Introduction

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is rare disease caused by polycystin mutations and is characterized by the development of slowly growing fluid-filled cysts in both kidneys that can lead to end-stage renal disease. Since kidney function declines late in the disease process, total kidney volume (TKV) has been approved as a surrogate endpoint for accelerated approval in autosomal dominant polycystic kidney disease. Preclinical MRI can accurately quantify TKV and renal cyst development^1,2. We have previously published our approaches for high resolution anatomical mouse kidney MRI and automatic kidney segmentation for accurate TKV measurements³. Beyond TKV measurements, MRI offers access to a wide variety of parameters to quantify various changes in tissues associated with disease progression. Recently, T₁ and T₂ quantifications have been shown to correlate with disease progression in the closely related Autosomal Recessive Polycystic Kidney Disease, both in mouse models and clinically⁴. Here, we investigate the correlation between T₂ changes in two different mouse models of ADPKD with TKV and glomerular filtration rates (GFR) to evaluate if T₂ mapping can serve as a useful biomarker for ADPKD progression.

Methods

All animal procedures were approved by the IACUC and followed AAALAC guidelines. Animals received a SC injection of 0.4 mg/kg atropine approx. 20 minutes before imaging to slow down peristaltic motion. Mice were anesthetized with isoflurane (2%) and kept at 37±0.5°C. Imaging was performed with a 9.4T Bruker BioSpec (Bruker, Ettlingen, Germany). Animals were placed supine, with their abdomen on a 4-channel cardiac receiver array in a 86mm transmit/receive volume coil. We performed MRI on two models of polycystic kidney disease: Pkd1^RC/RC mice, a slowly progressing genetic model for ADPKD, and a doxycycline-induced conditional knockout model for PKD1: Pkd1^fl/fl;Pax8^rtTA;TetO-cre (courtesy of the Peter Jackson lab at Stanford), and some C57BL/6J mice as healthy controls. Anatomical images were acquired for TKV measurement with an improved resolution³ compared to previous publications. To acquire T₂ maps, a multi-SE-series was acquired (0.16×0.16×0.8 mm³, 5 slices, 10 TEs from 12.5 to 192.5 ms, TR=1.05s, acquisition time around 3 minutes). Automatic segmentation was used to mask kidneys in images, and T₂ maps were calculated by fitting a voxel wise nonlinear exponential decay using MATLAB.
Glomerular filtration rates (GFR) were determined by measuring the transdermal fluorescence signal of a dye (FITC-sininstrin) undergoing renal clearance (MediBeacon transdermal mini GFR monitor, MediBeacon, St. Louis, MO, USA). For the GFR measurement, the flank was shaved under isoflurane, the transcutaneous GFR monitor was taped to the flank, and background signal was recorded for 3 mins. Then, one drop of ophthalmic anesthetic (0.5% proparacaine hydrochloride ophthalmic solution) was administered to one eye, and 0.075 mg/g BW of FITC-sinistrin in approx. 50 ul of sterile PBS were injected retro-orbitally. Animals were placed in a clean cage each and data were recorded for 1.5-2 h. At the end of the recording, animals were anesthetized again for removal of the GFR monitor.
Recorded data were analyzed in the vendor’s software to obtain GFR estimates.

Results

Both ADPKD models showed strongly different rates of disease progression, indicated by TKV and T₂ increase (Fig. 1). T₂ quantification were utilized in two ways: by calculating mean T₂ for every kidney, and by measuring the fraction of the kidney volume where voxels showed elevated T₂ values (>80ms).

Mean kidney T₂ and elevated T₂ volume fraction were non-linearly correlated for both ADPKD models and wildtype control mice (Fig 2 A). Progression to high T₂ values was particularly prevalent in Pkd1^fl/fl mice. A good correlation was observed between elevated T₂ fraction and cystic area determined from histological slides (R²=0.84). Elevated T₂ fraction has systematic offset when compared to cystic area estimates from histology (Fig. 2 B). Mean T₂ increased with increasing TKV in both models (Fig 2 C,D), with higher variability in the Pkd1^RC/RC mice (R²=0.51), and mean T₂ reaching a plateau above TKVs of over 2000 mm³ in the Pkd1^fl/fl model (R²=0.86). GFR measurements showed high variability, but were correlated with both elevated T₂ fraction and mean T₂ (R2=0.49 and 0.47, respectively, Fig 2 E,F). GFR appeared normal approximately up to a mean T₂ of 80 ms or less than 40% of elevated T₂ fraction, respectively, and declined for higher values.

Discussion and Conclusion

Cyst load measured from histology and kidney T₂ measurements showed good correlation, indicating that kidney cyst load can be accurately predicted by in vivo T₂ mapping. Volume fraction of the kidneys with elevated T₂ were larger than cystic fractions measured by histology, which is likely due to partial volume effects overestimating cystic load. Measured GFR decline was correlated with increased mean T₂ and higher elevated T₂ fractions. Kidney T₂ measurements are a promising biomarker for ADPKD progression in mouse models.