Antiphospholipid Syndrome and Kidney Involvement.

BACKGROUND: Antiphospholipid syndrome (APS) is an autoimmune disease characterized by the development of autoantibodies and the impairment of the coagulation system. Knowledge about this syndrome is increasing over time, but kidney involvement, especially APS nephropathy, still represents a challenge for physicians. SUMMARY: A "two hit" model has been hypothesized to explain APS pathophysiology, and the role played by some factors, such as the complement system, is becoming more and more clear. From a clinical point of view, along with thrombosis in any site and/or obstetric morbidities, that are the hallmarks of APS, a constellation of several other clinical symptoms is related to APS. These symptoms alone are not sufficient to fulfill Sydney criteria for APS and this could potentially lead to omitting some diagnoses. The mainstay of management of APS is antithrombotic therapy, but there are expectations for new drugs that regulate the immune system. APS could affect the kidneys in many ways and among them, APS nephropathy is an intriguing entity that has been overlooked in recent years. Novel studies on APS nephropathy are lacking. KEY MESSAGES: In this review, we discuss what we currently know about APS and its relationship with the kidney, with an eye toward the future perspectives. Multicenter studies on APS nephropathy are necessary in order to develop targeted therapies.

How the Availability of Anti-C5a Agents Could Change the Management of Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

BACKGROUND: Antineutrophil cytoplasmic antibody-associated vasculitis (AAV) is a cluster of potentially life-threatening disorders, often involving the kidney with a necrotizing crescentic glomerulonephritis with scanty deposition of immunoglobulins and complement. Historically the role of complement has been considered ancillary. Recently, an anti-myeloperoxidase (MPO) AAV model in complement-deficient mice has shown an involvement for the complement cascade in the development of the renal injuries. Further animal studies showing that in contrast to mice deficient for factor B and C5 animals deficient for C4 were susceptible to AAV development by injection of anti-MPO antibodies emphasized the specific involvement of the alternative pathway. Consonantly, the C5a receptor (Cd88) blockade was found to protect mice from MPO-AAV. CCX168, i.e., avacopan, a powerful inhibitor of C5a receptor that can be administered orally, was shown to reduce the proinflammatory effects of C5a and abolish the activation of neutrophils, their migration and adherence to endothelium, and the vascular endothelial cell retraction that increases permeability. SUMMARY: Avacopan was found to be safe in healthy volunteers given a wide range of doses in a phase 1 clinical trial. The phase 2 trial CLEAR assessed the possibility to decrease dose or entirely replace glucocorticosteroids in the standard-of-care therapy of AAV. Avacopan, added to CYC or RTX either in combination with GCs or not, shortened the time to remission in patients with either newly diagnosed or relapsing AAV. The phase 3 ADVOCATE study compared the ability of an avacopan-associated regimen to induce and sustain remission in AAV patients versus a conventional GC-associated scheme. Remission at week 26 was observed in 72.3% of patients given avacopan and in 70.1% of those given prednisone. Sustained remission at week 52 (second primary endpoint) was obtained in 65.7% of patients given avacopan and in 54.9% receiving prednisone. The avacopan-associated regimen was noninferior at week 26 and superior at week 52 in sustaining remission as compared to the GC-based scheme. KEY MESSAGES: The results of the ADVOCATE trial opened new prospects for the treatment of AAV and also other immune-mediated diseases with renal involvement. The possible position of avacopan in a routine clinical setting and its possible indications in specific subsets of patients with AAV are extensively discussed.

New Onset Biopsy-Proven Nephropathies after COVID Vaccination.

INTRODUCTION: To date, almost 7 billion doses of the different types of vaccine against SARS-CoV-2 have been administered worldwide. Although the severity of new cases of SARS-CoV-2 has progressively decreased, and the pressure on national health systems has declined, the development of de novo glomerular injuries has been suggested. METHODS: This study aimed to examine the patients who were hospitalized in our Unit between April and November 2021 and underwent renal biopsy for new-onset urinary abnormalities (UA) and/or renal impairment within 3 months of SARS-CoV-2 vaccination. RESULTS: We identified 17 patients who developed UA and/or renal insufficiency within 3 months of vaccination. Minimal change disease was the most common disease in our cohort (5 patients, 29.4%) followed by acute tubulointerstitial nephritis (TIN; 3 patients, 17.6%), membranous nephropathy (3 patients, 17.6%), and rapidly progressive IgA nephropathy (2 patients, 11.8%). The other 4 patients had a diagnosis of membranoproliferative glomerulonephritis (1 patient), systemic lupus erythematosus (1 patient), ANCA-associated vasculitis (1 patient), and tip-variant focal segmental glomerulosclerosis (1 patient), respectively. Eight out of the 17 patients (47.1%) developed acute kidney injury. Two patients with acute TIN had to start hemodialysis that was discontinued after 1 and 2 months, respectively, due to the recovery of renal function. All patients underwent treatment with corticosteroids and/or immunosuppressants. DISCUSSION: Although it is not possible to conclusively determine whether there is a causal relationship between SARS-CoV-2 vaccination and new-onset nephropathies, based on the appearance of UA and/or renal insufficiency shortly after vaccination, we hypothesize that the immune response to the COVID-19 vaccine may be a trigger of nephropathies. Therefore, our results highlight the need for pharmacovigilance. However, this report should not lead to vaccine hesitation during this pandemic as the benefits of vaccination strongly outweigh the potential risks.

Pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe COVID-19.

OBJECTIVES: No agent has yet been proven to be effective for the treatment of patients with severe COVID-19. METHODS: We conducted a pilot prospective open, single-arm multicentre study on off-label use of tocilizumab (TCZ) involving 63 hospitalised adult patients (56 males, age 62.6±12.5) with severe COVID-19. Clinical and laboratory parameters were prospectively collected at baseline, day 1, 2, 7 and 14. No moderate-to-severe adverse events attributable to TCZ were recorded. RESULTS: We observed a significant improvement in the levels of ferritin, C-reactive protein, D-dimer. The ratio of the partial pressure of oxygen (Pa02) to the fraction of inspired oxygen (Fi02) improved (mean±SD Pa02/Fi02 at admission: 152±53; at day 7: 283.73±115.9, at day 14: 302.2±126, p<0.05). The overall mortality was 11%; D-dimer level at baseline, but not IL-6 levels were predictors of mortality. TCZ administration within 6 days from admission in the hospital was associated with an increased likelihood of survival (HR 2.2 95%CI 1.3-6.7, p<0.05). CONCLUSIONS: In hospitalised adult patients with severe COVID-19, TCZ could be a safe option. An improvement in respiratory and laboratory parameters was observed. Future controlled trials in patients with severe illness are urgently needed to confirm the definite benefit with IL-6 target therapy.

Immunogenetics of complement in mixed cryoglobulinaemia.

OBJECTIVES: A low C4 level is one of the hallmarks of mixed cryoglobulinaemia (MC). However, several reports suggest that other factors may be involved in C4 depletion. The C4 gene is located in a multiallelic CNV locus in the human MHC region. We studied the C4 gene copy number (GCN) and both C4A and C4B isotypes, as well as the presence of the hypofunctional C4A6 allotype (rs41315824) and C4A0 allotype (rs367709216) in 41 MC patients, 16 SLE patients and 78 healthy controls. METHODS: GCN of the C4 gene were evaluated by real time PCR. C4A6 allotype (p.Arg458Trp) and ins 2-bp mutation in exon 29 were screened by primer extension. Correlation with clinical signs of the disease (cutaneous ulcers, peripheral neuropathy, GN, purpura, hepatitis) have been performed by cluster analysis, (K-means algorithm). RESULTS: C4 GCN analysis showed that fewer MC patients had more than 2 copies of the C4A gene as well as a lower C4A gene-copy index (1.90 ± 0.54 vs. 2.21 ± 0.78) as compared to healthy controls. SNP rs41315824 analysis showed a significant increase in the frequency of the p.Arg458Trp (C4A6) variant in cryoglobulinaemic patients. Lastly, cluster analysis allowed us to identify two separate clusters of patients. The cluster that included patients with three or less C4 gene copies was found to have a greater prevalence of the most severe complications such as glomerulonephritis, neuropathy and severe cutaneous ulcers. CONCLUSIONS: These data suggest there may be a relationship between polymorphisms of the C4 gene and clinical presentation.

A HPLC-MS method for the simultaneous quantification of fourteen antiretroviral agents in peripheral blood mononuclear cell of HIV infected patients optimized using medium corpuscular volume evaluation.

A sensitive and accurate high performance liquid chromatography-mass spectrometric (HPLC-MS) method for the intracellular determination of 14 antiretroviral drugs in peripheral blood mononuclear cells (PBMCs) for HIV+ patients was validated. PBMCs are isolated by Ficoll density gradient centrifugation and cells count and the relative mean volume is performed with a Coulter(®) instrument. Extraction of drugs from PBMCs pellets was obtained with methanol:water (70:30, v/v), with quinoxaline added as internal standard, after a sonication step. Supernatant was dried and then dissolved in water/acetonitrile (60/40, v/v), before injection into a 2.1 mm×150 mm Atlantis(®) T3 3µ column. Chromatographic separations were performed using a gradient program with a mixture of water (0.05% formic acid), as mobile phase A and acetonitrile (0.05% formic acid), as mobile phase B. Analytes quantification was performed by electro-spray ionisation-single quadrupole mass spectrometry using the selected ion recording (SIR) detection mode. The positive ionization was used for the HIV protease inhibitors (PIs) indinavir, saquinavir, nelfinavir, nelfinavir M8 metabolite, amprenavir, darunavir, atazanavir, ritonavir, lopinavir, tipranavir, the integrase inhibitor (II) raltegravir and the non-nucleoside reverse transcriptase inhibitors (NNRTIs) nevirapine and etravirine, while the negative ionization is applied for efavirenz. The calibration curves were built using blank PBMCs spiked with antiretroviral drugs at concentrations ranging from 0.1 to 32 ng/mL (1-320 ng/mL for tipranavir) and fitted to a quadratic regression model weighted by 1/X. The mean extraction recovery for all PIs, II and NNRTIs was always above 82%. The method was precise, with a range of intra/inter-day percent standard deviation within 2.6-14.8%, and accurate with mean of percent coefficient of variation (CV%) from nominal values -7.85 to +9.7%. Each drug concentration evaluated was expressed in ng/mL and optimized using each patient medium corpuscolar volume and cell number. This analytical method is routinely used in our clinical research center for the assessment of intracellular levels of all PIs, raltegravir and NNRTIs commercially available at present.

An HPLC-PDA method for the simultaneous quantification of the HIV integrase inhibitor raltegravir, the new nonnucleoside reverse transcriptase inhibitor etravirine, and 11 other antiretroviral agents in the plasma of HIV-infected patients.

A new method using high-performance liquid chromatography coupled with photo diode array detection was developed and validated for the quantification of plasma concentrations of the human immunodeficiency syndrome integrase inhibitor raltegravir (RGV), the new nonnucleoside reverse transcriptase inhibitor etravirine (ETV), and 11 other antiretroviral agents: ritonavir, atazanavir, lopinavir, nevirapine, efavirenz, saquinavir, indinavir, nelfinavir, and its metabolite M-8, amprenavir, and darunavir. A simple solid phase extraction procedure was applied to 500 microL aliquots of plasma, and chromatographic separation of the drugs and internal standard (quinoxaline) was achieved with a gradient (acetonitrile and phosphate buffer) on an C-18 reverse-phase analytical column with a 28-minute analytical run time. Calibration curves were optimized according to expected ranges of drug concentrations in patients, and the coefficient of determination (r) was higher than 0.998 for all analytes. Mean intraday and interday precisions (percent relative SD) for all compounds were 3.67% and 6.39%, respectively, and mean accuracy (percent deviation from nominal concentration) was -1.17%. Extraction recovery ranged within 75% and 83% for all drugs analyzed. The solid phase extraction and high-performance liquid chromatography coupled with photo diode array method described allow a specific, sensitive, and reliable simultaneous determination of RGV, ETV, and 11 antiretroviral agents in plasma by a single assay. Good extraction efficiency and low limit of quantification make this a suitable method for use in clinical trials and for therapeutic drug monitoring of RGV, protease inhibitors, and nonnucleoside reverse transcriptase inhibitors, including ETV.