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.

1 |. INTRODUCTION

There is a known discrepancy between the number of patients requiring organ transplantation each year and the number of deceased human organs that become available for transplantation. Pig xenografts might provide a practical alternative source of organs for transplantation. The combination of increasingly sophisticated genetic engineering of the organ-source pig with an immunosuppressive regimen based on blockade of the CD40/CD154 co-stimulation pathway has allowed pig kidney graft survival in nonhuman primates (NHPs) for >1 year without graft rejection.^1–3 As clinical trials draw closer, it becomes important to measure the function of pig organ xenografts in the “foreign” environment of the NHP host, and consider whether there are abnormalities in the function that need to be addressed.^4–9

The common global clinical measurement of renal function is the glomerular filtration rate (GFR).¹⁰ In progressive renal injury, compensation from remaining nephrons will maintain homeostatic electrolyte balances, but the overall GFR diminishes. In xenotransplantation, it becomes vital to know whether renal filtration and secretory functions are adequate in the recipient and whether they change with time after the transplantation of a pig kidney into a NHP. Some groups have studied the filtration of pig kidneys in isolation, which can provide important baseline information, but fails to capture the multitude of factors that influence renal filtration after transplantation.¹¹ To our knowledge, this is first study to report the GFR and tubular secretory function of pig renal grafts in NHP recipients.

There are several methods of measuring filtration and secretory functions in humans, but these methods have not been validated in an NHP model of xenotransplantation. GFR is typically measured using creatinine clearance, estimated from the serum creatinine. But creatinine undergoes some secretion and does not provide true filtration capacity of the kidney. Inulin, iothalamate and iohexol are exogenous probes that provide better measures of GFR and have been used in humans.¹² Renal secretory functions are normally mediated by transporters expressed in the renal proximal tubules. They can be broadly classified as anionic and cationic transporters and are involved in active secretion of several endogenous and exogenous compounds.

The aims of this study were (i) to compare GFR measured using two inulin-based infusion methods, (a) a single-short inulin infusion, and (b) the “gold standard” steady-state infusion; and (ii) to assess renal anionic secretory function using cefoxitin as a probe substrate^13–16 in healthy baboons, healthy pigs, and baboons after bilateral native nephrectomy and transplantation of a single genetically-engineered pig kidney.

2 |. METHODS

2.1 |. Animals

The study involved eight baboons (Papio anubis) and three pigs (Sus scrofa). Baseline data obtained from studies in healthy baboons (n = 7), healthy pigs (n = 3) and five baboons after bilateral nephrectomy and pig kidney transplantation is presented in Table 1. Two baboons had GFR measured prior to and at various time-points after pig kidney xenotransplantation. We were hesitant to utilize the scarce genetically engineered pigs for the control studies because of the risk of complications, for example, infection, and so two of the “control” pigs were wild-type (i.e., genetically unmodified) and only one was genetically modified. All three pigs tolerated inulin and cefoxitin administration well without complications. All the kidneys transplanted into baboons were from gene-edited pigs.

Table 1:

Baboons and Pigs studied.

Animal (Sex)	Age (Mo)	Timing Date	Weight (kg)	Inulin		Cr CL/ GFR (ml/min)	Cefoxitin
				Bolus (mg)	Infusion (mg/kg/hr)		Bolus (mg)	Clearance (ml/min)
Baboons
B1717 (M)	44	Naïve 121720	10.5	410	158	135	N/A	N/A
		Naïve 011221	10.5	410	158	102	N/A	N/A
B4017 (M)	41	Naïve 021821	11.75	468	176	15	N/A	N/A
B4218 (M)	32	Naïve 040621	9.5	371	143	58	48	86.4
B5618 (F)	29	POD10 031821	7.5	293	113	34	38	23.6
B14016 (F)	52	POD21 032921	13.5	526	203	11	68	67.4
B1318 (M)	34	POD188 011121	6.8	265	102	1.5	N/A	N/A
B7018 (M)	30	Naïve 041321	10.1	394	151	57	50	3.6
		POD7 051021	9.4	366	141	14	45	54.4
		POD31 060321	9.4	366	141	12	45	66.3
		POD38 061021	9.4	366	141	31	45	34.1
B4918 (M)	30	Naïve 042221	9.1			65	45	84.1
		POD9 051221	9.1	354.9	137	30	45	77.3
		POD37 060921	9.1	354.9	137	67	45	-3.6
		POD44 061621	9.1	354.9	137	52	45	127.6
Pigs
355399	1.5	Naïve 042021	41.0	1599	615	127	200	453.5
355400	2	Naïve 042621	41.0	1599	615	99	200	160.0
A340-5	5	GE 052621	73.9	2882	1109	178	370	269.4

Notes: Control animals (healthy baboons and healthy pigs) are indicated as naïve. M indicates male sex; F indicates female sex. Age is listed at the time of the first study. Bilaterally nephrectomized baboons with a life-supporting pig kidney graft are indicated as naïve pre-transplant and by post-operative day (POD) following transplantation. Weight refers to the weight (in kg) of the animal at the time of the study. Secretory clearance was determined as renal clearance of cefoxitin minus creatinine clearance and is reported as milliliter perminute. N/Ameans the experimentwas not performed (mainly due to lack of availability of cefoxitin for research purposes during the initial COVID-19 pandemic). Genotype of source pig for renal xenotransplant to B7018 and B4918 was GTKO.CD46.TBM. For B5618 and B14016 it was GTKO.CD46.DAF.TBM.EPCR. For B1318 it was GTKO.β4GalNT2KO.CD46.TBM.EPCR.CD47.GHRKO. The genotype of A340-5was 9-gene GTKO.CMAHKO.B4KO.CD46.DAF.TBM.EPCR.CD47.HO1.

Abbreviations: GE, refers to genetically engineered pig; GFR, glomerular filtration rate.

In the baboons with pig kidneys, the studies were carried out when they were clinically stable with features suggesting satisfactory renal function in the absence of rejection (e.g., consistent urine output, minimal proteinuria, good blood flow on renal ultrasound, and normal serum creatinine). The earliest study was conducted 7 days following transplant and on average the first renal function study was conducted approximately 2 weeks following transplantation (Table 1). The length of the kidneys transplanted into baboons was measured at different times during the study.

2.2 |. Pig kidney transplantation and immunosuppressive therapy

Details of transplantation and immunosuppressive drug therapy used have been reported previously^6,17 and will only be briefly summarized here. Pig kidney transplantation involved end-to-side anastomoses of the pig renal artery to the baboon abdominal aorta and of the pig renal vein to the baboon inferior vena cava. The pig ureter was implanted into the baboon bladder. Both baboon native kidneys were removed at the time of pig kidney transplantation. Immunosuppressive therapy consisted of induction with anti-thymocyte globulin, an anti-CD20mAb, and a C1-esterase inhibitor. Maintenance therapy was based on an anti-CD40mAb, rapamycin (administered intramuscularly and titrated to a through concentration of 6–10 ng/mL), and corticosteroids.¹⁷

All animal care was in accordance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals prepared by National Academy of Sciences. (Guide for the Care and Use of Laboratory Animals. 8th ed. Washington (DC): National Academies Press (US); 2011) Protocols were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee (protocol #22142).

3 |. ADMINISTRATION OF INULIN AND CEFOXITIN AND COLLECTION OF BLOOD

3.1 |. Baboons

After induction of inhalational anesthesia with isoflurane, a urinary catheter (6 Fr, Medline, Mundelein, IL) was placed. Probe substrates were infused through a peripheral venous catheter, and blood was sampled from an arterial line (if present) or a central venous line (to prevent contaminating the sample with the infusate). Once vascular and urinary access were secured, the bladder was emptied and a 39 mg/kg inulin (BioPAL, Worcester, MA) in normal (0.9%) saline was infused intravenously (IV) over 10 min. In selected experiments, 5 mg/kg cefoxitin (Sagent, Schaumburg, IL) in normal saline was also administered as a bolus at this time.

Blood collection began 7.5 min following the completion of the short infusion. Blood was collected at frequent time-intervals for one hour after the short infusion to generate a serum concentration versus time curve for inulin (Figure 1).¹⁸ At this point, the urine was again collected. Thereafter, a constant IV infusion of inulin at 15 mg/kg/h was initiated with the intent of reaching steady-state inulin levels in serum. After an equilibration period of 1 h, blood sample was collected every hour, and urine was collected every 2 h, for the next 4 h (Figure 1).^13,15

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.

Throughout the course of the experiment, a systolic blood pressure of 70–90 mm Hg was maintained by manipulation of the inhaled isoflurane anesthetic, titration of the infusionrate of maintenance fluid, and intermittent intravenous fluid boluses. There was one episode of hypotension in a baboon with a pig kidney graft following inulin bolus which was corrected by IV fluid administration. Hypotension was not seen in the remaining cases when the inulin was diluted and administered.

3.2 |. Pigs

The procedures were very similar to those in baboons. After inhalational anesthesia with isoflurane, catheters were placed in the external jugular vein and in the carotid artery under direct vision. The venous line was used for medication administration, and the arterial line for blood sampling. Given the known difficulty of urethral catheterization in pigs, a urinary catheter was placed into the bladder under direct visualization through a small suprapubic midline incision.¹⁹ Probe administration and sample collection time-points were identical to those described in the baboon (Figure 1). Fluids and isoflurane were titrated to maintain a systolic blood pressure of 70–90 mm Hg. immediately prior to inulin and cefoxitin administration.

4 |. MEASUREMENT OF INULIN AND CEFOXITIN CONCENTRATIONS

Inulin and cefoxitin concentrations in serum from both baboons and pigs were measured using a validated high-performance liquid chromatography (HPLC) assay. Inulin concentrations in urine samples were measured by a validated spectrophotometric assay.

4.1 |. Plasma high-performance liquid chromatography (HPLC)

Baboon and pig plasma samples were diluted 5-fold with deionized water. The diluted plasma (200 μL) was mixed with 100 μL of 30% perchloric acid in a microcentrifuge tube and centrifuged at 21 000 × g for 12 min. The supernatant was transferred into another microcentrifuge tube and heated in a boiling water bath (at 100°C) for 60 min. The tubes were transferred to a room temperature water bath and cooled for 5 min, and centrifuged at 21 000 × g for 5 min. Supernatant was transferred to HPLC vials and 50 μL was injected on to a column and analyzed using a Waters 2695 HPLC system equipped with Waters 2489 UV detector (Waters corporation, Milford, MA). The samples were separated on Waters Symmetry C18, 4.6 × 250 mm reverse phase column using an isocratic mobile phase consisting of 6% acetonitrile, 10 mM potassium dihydrogen phosphate, and 20 mM tetramethyl ammonium chloride (pH 3.0). The flowrate was set at 1.2 mL/min and absorbance at 285 nm was measured. The concentration of inulin in serum was determined using standard curve established in baboon or pig serum, as necessary.

4.2 |. Urine analysis using spectrophotometry

A urine sample (100 μL) was mixed with 150 μL indole acetic acid solution (50 mM) and 3 mL concentrated hydrochloric acid (HCl) in a glass tube and incubated in a water bath maintained at 60°C for 20 min. The tubes were then cooled in a room temperature water bath for 40 min and 3 mL of deionized water was added. The resultant mixture (200 μL) was transferred into a microtiter plate and absorbance was read at 530 nm using a SpectraMax M5 microplate reader (Molecular Devices, Sunnyvale, CA). A calibration curve and quality control samples were also analyzed with each batch of samples.

5 |. CALCULATIONS OF GFR AND SECRETION

5.1 |. GFR

GFR was assessed by estimating Inulin clearance using three methods.

5.1.1 |. Short infusion

The elimination rate constant after the short infusion was obtained from the slope of the log serum inulin concentration versus time (10–60 min) plot. The area under the inulin serum concentration (AUC_{0–60 min}) versus time curve after IV short infusion dose was calculated. The AUC_60-inf was obtained by dividing concentration at 60 min by the elimination rate constant.

Inulin total body clearance after the bolus dose was calculated using the formula:

$$C{L}_{IVBlous}=\frac{Dose}{AU{C}_{0-inf}}$$

5.1.2 |. IV infusion to steady state

Inulin clearance during the steady-state was calculated from the inulin infusion rate and steady-state serum levels during infusion (2–6 h) using the formula:

$$C{L}_{IVinfusion}=\frac{\mathit{Infusion\; rate}}{C_{\mathit{ss}}}$$

5.1.3 |. IV bolus ± IV infusion

The area under inulin serum concentration (AUC_{0–360 min}) versus time curve after IV bolus and IV infusion doses was calculated. The AUC_360-inf was obtained by dividing the concentration at 360 min by the elimination rate constant obtained from the slope of the log serum inulin concentration versus time (10–60 min). Total Body Clearance of inulin after IV bolus and IV infusion was estimated using the formula:

$$C{L}_{TBC}=\frac{Dos{e}_{(\mathit{IV}\mathit{bolous}+\mathit{IV}\mathit{infusion})}}{TotalAU{C}_{0-inf}}$$

The renal clearance was estimated by two-methods: (i) The renal clearance of inulin after IV infusion was estimated as the amount of inulin excreted in urine between 2 and 6 h/AUC in serum over 2–6 h, and (ii) the urinary excretion rate over the steady-state concentrations in the serum.

5.2 |. Creatinine clearance

Creatinine levels in serum and urine were measured in the central laboratory at the University of Pittsburgh Medical Center (UPMC). The creatinine excretion rate in the urine was calculated using the formula:

$$Creatinineexcretionrate=\frac{Creatininecon{c}_{urine}\times Volumeofurineexcreted0-6h}{Durationofstudy6h}$$

Creatinine clearance was estimated from the serum levels and the creatinine excretion rate in the urine during the study duration using the formula:

$$CreatinineClearance=\frac{Creatinineexcretionrateinurine}{Creatininecon{c}_{serum}}$$

5.3 |. Secretion

Total body clearance of cefoxitin was estimated from the dose of cefoxitin and area under the plasma concentration versus time curve from time 0-infinity. Renal clearance of cefoxitin was estimated from the amount of cefoxitin excreted in the urine and the area under the plasma concentration versus time curve for the duration of the study. The secretory clearance of cefoxitin was calculated as the difference between the renal clearance of cefoxitin and the creatinine clearance.

6 |. RESULTS

Each pig underwent one renal function study whereas baboons underwent between one and four studies (Table 1 and Supplemental Data). In all cases in both species, following the short infusion there was a curvilinear decline in serum inulin (Figure 2A) and cefoxitin (Figure 2B) concentrations with time. A steady-state serum concentration for inulin was achieved in most animals with a constant infusion (Figure 2A).

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.

6.1 |. Comparison of results of inulin/cefoxitin infusions in healthy pigs and baboons

Intraprocedural complications included occasional hypotension following inulin which was corrected by fluid administration. This may have been related to the inulin bolus concentration as this phenomenon was not seen in later experiments when the inulin was diluted (i.e., the same quantity of inulin was given in approximately 250 mL normal saline as opposed to approximately 100 mL normal saline). One study in a post-transplant baboon had to be stopped early at 4 h due to persistent hypotension. Otherwise, no major differences were noted between the responses of the pigs and baboons to the short infusion of inulin or cefoxitin.

Baboons had highly variable hourly urine outputs during the procedure (resulting in variable urine concentrations for inulin and cefoxitin). Pigs, perhaps because of their larger size, had more consistent and higher volume of urine output and also revealed a steady state of urine excretion of inulin during constant infusion.

6.2 |. Comparison of bolus versus steady-state inulin infusion

Inulin clearance estimated after the short infusion and during continuous infusion was similar in naïve baboons, naïve pigs, and baboons transplanted with pig kidneys. Inulin clearance values correlated well between both methods in native and transplanted animals with correlation coefficients of 0.8985 and 0.8843, respectively (Figure 3). The calculated inulin clearance after the short infusion dose in naïve baboons was ~19.5 ± 7.8 mL/min/kg, whereas inulin clearance estimated during the steady-state infusion was 16.6 ± 6.2 mL/min/kg. In naïve pigs, inulin clearances calculated after a short infusion and a steady-state infusion were 17.9 ± 2.6 and 13.4 ± 1.9 mL/min/kg, respectively. The inulin clearance in baboons transplanted with pig kidneys varied with time after transplant, but the estimations correlated well between short and steady-state infusions. The inulin clearance, which was used to estimate filtration capacity in baboons, was sufficient to maintain filtration needs in the baboons (Figure 6A).

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 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.

The plasma clearance of inulin estimated after bolus dose and continuous infusion correlated well with a correlation coefficient of 0.8691 (data not shown). The mean total body clearance for inulin was 14.84 ± 4.57 mL/min/kg and the mean renal clearance was 10.3 ± 5.1 mL/min/kg (naïve and transplanted animals together). The renal clearance values obtained by both methods were similar and showed a good correlation (R² = 0.92; Figure 4A). The inulin clearance after short infusion dose and renal clearance of inulin correlated with a correlation coefficient of .61, indicating that renal excretion is the major elimination pathway for inulin (Figure 4B). Urine output during the first hour was inconsistent in the baboons and we were unable to calculate the renal clearance of inulin during the initial 60 min of the study. But, by the end of 6-h study, the estimated renal clearance correlated well with the plasma clearance in both baboons and pigs (Figure 4B). Renal excretion contributed to elimination of >52% of the inulin dose. The lower renal clearance compared to total body clearance may be related to incomplete collection of the urine in this study.

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.

The plasma clearance of inulin estimated after bolus dose and during steady state infusion was similar in transplanted baboons and values obtained by the two methods showed a good correlation with a coefficient of .8843 indicating that the clearance values obtained from short infusion study reflects the function of the kidney.

6.3 |. Function of pig kidneys after transplantation into baboons

The mean total body clearance of inulin in baboons was 143.4 ± 50.1 mL/min, whereas pigs showed a mean total body inulin clearance of 723.8 ± 211.4 mL/min. The weight normalized inulin clearance in baboons and pigs was 14.98 ± 4.99 and 14.84 ± 4.6 mL/min/kg, respectively. Despite a large difference in absolute values for inulin clearance between baboons and pigs, the body weight normalized inulin clearance was similar in both animals, indicating that the pig kidneys can support the requirements for kidney function in baboons.

We transplanted one pig kidney into each of two bilaterally-nephrectomized baboons and performed longitudinal functional assessments over 6 weeks. The serum levels of inulin and cefoxitin observed on different days after transplantation are presented in Figure 5. Panels A and B indicate that plasma levels of inulin show a curvilinear decline after the short infusion dose and a pseudo steady state during the infusion at different phases of the study. The clearance of inulin was consistent, and the functional capacity of the pig kidneys was maintained well for 6 weeks following transplantation (Figure 6A). Additionally, the serum levels of creatinine, an endogenous marker for renal function, were maintained <1.5 mg/dL, indicating good renal function (Figure 6B). The serum levels of creatinine and inulin clearly indicate that the genetically modified pig kidneys transplanted into baboons were able to sustain relatively normal renal function in baboons. Although the clearance values for inulin declined over time in one baboon, the remaining kidney function was enough to provide filtration support.

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.

The secretory function of the kidneys was assessed by the difference between the renal clearance of cefoxitin and the creatinine clearance. The longitudinal assessment for estimated secretory clearance of the kidney in two baboons transplanted with genetically modified pig kidneys is presented in Figure 7. The secretory function of the kidneys in naïve baboons and pigs was 85 ± 1 and 294 ± 148 mL/min, respectively. Despite a big difference in absolute numbers, the weight normalized secretory clearance was 4.58 and 3.1 mL/min/kg for each kidney in naïve baboons and pigs, respectively.

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.

Following transplant into a baboon, a pig kidney maintains a mean cefoxitin secretory clearance of 6.73 + 3.71 mL/min/kg. The baboons remained clinically active. Both baboons eventually developed evidence of cystitis (with increased urinary leukocytosis). One baboon (B7018) required euthanasia as a result of suspected pyelonephritis in the graft. The other baboon (B4918) was successfully treated with IV antibiotics and remained well for two months on single-agent immunosuppressive therapy until electively euthanized.²⁰ Repeated bladder catheterization was suspected as the likely source of urinary tract infection (which is otherwise rare after pig kidney transplantation in baboons).

6.4 |. Growth of donor kidney in baboons

The length of donor kidney was measured at different times during the study. The data presented in Figure 8 shows that donor kidneys grew gradually post-transplant and showed a 2-fold increase in length by 6 weeks. Two baboons (B7018 and B-4918) followed till 6 and 15 weeks respectively showed gradual growth in kidneys during the study.

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.

7 |. DISCUSSION

7.1 |. Short inulin infusion versus steady-state inulin infusion

To our knowledge, this is the first study to validate a single short inulin infusion technique to measure GFR and renal secretory function in a pig-to-NHP renal xenotransplant model. The serum concentration versus time curves generated during this experiment followed the predicted time-course and revealed a curvilinear decline in serum inulin following a short infusion. The clearance of inulin estimated by using the “gold standard” steady-state method correlated well with that of a bolus infusion in naïve baboons, pigs, and baboons with pig kidneys, with a correlation coefficient of 0.8985 (Figure 3A). We conclude that the single short inulin infusion method is a simpler method to precisely and rapidly measure GFR in the pig-to-baboon renal xenotransplant model. This not only minimizes the study time period but also minimizes inconvenience to the animals.

The total body clearance and renal clearance of inulin observed in naïve baboons and pigs and baboons transplanted with pig kidneys correlated well with a correlation coefficient of .61. A good correlation indicates that total body clearance for inulin could be used to predict the renal clearance simply with serum samples, without the need for collecting urine. This avoids prolonged studies under general post-xenotransplantation in the same animals. anesthesia and the need for cumbersome catheterization of the bladder to collect urine samples.

7.2 |. GFR in healthy pigs and baboons

GFR and renal secretion function were measured in healthy pigs and baboons (Figures 6 and 7), and were found to be comparable to historical controls.^21,22 The mean total body clearance of inulin, which is a measure of GFR, in naïve baboons and pigs with two kidneys was 16.91 ± 6.22 and 14.2 ± 2.01 mL/min/kg. At baseline, each kidney in baboons and pigs showed a GFR of 8.5 and 7.1 mL/min/kg, respectively. After bilateral nephrectomy and transplantation of one pig kidney, the mean GFR observed in baboons on different days was 6.95 mL/min/kg, indicating that the filtration capacity of the pig kidney was intact and continued to provide life support in baboons.

We have conducted studies in two wild-type pigs and one genetically modified pig. The GFR was 14.84 and 12.92 mL/min/kg in wild-type pig and genetically modified pig, respectively. This suggests that genetic manipulation per se may not have an impact on the GFR. As expected, larger pigs had higher GFR due to increased renal mass. Although the studies were conducted in a small number of animals, the GFR values are similar in baboons and pigs when normalized to body weight. While this study was not designed to estimate pig GFR based on weight, it is conceivable that this association could be used to guide an estimate of the weight of the source-pig needed to provide a kidney that would ensure adequate renal function in a recipient of a certain weight.

7.3 |. Creatinine clearance versus inulin total body/renal clearance

The creatinine clearance calculated based on serum creatinine and amount excreted in urine for the duration of the study correlated reasonably well with the total body inulin clearance measured (R² = 0.5403). Similarly, creatinine clearance correlated with renal clearance with a correlation coefficient of 0.5139 (data not shown). The incomplete urine collection significantly effects the creatinine clearance values calculated and result in substantial underestimation of GFR based on values for creatinine and inulin renal clearance.

7.4 |. Pig kidney function after transplantation into bilaterally-nephrectomized baboons

To our knowledge, this is the first study to measure function of a pig kidney after its transplantation into an immunosuppressed NHP. In the absence of rejection, GFR as measured by inulin clearance decreased with time post-transplant but remained between 60 and 105 mL/min for at least 5 weeks after transplantation. In healthy baboons and healthy pigs, the % inulin dose excreted in urine was 47% and 74%, respectively. The mean % inulin dose excreted in urine in baboons transplanted with pig kidneys was 48%. Additionally, 81% of the cefoxitin dose was excreted in the urine from healthy baboons and pigs while 76% of the cefoxitin dose was excreted in the urine in baboons transplanted with pig kidneys. The filtration and secretory functions of the kidneys were maintained for the duration of the study. This might be expected when one pig kidney replaces two baboon kidneys, although the pig kidneys were derived from larger animals. Interestingly, the body weight-normalized secretory function in baboons seems to be higher than in pigs. Post transplantation, the secretory function in baboons was higher than the values seen in naïve baboons. Secretion function remained relatively preserved following xenotransplantation.

Long-term follow-up of renal function was limited to two baboons by (i) euthanasia for pyelonephritis in one (B7018), and (ii) elective euthanasia at the conclusion of the IACUC-approved experiment in the other (B4918). Other limitations of the study included (i) the small number of animals in each group, and (ii) the variable urine output for baboons possibly associated with their small sizes. Also (iii), in the experimental baboons, the native kidneys were removed to be certain that life was supported by the transplanted pig kidney. However, in patients with end-stage renal disease undergoing pig kidney transplantation, the native kidneys will likely be left in situ, and there may still be some minimal residual filtration function. This, however, is anticipated to be <10% of overall GFR. In addition, the baboons may be subject to further physiological derangement as a result of severing the renal nerves as part of the pig nephrectomies, which affects the sympathetic stimulation involved in several renal pathways.²³ Although the number of experiments was limited, the study points to the importance of learning more about the function of pig kidneys to support the physiologic needs of a human recipient.

In summary, this study indicates that (i) a short infusion of inulin is simpler than the steady-state infusion method, and gives comparable results, (ii) total body clearance of inulin after a short infusion appears to be a good measure of kidney function, (iii) the GFR in baboons and pigs is comparable when normalized for body weight, and (iv) secretion clearance in baboons transplanted with pig kidneys is similar to the secretory clearance observed in naïve pigs and baboons. In addition, (v) our data suggest that, in the absence of rejection, pig kidney grafts in their current form would provide life-sustaining renal filtration and secretory function for humans for at least several months, despite some reduction in renal function. Further studies are needed to determine the longevity of adequate pig kidney graft function.

Abbreviations:

GFR

glomerular filtration rate

IV

intravenous

NHP

nonhuman primate