In situ multiplex immunofluorescence analysis of the inflammatory burden in kidney allograft rejection: A new tool to characterize the alloimmune response.

The exact composition of leukocyte infiltration during kidney allograft rejection is difficult to comprehend and visualize on the same biopsy slide. Using an innovative technology of multiplex immunofluorescence (mIF), we were able to detect simultaneously NK cells, macrophages, and T cells and to determine their intra- or extravascular localization using an endothelial marker. Twenty antibody-mediated rejection (ABMR), 20 T cell-mediated rejection (TCMR), and five normal biopsies were labeled, with automatic leukocyte quantification and localization. This method was compared to a classic NKp46 immunohistochemistry (IHC) with manual quantification and to mRNA quantification. mIF automatic quantification was strongly correlated to IHC (r = .91, P < .001) and to mRNA expression levels (r > .46, P < .021). T cells and macrophages were the 2 predominant populations involved in rejection (48.0 ± 4.4% and 49.3 ± 4.4%, respectively, in ABMR; 51.8 ± 6.0% and 45.3 ± 5.8% in TCMR). NK cells constituted a rare population in both ABMR (2.7 ± 0.7%) and TCMR (2.9 ± 0.6%). The intravascular compartment was mainly composed of T cells, including during ABMR, in peritubular and glomerular capillaries. However, NK cell and macrophage densities were significantly higher during ABMR in glomerular and peritubular capillaries. To conclude, this study demonstrates the feasibility and utility of mIF imaging to study and better understand the kidney allograft rejection process.

Development and validation of an optimized integrative model using urinary chemokines for noninvasive diagnosis of acute allograft rejection.

The urinary chemokines CXCL9 and CXCL10 are promising noninvasive diagnostic markers of acute rejection (AR) in kidney recipients, but their levels might be confounded by urinary tract infection (UTI) and BK virus (BKV) reactivation. Multiparametric model development and validation addressed these confounding factors in a training set of 391 samples, optimizing the diagnostic performance of urinary chemokines. CXCL9/creatinine increased in UTI and BKV viremia with or without nephropathy (BKVN) (no UTI/leukocyturia/UTI: -0.10/1.61/2.09, P = .0001 and no BKV/viremia/BKVN: -0.10/1.90/2.29, P < .001) as well as CXCL10/creatinine (1.17/2.09/1.98, P < .0001 and 1.13/2.21/2.51, P < .001, respectively). An optimized 8-parameter model (recipient age, sex, estimated glomerular filtration rate, donor specific antibodies, UTI, BKV blood viral load, CXCL9, and CXCL10) diagnosed AR with high accuracy (area under the curve [AUC]: 0.85, 95% confidence interval [CI]: 0.80-0.89) and remained highly accurate at the time of screening (AUC: 0.81, 95% CI: 0.48-1) or indication biopsies (AUC: 0.85, 95% CI: 0.81-0.90) and within the first year (AUC: 0.86, 95% CI: 0.80-0.91) or later (AUC: 0.90, 95% CI: 0.84-0.96), achieving AR diagnosis with an AUC of 0.85 and 0.92 (P < .0001) in 2 external validation cohorts. Decision curve analyses demonstrated the clinical utility of the model. Considering confounding factors rather than excluding them, we optimized a noninvasive multiparametric diagnostic model for AR of kidney allografts with unprecedented accuracy.

Adult onset tubulo-interstitial nephropathy in MT-ND5-related phenotypes.

Kidney is a highly adenosine triphosphate dependent organ in human body. Healthy and functional mitochondria are essential for normal kidney function. Clinical and genetic variability are the hallmarks of mitochondrial disorders. We report here the involvement of two MT-ND5 pathogenic variants encoding for ND5 subunit of respiratory chain complex I, the m.13513G>A and the m.13514A>G, in adult-onset kidney disease in three unrelated patients. The first patient had myopathy encephalopathy lactic acidosis and stroke syndrome, left ventricular hypertrophy with Wolff-Parkinson-White syndrome and tubulo-interstitial kidney disease. The second presented Leber hereditary optic neuropathy associated with tubulo-interstitial kidney disease. The third presented with an isolated chronic tubulo-interstitial kidney disease. These mutations have never been associated with adulthood mitochondrial nephropathy. These case reports highlight the importance to consider mitochondrial dysfunction in tubulo-interstitial kidney disease.

No clinical benefit of rapid versus gradual tapering of immunosuppression to treat sustained BK virus viremia after kidney transplantation: a single-center experience.

Immunosuppressive drug tapering is currently the recommended treatment of BK virus (BKV) viremia after kidney transplantation; however, its exact modalities remain unclear. We retrospectively compared two consecutive strategies in 111 patients with sustained viremia: a gradual monitoring/tapering group (GT, n = 57) before 2012 and a rapid monitoring/tapering group (RT, n = 54) after 2012. At viremia diagnosis, the dose of mycophenolic acid (MPA) and tacrolimus levels (T0 ) were similar among patient groups. However, following onset, the dose of MPA at 1 month (P = 0.002) and 3 months (P = 0.005) and Tac T0 at 1 month (P = 0.030) and 3 months (P = 0.006) were lower in the RT group. This rapid minimization shortened BKV viremia (P < 0.001) and resulted in a better protection of graft function in patients with confirmed BKV-associated nephropathy (P = 0.033) without impacting 5-year graft survival. Survival without rejection was similar (P = 0.571), but the RT group had increased the development of de novo donor-specific antibodies (dnDSAs; P < 0.001). Multivariate Cox analysis identified basiliximab versus Thymoglobulin® induction [hazard ratio (HR), 3.090; P = 0.001] and the RT strategy (HR, 6.021; P = 0.002) as independently associated with dnDSAs. Compared to a gradual tapering, rapid immunosuppression tapering to treat sustained BKV viremia does not improve medium-term clinical outcome but increases the risk of developing dnDSAs.

Renal involvement is frequent in adults with primary mitochondrial disorders: an observational study.

Background: Mitochondrial functions are controlled by genes of both mitochondrial and nuclear DNA. Pathogenic variants affecting any of these are responsible for primary mitochondrial disorders (MIDs), which can be diagnosed during adulthood. Kidney functions are highly dependent on mitochondrial respiration. However, the prevalence of MID-associated nephropathies (MIDANs) is unknown in the adult population. We aimed to address this point and to provide a full characterization of MIDANs in this population. Methods: We retrospectively included for observational study adults (≥16 years of age) with genetically diagnosed MID between 2000 and 2020 in our tertiary care academic centre when they had a chronic kidney disease (CKD) evaluation. MIDANs were ascertained by CKD occurring in MIDs. The phenotypic, biological, histopathological and genotypic characteristics were recorded from the medical charts. Results: We included 80 MID-affected adults and ascertained MIDANs in 28/80 (35%). Kidney diseases under the care of a nephrologist occurred in only 14/28 (50%) of the adults with MIDAN. MIDANs were tubulointerstitial nephropathy in 14/28 patients (50%) and glomerular diseases in 9/28 (32.1%). In adults with MID, MIDAN was negatively associated with higher albumin levels {odds ratio [OR] 0.79 [95% confidence interval (CI) 0.67-0.95]} and vision abnormalities [OR 0.17 (95% CI 0.03-0.94)] and positively associated with hypertension [OR 4.23 (95% CI 1.04-17.17)]. Conclusion: MIDANs are frequent among adult MIDs. They are mostly represented by tubulointerstitial nephropathy or glomerular disease. Vision abnormalities, hypertension and albumin levels were independently associated with MIDANs. Our results pave the way for prospective studies investigating the prevalence of MIDANs among undetermined kidney disease populations.

Multisystem T-cell Chronic Active Epstein-Barr Virus Infection: From the Eye to the Kidney.

Chronic active Epstein-Barr virus (CAEBV) infection is usually a fatal disease associated with clonal proliferation of EBV-infected T or NK cells. We present the case of a 33-year-old Peruvian patient who developed a multisystem CAEBV, notably responsible for exceptional ophthalmological and renal damage. We describe the clinicopathological features of EBV-induced lymphoproliferative disorder.

Magnetic Resonance Elastography as Surrogate Marker of Interstitial Fibrosis in Kidney Transplantation: A Prospective Study.

Background: Fibrosis progression is a major prognosis factor in kidney transplantation. Its assessment requires an allograft biopsy, which remains an invasive procedure at risk of complications. Methods: We assessed renal stiffness by magnetic resonance elastography (MRE) as a surrogate marker of fibrosis in a prospective cohort of kidney transplant recipients compared with the histologic gold standard. Interstitial fibrosis was evaluated by three methods: the semi-quantitative Banff ci score, a visual quantitative evaluation by a pathologist, and a computer-assisted quantitative evaluation. MRE-derived stiffness was assessed at the superior, median, and inferior poles of the allograft. Results: We initially enrolled 73 patients, but only 55 had measurements of their allograft stiffness by MRE before an allograft biopsy. There was no significant correlation between MRE-derived stiffness at the biopsy site and the ci score (ρ=-0.25, P=0.06) or with the two quantitative assessments (pathologist: ρ=-0.25, P=0.07; computer assisted: ρ=-0.21, P=0.12). We observed negative correlations between the stiffness of both the biopsy site and the whole allograft, with either the glomerulosclerosis percentage (ρ=-0.32, P=0.02 and ρ=-0.31, P=0.02, respectively) and the overall nephron fibrosis percentage, defined as the mean of the percentages of glomerulosclerosis and interstitial fibrosis (ρ=-0.30, P=0.02 and ρ=-0.28, P=0.04, respectively). At patient level, mean MRE-derived stiffness was similar across the three poles of the allograft (±0.25 kPa). However, a high variability of mean stiffness was found between patients, suggesting a strong influence of confounding factors. Finally, no significant correlation was found between mean MRE-derived stiffness and the slope of eGFR (P=0.08). Conclusions: MRE-derived stiffness does not directly reflect the extent of fibrosis in kidney transplantation.

WARS1, TYMP and GBP1 display a distinctive microcirculation pattern by immunohistochemistry during antibody-mediated rejection in kidney transplantation.

Antibody-mediated rejection (ABMR) is the leading cause of allograft failure in kidney transplantation. Defined by the Banff classification, its gold standard diagnosis remains a challenge, with limited inter-observer reproducibility of the histological scores and efficient immunomarker availability. We performed an immunohistochemical analysis of 3 interferon-related proteins, WARS1, TYMP and GBP1 in a cohort of kidney allograft biopsies including 17 ABMR cases and 37 other common graft injuries. Slides were interpreted, for an ABMR diagnosis, by four blinded nephropathologists and by a deep learning framework using convolutional neural networks. Pathologists identified a distinctive microcirculation staining pattern in ABMR with all three antibodies, displaying promising diagnostic performances and a substantial reproducibility. The deep learning analysis supported the microcirculation staining pattern and achieved similar diagnostic performance from internal validation, with a mean area under the receiver operating characteristic curve of 0.89 (± 0.02) for WARS1, 0.80 (± 0.04) for TYMP and 0.89 (± 0.04) for GBP1. The glomerulitis and peritubular capillaritis scores, the hallmarks of histological ABMR, were the most highly correlated Banff scores with the deep learning output, whatever the C4d status. These novel immunomarkers combined with a CNN framework could help mitigate current challenges in ABMR diagnosis and should be assessed in larger cohorts.

The Proteome of Antibody-Mediated Rejection: From Glomerulitis to Transplant Glomerulopathy.

Antibody-mediated rejection (ABMR) is the leading cause of allograft failure in kidney transplantation. Its histological hallmark is represented by lesions of glomerulitis i.e., inflammatory cells within glomeruli. Current therapies for ABMR fail to prevent chronic allograft damage i.e., transplant glomerulopathy, leading to allograft loss. We used laser microdissection of glomeruli from formalin-fixed allograft biopsies combined with mass spectrometry-based proteomics to describe the proteome modification of 11 active and 10 chronic active ABMR cases compared to 8 stable graft controls. Of 1335 detected proteins, 77 were deregulated in glomerulitis compared to stable grafts, particularly involved in cellular stress mediated by interferons type I and II, leukocyte activation and microcirculation remodeling. Three proteins extracted from this protein profile, TYMP, WARS1 and GBP1, showed a consistent overexpression by immunohistochemistry in glomerular endothelial cells that may represent relevant markers of endothelial stress during active ABMR. In transplant glomerulopathy, 137 proteins were deregulated, which favor a complement-mediated mechanism, wound healing processes through coagulation activation and ultimately a remodeling of the glomerular extracellular matrix, as observed by light microscopy. This study brings novel information on glomerular proteomics of ABMR in kidney transplantation, and highlights potential targets of diagnostic and therapeutic interest.

Selectins impair regulatory T cell function and contribute to systemic lupus erythematosus pathogenesis.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by a loss of tolerance toward self-nucleic acids, autoantibody production, interferon expression and signaling, and a defect in the regulatory T (Treg) cell compartment. In this work, we identified that platelets from patients with active SLE preferentially interacted with Treg cells via the P-selectin/P-selectin glycoprotein ligand-1 (PSGL-1) axis. Selectin interaction with PSGL-1 blocked the regulatory and suppressive properties of Treg cells and particularly follicular Treg cells by triggering Syk phosphorylation and an increase in intracytosolic calcium. Mechanistically, P-selectin engagement on Treg cells induced a down-regulation of the transforming growth factor-ß axis, altering the phenotype of Treg cells and limiting their immunosuppressive responses. In patients with SLE, we found an up-regulation of P- and E-selectin both on microparticles and in their soluble forms that correlated with disease activity. Last, blocking P-selectin in a mouse model of SLE improved cardinal features of the disease, such as anti-dsDNA antibody concentrations and kidney pathology. Overall, our results identify a P-selectin-dependent pathway that is active in patients with SLE and validate it as a potential therapeutic avenue.

Deciphering the Prognostic and Predictive Value of Urinary CXCL10 in Kidney Recipients With BK Virus Reactivation.

BK virus (BKV) replication increases urinary chemokine C-X-C motif ligand 10 (uCXCL10) levels in kidney transplant recipients (KTRs). Here, we investigated uCXCL10 levels across different stages of BKV replication as a prognostic and predictive marker for functional decline in KTRs after BKV-DNAemia. uCXCL10 was assessed in a cross-sectional study (474 paired urine/blood/biopsy samples and a longitudinal study (1,184 samples from 60 KTRs with BKV-DNAemia). uCXCL10 levels gradually increased with urine (P-value < 0.0001) and blood BKV viral load (P < 0.05) but were similar in the viruria and no BKV groups (P > 0.99). In viremic patients, uCXCL10 at biopsy was associated with graft functional decline [HR = 1.65, 95% CI (1.08-2.51), P = 0.02], irrespective of baseline eGFR, blood viral load, or BKVN diagnosis. uCXL10/cr (threshold: 12.86 ng/mmol) discriminated patients with a low risk of graft function decline from high-risk patients (P = 0.01). In the longitudinal study, the uCXCL10 and BKV-DNAemia trajectories were superimposable. Stratification using the same uCXCL10/cr threshold at first viremia predicted the subsequent inflammatory response, assessed by time-adjusted uCXCL10/cr AUC (P < 0.001), and graft functional decline (P = 0.03). In KTRs, uCXCL10 increases in BKV-DNAemia but not in isolated viruria. uCXCL10/cr is a prognostic biomarker of eGFR decrease, and a 12.86 ng/ml threshold predicts higher inflammatory burdens and poor renal outcomes.

Idiopathic Nephrotic Syndrome: Characteristics and Identification of Prognostic Factors.

There are various histopathological forms of idiopathic nephrotic syndrome, including minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS). Whereas some relapse predictor factors have been identified in renal transplantation, the clinical future of idiopathic nephrotic syndrome in the native kidney remains uncertain. We designed a multicentric retrospective descriptive cohort study including all patients aged 15 years and over whose renal biopsy confirmed MCD or FSGS between January 2007 and December 2014. We studied 165 patients with idiopathic nephrotic syndrome; 97 with MCD and 68 with FSGS. In the MCD cohort, 91.7% of patients were treated with corticosteroids for a median total duration of 13 months. During 45 months of follow-up, 92.8% of patients achieved remission and 45.5% experienced relapse. In this cohort, 5% of patients experienced terminal kidney disease. With respect to FSGS patients, 51.5% were treated with corticosteroids for a median total duration of 15 months. During 66 months of follow-up, 73.5% of patients achieved remission and 20% experienced relapse. In this cohort, 26.5% of patients experienced terminal kidney disease. No statistical association was observed between clinical and biological initial presentation and relapse occurrence. This study describes the characteristics of a cohort of patients with the nephrotic idiopathic syndromes of MCD and FSGS from the time of renal biopsy and throughout follow-up.