Capacity for the management of kidney failure in the International Society of Nephrology Africa region: report from the 2023 ISN Global Kidney Atlas (ISN-GKHA).

The burden of chronic kidney disease and associated risk of kidney failure are increasing in Africa. The management of people with chronic kidney disease is fraught with numerous challenges because of limitations in health systems and infrastructures for care delivery. From the third iteration of the International Society of Nephrology Global Kidney Health Atlas, we describe the status of kidney care in the ISN Africa region using the World Health Organization building blocks for health systems. We identified limited government health spending, which in turn led to increased out-of-pocket costs for people with kidney disease at the point of service delivery. The health care workforce across Africa was suboptimal and further challenged by the exodus of trained health care workers out of the continent. Medical products, technologies, and services for the management of people with nondialysis chronic kidney disease and for kidney replacement therapy were scarce due to limitations in health infrastructure, which was inequitably distributed. There were few kidney registries and advocacy groups championing kidney disease management in Africa compared with the rest of the world. Strategies for ensuring improved kidney care in Africa include focusing on chronic kidney disease prevention and early detection, improving the effectiveness of the available health care workforce (e.g., multidisciplinary teams, task substitution, and telemedicine), augmenting kidney care financing, providing quality, up-to-date health information data, and improving the accessibility, affordability, and delivery of quality treatment (kidney replacement therapy or conservative kidney management) for all people living with kidney failure.

Real-time monitoring of mitochondrial oxygenation during machine perfusion using resonance Raman spectroscopy predicts organ function.

Organ transplantation is a life-saving procedure affecting over 100,000 people on the transplant waitlist. Ischemia reperfusion injury (IRI) is a major challenge in the field as it can cause post-transplantation complications and limit the use of organs from extended criteria donors. Machine perfusion technology has the potential to mitigate IRI; however, it currently fails to achieve its full potential due to a lack of highly sensitive and specific assays to assess organ quality during perfusion. We developed a real-time and non-invasive method of assessing organs during perfusion based on mitochondrial function and injury using resonance Raman spectroscopy. It uses a 441 nm laser and a high-resolution spectrometer to quantify the oxidation state of mitochondrial cytochromes during perfusion. This index of mitochondrial oxidation, or 3RMR, was used to understand differences in mitochondrial recovery of cold ischemic rodent livers during machine perfusion at normothermic temperatures with an acellular versus cellular perfusate. Measurement of the mitochondrial oxidation revealed that there was no difference in 3RMR of fresh livers as a function of normothermic perfusion when comparing acellular versus cellular-based perfusates. However, following 24 h of static cold storage, 3RMR returned to baseline faster with a cellular-based perfusate, yet 3RMR progressively increased during perfusion, indicating injury may develop over time. Thus, this study emphasizes the need for further refinement of a reoxygenation strategy during normothermic machine perfusion that considers cold ischemia durations, gradual recovery/rewarming, and risk of hemolysis.

Real-time monitoring of mitochondrial oxygenation during machine perfusion using resonance Raman spectroscopy predicts organ function.

Organ transplantation is a life-saving procedure affecting over 100,000 people on the transplant waitlist. Ischemia reperfusion injury is a major challenge in the field as it can cause post-transplantation complications and limits the use of organs from extended criteria donors. Machine perfusion technology is used to repair organs before transplant, however, currently fails to achieve its full potential due to a lack of highly sensitive and specific assays to predict organ quality during perfusion. We developed a real-time and non-invasive method of assessing organ function and injury based on mitochondrial oxygenation using resonance Raman spectroscopy. It uses a 441 nm laser and a high-resolution spectrometer to predict the oxidation state of mitochondrial cytochromes during perfusion, which vary due to differences in storage compositions and perfusate compositions. This index of mitochondrial oxidation, or 3RMR, was found to predict organ health based on clinically utilized markers of perfusion quality, tissue metabolism, and organ injury. It also revealed differences in oxygenation with perfusates that may or may not be supplemented with packed red blood cells as oxygen carriers. This study emphasizes the need for further refinement of a reoxygenation strategy during machine perfusion that is based on a gradual recovery from storage. Thus, we present a novel platform that provides a real-time and quantitative assessment of mitochondrial health during machine perfusion of livers, which is easy to translate to the clinic.

Partial freezing of rat livers extends preservation time by 5-fold.

The limited preservation duration of organs has contributed to the shortage of organs for transplantation. Recently, a tripling of the storage duration was achieved with supercooling, which relies on temperatures between -4 and -6 °C. However, to achieve deeper metabolic stasis, lower temperatures are required. Inspired by freeze-tolerant animals, we entered high-subzero temperatures (-10 to -15 °C) using ice nucleators to control ice and cryoprotective agents (CPAs) to maintain an unfrozen liquid fraction. We present this approach, termed partial freezing, by testing gradual (un)loading and different CPAs, holding temperatures, and storage durations. Results indicate that propylene glycol outperforms glycerol and injury is largely influenced by storage temperatures. Subsequently, we demonstrate that machine perfusion enhancements improve the recovery of livers after freezing. Ultimately, livers that were partially frozen for 5-fold longer showed favorable outcomes as compared to viable controls, although frozen livers had lower cumulative bile and higher liver enzymes.

The role of antifreeze glycoprotein (AFGP) and polyvinyl alcohol/polyglycerol (X/Z-1000) as ice modulators during partial freezing of rat livers.

Introduction: The current liver organ shortage has pushed the field of transplantation to develop new methods to prolong the preservation time of livers from the current clinical standard of static cold storage. Our approach, termed partial freezing, aims to induce a thermodynamically stable frozen state at high subzero storage temperatures (-10°C to -15°C), while simultaneously maintaining a sufficient unfrozen fraction to limit ice-mediated injury. Methods and results: Using glycerol as the main permeating cryoprotectant agent, this research first demonstrated that partially frozen rat livers showed similar outcomes after thawing from either -10°C or -15°C with respect to subnormothermic machine perfusion metrics. Next, we assessed the effect of adding ice modulators, including antifreeze glycoprotein (AFGP) or a polyvinyl alcohol/polyglycerol combination (X/Z-1000), on the viability and structural integrity of partially frozen rat livers compared to glycerol-only control livers. Results showed that AFGP livers had high levels of ATP and the least edema but suffered from significant endothelial cell damage. X/Z-1000 livers had the highest levels of ATP and energy charge (EC) but also demonstrated endothelial damage and post-thaw edema. Glycerol-only control livers exhibited the least DNA damage on Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining but also had the lowest levels of ATP and EC. Discussion: Further research is necessary to optimize the ideal ice modulator cocktail for our partial-freezing protocol. Modifications to cryoprotective agent (CPA) combinations, including testing additional ice modulators, can help improve the viability of these partially frozen organs.

The effect of blood cells retained in rat livers during static cold storage on viability outcomes during normothermic machine perfusion.

In transplantation, livers are transported to recipients using static cold storage (SCS), whereby livers are exposed to cold ischemic injury that contribute to post-transplant risk factors. We hypothesized that flushing organs during procurement with cold preservation solutions could influence the number of donor blood cells retained in the allograft thereby exacerbating cold ischemic injury. We present the results of rat livers that underwent 24 h SCS after being flushed with a cold University of Wisconsin (UW) solution versus room temperature (RT) lactated ringers (LR) solution. These results were compared to livers that were not flushed prior to SCS and thoroughly flushed livers without SCS. We used viability and injury metrics collected during normothermic machine perfusion (NMP) and the number of retained peripheral cells (RPCs) measured by histology to compare outcomes. Compared to the cold UW flush group, livers flushed with RT LR had lower resistance, lactate, AST, and ALT at 6 h of NMP. The number of RPCs also had significant positive correlations with resistance, lactate, and potassium levels and a negative correlation with energy charge. In conclusion, livers exposed to cold UW flush prior to SCS appear to perform worse during NMP, compared to RT LR flush.

Conventional peritoneal cytology lacks the prognostic significance of detecting local or peritoneal recurrence in colorectal cancer: An Egyptian experience.

BACKGROUND AND AIM: Colorectal cancer (CRC) accounts for over 8% of all deaths each year, with 1.2 million new cases diagnosed annually worldwide. It represents the seventh most common cancer in Egypt. Early detection of peritoneal metastasis is a major challenge in such cases. It helps with the decision of the immediate application of intraperitoneal chemotherapy after resection. Meta-analysis studies reported contrast evidence for a possible prognostic role of intraperitoneal free cancer cells (IPCCs) in peritoneal recurrence and survival after curative resection. In this work, we aim to evaluate the prevalence and impact of detecting free malignant cells in peritoneal fluid on survival and local recurrence and to estimate the incidence of peritoneal carcinomatosis (PC) during follow up. METHODS: Design: This was a prospective cohort study. Settings: From June 2016 to December 2018, samples were collected from 104 patients who underwent abdominal surgery for colorectal cancer in the Egyptian National Cancer Institute. A total of 96 Egyptian CRC patients who underwent curative resection were enrolled. Intraoperative peritoneal lavage was performed to detect IPCC by conventional cytology. Patients with no residual tumor after surgery and no evidence of PC were followed up for a median 14 months. The cumulative 12-month overall survival rate for patients with IPCC was 100% versus 86% for patients with negative cytology. RESULTS: Our results demonstrated that the prevalence of IPCC in the peritoneal lavage was 11.5%. Peritoneal and local recurrence occurred at a higher rate in patients with cytology positive lavage (9.1% vs 6.3% and 9.1% vs 3.8%, respectively), although this was statistically insignificant. Distant metastasis occurred significantly in patients with positive cytology (45.5% vs 8.9%) with P-value <0.001.The conventional cytology technique has a high specificity but less sensitivity. CONCLUSIONS: The presence of IPCC using conventional cytology was not an independent prognostic factor for the development of PC or survival.

Development of liver microtissues with functional biliary ductular network.

Liver tissue engineering aims to create transplantable liver grafts that can serve as substitutes for donor's livers. One major challenge in creating a fully functional liver tissue has been to recreate the biliary drainage in an engineered liver construct through integration of bile canaliculi (BC) with the biliary ductular network that would enable the clearance of bile from the hepatocytes to the host duodenum. In this study, we show the formation of such a hepatic microtissue by coculturing rat primary hepatocytes with cholangiocytes and stromal cells. Our results indicate that within the spheroids, hepatocytes maintained viability and function for up to 7 days. Viable hepatocytes became polarized by forming BC with the presence of tight junctions. Morphologically, hepatocytes formed the core of the spheroids, while cholangiocytes resided at the periphery forming a monolayer microcysts and tubular structures extending outward. The spheroids were subsequently cultured in clusters to create a higher order ductular network resembling hepatic lobule. The cholangiocytes formed functional biliary ductular channels in between hepatic spheroids that were able to collect, transport, and secrete bile. Our results constitute the first step to recreate hepatic building blocks with biliary drainage for repopulating the whole liver scaffolds to be used as transplantable liver grafts.

Subzero non-frozen preservation of human livers in the supercooled state.

Preservation of human organs at subzero temperatures has been an elusive goal for decades. The major complication hindering successful subzero preservation is the formation of ice at temperatures below freezing. Supercooling, or subzero non-freezing, preservation completely avoids ice formation at subzero temperatures. We previously showed that rat livers can be viably preserved three times longer by supercooling as compared to hypothermic preservation at +4 °C. Scalability of supercooling preservation to human organs was intrinsically limited because of volume-dependent stochastic ice formation at subzero temperatures. However, we recently adapted the rat preservation approach so it could be applied to larger organs. Here, we describe a supercooling protocol that averts freezing of human livers by minimizing air-liquid interfaces as favorable sites of ice nucleation and uses preconditioning with cryoprotective agents to depress the freezing point of the liver tissue. Human livers are homogeneously preconditioned during multiple machine perfusion stages at different temperatures. Including preparation, the protocol takes 31 h to complete. Using this protocol, human livers can be stored free of ice at -4 °C, which substantially extends the ex vivo life of the organ. To our knowledge, this is the first detailed protocol describing how to perform subzero preservation of human organs.

Split-Liver Ex Situ Machine Perfusion: A Novel Technique for Studying Organ Preservation and Therapeutic Interventions.

Ex situ machine perfusion is a promising technology to help improve organ viability prior to transplantation. However, preclinical studies using discarded human livers to evaluate therapeutic interventions and optimize perfusion conditions are limited by significant graft heterogeneity. In order to improve the efficacy and reproducibility of future studies, a split-liver perfusion model was developed to allow simultaneous perfusion of left and right lobes, allowing one lobe to serve as a control for the other. Eleven discarded livers were surgically split, and both lobes perfused simultaneously on separate perfusion devices for 3 h at subnormothermic temperatures. Lobar perfusion parameters were also compared with whole livers undergoing perfusion. Similar to whole-liver perfusions, each lobe in the split-liver model exhibited a progressive decrease in arterial resistance and lactate levels throughout perfusion, which were not significantly different between right and left lobes. Split liver lobes also demonstrated comparable energy charge ratios. Ex situ split-liver perfusion is a novel experimental model that allows each graft to act as its own control. This model is particularly well suited for preclinical studies by avoiding the need for large numbers of enrolled livers necessary due to the heterogenous nature of discarded human liver research.

Metabolic and lipidomic profiling of steatotic human livers during ex situ normothermic machine perfusion guides resuscitation strategies.

There continues to be a significant shortage of donor livers for transplantation. One impediment is the discard rate of fatty, or steatotic, livers because of their poor post-transplant function. Steatotic livers are prone to significant ischemia-reperfusion injury (IRI) and data regarding how best to improve the quality of steatotic livers is lacking. Herein, we use normothermic (37°C) machine perfusion in combination with metabolic and lipidomic profiling to elucidate deficiencies in metabolic pathways in steatotic livers, and to inform strategies for improving their function. During perfusion, energy cofactors increased in steatotic livers to a similar extent as non-steatotic livers, but there were significant deficits in anti-oxidant capacity, efficient energy utilization, and lipid metabolism. Steatotic livers appeared to oxidize fatty acids at a higher rate but favored ketone body production rather than energy regeneration via the tricyclic acid cycle. As a result, lactate clearance was slower and transaminase levels were higher in steatotic livers. Lipidomic profiling revealed ω-3 polyunsaturated fatty acids increased in non-steatotic livers to a greater extent than in steatotic livers. The novel use of metabolic and lipidomic profiling during ex situ normothermic machine perfusion has the potential to guide the resuscitation and rehabilitation of steatotic livers for transplantation.

Cell release during perfusion reflects cold ischemic injury in rat livers.

The global shortage of donor organs has made it crucial to deeply understand and better predict donor liver viability. However, biomarkers that effectively assess viability of marginal grafts for organ transplantation are currently lacking. Here, we showed that hepatocytes, sinusoidal endothelial, stellate, and liver-specific immune cells were released into perfusates from Lewis rat livers as a result of cold ischemia and machine perfusion. Perfusate comparison analysis of fresh livers and cold ischemic livers showed that the released cell profiles were significantly altered by the duration of cold ischemia. Our findings show for the first time that parenchymal cells are released from organs under non-proliferative pathological conditions, correlating with the degree of ischemic injury. Thus, perfusate cell profiles could serve as potential biomarkers of graft viability and indicators of specific injury mechanisms during organ handling and transplantation. Further, parenchymal cell release may have applications in other pathological conditions beyond organ transplantation.

Subnormothermic Machine Perfusion of Steatotic Livers Results in Increased Energy Charge at the Cost of Anti-Oxidant Capacity Compared to Normothermic Perfusion.

There continues to be significant debate regarding the most effective mode of ex situ machine perfusion of livers for transplantation. Subnormothermic (SNMP) and normothermic machine perfusion (NMP) are two methods with different benefits. We examined the metabolomic profiles of discarded steatotic human livers during three hours of subnormothermic or normothermic machine perfusion. Steatotic livers regenerate higher stores of ATP during SNMP than NMP. However, there is a significant depletion of available glutathione during SNMP, likely due to an inability to overcome the high energy threshold needed to synthesize glutathione. This highlights the increased oxidative stress apparent in steatotic livers. Rescue of discarded steatotic livers with machine perfusion may require the optimization of redox status through repletion or supplementation of reducing agents.

Supercooling extends preservation time of human livers.

The inability to preserve vascular organs beyond several hours contributes to the scarcity of organs for transplantation1,2. Standard hypothermic preservation at +4 °C (refs. 1,3) limits liver preservation to less than 12 h. Our group previously showed that supercooled ice-free storage at -6 °C can extend viable preservation of rat livers4,5 However, scaling supercooling preservation to human organs is intrinsically limited because of volume-dependent stochastic ice formation. Here, we describe an improved supercooling protocol that averts freezing of human livers by minimizing favorable sites of ice nucleation and homogeneous preconditioning with protective agents during machine perfusion. We show that human livers can be stored at -4 °C with supercooling followed by subnormothermic machine perfusion, effectively extending the ex vivo life of the organ by 27 h. We show that viability of livers before and after supercooling is unchanged, and that after supercooling livers can withstand the stress of simulated transplantation by ex vivo normothermic reperfusion with blood.

Endothelial Dysfunction in Steatotic Human Donor Livers: A Pilot Study of the Underlying Mechanism During Subnormothermic Machine Perfusion.

BACKGROUND: Steatosis is a major risk factor for primary nonfunction in liver transplantations. Steatotic livers recover poorly from ischemia reperfusion injury, in part due to alterations in the microcirculation, although the exact mechanism is unclear. In this study, we tested if there were any alterations in the shear stress sensing Kruppel-like factor 2 (KLF2) and its likely downstream consequences in the ex vivo perfused human liver endothelium, which would imply perturbations in microcirculatory flow in macrosteatotic livers disrupts laminar flow to evaluate if this is a potential therapeutic target for steatotic livers. METHODS: Using a subnormothermic machine perfusion system, 5 macrosteatotic and 4 nonsteatotic human livers were perfused for 3 hours. Flow, resistance, and biochemical profile were monitored. Gene expression levels of nitric oxide synthase 3 (eNOS), KLF2, and thrombomodulin were determined. Nitric oxide (NO) was measured in the perfusion fluid and activation of eNOS was measured with Western blotting. RESULTS: Flow dynamics, injury markers, and bile production were similar in both groups. Kruppel-like factor 2 expression was significantly higher in nonsteatotic livers. Western blotting analyses showed significantly higher levels of activated eNOS in nonsteatotic livers, consistent with an increase in NO production over time. Macrosteatotic livers showed decreased KLF2 upregulation, eNOS activity, and NO production during machine perfusion. CONCLUSIONS: These results indicate a perturbed KLF2 sensing in steatotic livers, which aligns with perturbed microcirculatory state. This may indicate endothelial dysfunction and contribute to poor posttransplantation outcomes in fatty livers, and further studies to confirm by evaluation of flow and testing treatments are warranted.