPM allows in vivo visualisation of intact kidney tissue at the single cell resolution, which is solved a long-standing critical technical barrier in renal research to study several complex and inaccessible cell types and anatomical structures. In this study, MPM was employed to visualise morphology changes and study the functions of acute kidney injury (AKI).

Methods: Glycerol–induced AKI model (elevated serum creatinine) can be used to mimic rhabdomyolysis, which was developed in male C57BL/6 mice (8-12 weeks) by intramuscular injection of 50% (v/v) glycerol /PBS (10 ml/kg) into the left hind leg. The control mice were injected by PBS using the same method. After 24 hours, kidneys were exposed to image using MPM for morphology examination in both groups. Since Rhodamine 123 (RH123) is a marker to measure P-glycoprotein (P-gp) transporter function, it was intravenously injected into control and AKI mice and imaged by MPM to evaluate the P-gp transporter function. Using a bolus injection of FITC labelled inulin, rapid quantification of glomerular filtration rate (GFR) was determined in both groups by directly imaging of inulin clearance from glomerulus. A LaVision Biotec Nikon MPM was used to image RH123 excretion from tubules. GFR was measured using a DermanInspect system equipped with the ultrashort (85 fs pulse width, 80 MHz repetition rate) pulsed mode-locked tunable Ti:sapphire laser (Mai Tai, Spectra Physics, 25 Mountain View, USA).

Results: The MPM images directly showed morphology changes in AKI mice compared to control group, where acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation (Fig.1). Intravital imaging showed that RH123 was rapidly excreted from tubules in control group but slowly eliminated from AKI model (Fig. 2), indicating P-gp transporter dysfunction in glycerol-induced AKI. As shown in Fig, 3, reduced glomerular permeability was also observed in AKI model, where FITC-labelled inulin (red color) was quickly cleared from glomerular at 30 mins after injection in control group, while fluorescence from FITC-labelled inulin was retained in glomerular in AKI model.

Conclusion: This advanced imaging technique can be used to rapid diagnosis and quantification of AKI induced morphology and functional changes.

Key words: Intravital imaging, Multiphoton microscopy, Morphology, Renal function, AKI

Figures:

Fig. 1 Acute injury of tubular cells as indicated by reduced autofluorescence and cellular vacuolation (red circle) scale bar: 40 µm. Image recorded at λExc/λEm: 740/350 to 450 nm.

Fig. 2 MPM images of RH123 in representative mouse kidney after IV injection. Image recorded at λExc/λEm: 920/520 to 580 nm.

Fig. 3 GFR changes in AKI model compared to control group. Green: autofluorescence of renal tubules; Red: FITC-inulin fluorescence; G: glomerulus; T: tubules (scale bar: 40 µm).

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EMC Abstracts - https://emc-proceedings.com/abstract/novel-in-vivo-imaging-techniques-to-visualise-renal-morphology-and-function-in-acute-kidney-injury/