Risk Stratification to Predict Renal Survival in Anti-Glomerular Basement Membrane Disease.

SIGNIFICANCE STATEMENT: Most patients with anti-glomerular basement membrane (GBM) disease present with rapidly progressive glomerulonephritis, and more than half develop ESKD. Currently, no tools are available to aid in the prognostication or management of this rare disease. In one of the largest assembled cohorts of patients with anti-GBM disease (with 174 patients included in the final analysis), the authors demonstrated that the renal risk score for ANCA-associated vasculitis is transferable to anti-GBM disease and the renal histology is strongly predictive of renal survival and recovery. Stratifying patients according to the percentage of normal glomeruli in the kidney biopsy and the need for RRT at the time of diagnosis improves outcome prediction. Such stratification may assist in the management of anti-GBM disease. BACKGROUND: Prospective randomized trials investigating treatments and outcomes in anti-glomerular basement membrane (anti-GBM) disease are sparse, and validated tools to aid prognostication or management are lacking. METHODS: In a retrospective, multicenter, international cohort study, we investigated clinical and histologic parameters predicting kidney outcome and sought to identify patients who benefit from rescue immunosuppressive therapy. We also explored applying the concept of the renal risk score (RRS), currently used to predict renal outcomes in ANCA-associated vasculitis, to anti-GBM disease. RESULTS: The final analysis included 174 patients (out of a total of 191). Using Cox and Kaplan-Meier methods, we found that the RRS was a strong predictor for ESKD. The 36-month renal survival was 100%, 62.4%, and 20.7% in the low-risk, moderate-risk, and high-risk groups, respectively. The need for renal replacement therapy (RRT) at diagnosis and the percentage of normal glomeruli in the biopsy were independent predictors of ESKD. The best predictor for renal recovery was the percentage of normal glomeruli, with a cut point of 10% normal glomeruli providing good stratification. A model with the predictors RRT and normal glomeruli ( N ) achieved superior discrimination for significant differences in renal survival. Dividing patients into four risk groups led to a 36-month renal survival of 96.4% (no RRT, N ≥10%), 74.0% (no RRT, N <10%), 42.3% (RRT, N ≥10%), and 14.1% (RRT, N <10%), respectively. CONCLUSIONS: These findings demonstrate that the RRS concept is transferrable to anti-GBM disease. Stratifying patients according to the need for RRT at diagnosis and renal histology improves prediction, highlighting the importance of normal glomeruli. Such stratification may assist in the management of anti-GBM disease. PODCAST: This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/JASN/2023_02_27_JASN0000000000000060.mp3.

A Case of IgG4-Related Disease and Membranous Nephropathy Associated with Thrombospondin Type-1 Domain-Containing 7A.

Background: IgG4-related disease (IgG4-RD) is a systemic multi-organ inflammatory disorder which affects the kidney 20% of the time. Patients with intrinsic IgG4-related kidney disease (IgG4-RKD) often have tubulointerstitial nephritis (TIN) whereas glomerular lesions like membranous nephropathy (MN) are less common. Antibodies to thrombospondin type-1 domain-containing 7A (THSD7A) have been described in primary MN, but never in association with IgG4-RKD. Case Report: We report the first case of IgG4-MN associated with THSD7A antibodies in serum and positivity on glomerular staining, in a 57-year-old Caucasian male with IgG4-RD affecting the pancreas, liver, lacrimal glands, extraocular muscles, and kidneys. This patient presented initially with glomerular disease including significant proteinuria consistent with MN. Glomerular staining for THSD7A antigen and serum THSD7A antibody titres was positive. Treatment with corticosteroids and cyclophosphamide successfully induced remission with resolution of proteinuria, and improvement in renal function. However, despite maintenance azathioprine, the patient relapsed 39 months later. On relapse, there was minimal proteinuria but a significant rise in creatinine. Subsequent renal biopsy showed less glomerular disease and instead a TIN pattern. Subsequent treatment with Rituximab and corticosteroids successfully induced remission. Conclusion: The role of THSD7A autoantibodies in MN is emerging, and as both IgG4-MN and presence of THSD7A antibody are rare occurrences in themselves, we speculate that there may be an undiscovered association between THSD7A and IgG4-MN. Routine testing for THSD7A in IgG4-MN may help to identify the link.

Gene Expression Profiling in Kidney Transplants with Immune Checkpoint Inhibitor-Associated Adverse Events.

BACKGROUND AND OBJECTIVES: Immune checkpoint inhibitors are increasingly used to treat various malignancies, but their application in patients with kidney transplants is complicated by high allograft rejection rates. Immune checkpoint inhibitor-associated rejection is a novel, poorly understood entity demonstrating overlapping histopathologic features with immune checkpoint inhibitor-associated acute interstitial nephritis, which poses a challenge for diagnosis and clinical management. We sought to improve the understanding of these entities through biopsy-based gene expression analysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS: NanoString was used to measure and compare the expression of 725 immune-related genes in 75 archival kidney biopsies, including a 25-sample discovery cohort comprising pure T cell-mediated rejection and immune checkpoint inhibitor-associated acute interstitial nephritis and an independent 50-sample validation cohort comprising immune checkpoint inhibitor-associated acute interstitial nephritis, immune checkpoint inhibitor-associated T cell-mediated rejection, immune checkpoint inhibitor-associated crescentic GN, drug-induced acute interstitial nephritis, BK virus nephropathy, and normal biopsies. RESULTS: Significant molecular overlap was observed between immune checkpoint inhibitor-associated acute interstitial nephritis and T cell-mediated rejection. Nevertheless, IFI27, an IFN-α-induced transcript, was identified and validated as a novel biomarker for differentiating immune checkpoint inhibitor-associated T cell-mediated rejection from immune checkpoint inhibitor-associated acute interstitial nephritis (validation cohort: P<0.001, area under the receiver operating characteristic curve =100%, accuracy =86%). Principal component analysis revealed heterogeneity in inflammatory gene expression patterns within sample groups; however, immune checkpoint inhibitor-associated T cell-mediated rejection and immune checkpoint inhibitor-associated acute interstitial nephritis both demonstrated relatively more molecular overlap with drug-induced acute interstitial nephritis than T cell-mediated rejection, suggesting potential dominance of hypersensitivity mechanisms in these entities. CONCLUSIONS: These results indicate that, although there is significant molecular similarity between immune checkpoint inhibitor-associated rejection and acute interstitial nephritis, biopsy-based measurement of IFI27 gene expression represents a potential biomarker for differentiating these entities.

Intragraft gene expression in native kidney BK virus nephropathy versus T cell-mediated rejection: Prospects for molecular diagnosis and risk prediction.

Novel tools are needed to improve diagnostic accuracy and risk prediction in BK virus nephropathy (BKVN). We assessed the utility of intragraft gene expression testing for these purposes. Eight hundred genes were measured in 110 archival samples, including a discovery cohort of native kidney BKVN (n = 5) vs pure T cell-mediated rejection (TCMR; n = 10). Five polyomavirus genes and seven immune-related genes (five associated with BKVN and two associated with TCMR) were significantly differentially expressed between these entities (FDR < 0.05). These three sets of genes were further evaluated in samples representing a spectrum of BK infection (n = 25), followed by a multicenter validation cohort of allograft BKVN (n = 60) vs TCMR (n = 10). Polyomavirus 5-gene set expression reliably distinguished BKVN from TCMR (validation cohort AUC = 0.992), but the immune gene sets demonstrated suboptimal diagnostic performance (AUC ≤ 0.720). Within the validation cohort, no significant differences in index biopsy gene expression were identified between BKVN patients demonstrating resolution (n = 35), persistent infection (n = 14) or de novo rejection (n = 11) 6 months following a standardized reduction in immunosuppression. These results suggest that, while intragraft polyomavirus gene expression may be useful as an ancillary diagnostic for BKVN, assessment for concurrent TCMR and prediction of clinical outcome may not be feasible with current molecular tools.

The role of Thr268 and Phe393 in cytochrome P450 BM3.

In flavocytochrome P450 BM3 there are several active site residues that are highly conserved throughout the P450 superfamily. Of these, a phenylalanine (Phe393) has been shown to modulate heme reduction potential through interactions with the implicitly conserved heme-ligand cysteine. In addition, a distal threonine (Thr268) has been implicated in a variety of roles including proton donation, oxygen activation and substrate recognition. Substrate binding in P450 BM3 causes a shift in the spin state from low- to high-spin. This change in spin-state is accompanied by a positive shift in the reduction potential (DeltaE(m) [WT+arachidonate (120 microM)]=+138 mV). Substitution of Thr268 by an alanine or asparagine residue causes a significant decrease in the ability of the enzyme to generate the high-spin complex via substrate binding and consequently leads to a decrease in the substrate-induced potential shift (DeltaE(m) [T268A+arachidonate (120 microM)]=+73 mV, DeltaE(m) [T268N+arachidonate (120 microM)]=+9 mV). Rate constants for the first electron transfer and for oxy-ferrous decay were measured by pre-steady-state stopped-flow kinetics and found to be almost entirely dependant on the heme reduction potential. More positive reduction potentials lead to enhanced rate constants for heme reduction and more stable oxy-ferrous species. In addition, substitutions of the threonine lead to an increase in the production of hydrogen peroxide in preference to hydroxylated product. These results suggest an important role for this active site threonine in substrate recognition and in maintaining an efficiently functioning enzyme. However, the dependence of the rate constants for oxy-ferrous decay on reduction potential raises some questions as to the importance of Thr268 in iron-oxo stabilisation.

Redox behaviour of the haem domain of flavocytochrome c3 from Shewanella frigidimarina probed by NMR.

Flavocytochrome c3 from Shewanella frigidimarina (fcc3) is a tetrahaem periplasmic protein of 64 kDa with fumarate reductase activity. This work reports the first example of NMR techniques applied to the assignment of the thermodynamic order of oxidation of the four individual haems for such large protein, expanding its applicability to a wide range of proteins. NMR data from partially and fully oxidised samples of fcc3 and a mutated protein with an axial ligand of haem IV replaced by alanine were compared with calculated chemical shifts, allowing the structural assignment of the signals and the unequivocal determination of the order of oxidation of the haems. As oxidation progresses the fcc3 haem domain is polarised, with haems I and II much more oxidised than haems III and IV, haem IV being the most reduced. Thus, during catalysis as an electron is taken by the flavin adenosine dinucleotide from haem IV, haem III is eager to re-reduce haem IV, allowing the transfer of two electrons to the active site.

Expression, purification and spectroscopic characterization of the cytochrome P450 CYP121 from Mycobacterium tuberculosis.

The CYP121 gene from the pathogenic bacterium Mycobacterium tuberculosis has been cloned and expressed in Escherichia coli, and the protein purified to homogeneity by ion exchange and hydrophobic interaction chromatography. The CYP121 gene encodes a cytochrome P450 enzyme (CYP121) that displays typical electronic absorption features for a member of this superfamily of hemoproteins (major Soret absorption band at 416.5 nm with alpha and beta bands at 565 and 538 nm, respectively, in the oxidized form) and which binds carbon monoxide to give the characteristic Soret band shift to 448 nm. Resonance Raman, EPR and MCD spectra show the protein to be predominantly low-spin and to have a typical cysteinate- and water-ligated b-type heme iron. CD spectra in the far UV region describe a mainly alpha helical conformation, but the visible CD spectrum shows a band of positive sign in the Soret region, distinct from spectra for other P450s recognized thus far. CYP121 binds very tightly to a range of azole antifungal drugs (e.g. clotrimazole, miconazole), suggesting that it may represent a novel target for these antibiotics in the M. tuberculosis pathogen.