Dynamics of HLA and angiotensin II type 1 receptor antibodies during pregnancy.

BACKGROUND: Alloantibodies, especially anti-human leukocyte antigen antibodies (HLA antibodies), and autoantibodies, as angiotensin II type 1 receptor antibodies (AT1R antibodies), may complicate the access and the course of transplantation. Pregnancy is a known source of HLA antibodies, with most studies evaluating pregnancy-induced sensitization by complement-dependent cytotoxicity assays, mainly after childbirth. AT1R antibodies have been evaluated in the context of preeclampsia. We aimed to evaluate pregnancy as a natural source of HLA antibodies and AT1R antibodies, their dynamics along gestation and the potential factors involved in antibody appearance. METHODS: Serum samples from pregnant women were collected during the three trimesters of pregnancy (1T, 2T, 3T). Presence of HLA antibodies was assessed by screening beads on Luminex and AT1R antibodies by ELISA. RESULTS: A cohort of 138 pregnant women were included. Samples from all were tested in 1T, 127 in 2T and 102 in 3T. HLA antibodies increased from 29.7 % (1T) to 38.2 % (3T). AT1R antibodies were stable around 30 % along pregnancy. Up to 43.2 % multiparous women had HLA antibodies, with a similar proportion of class I and class II antibodies. In primiparous women HLA antibodies increased along pregnancy (from 17.6 % to 34.1 %), with predominance of class II HLA antibodies. AT1R antibodies were not different in primiparous and multiparous women. CONCLUSIONS: Pregnancy is a relevant source of HLA antibodies sensitization, but not of AT1R antibodies. HLA antibodies increased clearly in primiparous women with predominance of class II. The use of newer solid-phase techniques on Luminex evidence a higher degree of HLA sensitization during pregnancy.

Evolution of kidney allograft loss causes over 40 years (1979-2019).

INTRODUCTION: The improvement of kidney allograft recipient and graft survival showed a decrease over the last 40 years. Long-term graft loss rate remained stable during a 25-year time span. Knowing the changing causes and the risk factors associated with graft loss requires special attention. The present study aimed to assess the causes of graft loss and kidney allograft recipient death. Also, we aimed to compare two different periods (1979-1999 and 2000-2019) to identify changes in the characteristics of the failed allografts and recipient and donors profile. METHODS AND PATIENTS: We performed a single-center cohort study. We included all the kidney transplant recipients at the Hospital del Mar (Barcelona) between May 1979 and December 2019. Graft loss was defined as recipient death with functioning graft and as loss of graft function (return to dialysis or retransplantation). We assessed the causes of graft loss using clinical and histological information. We also analyzed the results of the two different transplant periods (1979-1999 and 2000-2019). RESULTS: Between 1979 and 2019, 1522 transplants were performed. The median follow-up time was 56 (IQR 8-123) months. During follow-up, 722 (47.5%) grafts were lost: 483 (66.9%) due to graft failure and 239 (33.1%) due to death with functioning graft. The main causes of death were cardiovascular (25.1%), neoplasms (25.1%), and infectious diseases (21.8%). These causes were stable between the two periods of time. Only the unknown cause of death has decreased in the last period. The main cause of graft failure (loss of graft function) was the allograft chronic dysfunction (75%). When histologic information was available, antibody-mediated rejection (ABMR) and interstitial fibrosis/tubular atrophy (IF/TA) were the most frequent specific causes (15.9% and 12.6%). Of the graft failures, 213 (29.5%) were early (<1 year of transplantation). Vascular thrombosis was the main cause of early graft failure in the second period (2000-2019) (46.7%) and T-cell-mediated rejection (TCMR) was the main cause (31.3%) in the first period (1979-1999). The causes of late graft loss were similar between the two periods. CONCLUSIONS: The causes of kidney allograft recipient death are still due to cardiovascular and malignant diseases. Vascular thrombosis has emerged as a frequent cause of early graft loss in the most recent years. The evaluation of the causes of graft loss is necessary to improve kidney transplantation outcomes.

Microvascular inflammation in the absence of human leukocyte antigen-donor-specific antibody and C4d: An orphan category in Banff classification with cytotoxic T and natural killer cell infiltration.

Isolated microvascular inflammation (iMVI) without HLA donor-specific antibodies or C4d deposition in peritubular capillaries remains an enigmatic phenotype that cannot be categorized as antibody-mediated rejection (ABMR) in recent Banff classifications. We included 221 kidney transplant recipients with biopsies with ABMR (n = 73), iMVI (n = 32), and normal (n = 116) diagnoses. We compared peripheral blood leukocyte distribution by flow cytometry and inflammatory infiltrates in kidney transplant biopsies among groups. Flow cytometry showed fewer lymphocytes and total, CD4+, and CD8+ peripheral T cells in iMVI compared with ABMR and normal cases. ABMR and iMVI had fewer total natural Killer (NK) cells but more NKG2A+ NK cells. Immunohistochemistry indicated that ABMR and iMVI had greater CD3+ and CD68+ glomerular infiltration than normal biopsies, whereas CD8+ and TIA1+ cells showed only increased iMVI, suggesting they are cytotoxic T cells. Peritubular capillaries displayed more CD3+, CD56+, TIA1+, and CD68+ cells in both ABMR and iMVI. In contrast, iMVI had less plasma cell infiltration in peritubular capillaries and interstitial aggregates than ABMR. iMVI displayed decreased circulating T and NK cells mirrored by T cell and NK cell infiltration in the renal allograft, similar to ABMR. However, the lesser plasma cell infiltration in iMVI may suggest an antibody-independent underlying stimulus.

Dynamics of Humoral and Cellular Responses in Renal Transplant Recipients Receiving 3 Doses of SARS-CoV-2 mRNA Vaccine.

BACKGROUND: The original SARS-CoV-2 vaccination regimen (2 doses) induces insufficient short-term response in kidney transplant (KT) recipients. This study assessed the response to a third dose and the long-term immunogenicity after 2 doses in KT. METHODS: We analyzed the dynamics of the humoral and cellular response by monitoring SARS-CoV-2 IgG antibodies against the Spike-protein (IgG-Spike) and QuantiFERON SARS-CoV-2 IFN-γ release assay 6 mo after the second dose (T2) and 28 d after the third dose of mRNA vaccines (T3) to KT and controls (dialysis patients and healthy individuals). RESULTS: At T2, the percentage of IgG-Spike+ KT and dialysis patients decreased (KT 65.8%-52.6%, hemodialysis 92.6-81.5%, and peritoneal dialysis 100%-90%), whereas 100% of healthy controls remained positive. About the cellular response, the percentage of responders decreased in all groups, especially in KT (22.4%-9.2%, P = 0.081). At T3, 92% of KT, 94%-98% of dialysis patients, and 100% of healthy controls were IgG-Spike+. In terms of antibody titers, patients and controls showed a reduction between T2 and T3 and about 80% of dialysis patients and 100% of controls achieved high titers after the third dose (>1479.5 Binding Antibody Units/mL), whereas this percentage was only 50% in KT. With respect to the cellular response, only KT displayed a significant rise after the third dose. CONCLUSIONS: The third dose of mRNA vaccine improves both humoral and cellular responses, but less effectively in KT compared with dialysis patients and healthy controls.

Clinical and genetic characteristics of Dent's disease type 1 in Europe.

BACKGROUND: Dent's disease type 1 (DD1) is a rare X-linked nephropathy caused by CLCN5 mutations, characterized by proximal tubule dysfunction, including low molecular weight proteinuria (LMWP), hypercalciuria, nephrolithiasis-nephrocalcinosis, progressive chronic kidney disease (CKD) and kidney failure (KF). Current management is symptomatic and does not prevent disease progression. Here we describe the contemporary DD1 picture across Europe to highlight its unmet needs. METHODS: A physician-based anonymous international e-survey supported by several European nephrology networks/societies was conducted. Questions focused on DD1 clinical features, diagnostic procedure and mutation spectra. RESULTS: A total of 207 DD1 male patients were reported; clinical data were available for 163 with confirmed CLCN5 mutations. Proteinuria was the most common manifestation (49.1%). During follow-up, all patients showed LMWP, 66.4% nephrocalcinosis, 44.4% hypercalciuria and 26.4% nephrolithiasis. After 5.5 years, ≈50% of patients presented with renal dysfunction, 20.7% developed CKD stage ≥3 and 11.1% developed KF. At the last visit, hypercalciuria was more frequent in paediatric patients than in adults (73.4% versus 19.0%). Conversely, nephrolithiasis, nephrocalcinosis and renal dysfunction were more prominent in adults. Furthermore, CKD progressed with age. Despite no clear phenotype/genotype correlation, decreased glomerular filtration rate was more frequent in subjects with CLCN5 mutations affecting the pore or CBS domains compared with those with early-stop mutations. CONCLUSIONS: Results from this large DD1 cohort confirm previous findings and provide new insights regarding age and genotype impact on CKD progression. Our data strongly support that DD1 should be considered in male patients with CKD, nephrocalcinosis/hypercalciuria and non-nephrotic proteinuria and provide additional support for new research opportunities.

Isolation and characterization of exosome-enriched urinary extracellular vesicles from Dent's disease type 1 Spanish patients.

BACKGROUND AND OBJECTIVES: Dent's disease type 1 (DD1) is a rare X-linked hereditary pathology caused by CLCN5 mutations that is characterized mainly by proximal tubule dysfunction, hypercalciuria, nephrolithiasis/nephrocalcinosis, progressive chronic kidney disease, and low-weight proteinuria, the molecular hallmark of the disease. Currently, there is no specific curative treatment, only symptomatic and does not prevent the progression of the disease. In this study we have isolated and characterized urinary extracellular vesicles (uEVs) enriched in exosomes that will allow us to identify biomarkers associated with DD1 progression and a better understanding of the pathophysiological bases of the disease. MATERIALS AND METHODS: Through a national call from the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP), urine samples were obtained from patients and controls from different Spanish hospitals, which were processed to obtain the uEVS. The data of these patients were provided by the respective nephrologists and/or extracted from the RENALTUBE registry. The uEVs were isolated by ultracentrifugation, morphologically characterized and their protein and microRNA content extracted. RESULTS: 25 patients and 10 controls were recruited, from which the urine was processed to isolate the uEVs. Our results showed that the relative concentration of uEVs/mL is lower in patients compared to controls (0.26 × 106 uEVs/mL vs 1.19 × 106 uEVs/mL, p < 0.01). In addition, the uEVs of the patients were found to be significantly larger than those of the control subjects (mean diameter: 187.8 nm vs 143.6 nm, p < 0.01). Finally, our data demonstrated that RNA had been correctly extracted from both patient and control exosomes. CONCLUSIONS: In this work we describe the isolation and characterization of uEVs from patients with Dent 1 disease and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology.

Increased mortality after kidney transplantation in mildly frail recipients.

Background: Physical Frailty Phenotype (PFP) is the most used frailty instrument among kidney transplant recipients, classifying patients as pre-frail if they have 1-2 criteria and as frail if they have ≥3. However, different definitions of robustness have been used among renal patients, including only those who have 0 criteria, or those with 0-1 criteria. Our aim was to determine the impact of one PFP criterion on transplant outcomes. Methods: We undertook a retrospective study of 296 kidney transplant recipients who had been evaluated for frailty by PFP at the time of evaluating for transplantation. Results: Only 30.4% of patients had 0 criteria, and an additional 42.9% showed one PFP criterion. As PFP score increased, a higher percentage of women and cerebrovascular disease were found. Recipients with 0-1 criteria had lower 1-year mortality after transplant than those with ≥2 (1.8% vs 10.1%), but this difference was already present when we only considered those who scored 0 (mortality 1.1%) and 1 (mortality 2.4%) separately. The multivariable analysis confirmed that one PFP criterion was associated to a higher risk of patient death after kidney transplantation [hazard ratio 3.52 (95% confidence interval 1.03-15.9)]. Conclusions: Listed kidney transplant candidates frequently show only one PFP frailty criterion. This has an independent impact on patient survival after transplantation.

The ulcerative colitis-associated gene FUT8 regulates the quantity and quality of secreted mucins.

Mucins are the main macrocomponents of the mucus layer that protects the digestive tract from pathogens. Fucosylation of mucins increases mucus viscoelasticity and its resistance to shear stress. These properties are altered in patients with ulcerative colitis (UC), which is marked by a chronic inflammation of the distal part of the colon. Here, we show that levels of Fucosyltransferase 8 (FUT8) and specific mucins are increased in the distal inflamed colon of UC patients. Recapitulating this FUT8 overexpression in mucin-producing HT29-18N2 colonic cell line increases delivery of MUC1 to the plasma membrane and extracellular release of MUC2 and MUC5AC. Mucins secreted by FUT8 overexpressing cells are more resistant to removal from the cell surface than mucins secreted by FUT8-depleted cells (FUT8 KD). FUT8 KD causes intracellular accumulation of MUC1 and alters the ratio of secreted MUC2 to MUC5AC. These data fit well with the Fut8-/- mice phenotype, which are protected from UC. Fut8-/- mice exhibit a thinner proximal colon mucus layer with an altered ratio of neutral to acidic mucins. Together, our data reveal that FUT8 modifies the biophysical properties of mucus by controlling levels of cell surface MUC1 and quantity and quality of secreted MUC2 and MUC5AC. We suggest that these changes in mucus viscoelasticity likely facilitate bacterial-epithelial interactions leading to inflammation and UC progression.

Treatment with sotrovimab for SARS-CoV-2 infection in a cohort of high-risk kidney transplant recipients.

Background: Sotrovimab is a neutralizing monoclonal antibody (mAb) that seems to remain active against recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. The evidence on its use in kidney transplant (KT) recipients, however, is limited. Methods: We performed a multicenter, retrospective cohort study of 82 KT patients with SARS-CoV-2 infection {coronavirus disease 2019 [COVID-19]} treated with sotrovimab. Results: Median age was 63 years. Diabetes was present in 43.9% of patients, and obesity in 32.9% of patients; 48.8% of patients had an estimated glomerular filtration rate under 30 mL/minute/1.73 m2. Additional anti-COVID-19 therapies were administered to 56 patients, especially intravenous steroids (65.9%). Sotrovimab was administered early (<5 days from the onset of the symptoms) in 46 patients (56%). Early-treated patients showed less likely progression to severe COVID-19 than those treated later, represented as a lower need for ventilator support (2.2% vs 36.1%; P < .001) or intensive care admission (2.2% vs 25%; P = .002) and COVID-19-related mortality (2.2% vs 16.7%; P = .020). In the multivariable analysis, controlling for baseline risk factors to severe COVID-19 in KT recipients, early use of sotrovimab remained as a protective factor for a composite outcome, including need for ventilator support, intensive care, and COVID-19-related mortality. No anaphylactic reactions, acute rejection episodes, impaired kidney function events, or non-kidney side effects related to sotrovimab were observed. Conclusions: Sotrovimab had an excellent safety profile, even in high-comorbidity patients and advanced chronic kidney disease stages. Earlier administration could prevent progression to severe disease, while clinical outcomes were poor in patients treated later. Larger controlled studies enrolling KT recipients are warranted to elucidate the true efficacy of monoclonal antibody therapies.

Outcomes of Frail Patients While Waiting for Kidney Transplantation: Differences between Physical Frailty Phenotype and FRAIL Scale.

Frailty is associated with poorer outcomes among patients waiting for kidney transplantation (KT). Several different tools to measure frailty have been used; however, their predictive value is unknown. This is a prospective longitudinal study of 449 KT candidates evaluated for frailty by the Physical Frailty Phenotype (PFP) and the FRAIL scale. During the study period, 296 patients received a KT, while 153 remained listed. Patients who did not get receive a transplant were more frequently frail according to PFP (16.3 vs. 7.4%, p = 0.013). Robust patients had fewer hospital admissions during the 1st year after listing (20.8% if PFP = 0 vs. 43.4% if ≥1, and 27.1% if FRAIL = 0 vs. 48.9% if ≥1) and fewer cardiovascular events (than FRAIL ≥ 1) or major infectious events (than PFP ≥ 1). According to PFP, scoring 1 point had an impact on patient survival and chance of transplantation in the univariate analysis. The multivariable analysis corroborated the result, as candidates with PFP ≥ 3 had less likelihood of transplantation (HR 0.45 [0.26-0.77]). The FRAIL scale did not associate with any of these outcomes. In KT candidates, pre-frailty and frailty according to both the PFP and the FRAIL scale were associated with poorer results while listed. The PFP detected that frail patients were less likely to receive a KT, while the FRAIL scale did not.

Frailty in kidney transplant candidates: a comparison between physical frailty phenotype and FRAIL scales.

BACKGROUND: Frailty is common among advanced chronic kidney disease (CKD) patients who are kidney transplant (KT) candidates, and predisposes to poor outcomes after transplantation. However, frailty is not routinely measured during pretransplant work-up and it is unknown which metric should be used in this specific population. Our aim was to establish frailty prevalence in KT candidates according to different frailty scales. METHODS: Prospective longitudinal study of 451 KT candidates evaluated for frailty by both Physical Frailty Phenotype (PFP) and FRAIL scale at the time of inclusion on the KT waiting list. Clinical and functional characteristics including sociodemographics, comorbidities, disability and nutritional status were recorded. Agreement between PFP and FRAIL scales as well as dissonant patients were analyzed. RESULTS: Mean age was 60.9 years and 31.7% were female. Comorbidity burden among patients was high, with 36.9% and 16.2% presenting with diabetes and ischemic coronary disease, respectively. Disabilities were also frequent. More than 70% of patients presented with ≥ 1 PFP criteria while this percentage for ≥ 1 FRAIL criteria was 45.4%. Agreement between PFP and FRAIL was not good (kappa index 0.317). There were 132 patients who were pre-frail or frail according to PFP but non-frail according to the FRAIL scale and they presented with fewer comorbidities and less disability. CONCLUSIONS: Frailty is frequent in advanced CKD patients, although its prevalence may vary according to different scales. Agreement between PFP and FRAIL scale is not good, and FRAIL scale might misclassify as robust patients those frail/prefrail patients who are in better health conditions.

Early Hypertransaminasemia after Kidney Transplantation: Significance and Evolution According to Donor Type.

Early hypertransaminasemia after kidney transplantation (KT) is frequent. It has been associated with the crosstalk produced between the liver and the kidney in ischemia-reperfusion situations. However, the influence of the donor type has not been evaluated. We present a retrospective study analyzing the increase in serum aspartate aminotransferase/alanine aminotransferase (AST/ALT) during the first three months post-KT in 151 recipients who received thymoglobulin as induction therapy, either from brain-death donors (DBD, n = 75), controlled circulatory death donors (cDCD, n = 33), or uncontrolled DCD (uDCD, n = 43). Eighty-five KT recipients from DBD who received basiliximab were included as controls. From KT recipients who received thymoglobulin, 33.6/43.4% presented with an increase in AST/ALT at 72 h post-KT, respectively. Regarding donor type, the percentage of recipients who experienced 72 h post-KT hypertransaminasemia was higher in uDCD group (65.1/83.7% vs. 20.3/26% in DBD and 20.7/27.6% in cDCD, p < 0.001). Within the control group, 9.4/12.9% of patients presented with AST/ALT elevation. One month after transplant, AST/ALT values returned to baseline in all groups. The multivariate analysis showed that uDCD recipients had 6- to 12-fold higher risk of developing early post-KT hypertransaminasemia. Early post-KT hypertransaminasemia is a frequent and transient event related to the kidney donor type, being more frequent in uDCD recipients.

Recurrence of IgA Nephropathy after Kidney Transplantation in Adults.

BACKGROUND AND OBJECTIVES: In patients with kidney failure due to IgA nephropathy, IgA deposits can recur in a subsequent kidney transplant. The incidence, effect, and risk factors of IgA nephropathy recurrence is unclear, because most studies have been single center and sample sizes are relatively small. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: We performed a multicenter, international, retrospective study to determine the incidence, risk factors, and treatment response of recurrent IgA nephropathy after kidney transplantation. Data were collected from all consecutive patients with biopsy-proven IgA nephropathy transplanted between 2005 and 2015, across 16 "The Post-Transplant Glomerular Disease" study centers in Europe, North America, and South America. RESULTS: Out of 504 transplant recipients with IgA nephropathy, recurrent IgA deposits were identified by kidney biopsy in 82 patients; cumulative incidence of recurrence was 23% at 15 years (95% confidence interval, 14 to 34). Multivariable Cox regression revealed a higher risk for recurrence of IgA deposits in patients with a pre-emptive kidney transplant (hazard ratio, 3.45; 95% confidence interval, 1.31 to 9.17) and in patients with preformed donor-specific antibodies (hazard ratio, 2.59; 95% confidence interval, 1.09 to 6.19). After kidney transplantation, development of de novo donor-specific antibodies was associated with subsequent higher risk of recurrence of IgA nephropathy (hazard ratio, 6.65; 95% confidence interval, 3.33 to 13.27). Immunosuppressive regimen was not associated with recurrent IgA nephropathy in multivariable analysis, including steroid use. Graft loss was higher in patients with recurrence of IgA nephropathy compared with patients without (hazard ratio, 3.69; 95% confidence interval, 2.04 to 6.66), resulting in 32% (95% confidence interval, 50 to 82) graft loss at 8 years after diagnosis of recurrence. CONCLUSIONS: In our international cohort, cumulative risk of IgA nephropathy recurrence increased after transplant and was associated with a 3.7-fold greater risk of graft loss.

Non-HLA Antibodies and Epitope Mismatches in Kidney Transplant Recipients With Histological Antibody-Mediated Rejection.

Background: Correlation between antibody-mediated rejection (ABMR) and circulating HLA donor-specific antibodies (HLA-DSA) is strong but imperfect in kidney transplant (KT) recipients, raising the possibility of undetected HLA-DSA or non-HLA antibodies contributing to ABMR. Detailed evaluation of the degree of HLA matching together with the identification of non-HLA antibodies in KT may help to decipher the antibody involved. Methods: We retrospectively assessed patients with transplant biopsies scored following Banff'15 classification. Pre- and post-transplant serum samples were checked for HLA and non-HLA antibodies [MICA-Ab, angiotensin-II type-1-receptor (AT1R)-Ab, endothelin-1 type-A-receptor (ETAR)-Ab and crossmatches with primary aortic endothelial cells (EC-XM)]. We also analyzed HLA epitope mismatches (HLA-EM) between donors and recipients to explore their role in ABMR histology (ABMRh) with and without HLA-DSA. Results: One-hundred eighteen patients with normal histology (n = 19), ABMRh (n = 52) or IFTA (n = 47) were studied. ABMRh patients were HLA-DSApos (n = 38, 73%) or HLA-DSAneg (n = 14, 27%). Pre-transplant HLA-DSA and AT1R-Ab were more frequent in ABMRh compared with IFTA and normal histology cases (p = 0.006 and 0.003), without differences in other non-HLA antibodies. Only three ABMRhDSAneg cases showed non-HLA antibodies. ABMRhDSAneg and ABMRhDSApos cases showed similar biopsy changes and graft-survival. Both total class II and DRB1 HLA-EM were associated with ABMRhDSApos but not with ABMRhDSAneg. Multivariate analysis showed that pre-transplant HLA-DSA (OR: 3.69 [1.31-10.37], p = 0.013) and AT1R-Ab (OR: 5.47 [1.78-16.76], p = 0.003) were independent predictors of ABMRhDSApos. Conclusions: In conclusion, pre-transplant AT1R-Ab is frequently found in ABMRhDSApos patients. However, AT1R-Ab, MICA-Ab, ETAR-Ab or EC-XM+ are rarely found among ABMRhDSAneg patients. Pre-transplant AT1R-Ab may act synergistically with preformed or de novo HLA-DSA to produce ABMRhDSApos but not ABMRhDSAneg. HLA epitope mismatch associates with ABMRhDSApos compared with ABMRhDSAneg, suggesting factors other than HLA are responsible for the damage.

Novel Dent disease 1 cellular models reveal biological processes underlying ClC-5 loss-of-function.

Dent disease 1 (DD1) is a rare X-linked renal proximal tubulopathy characterized by low molecular weight proteinuria and variable degree of hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressing to chronic kidney disease. Although mutations in the electrogenic Cl-/H+ antiporter ClC-5, which impair endocytic uptake in proximal tubule cells, cause the disease, there is poor genotype-phenotype correlation and their contribution to proximal tubule dysfunction remains unclear. To further discover the mechanisms linking ClC-5 loss-of-function to proximal tubule dysfunction, we have generated novel DD1 cellular models depleted of ClC-5 and carrying ClC-5 mutants p.(Val523del), p.(Glu527Asp) and p.(Ile524Lys) using the human proximal tubule-derived RPTEC/TERT1 cell line. Our DD1 cellular models exhibit impaired albumin endocytosis, increased substrate adhesion and decreased collective migration, correlating with a less differentiated epithelial phenotype. Despite sharing functional features, these DD1 cell models exhibit different gene expression profiles, being p.(Val523del) ClC-5 the mutation showing the largest differences. Gene set enrichment analysis pointed to kidney development, anion homeostasis, organic acid transport, extracellular matrix organization and cell-migration biological processes as the most likely involved in DD1 pathophysiology. In conclusion, our results revealed the pathways linking ClC-5 mutations with tubular dysfunction and, importantly, provide new cellular models to further study DD1 pathophysiology.

Kidney transplantation outcomes from elderly donors after circulatory death: a comparison with elderly brain-dead donors.

BACKGROUND: The use of kidneys from elderly controlled donation after circulatory death (cDCD) donors has increased significantly in recent years. Concerns about outcomes achieved with these elderly cDCD kidneys have arisen. We aimed to compare outcomes from elderly cDCD kidney transplant recipients (KTrs) and elderly donation after brain death donors (DBDs) in KTrs. METHODS: We conducted a single-centre retrospective study including 87 cDCD-KTrs (46 from donors ≥65 years of age and 41 from <65 years) and 126 DBD-KTrs from donors ≥65 years of age from 2013 through 2017). Young cDCD-KTrs were used as controls. The median follow-up was 27.1 months for all cDCD-KTrs and 29.7 months for DBD-KTrs ≥65 years of age. RESULTS: Donors >65 years of age represented more than half of our global cDCD cohort (52.9%). KTs from elderly cDCDs had similar rates of delayed graft function, primary non-function and vascular complications compared with young cDCD-KTrs and elderly DBD-KTrs. Short and medium-term graft survival from elderly cDCD kidneys are excellent and are comparable to those from young cDCD and elderly DBD kidneys (90% young cDCD versus 88% elderly cDCD versus 80% elderly DBD at 36 months, P = 0.962 and 0.180, respectively). Although recipients from cDCDs ≥65 years of age showed lower 3-year patient survival (78% versus 87% in elderly DBD-KTrs; P = 0.01), recipient age was the only determinant of patient survival [hazard ratio 1.10 (95% confidence interval 1.02-1.17); P < 0.01], without any influence of donor characteristics. CONCLUSIONS: The use of kidneys from elderly cDCDs is increasing in Spain. Short- and medium-term graft outcomes are similar when comparing kidneys from elderly cDCDs and DBDs. Recipient age is the only determinant of patient survival. Additional studies are needed to assess long-term outcomes.

Activation of final complement components after kidney transplantation as a marker of delayed graft function severity.

BACKGROUND: Ischaemia-reperfusion (I/R) damage is a relevant cause of delayed graft function (DGF). Complement activation is involved in experimental I/R injury, but few data are available from kidney transplant (KT) patients. We studied the dynamics of membrane attack complex (C5b-9) as a soluble fraction (SC5b-9) and the histological deposit pattern of C3b, complement Factor H (FH) and C5b-9 in DGF patients. METHODS: We evaluated SC5b-9 levels in 59 recipients: 38 with immediate graft function and 21 with DGF. The SC5b-9 was measured at admission for KT and 7 days after KT. DGF-kidney biopsies (n = 12) and a control group of 1-year protocol biopsies without tissue damage (n = 4) were stained for C5b-9, C3b and FH. RESULTS: SC5b-9 increased significantly in DGF patients (Day 0: 6621 ± 2202 mAU/L versus Day 7: 9626 ± 4142 mAU/L; P = 0.006), while it remained stable in non-DGF patients. Days 0-7 increase >5% was the better cut-off associated with DGF versus non-DGF patient discrimination (sensitivity = 81%). In addition, SC5b-9 increase was related to DGF duration and worse graft function, and independently associated with DGF occurrence. SC5b-9, C3b and FH stains were observed in tubular epithelial cells basal membrane. DGF-kidney biopsies showed a more frequently high-intensity stain, a higher number of tubules with positive stain and larger perimeter of tubules with positive stains for SC5b-9, C3b and FH than control patients. CONCLUSIONS: Both SC5b-9 levels and SC5b-9, C3b and FH deposits in tubular epithelial cells basal membrane are highly expressed in patients experiencing DGF. SC5b-9 levels increase could be useful as a marker of DGF severity.

Role of C5aR1 and C5L2 Receptors in Ischemia-Reperfusion Injury.

The role of C5a receptors (C5aR1 and C5L2) in renal ischemia-reperfusion injury (IRI) is uncertain. We generated an in vitro model of hypoxia/reoxygenation with human proximal tubule epithelial cells to mimic some IRI events. C5aR1, membrane attack complex (MAC) and factor H (FH) deposits were evaluated with immunofluorescence. Quantitative polymerase chain reaction evaluated the expression of C5aR1, C5L2 genes as well as genes related to tubular injury, inflammation, and profibrotic pathways. Additionally, C5aR1 and C5L2 deposits were evaluated in kidney graft biopsies (KB) from transplant patients with delayed graft function (DGF, n = 12) and compared with a control group (n = 8). We observed higher immunofluorescence expression of C5aR1, MAC and FH as higher expression of genes related to tubular injury, inflammatory and profibrotic pathways and of C5aR1 in the hypoxic cells; whereas, C5L2 gene expression was unaffected by the hypoxic stimulus. Regarding KB, C5aR1 was detected in the apical and basal membrane of tubular epithelial cells, whereas C5L2 deposits were observed in endothelial cells of peritubular capillaries (PTC). DGF-KB showed more frequently diffuse C5aR1 staining and C5L2 compared to controls. In conclusion, C5aR1 expression is increased by hypoxia and IRI, both in vitro and in human biopsies with an acute injury. C5L2 expression in PTC could be related to endothelial cell damage during IRI.

Heterogeneity is a common ground in familial hypomagnesemia with hypercalciuria and nephrocalcinosis caused by CLDN19 gene mutations.

BACKGROUND: Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC) is a rare tubulopathy caused by mutations in the CLDN16 or CLDN19 genes. Patients usually develop hypomagnesemia, hypercalciuria, nephrocalcinosis and renal failure early in life. Patients with CLDN19 mutations may also have ocular abnormalities. Despite clinical variability, factors associated with kidney function impairment, especially in patients with CLDN19 mutations, have not been addressed. METHODS: Retrospective multicenter study of 30 genetically confirmed FHHNC Spanish patients. We analyzed kidney function impairment considering as outcomes chronic kidney disease (CKD) stage 3 and annual estimated glomerular filtration rate (eGFR) decline, to identify factors associated with the different phenotypes. RESULTS: Of thirty patients, 27 had mutations in the CLDN19 gene (20 homozygous for the p.G20D mutation) and 3 in the CLDN16. Age at diagnosis was 1.71 (0.67-6.04) years and follow-up time was 8.34 ± 4.30 years. No differences in CKD stage 3-free survival based on CLDN19 mutation (p.G20D homozygous vs. other mutations) or gender were found, although females seemed to progress faster than males. Patients with more pronounced eGFR decline had higher PTH levels at diagnosis than those with stable kidney function, despite similar initial eGFR. Approximately 60% of CLDN19 patients presented ocular abnormalities. Furthermore, we confirmed high phenotypic intrafamilial variability. CONCLUSIONS: In a contemporary cohort of FHHNC patients with CLDN19 mutations, females seemed to progress to CKD-stage 3 faster than males. Increased PTH levels at baseline may indicate a more severe renal course. There was high phenotype variability among patients with CLDN19 mutations and kidney function impairment  differed even between siblings.