Assessment of glomerular filtration and tubular secretion in baboons with life-supporting pig kidney grafts

Abstract

With pig kidney xenotransplantation nearing clinical reality, it is imperative to measure pig kidney function in the graft recipients. Our aims were (i) to compare inulin clearance after a short intravenous (IV) bolus with steady-state inulin IV infusion, (ii) to use this method to measure the glomerular filtration rate (GFR), and (iii) to determine the tubular secretory function using cefoxitin in a pig-to-baboon renal transplant model. A short IV infusion of inulin and cefoxitin were followed by a maintenance IV infusion of inulin over 5 h in seven healthy baboons, three healthy pigs, and five baboons after bilateral native nephrectomy and intra-abdominal pig renal transplantation. Blood and urine samples were collected. Serum and urinary inulin and serum cefoxitin concentrations measured by validated assays were used to calculate GFR and renal secretion. GFR calculated were similar by both methods. The body weight normalized total body clearance of inulin was similar in pigs and baboons despite differences in absolute clearances. Pig kidney transplanted into baboons provided similar clearance in baboons when normalized to baboon body weight and sustained filtration and secretory functions. The study documented that pig kidneys support the physiologic needs of baboons and are likely to support human recipients as well.

Keywords: baboon; glomerular filtration rate; kidney function; pig; tubular secretion; xenotransplantation.

Figure 1:

Timeline for administration of agents and collection of blood and urine during the inulin and cefoxitin clearance experiments. Blood was collected at all marked time-points. The first blood collection time-point (time = 0 minutes) was immediately prior to inulin and cefoxitin (when used) bolus administration. Urine was collected each hour for the first two hours and every two hours for four hours. The bladder was emptied immediately prior to inulin and cefoxitin administration.

Figure 2:

Mean serum levels of inulin (A) and cefoxitin (B) following bolus injection (39 mg/Kg) and maintenance infusion of inulin (15 mg/Kg/h) starting at 60 min in baboons and pigs. Open circles represent mean serum levels in naïve baboons. Open squares represent mean serum levels in naïve pigs. Data are presented as mean ± SE.

Figure 3:

(A) Inulin clearance estimated in naïve baboons and pigs after bolus injection (CL _bolus) compared to total body clearance estimated after bolus and continuous infusion (CL _{bolus + infusion}) of inulin. (B) Inulin clearance estimated in baboons transplanted with pig kidneys after bolus injection (CL _bolus) compared to clearance during steady state infusion (CL _ss) of inulin. Open circles represent clearance values from naïve baboons, naïve pigs, and longitudinal assessments from baboons transplanted with pig kidneys. Each point represents values obtained from each phase of the study. Dotted lines and R² represent the linear regression lines and the spearman correlation co-efficient. Correlation coefficients of 0.8985 and 8843 suggests that inulin clearance values correlated well between both methods.

Figure 4:

(A) Inulin clearance estimated by method (i) as the total amount excreted in urine over the area under the serum concentration - time curve compared to method (ii) renal excretion rate over steady-state serum levels. Data from naïve and transplanted animals were used for correlation. Dotted line represents the linear regression, equation shows slope and intercept for regression line and R² is the Spearman correlation co-efficient. Each point represents clearance values obtained from different phases of the study. Renal clearance as calculated by an area under the curve model based on serum values correlates very well with renal clearance calculated by the gold standard steady-state model. As such, serum clearance of inulin in this model can accurately reflect renal clearance. Correlation coefficient of 0.9166 suggests that inulin renal clearance values correlated well between both methods. (B) Clearance after bolus dose compared to renal clearance of inulin. The inulin clearance in naïve baboons, pigs, and baboons transplanted with pig kidneys was used for correlation. Filled circles represent clearances observed in transplanted baboons and open circles show data from naïve animals. The equation shows the slope and intercept for regression line and R² is Spearman correlation coefficient. Each point represents clearance values obtained from different phases of the study. The results show that the main mechanism of inulin clearance is via renal excretion which can be measured using serum clearance of inulin in this model.

Figure 5:

Mean serum levels of inulin and cefoxitin and time following bolus injection of inulin and cefoxitin, and maintenance infusion of inulin in baboons B4918 (A, C) and B7018 (B, D), respectively. B4918 and B7018 were transplanted with pig kidneys and longitudinal assessments were performed. POD is post-transplant day. The animals were given inulin at 39 mg/Kg bolus dose and 15 mg/Kg IV infusion initiated at 60 min after bolus dose. Cefoxitin (5 mg/Kg) was administered along with the inulin bolus dose. Each curve represents one study and details are presented in respective panels. Serum inulin and cefoxitin levels during the renal function experiment reproducibly follow predicted values pre- and post-xenotransplantation in the same animals.

Figure 6:

(A) Estimated Inulin clearance in B4918 and B7018 before transplantation and on different days following transplantation with a pig kidney. (B) Serum creatinine levels in B4918 and B7018 before transplantation and on different days following transplantation with a pig kidney. Total body clearance of inulin was used. Serum creatinine levels were measured using clinical assay methodology. The results show that inulin clearance (as a marker for GFR) decreases slightly and serum creatinine increases slightly over 2 months following pig-to-baboon renal transplantation.

Figure 7:

Estimated secretory clearance of cefoxitin in B4918 and B7018 before transplantation and on different days following transplantation with a pig kidney. Secretory clearance was estimated as the difference between GFR and creatinine clearance. The results show that secretory clearance decreases slightly over 2 months following pig-to-baboon renal transplantation.

Figure 8:

Kidney length measured by ultrasound in baboons transplanted with genetically engineered kidneys at different times during the study. The results show growth of kidneys in baboons with almost 2-fold increase in organ length over 6 weeks.