Correlation of histopathologic and dynamic tissue perfusion measurement findings in transplanted kidneys

Background Cortical perfusion of the renal transplant can be non-invasively assessed by color Doppler ultrasonography. We performed the Dynamic Tissue Perfusion Measurement (DTPM) of the transplant’s renal cortex using color Doppler ultrasonography (PixelFlux technique), and compared the results with the histopathological findings of transplant biopsies. Methods Ninety-six DTPM studies of the renal transplant’s cortex followed by transplant biopsies were performed in 78 patients. The cortical perfusion data were compared with the parameter of peritubular inflammatory cell accumulation (PTC 0 to 3) based on Banff-classification system. Results A significant decrease of cortical perfusion could be demonstrated as the inflammatory cells accumulation in peritubular capillaries increased. Increasing peritubulitis caused a perfusion loss from central to distal layers of 79% in PTC 0, of 85% in PTC 1, of 94% in PTC 2, and of 94% in PTC 3. Furthermore, the perfusion loss due to peritubular inflammation was more prominent in the distal cortical layer. The extent of perfusion decline with increasing peritubulitis (from PTC 0 to PTC 3) was 64% in proximal 20% cortical layer (p20), 63% in proximal 50% cortical layer (p50), increased to 76% in distal 50% cortical layer (d50), and peaked at 90% in the distal 20% cortical layer (d20). For those without peritubulitis (PTC 0), the increase in the the Interstitial Fibrosis/Tubular Atrophy (IF/TA) score was accompanied by a significantly increased cortical perfusion. A Polyomavirus infection was associated with an increased cortical perfusion. Conclusions Our study demonstrated that the perfusion of the renal transplant is associated with certain pathological changes within the graft. DTPM showed a significant reduction of cortical perfusion in the transplant renal cortex related to peritubular capillary inflammation.

Results: A significant decrease of cortical perfusion could be demonstrated as the inflammatory cells accumulation in peritubular capillaries increased. Increasing peritubulitis caused a perfusion loss from central to distal layers of 79% in PTC 0, of 85% in PTC 1, of 94% in PTC 2, and of 94% in PTC 3. Furthermore, the perfusion loss due to peritubular inflammation was more prominent in the distal cortical layer. The extent of perfusion decline with increasing peritubulitis (from PTC 0 to PTC 3) was 64% in proximal 20% cortical layer (p20), 63% in proximal 50% cortical layer (p50), increased to 76% in distal 50% cortical layer (d50), and peaked at 90% in the distal 20% cortical layer (d20). For those without peritubulitis (PTC 0), the increase in the the Interstitial Fibrosis/Tubular Atrophy (IF/TA) score was accompanied by a significantly increased cortical perfusion. A Polyomavirus infection was associated with an increased cortical perfusion.
Conclusions: Our study demonstrated that the perfusion of the renal transplant is associated with certain pathological changes within the graft. DTPM showed a significant reduction of cortical perfusion in the transplant renal cortex related to peritubular capillary inflammation.

Background
Acute and chronic damage to renal transplants requires an early and accurate diagnosis to start the appropriate treatment. Renal transplant biopsies are an established method to evaluate such changes. These biopsies are performed as routine protocol biopsies in regular intervals in asymptomatic recipients or are performed when a clinical demand emerges. Biopsies are invasive and therefore a need for less potentially harmful procedures exists. Sonography is widely used to describe morphological changes of the transplant. Doppler sonography is used to describe changes of the blood flow to transplants. Today, only limited information can be drawn from Doppler sonographic measurements. To refine the impression of a transplant's perfusion contrast enhanced sonography was introduced. Nevertheless, a need for a more differentiated evaluation remains. Dynamic tissue perfusion measurement (DTPM) has demonstrated the potential to describe perfusion in renal cortex in thin cortical slices with more than 50 parameters [1][2][3][4][5] as well as in other organs [6][7][8][9][10].

Aim
Our aim was to compare histopathological descriptions of renal transplants with DTPM data to plumb the potential of this new tool.

Patient consent
Informed consent was obtained from every patient before the study.

Ethical approval
This study was supported by research grants 96-2321-B-075-009 and 97-2314-B-075-052 from the National Science Council, Taiwan and was approved by the Institutional Review Board, Taipei Veterans Genereal Hospital, Taipei, Taiwan. Step 1 The software opened the video and calibrated the distances automatically Step 2 The software read out the color bar and all hues were calibrated Step 3 Maximal velocities encoded by the colors were registered Step 4 Selection of the region of interest (ROI) a. The ROI consisted of a parallelogram which included an entire cortical segment in the center of the transplant, fed by one interlobar artery running straight towards the transducer.
b. The four corners of the parallelogram were set as follows: i. At the center of the outer edge of a medullary pyramid (1) ii. At the center of the outer edge of the neighboring medullary pyramid (2) iii. At the renal surface perpendicular to the first two corners (3 and 4) c. The ROI was sliced into defined horizontal slices i. Each slice stretched from the left to the right border of the ROI and had a height which encompassed a certain percentage of the ROI's entire height (10%, 20% , 50% or 100%). So the sub-ROI encompassing the proximal 20% (resp. 50%) was labeled p20 (resp. p50) and those encompassing the distal 20% (resp. 50%) was labeled d20 (resp. d50). (Figures 1 and 2) ii. These slices were arranged to cover adjacent horizontal strips of the ROI.
Step 5 Dynamic tissue perfusion measurement was initiated. All steps (a-f) were carried out automatically by the PixelFlux-software a. Each pixel (colored or grey) was evaluated with respect to its size and coloration.
b. The color bar's hues were assigned a velocity value according to its relative distance from zero to the maximum velocity value.
c. The relative distance value (from 0 to 1) was then multiplied by the maximum velocity indicated to calculate a specific velocity value for each color hue. Colorless pixels were assigned the velocity of zero.
d. The mean velocity value of all colored pixels inside the ROI and the area of all colored pixels were calculated.
e. The measurements were repeated for all consecutive images of the video.
f. The software recognized beginning and end of any heart cycle inside the video automatically and restricted all consecutive calculations to a full heart cycle.
g. Then the mean perfusion velocity of each image was multiplied by the mean perfused area. This product was divided by the area of the entire ROI resulting in the mean flow intensity of the ROI during a complete heart cycle.      Table 2).

Results
In those without peritubulitis (PTC 0), perfusion intensity was 0.272 in IF/TA 0, 0.535 in IF/TA 1 (p < 0.001 vs. IF/TA 0), 0.726 in IF/TA 2 (p < 0.001 vs. IF/TA 0) ( Figure 5). IF/TA dependent changes were significantly higher than PTCs influences if PTCs reached values of 1 or 2 and IF/TA remained at zero-level compared to the mirror situation with IF/TA reaching levels of 1 and 2 and no PTCs (PTCs 0). Polyomavirus infection was accompanied by a significant increase in cortical perfusion in the outer cortical layer (distal 20%), albeit less pronounced in stage B than stage A ( Figure 6).

Discussion
Renal transplant biopsies are evaluated according to the Banff scoring system since it was shown that a high Banff score is predictive for the graft survival [13].
Color Doppler ultrasound of the outer renal cortex provides the basis for a quantitative measurement of the perfusion of renal transplants [1,2,14]. Vascular changes, which are mirrored by perfusion changes, may precede  Figure 4 Decline of cortical perfusion (y-axis) from proximal to distal cortical layers (x-axis) is different in the various Banff classes for peritubular inflammation (no significant cortical peritubular inflammatory changes (PTC 0) to cortical peritubular capillary with >10 luminal inflammatory cells (PTC 3)). Cortical layers are p20: proximal 20% of the renal cortex peripheral to the medullary pyramids, p50: proximal 50%, d50: distal 50% and d20: outer 20% of the cortex.
manifest function loss of the kidney and can be described histologically. Peritubular inflammation is a criterion of humoral rejection in renal transplants. According to the Banff classification, this feature is scored as peritubular capillaritis (PTCs) [15]. It might be important to detect peritubular capillaritis in renal transplants early since it can precede the development of a full blown chronic antibodymediated rejection (CAMR). Others propose to assume a chronic antibody-mediated rejection (CAMR) even in the absence of C4d deposition in cases with peritubular capillaritis, transplant glomerulitis, thickening of the peritubular capillaries basement membrane, and circulating anti-HLA antibodies [16,17]. Detection of peritubular inflammation might be significant for indicating the subsequent poor prognosis [18,19] and thus could be regarded an early sign of graft deterioration. Our study showed a significant decrease of cortical perfusion as the inflammatory cells accumulation in peritubular capillaries increased. The reduction of tissue perfusion due to peritubular inflammation depended on the position of the cortical layer too. The more distal the layer the stronger was the effect of peritubular inflammation. Cortical perfusion was also influenced by interstitial fibrosis and tubular atrophy (IF/TA). Unexpectedly, increasing IF/TA scores were connected to an increased perfusion. This might be explained by the short-circuiting of blood via shunts connecting the afferent and efferent vascular pools in juxtamedullary glomeruli [20,21]. Recently, in a mouse model the overexpression of EphB4 receptor tyrosine kinase in the kidney was shown to promote the development of such shunts [22]. Conventional color Doppler sonography with calculation of Resistance Index (RI) and Pulsatility Index (PI) were used for many years to evaluate the perfusion of renal transplants. RI measurements reflect a complex interaction of renal as well as systemic vascular influences and are therefore disregarded to be suitable for evaluation of the renal vascular state by some investigators [23,24]. An elevated RI alone cannot reliably differentiate the causes of transplanted kidney dysfunction and it is considered a nonspecific marker [25].
RI does not inform about the real blood flow velocities, since a ratio of two velocities (systolic and diastolic) is calculated. It represents spatially restricted information from a single vessel and gives no data on the width of the vessel, let alone the perfused area in a certain tissue. Thus RI is basically less suitable for describing the perfusion status of the renal parenchyma. Furthermore, the pathological changes inside the kidney can be distributed inhomogeneously [26], and RI at a single point of an artery may not represent the status of perfusion in the region of interest. In contrast, Dynamic Tissue Perfusion Measurement (DTPM) is based upon a heart cycle triggered measurement of a tissue's mean flow velocity of all vessels inside the region of interest multiplied by the mean perfused area (respecting changing width of all vessels during the heart cycle) [3,4]. Thus, DTPM offers a more sophisticated investigation of renal perfusion by using velocity as well as perfused area information, referring all measurements to all vessels inside the tissue and is capable in differentiating all perfusion data between millimeter-thin slices. It reflects histological changes as PTCs better than conventional RI (Figure 3: left diagram compared to the right one). DTPM thus has the potential to go beyond the limitations of usual vascular resistance calculations by RI measurements. The detection of subtle perfusion changes simultaneously to a polyomavirus infection ( Figure 6) clearly demonstrates that the slice wise perfusion measurement in consecutive layers of renal microvasculature offers a sophisticated access to relevant histological changes even beyond the Banff-classification. It was demonstrated in earlier investigations that with time elapsed after transplantation the cortical perfusion in renal transplants of children decreased significantly [2] and that simultaneously the pulsatility of renal transplants' microvascular perfusion increased significantly [14]. Reduction of smaller subcapsular transplant vessels occurs in acute as well as chronic rejected organs [27]. Cortical vessel loss, as determined visually in power Doppler images, was predictive in contrary to RI as for the transplant function 1 year after transplantation [28]. Recent investigations also showed that microvascular damage is the key process linked to the loss of transplants' function [29,30]. Our findings might be especially noteworthy since we  Table 2). demonstrated a significant and gradual decline of cortical perfusion along with increasing peritubular capillaritis using DTPM. DTPM clearly showed the potential to measure the progressive obliteration of the transplant's microvessels. This process starts in the periphery of renal cortex where the vessel diameters are naturally small. Our study did not investigate the relationship of DTPM with the clinical outcome of the graft. Further studies are needed to elucidate if specific histological findings or the destruction of the microvasculature are more predictive for the transplant's function.
In the long run many renal disorders lead to a gradual decline of cortical perfusion [31][32][33]. DTPM might thus be useful in other chronic diseases of the kidney too.
One limitation of our study is that we only assess the relationship between cortical perfusion and the histopathological changes of transplant kidney. Future studies are needed to confirm the role of DTPM as a surrogate of outcome of renal transplant. Then DTPM could become a non-invasive tool to reduce the need for invasive procedures such as biopsies.

Conclusion
Microvascular perfusion changes in renal transplants can be demonstrated by DTPM. A significant reduction of cortical perfusion in the transplant kidney, especially in its peripheral layers, is related to peritubular capillary inflammation.