Sandrine Florquin

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Background: Histopathological diagnoses in kidney transplant biopsies, as described by the Banff classification, suffer from substantial inter-observer variability and misinterpretation, making them prone to diagnostic errors. Continuous indices based on BanffNET automated lesion scores are proposed to quantify the continuous phenotypic spectrum of kidney transplant biopsies, complementary to pathologist assessment of biopsies. Methods: The BanffNET model was used to obtain continuous lesion scoring for a training cohort of 2544 biopsies, a large validation cohort of 3863 biopsies, and a multi-reader cohort of 36 biopsies scored by 67 pathologists. A penalized regression approach was used to condense 17 automated lesion scores into four indices, representing the most important dimensions of graft injury: a TCMR/TI Index, an AMR/MVI Index, an Activity Index and a Chronicity Index. The BanffNET Indices were evaluated against Banff diagnoses and histological indices in the training cohort and validation cohort. Since pathologist-assigned Banff lesions and diagnoses suffer from inter-observer variability, associations were also assessed with diagnoses in a multi-reader study, with time to kidney graft failure in the training and validation cohort and with biopsy-based molecular signatures in the validation cohort. Results: The BanffNET TCMR/TI Index, AMR/MVI Index and Activity Index showed excellent discrimination of Banff TCMR, Banff MVI and Banff Any Diagnosis respectively, with validation AUCs ranging from 0.80 to 0.92. In addition, the BanffNET Indices explained large amounts of variability in multi-observer reference standards, time to graft failure and molecular biopsy-based diagnostics. In the large majority of cases, variability explained by BanffNET Indices matched or exceeded the variability explained by pathologist-assigned diagnoses and histological indices. Conclusions: BanffNET Indices are reproducible descriptors of whole slide images that could complement and augment kidney transplant biopsy evaluation by pathologists. Associations of BanffNET Indices with multi-observer diagnoses and related outcomes suggest that they capture additional information on tissue injury type and severity on top of pathologist-assigned Banff diagnoses and histological indices.

Key Points: Repeated measurements of plasma tacrolimus concentrations were obtained retrospectively from 295 kidney transplant recipients. Plasma tacrolimus concentration was negatively associated with later eGFR, whereas whole blood concentration was not. Plasma tacrolimus concentration was associated with arteriolar hyalinosis in protocol biopsies, whereas whole blood concentration was not. Background: Tacrolimus is the cornerstone of immunosuppressive treatment after kidney transplantation. Its application requires therapeutic drug monitoring, which is routinely performed by assessment of whole blood tacrolimus concentrations (WBTC) that largely comprises a pharmacologically inactive fraction. Monitoring tacrolimus exposure using the plasma tacrolimus concentration (PTC) may better reflect the pharmacologic active fraction. We hypothesized that PTC better reflects effectivity and toxicity than WBTC. Methods: The Amsterdam, LEiden, GROningen trial randomized 295 kidney transplant recipients (KTR) to standard triple immunosuppression, lower tacrolimus exposure, or withdrawal of prednisolone. PTC was measured using liquid chromatography–tandem mass spectrometry. Associations between tacrolimus concentrations and eGFR were analyzed using linear regression, with bootstrap resampling used to compare PTC and WBTC. Associations with biopsy results and rejection were evaluated using logistic regression, and infection risk was assessed with joint modeling. Results: The mean age was 56.5±13.2 years, and 95 (32%) were female. The mean PTC during the first month post-transplantation (0.31±0.17 ng/ml) was negatively associated with eGFR at 4 weeks (st. β =−0.17 [−0.29 to −0.05], P = 0.006), whereas mean WBTC was not (st. β =0.09 [−0.03 to 0.22], P = 0.15), and the difference between these associations was statistically significant ( P = 0.004). Late mean PTC (6.5–24 months; 0.15±0.07 ng/ml) was negatively associated with eGFR at 2 years (st. β =−0.19 [−0.35 to −0.03], P = 0.02), whereas mean WBTC was not (st. β =0.04 [−0.13 to 0.20], P = 0.66), and this difference was also statistically significant ( P = 0.02). At 2 years, 10 (14%) of the 71 protocol biopsies showed moderate-to-severe arteriolar hyalinosis. Although in a limited number of events, late mean PTC was significantly associated with a risk of arteriolar hyalinosis (odds ratio=1.96 [1.07–3.80], P = 0.03), WBTC was not (odds ratio=1.65 [0.83–3.53], P = 0.17). Neither PTC nor WBTC was significantly associated with a risk of infection in univariable analysis. Twenty-two KTR (7%) experienced biopsy-proven rejection; neither PTC nor WBTC was associated with rejection. Conclusion: Larger studies are needed to determine whether late PTC is a better marker than WBTC to detect potential tacrolimus-related nephrotoxicity in KTR. Clinical Trial registry name and registration number: NCT01560572.

Background: The presence of lymphoid tissues and their impact on long-term kidney graft outcome remains unclear. This study investigates the occurrence, molecular and phenotypic characteristics of lymphoid aggregates (LA) in protocol biopsies of clinically stable kidney transplant recipients, and their correlation with long-term clinical outcome. Methods: We retrospectively analyzed 414 protocol biopsies from a multicenter cohort of 278 stable kidney transplant recipients taken at either 6-, 12- or 24-months post-transplant. On a subset of 16 biopsies at 6 months post-transplant, mRNA gene expression analysis was performed using Nanostring B-HOT panel alongside phenotypic analysis with immunostaining for developmental staging of LA. Finally, the presence of LA was correlated to various clinical kidney allograft outcomes. Results: Stage I LA were characterized by upregulation of genes linked to immune regulation (LAG3, CTLA4, FOXP3), T-cell signaling, and chemotaxis pathways, with enrichment of regulatory T cells (Tregs), memory T cells, T follicular helper cells (Tfh), and dendritic cells. Immunohistochemistry confirmed higher numbers of FOXP3+ Tregs and dendritic cells in stage I LA, while high endothelial venules were absent. Over follow-up, participants with stage I LA showed slightly higher eGFR values and a trend toward better graft survival compared with those without LA or with stage II LA, although most differences did not reach statistical significance. Conclusions: In stable kidney transplant recipients, stage I LA displayed molecular and phenotypic features consistent with a regulated immune environment. The molecular and immunohistochemical analysis of stage I LA suggests a more regulatory molecular phenotype and composition, which may contribute to better long-term graft survival observed in this cohort of functionally stable transplant recipients. Observed trends toward more favorable outcomes suggest that early LA may not be deleterious and could represent local immune regulation, though larger prospective studies are needed to confirm these findings.

Background.: Kidney preservation techniques, such as hypothermic machine perfusion (HMP), improve graft outcomes in deceased-donor kidney transplantation by pausing graft metabolism and ameliorated ischemia–reperfusion injury (IRI) but do not completely eliminate injury. Alkaline phosphatase (ALP) has been postulated to reduce IRI-induced kidney injury through the conversion of extracellular adenosine triphosphate into adenosine. This study aimed to evaluate whether ALP offers protection during deceased-donor kidney storage. Methods.: Sixteen abattoir porcine kidneys (8 ALP and 8 with placebo) were procured after euthanization and exposed to 30 min of warm ischemia followed by 24 h of HMP or static cold storage (SCS). Reperfusion was partly simulated by 240 min of normothermic machine perfusion (NMP). Throughout NMP, we obtained functional, biochemical, and histological parameters. Results.: Significantly lower urine production accompanied by lower perfusate pCO ₂ and higher pH were observed in the ALP group throughout NMP. At the end of NMP, intracellular ATP reserves and oxygen consumption were significantly higher in the ALP-treated group. Metabolomics analysis with principal component analysis demonstrated significant differences between the ALP and placebo groups in glycolysis and mitochondrial metabolites, along with a significantly attenuated rise in perfusate lactate dehydrogenase levels. Conclusions.: ALP supplementation during HMP was associated with lower urine production and energetic stress, with a shift toward less metabolic dysfunction and graft injury by the end of NMP. Our findings suggest an improvement in the early metabolic incompetency that characterizes delayed graft function in humans. Further research should elucidate whether these findings result in enhancement of graft functionality after transplantation.

A bstract: Background: Banff histological diagnoses do not capture how a graft’s injury has evolved over time. Repeated injury is common, but the consequences of repeated histological injury are currently not known, especially when observed in the absence of clinical signs. Methods: In a single-center observational cohort of 1818 kidney transplantations performed between 2004 and 2021, with 5218 post-transplant biopsies of which 82% were pre-planned protocol biopsies, transitions between six Banff histological diagnoses were described as recurrent events, with landmark Aalen-Johansen estimates and unadjusted Andersen-Gill rate ratios. A multistate model, with states for a first and a second diagnosis of rejection-related injury (RRI, comprising TCMR, AMR and MVI _DSA-/C4d- ), estimated cause-specific hazards of death-censored graft failure, in all biopsies and in protocol biopsies only. Follow-up ran from day 90 to five years after transplantation. Results: Each diagnosis was more frequent after an earlier episode of the same diagnosis, and several diagnoses also made a different diagnosis more or less frequent: TCMR was more frequent after AMR, MVI _DSA-/C4d- and BKPyVAN, and AMR less frequent after MVI _DSA-/C4d- . The hazard of graft failure was increased after a first RRI diagnosis (HR 2.18, 95% CI 1.35–3.51) and substantially further after a second (HR 9.31, 95% CI 5.84–14.85). The association persisted when both diagnoses were made on protocol biopsy, in the absence of clinical signs (HR 1.76, 95% CI 1.09–2.84 and HR 2.58, 95% CI 1.31–5.08), although the difference between a second and a first diagnosis was not statistically significant. Conclusions: A prior Banff diagnosis strongly predicts that same diagnosis recurring, and in several cases also changes the expected incidence of a different diagnosis. A second diagnosis of rejection-related injury identifies a markedly higher-risk graft than a first, independently of baseline risk factors, and subclinical episodes detected on protocol biopsy also mark increased risk. This defines both an unmet therapeutic need and a high-risk population for trial enrichment. K ey learning points: What was known: A Banff diagnosis is rendered from a single biopsy, yet alloimmune injury often persists, recurs or changes over time. Repeated T-cell mediated rejection adds risk of graft failure, but evidence comes almost entirely from indication biopsies. How other diagnoses recur, and whether one predicts another, was largely unknown. This study adds: In 1818 transplants with 5218 biopsies (82% protocol), a prior diagnosis strongly predicted its recurrence and altered the incidence of others. A second rejection-related episode carried a significantly higher hazard of death-censored graft failure than a first (9.31 versus 2.18). Subclinical protocol-biopsy episodes also carried risk (2.58 versus 1.76). Potential impact: A first episode marks a graft at elevated risk, not a resolved problem. Biopsies are best read alongside the graft’s diagnostic history. Protocol biopsies detect prognostically relevant injury in apparently well recipients. Recipients with a second episode form a high-risk group suited to trials of more durable immune modulation.

Community-acquired pneumonia (CAP) is a leading cause of death, with mortality linked to an unbalanced host response. Sirtuin (Sirt)1, a histone deacetylase, regulating metabolism and epigenetics, may be fundamental in activating the innate immune response. Sirt1 mRNA expression was significantly reduced in monocytes from CAP patients (n = 76) upon admission compared to healthy controls (n = 42), with levels returning to normal after 30 days. Pharmacological activation of Sirt1 with SRT1720 decreased LPS- and K. pneumoniae-induced IL-6 release in primary human monocytes and decreased NF-κB activation in THP1 cells. In a mouse K. pneumoniae pneumosepsis model, SRT1720 strongly reduced neutrophil influx and degranulation markers in bronchoalveolar lavage fluid, lowered pulmonary concentrations of IL-6 and TNF-α, and reduced lung pathology scores. Simultaneously, it reduced neutrophil content in liver tissue and plasma transaminase levels, alongside a trend toward reduced liver necrosis. Plasma IL-6 and TNF-α were significantly lower in SRT1720-treated mice at 42 h. Finally, while SRT1720 did not impact bacterial loads in the lungs, it reduced bacterial burden in blood, with a similar trend observed in liver homogenates. In conclusion, the Sirt1 activator SRT1720 exerts anti-inflammatory effects on human monocytes, reduces local and systemic inflammation and organ injury, and diminishes bacterial dissemination in murine pneumosepsis.

Background: Plasma cell rich rejection (PCRR) is an uncommon, ill-defined type of renal allograft rejection in the current literature considered a subtype of T cell-mediated rejection (TCMR). PCRR has poorer clinical outcome and is often refractory to classic immunosuppressive therapy. Our study analyzed clinical course, Banff lesion scores and mRNA expression of PCRR compared to (late) rejection. Methods: We retrospectively scored and reclassified the last known biopsy of 263 renal transplant recipients, morphologically classified as rejection according to the 2019 Banff classification. mRNA expression analysis was performed using the Nanostring B-HOT panel on a subset of cases. PCRR was compared to (late) TCMR, ABMR and mixed rejection for renal function follow-up and graft survival. Results: mRNA analysis revealed uniquely expressed genes in PCRR including LOX , CPA3 , IL4 , IL17F, and MMP12 . PCRR is enriched for genes related to mast cells, memory B- and T-cells and transcripts involved in NK cells and allograft fibrosis with heterogeneity in gene expression in biopsies with PCRR. PCRR might be a late event compared to late TCMR and ABMR, with a higher degree of total inflammation and fibrosis. Graft survival and renal function was similar to late TCMR and ABMR during a 5-year follow-up period after renal biopsy. Conclusion: PCRR represents a distinct late-onset stage of inflammation displaying diverse gene expression patterns, with presence of mainly mast cells, NK cells and transcripts involved in renal allograft fibrosis. Clinical outcomes in patients with PCRR appeared more similar to late TCMR and ABMR.

Chronic kidney disease (CKD) has emerged as a significant global public health concern. Recent epidemiological studies have highlighted the link between exposure to fine particulate matter (PM_2.5) and a decline in renal function. PM_2.5 exerts harmful effects on various organs through oxidative stress and inflammation. Acute kidney injury (AKI) resulting from ischaemia–reperfusion injury (IRI) involves biological processes similar to those involved in PM_2.5 toxicity and is a known risk factor for CKD. The objective of this study was to investigate the impact of PM_2.5 exposure on IRI‐induced AKI. Through a unique environmentally controlled setup, mice were exposed to urban PM_2.5 or filtered air for 12 weeks before IRI followed by euthanasia 48 h after surgery. Animals exposed to PM_2.5 and IRI exhibited reduced glomerular filtration, impaired urine concentration ability, and significant tubular damage. Further, PM_2.5 aggravated local innate immune responses and mitochondrial dysfunction, as well as enhancing cyclic GMP–AMP synthase‐stimulator of interferon genes (cGAS–STING) pathway activation. This increased renal senescence and suppressed the anti‐ageing protein klotho, leading to early fibrotic changes. In vitro studies using proximal tubular epithelial cells exposed to PM_2.5 and hypoxia/reoxygenation revealed heightened activation of the STING pathway triggered by cytoplasmic mitochondrial DNA, resulting in increased tubular damage and a pro‐inflammatory phenotype. In summary, our findings imply a role for PM_2.5 in sensitising proximal tubular epithelial cells to IRI‐induced damage, suggesting a plausible association between PM_2.5 exposure and heightened susceptibility to CKD in individuals experiencing AKI. Strategies aimed at reducing PM_2.5 concentrations and implementing preventive measures may improve outcomes for AKI patients and mitigate the progression from AKI to CKD. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Background: Chronic kidney disease often leads to kidney dysfunction due to renal fibrosis, regardless of the initial cause of kidney damage. Macrophages are crucial players in the progression of renal fibrosis as they stimulate inflammation, activate fibroblasts, and contribute to extracellular matrix deposition, influenced by their metabolic state. Nucleotide-binding domain and LRR-containing protein X (NLRX1) is an innate immune receptor independent of inflammasomes and is found in mitochondria, and it plays a role in immune responses and cell metabolism. The specific impact of NLRX1 on macrophages and its involvement in renal fibrosis is not fully understood. Methods: To explore the specific role of NLRX1 in macrophages, bone-marrow-derived macrophages (BMDMs) extracted from wild-type (WT) and NLRX1 knockout (KO) mice were stimulated with pro-inflammatory and pro-fibrotic factors to induce M1 and M2 polarization in vitro. The expression levels of macrophage polarization markers (Nos2, Mgl1, Arg1, and Mrc1), as well as the secretion of transforming growth factor β (TGFβ), were measured using RT-PCR and ELISA. Seahorse-based bioenergetics analysis was used to assess mitochondrial respiration in naïve and polarized BMDMs obtained from WT and NLRX1 KO mice. In vivo, WT and NLRX1 KO mice were subjected to unilateral ureter obstruction (UUO) surgery to induce renal fibrosis. Kidney injury, macrophage phenotypic profile, and fibrosis markers were assessed using RT-PCR. Histological staining (PASD and Sirius red) was used to quantify kidney injury and fibrosis. Results: Compared to the WT group, an increased gene expression of M2 markers—including Mgl1 and Mrc1—and enhanced TGFβ secretion were found in naïve BMDMs extracted from NLRX1 KO mice, indicating functional polarization towards the pro-fibrotic M2 subtype. NLRX1 KO naïve macrophages also showed a significantly enhanced oxygen consumption rate compared to WT cells and increased basal respiration and maximal respiration capacities that equal the level of M2-polarized macrophages. In vivo, we found that NLRX1 KO mice presented enhanced M2 polarization markers together with enhanced tubular injury and fibrosis demonstrated by augmented TGFβ levels, fibronectin, and collagen accumulation. Conclusions: Our findings highlight the unique role of NLRX1 in regulating the metabolism and function of macrophages, ultimately protecting against excessive renal injury and fibrosis in UUO.