**3. Results**

## *3.1. Postoperative Outcomes*

Of the 511 grafts that were included in this analysis, 40 were found to have moderate (>30%) macrovesicular steatosis. The average steatosis percentage was 41.1% ± 15.8% in the moderate group, compared to 3.8% ± 5.8% in the non-steatotic group (*p* < 0.001) (Table 1, Figure 1A,B). For the entire cohort, the incidence of post-LT AKI was 19.6% (*n* = 100). In assessing clinical risk factors for kidney disease, 12.5% (*n* = 64) of LT recipients were diabetic, 12.7% (*n* = 65) had hypertension, and 13.5% (*n* = 69) were both diabetic and hypertensive. The distribution of diabetes and hypertension did not vary between the steatotic and non-steatotic grafts (*p* = 0.95). There were no differences in age (*p* = 0.39), sex (*p* = 0.18), ethnicity (*p* = 0.72), race (*p* = 0.57), the biologic Model for End-Stage Liver Disease (MELD) (*p* = 0.14) or indications for liver transplant (*p* = 0.16) between the recipients receiving moderately steatotic and non-steatotic grafts (Table 1).


**Table 1.** Pre- and Post-Liver Transplant Recipient Demographics.

**Figure 1.** Liver Graft Biopsy Findings Liver Biopsy Findings. (**A**) Representative biopsy of a >30% macrovesicular steatotic allograft. (**B**) Representative biopsy of a non-steatotic allograft. (**C**) Approximately 30% macrovesicular steatosis with a mixture of large (arrows) and small (arrowheads) droplet fat (hematoxylin and eosin (H&E), 200×). (**D**) Microvesicular steatosis characterized by diffuse deposition of fat droplets in the hepatocyte cytoplasm without any macrovesicular steatosis (H&E, 200×). (**E**) Approximately 40% macrovesicular steatosis seen on a pre-implantation biopsy (H&E frozen section, 400×). (**F**) Zonal distribution of macrovesicular steatosis with fat deposition accentuated in zone three (asterisks) around the central veins (H&E, frozen section, 100×). (**G**), Lipopeliosis characterized by the rupture of hepatocytes with coalescence of fat droplets (arrow) in the sinusoidal spaces (H&E, frozen section, 600×). (**H**), Lipopeliosis (arrow) in post-reperfusion biopsy (H&E, 600×).

Post-LT AKI was observed in 52.5% of patients receiving moderately steatotic grafts versus 16.8% in the non-steatotic cohort (*p* < 0.0001). No patients in the entire cohort had liver allograft primary non-function. Patients transplanted with moderately steatotic grafts had significantly more early allograft dysfunction immediately following surgery (AST: 3212 ± 2413 U/L vs. 1118 ± 1473 U/L, *p* < 0.0001). The rise in AST was four-fold higher for recipients of steatotic grafts that went on to develop post-LT AKI (*p* < 0.0001). There was a greater need for newly initiated temporary post-LT dialysis in the moderately steatotic group (10.0% vs. 1.1%, *p* = 0.003). There were no differences in intensive care unit (ICU) length of stay (2.0 ± 1.8 vs. 1.8 ± 2.6, *p* = 0.62) or total hospital length of stay (9.1 ± 10.3 vs. 9.9 ± 10.9, *p* = 0.67). At one-year post-LT, there were no observed differences in the need for new chronic (ongoing) post-LT dialysis (*p* > 0.99), serum creatinine (1.3 ± 0.3 vs. 1.3 ± 0.7, *p* = 0.97), or eGFR (53.1 ± 7.9 vs. 53.5 ± 10.1, *p* = 0.70) (Table 1).

#### *3.2. Moderately Steatotic Graft Subgroup Analysis*

In order to investigate postoperative outcomes in moderately steatotic livers allografts in further detail, we reviewed the characteristics of the 40 patients who were transplanted with such grafts (Table 2). In this cohort, there were no differences in age (*p* = 0.61), sex (*p* = 0.43), ethnicity (*p* = 0.60), race (*p* = 0.64), or indication for transplant (*p* = 0.53) among those who developed AKI versus those who did not (Table 2). Of those receiving steatotic grafts, 52.5% went on to develop AKI; the other 47.5% maintained normal renal function post-transplant. Recipients of steatotic grafts that went on to develop post-LT AKI had a higher biologic MELD (20.5 ± 8.9 vs. 15.3 ± 6.9, *p* = 0.04) (Table 2). There were no differences in pre-LT creatinine (*p* = 0.21) or eGFR (*p* = 0.88).


**Table 2.** Moderately Steatotic Graft Subgroup Analysis.

Recipients of moderately steatotic grafts were all noted to have early allograft dysfunction as demonstrated through a significantly elevated AST immediately following surgery (Figure 2). This occurred regardless of whether or not they developed AKI. The rise in AST was four-fold higher for recipients of steatotic grafts that went on to develop post-LT AKI as compared to non-steatotic grafts (40001.0 ± 2471.0 U/L vs. 1118.0 ± 1473.0, *p* < 0.0001). The rise in AST was also two-fold higher when comparing steatotic grafts of recipients with and without post-LT AKI (40001.0 ± 2471.0 U/L vs. 2339.0 ± 2074.0, *p* < 0.0001). There were no differences between the post-LT AKI and no AKI groups with regards to graft type (i.e., donation after brain death, DBD, vs. donation after cardiac death, DCD) (19.0% vs. 10.5%, *p* = 0.66), sex (female: 42.9% vs. 47.4%, *p* = 0.38), or BMI (32.8 ± 5.9 kg/m<sup>2</sup> vs. 32.6 ± 9.4 kg/m2, *p* = 0.92). Donor age was yonger (43.2 ± 12.6 vs. 52.8 ± 14.9%, *p* = 0.03) in steatotic grafts that went on to develop AKI. In addition, there were no differences in allograft cold ischemia time (CIT) (*p* = 0.28) or estimated operative blood loss (EBL) (*p* = 0.49) (Table 3).

**Figure 2.** Post-Liver Transplant Patterns in Steatotic and Non-Steatotic Grafts. (**A**) Post-LT aspartate aminotransferase (AST) levels. Compared to non-steatotic graft, AST levels were 2-times higher in steatotic grafts without post-LT AKI and 4-times higher for steatotic grafts with AKI. (**B**) Post-LT creatinine levels.


**Table 3.** Steatotic Graft Recipient Operative Variables.

When operative hemodynamics of AKI and non-AKI recipients receiving moderately steatotic grafts were compared, there were no differences observed in inotrope requirements (*p* = 0.45), systolic blood pressure (SBP) (*p* = 0.25) or mean arterial pressure (MAP) (*p* = 0.76) during the pre-anhepatic phase or anhepatic phase (inotrope score *p* = 0.58; SBP *p* = 0.67; MAP *p* = 0.71) of the operation (Table 3). Post-reperfusion, SBP (*p* = 0.87) and MAP (*p* = 0.69) were similar in patients who went on to develop post-LT AKI versus those who did not suggesting appropriate perfusion parameters were able to be maintained. The post-reperfusion inotrope requirements, however, were significantly higher in the post-LT AKI group (19.5 ± 20.0 vs. 3.8 ± 4.4, *p* = 0.03). Ten percent of the patients in the steatotic post-LT AKI group (*n* = 4) required initiation of new dialysis post-LT (*p* = 0.003) (Table 2). There were no differences in ICU length of stay (*p* = 0.62) and total hospital length of stay (*p* = 0.67) between steatotic AKI and no AKI groups.

At one-year post-LT, there were no observed differences between those receiving steatotic grafts that developed AKI versus those who did not with regards to serum creatinine (1.3 ± 0.2 vs. 1.2 ± 0.4, *p* = 0.27) and eGFR (52.7 ± 6.9 vs. 53.6 ± 9.1, *p* = 0.74) (Table 2). At five years, there were no differences for AKI vs. no AKI patient survival (HR 0.95, 95% CI 0.08–10.6, *p* = 0.95) or allograft survival (HR 1.73, 95% CI 0.23–13.23, *p* = 0.59) for those using steatotic grafts (Figure 3).

**Figure 3.** Survival. (**A**) Patient survival post-LT. Hazard ratio (HR); Confidence Interval (CI). (**B**) Liver allograft survival post-transplant. Moderately steatotic grafts with AKI (>30% Macro-AKI); Moderately steatotic grafts without AKI (>30% Macro-No AKI); Non-steatotic grafts (<30% Macro).

#### *3.3. Liver Graft Biopsy Findings*

In prospectively re-reviewing biopsies of all liver allografts with moderate (>30%) macrovesicular steatosis, the majority of the steatosis was found to be large droplet (Table 4) (Figure 1C,E). When comparing biopsies in patients with and without post-LT AKI, no differences were observed with regard to large droplet versus small droplet percentage composition (Figure 1C) (*p* = 0.41). Although microvesicular steatosis was minimal in both groups (Figure 1D) (0.0% vs. 21.1%), a higher frequency was observed in the no AKI group (*n* = 4, *p* = 0.04) (Table 4). No significant differences were observed in the histologic distribution of the steatosis (zonation) in the allograft (*p* = 0.75) (Figure 1F), inflammation (*p* = 0.73), ballooning (*p* = 0.65), or Mallory hyaline (Table 4). A larger percentage of biopsies in the post-LT AKI group contained lipopeliosis (61% vs. 31.6%, *p* = 0.04) (Figure 1G–H). When plotted against MELD at the time of transplant, recipients of moderately steatotic grafts with lipopeliosis with a MELD ≥ 20 were found to more likely to develop AKI (88.9%) than recipients of such grafts with MELD < 20 (40.0%; *p* = 0.04) (Figure 4). In using logistic regression, variables predictive of post-LT AKI included the finding of lipopeliosis on liver biopsy and donor age (Table 5).



**Figure 4.** Relationship between MELD, Lipopeliosis and Post-LT AKI.

**Table 5.** Predictors of AKI in Steatotic Grafts—Logistic Regression.

