EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: The aging kidney exhibits a progressive decline in renal function with characteristic histopathologic changes and is a risk factor for renal transplant. However, the degree to which the kidney exhibits this decline depends on several factors that vary from one individual to the next. Optical coherence tomography is an evolving noninvasive imaging technology that has recently been used to evaluate acute tubular necrosis of living-human donor kidneys before their transplant. With the increasing use of kidneys from older individuals, it is important to determine whether optical coherence tomography also can distinguish the histopathology associated with aging.

Materials and Methods: In this investigation, we used Munich-Wistar rats to evaluate the ability of optical coherence tomography to detect histopathologic changes associated with aging. Optical coherence tomography observations were correlated with renal function and conventional light microscopic evaluation of these same kidneys.

Results: With the onset of severe proteinuria at 10 to 12 months of age, optical coherence tomography revealed tubular necrosis/atrophy, interstitial fibrosis, tubular dilation, and glomerulosclerosis. With a further deterioration in kidney function at 16 to 18 months of age (as indicated by rising creatinine levels), optical coherence tomography revealed more extensive interstitial fibrosis and tubular atrophy, increased tubular dilation with cyst formation and more sclerotic glomeruli.

Conclusions: The foregoing observations suggest that optical coherence tomography can be used to detect the histopathology of progressive nephropathy associated with aging.

Key words : Optical Coherence Tomography (OCT), Histopathology, Aging, Renal cysts, Kidney, Transplantation

Optical coherence tomography (OCT) is a rapidly evolving imaging modality that can function as a type of “optical biopsy,” providing cross-sectional images of tissue morphology in situ and in real-time.^1,2 Optical coherence tomography is similar to ultrasound imaging, except that it uses the echo delay of light instead of sound to generate images. Optical coherence tomography is safer than radiographic technologies, is less expensive than magnetic resonance imaging (MRI) devices, and provides higher resolution images than ultrasound. By using broadband optical light sources, OCT can achieve axial resolutions of 1 to 10 μm, more than an order of magnitude above that obtainable by conventional ultrasound. As a result, OCT can provide high-resolution images of organs and tissues in a noninvasive manner. This potential has been demonstrated in several biomedical applications including ophthalmology,^3-5 cardiology,^6,7 gastroenterology,^8-11 dermatology,¹² dentistry,¹³ urology,¹⁴ and gynecology.¹⁵ In contrast to other forms of noninvasive light microscopy, OCT can image with longer working distances, with improved penetration depth, and without the need for tissue contact. Not only can it image depths up to 1 to 2 mm in most light-scattering tissues; OCT can provide 3-dimensional images in arbitrary planes. Finally, OCT can be performed using a thin flexible sterile endoscope or catheter^16-18 or even with a needle,^19,20 enabling ease of use and the possibility of imaging deep within a solid tissue or organ.

Recent studies have shown that OCT can noninvasively image the entire kidney surface in minutes and in real-time.^21-24 Conversely, conventional excisional biopsies are invasive, damaging, can only sample a small region of the kidney, take time to process, and result in severe artifacts that are difficult to distinguish from ischemia and other injuries.²⁵ Because the morphology of the proximal convoluted tubules imaged by OCT correlates with the extent of acute tubular necrosis (ATN),²⁶ the information obtained by OCT is potentially useful in evaluating the status of donor kidneys. In view of this potential use of OCT imaging in evaluating donor kidneys, it is important to determine the ability of OCT to distinguish pretransplant factors, in addition to ATN that might significantly affect donor kidney outcome.

The use of older kidneys from either cadavers or living donors is increasing as the population ages and due to an increasing need to expand the donor pool of kidneys.²⁷ However, this approach brings with it the risk of poor prognosis because of age-related deterioration of older donor kidneys.^28,29 For example, in living donor kidneys, there is a marked increase in the prevalence of glomerulosclerosis with aging³⁰ and a decrease in kidney weight resulting from loss of renal mass that begins in the fourth and fifth decades of life.^31,32 Also, studies of laboratory animals have indicated that the kidneys of senescent laboratory animals are more susceptible to ischemia and anoxia,33 which are major concerns for ATN during transplants.

Chronic progressive nephropathy is a common spontaneous disease in senescent laboratory rats. While the severity of progressive nephropathy varies, it is qualitatively similar among different strains with male rats being more susceptible.³³ In this study, we used Munich-Wistar rats to evaluate OCT imaging’s ability to reveal the histopathology associated with aging, as it is characterized by more superficial glomeruli than other strains^34,35 that are available for OCT imaging and that are more prone to sclerotic changes.³³

Materials and Methods

Fifteen adult male Munich-Wistar rats were divided into 3 groups: young adult rats (aged 3-5 mo), late middle age (10-12 mo), and old rats (age 16-18 mo). Every month until surgery, rats were weighed, and 24-hour urine volumes were collected in metabolic cages. At the time of surgery, fresh urine samples were tested for albuminuria (Albustix), and blood samples taken from the tail vein analyzed for serum creatinine (Beckman Coulter Creatinine Analyzer, Brea, CA, USA). Before surgery, the animals were anesthetized with isoflurane/O2 (4% induction, 1.5% during operation, O₂ 1 L/min). After induction of anesthesia, the abdominal cavity was opened through a midline incision, and the left kidney exposed and studied using OCT as described in previous publications.^21,22 Immediately after in vivo OCT evaluation, the kidneys were fixed in situ by flushing with warm (ie, 37°C) oxygenated saline, followed immediately by phosphate buffered 2% paraformaldehyde and 0.1% glutaraldehyde. The fixed kidneys were excised, and the rat killed by intracardiac injection of pentobarbital sodium to induce cardiac arrest. Blocks of fixed kidneys were embedded in paraffin, sectioned, stained with hematoxylin and eosin, and then examined and photographed with an Olympus light microscope (BH-2, Olympus Corp., Lake Success, NY, USA) equipped with a 35-mm digital camera. The OCT data were analyzed and correlated with the functional and light microscopic histopathologic data collected. Mean tubular diameters were determined by measuring a minimum of 50 representative tubules per age group.

A high-speed OCT system using swept source/Fourier domain detection was used in this study. The details of this system can be found in previous publications.^22,23 The light source is a wavelength-swept laser light source generating a 100 nm full width at half maximum sweep bandwidth at approximately 1310 nm. The laser operates at a sweep repetition rate of 16 kHz with an average output power of 12 mW. The imaging is performed using an OCT microscope and imaging engine, modified from a commercially available OCT system (Thorlabs, Inc., Newton, NJ, USA). Three percent of the laser output power is coupled to a Mach–Zehnder interferometer that is used to generate a clock signal for recalibration of the OCT signal on a uniformly-spaced optical frequency grid. The other 97% of the laser power is divided equally and delivered to the sample and reference arms of a Michelson interferometer. Imaging of the tissue samples is performed using a pair of mirrors mounted to XY scanning galvanometers (Cambridge Technology, Cambridge, MA, USA) and a microscope objective. A polarization controller and a spatial aperture are used to set the reference arm power. The system sensitivity is 93 dB with 6 mW of average power incident on the sample. The resolution for the imaging system is measured to be 10 μm (in tissue) in the axial (Z) direction and 6 μm in the transverse (XY) directions. Individual cross-sectional OCT images (XZ) consisting of 512 axial scans are generated at a rate of 30 frames per second over ranges of 1.4 mm in length (X, 1024 transverse pixels) and 1.8 mm in depth (Z, axial 512 pixels).

All the foregoing studies were approved by the University of Maryland IRB committee and were in conformity with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health 86-23, revised in 1985.

Table 1 shows the gradual decline in renal function over time. Before surgery, the 3- to 5-month-old group exhibited normal serum creatinine, proteinuria, and urine volumes (Table 1). Optical coherence tomography revealed the open lumens of proximal convoluted tubules that appeared rounded in cross-sectional profile. Although distal convoluted tubules are occasionally viewed, they are relatively rare in the superficial cortex of rats and difficult to discern from proximal convoluted tubules in OCT images. The superficial tubules had a mean diameter of 25.3 ± 8.9 (SD) microns (Figure 1A). The renal corpuscles appeared normal, with glomeruli filling the capsular space of Bowman (Figure 1A). Light microscopic examination of the same samples using conventional light microscopy of hematoxylin and eosin stained samples verified the normal appearance of the uriniferous tubules and renal corpuscles (Figure 1B). The serum creatinine and urine volumes in the 10- to 12-month group also were within the normal range, but these animals exhibited proteinuria (Table 1). Unlike the 3- to 5-month-old group, the mean body weights remained the same and did not increase during the last 2 months leading up to surgery. Optical coherence tomography imaging of the 10- to 12-month-old group revealed tubular dilation, tubular atrophy, and interstitial fibrosis (Figure 2A). The mean tubular diameter of the uriniferous tubules was 40.1 ± 17.5 (SD) microns. Also, some of the renal corpuscles appeared sclerotic, with the glomerular tuft appearing smaller, irregular in shape, and not filling the capsular space of Bowman (Figure 3A). The foregoing OCT observations were verified by conventional light microscopy of stained sections showing dilated tubules, regions of interstitial fibrosis (Figure 2B), and renal corpuscles exhibiting focal glomerular sclerosis (Figure 3B). All animals in the 16- to 18-month-old group exhibited significantly elevated serum creatinine, a significant decrease in mean body weights, polyuria, and proteinuria during 24 hours when accounting for increased urine volumes (Table 1). Optical coherence tomography revealed areas with extremely dilated tubules (Figure 4A), some of which formed cysts that were grossly visible on the surface of the kidneys (Figure 4B, inset). These cysts appeared to be composed of clusters of tubules (mainly proximal convoluted tubules) with dilated lumens. The mean tubular diameter of the uriniferous tubules was 101.6 ± 45.0 (SD) microns. The large standard deviation (ie, 45.0 μm) reflects the significant variability in the dimensions of the dilated tubules. Also evident were regions of tubular degeneration and associated interstitial fibrosis (Figure 4A). Finally, many of the glomeruli appeared shrunken and sclerotic, similar to those seen in the 10- to 12-month-old rats (Figure 3A and 3B). However, some glomeruli appear abnormally large, reflecting perhaps a compensatory response to the decreasing number of viable nephrons. Conventional light microscopy verified the same histopathology, including dilated tubules, interstitial fibrosis, degenerating tubules, and sclerotic and hypertrophic glomeruli (Figure 4B).

Recent studies have supported the use of OCT imaging to determine the status of donor kidneys by revealing the extent of ATN before transplant.^21-24 With the deterioration in renal function associated with aging being an important variable for determining suitability for transplant,^27,28 it is important to determine whether OCT also can access nephropathy associated with aging. In the present investigation, male Munich-Wistar rats exhibited characteristic age-related nephropathy over a 16- to 18-month lifespan. At 10 months of age, OCT revealed histopathologic changes of the aging rat kidney including tubular dilation, tubular atrophy, interstitial fibrosis, and glomerular sclerosis. These histopathologic features commonly occur together and constitute nephrosclerosis. The foregoing changes were evident with the onset of severe proteinuria and before a rise in serum creatinine values. This finding correlates with the previous observations of Goldstein and associates,³³ who reported that proteinuria characterizes the aging rat kidney and correlates well with the appearance of histopathologic features. The cysts that were an obvious feature in the 16- to 18-month-old rats also are a common feature of elderly patients.^36,37 Baert and Steg³⁸ suggest that these cysts might originate from diverticula of the distal tubules or collecting ducts. However, the cysts that we found appear to result from merging clusters of dilated proximal convoluted tubules. The distal tubules and collecting ducts, unlike proximal convoluted tubules, do not cluster in the cortex of the rat kidney. Optical coherence tomography also revealed sclerotic glomeruli, with glomeruli appearing shrunken, asymmetric in shape, and surrounded by a large capsular space of Bowman. This is in contrast to normal glomeruli, which appear rounded and nearly filling the entire space of Bowmen. It has been proposed that the mechanism of this sclerosis is hyperfiltration.³⁹ While some glomeruli were shrunken and sclerotic, others were abnormally enlarged. This observed heterogeneity in glomerular size also has been reported to be characteristic of an aging kidney.³⁰ The enlarged glomeruli are believed to reflect a compensatory response (ie, compensatory hypertrophy) of the remaining functional glomeruli because of the additional workload responding to a diminished population of glomeruli.^30,39,40 While the histopathology seen using OCT of the aging kidney correlates with similar changes seen in human kidneys,30 it must be noted that histopathologic features observed in the aging kidney also are characteristic of other renal insults including diabetes and hypertension.⁴¹ Nevertheless, the extent to which these changes occur is an indication of the future functioning of a given donor kidney and can therefore help determine the prognosis of a donor kidney and identify donor kidneys that are unsuitable for transplant.

In addition to analyzing potential donor kidneys, the ability of OCT to reveal the nephropathy as evidenced in the present investigation suggests another potential role of OCT as an adjunct procedure for use in conjunction with renal biopsies. Currently, percutaneous and surgical biopsies of the kidney are required not only for conventional microscopic evaluation, but for immunofluorescence and transmission electron microscope evaluation. However, these biopsies have the disadvantages of sampling small regions of the kidney only, are damaging to the kidney, increase the risk of bleeding, and display artifacts that are difficult to discern from ATN.²⁵ Because OCT can be performed at the end of a catheter or needle,^19,20 it can be used alongside of percutaneous biopsies to survey the kidney before obtaining needle plugs of tissue. In the case of surgical excisional biopsies, OCT may provide a similar preliminary scan before tissue excision. This would allow one to localize areas of interest for tissue extraction and obtain a better perspective of the global nature of the histopathologic changes occurring in the kidney.

Finally, we should add that, previously, OCT has been demonstrated to provide flow information at the microscopic level to evaluate individual glomerular blood flow.^22,42 Because intrarenal hemodynamic abnormalities are thought to be a primary factor associated with the onset and progression of acute injury,⁴³ this is another novel feature of this evolving technology supporting its application for evaluating potential donor kidneys.

In summary, the present investigation has demonstrated that OCT imaging can identify age-related nephropathy in a rat model of aging. Because age-related nephropathy can affect donor kidney outcomes,²⁷ this diagnostic feature is another variable to evaluate when using OCT to evaluate the status of donor kidneys before their transplant.

Volume: 14 Issue: 6 December 2016

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