Significance Statement: Lupus nephritis (LN) is the major cause of death among systemic lupus erythematosus patients, with heterogeneous phenotypes and different responses to therapy. Identifying genetic causes and finding potential therapeutic targets of LN is a major unmet clinical need. We identified a novel DDX58 pathogenic variant, R109C, that leads to RIG-I hyperactivation and type I IFN signaling upregulation by disrupting RIG-I autoinhibition, causing LN, which may respond to a JAK inhibitor. Genetic testing of families with multiple cases of LN that identifies this variant may lead to targeted therapy. Background: Lupus nephritis (LN) is one of the most severe complications of systemic lupus erythematosus, with heterogeneous phenotypes and different responses to therapy. Identifying genetic causes of LN can facilitate more individual treatment strategies. Methods: We performed whole-exome sequencing in a cohort of Chinese patients with LN and identified variants of a disease-causing gene. Extensive biochemical, immunologic, and functional analyses assessed the effect of the variant on type I IFN signaling. We further investigated the effectiveness of targeted therapy using single-cell RNA sequencing. Results: We identified a novel DDX58 pathogenic variant, R109C, in five unrelated families with LN. The DDX58 R109C variant is a gain-of-function mutation, elevating type I IFN signaling due to reduced autoinhibition, which leads to RIG-I hyperactivation, increased RIG-I K63 ubiquitination, and MAVS aggregation. Transcriptome analysis revealed an increased IFN signature in patient monocytes. Initiation of JAK inhibitor therapy (baricitinib 2 mg/d) effectively suppressed the IFN signal in one patient. Conclusions: A novel DDX58 R109C variant that can cause LN connects IFNopathy and LN, suggesting targeted therapy on the basis of pathogenicity.

Background: The PAXgene Blood RNA tube is used to protect RNA of whole blood, and the stability of RNA in this tube has already been reported. However, there are few reports on the quality of RNA from long-term preservation of these tubes. Materials and Methods: Our biobank conducted quality control of RNA extracted from the tubes after varying storage periods. A total of 300 blood samples of renal disease patients were randomly selected at each time point (from 1 month to 7 years). Results: Median RNA yields were 3.46 (2.65–4.87) μg, 4.34 (3.20–5.87) μg, 4.77 (2.88–6.29) μg, 4.19 (2.65–6.26) μg, 3.85 (2.43–6.13) μg, and 3.21 (1.85–6.61) μg at 1 month, 1, 2, 3, 6, and 7 years, respectively. There were no significant differences in RNA yields among all the storage periods. A _260/280 ratios were 2.02 ± 0.04, 2.05 ± 0.04, 2.05 ± 0.04, 2.08 ± 0.03, 2.12 ± 0.10, and 2.11 ± 0.05, all of which were ≥1.8. However, A _260/280 of the samples stored for 6 and 7 years had a rising trend, compared with the other time points ( p < 0.05). Median RNA integrity number (RIN) values were 8.4 (7.6–9.1), 8.3 (7.7–8.9), 8.3 (7.8–8.8), 8.5 (8.3–8.9), 7.6 (7.1–8.1), and 7.9 (7.2–8.3) at each time point. Lower RIN values were found at 6 and 7 years compared with the other storage periods ( p < 0.05). The rates of RIN values ≥7.0 were 92%, 84%, 96%, 92%, 86%, and 84%, which exhibited no differences across all the storage periods. In addition, the yields and RIN values of RNA from samples with blood clots were significantly lower than those without ( p < 0.001). Conclusions: The quantity and quality of RNA extracted from PAXgene Blood RNA tubes are stable throughout cryopreservation for 7 years.

Background: Patients who recover from acute kidney injury (AKI) have a 25% increase in the risk of chronic kidney disease (CKD) and a 50% increase in mortality after a follow-up of approximately 10 years. Circulating FGF-23 increases significantly early in the development of AKI, is significantly elevated in patients with CKD and has become a major biomarker of poor clinical prognosis in CKD. However, the potential link between fibroblast growth factor-23 levels and the progression of AKI to CKD remains unclear. Method: Serum FGF-23 levels in AKI patients and ischaemia‒reperfusion injury (IRI) mice were detected with ELISA. Cultured HK2 cells were incubated with FGF-23 and PD173074, a blocker of FGFR, and then TGFβ/Smad and Wnt/β-catenin were examined with immunofluorescence and immunoblotting. Quantitative real-time polymerase chain reaction was used to detect the expression of COL1A1 and COL4A1. Histologic staining confirmed renal fibrosis. Results: The level of serum FGF-23 was significantly different between AKI patients and healthy controls (P < 0.01). Moreover, serum FGF-23 levels in the CKD progression group were significantly higher than those in the non-CKD progression group of AKI patients (P < 0.01). In the AKI-CKD mouse model, serum FGF-23 levels were increased, and renal fibrosis occurred; moreover, the protein expression of β-catenin and p-Smad3 was upregulated. PD173074 downregulated the expression of β-catenin and p-Smad3 and reduced fibrosis in both mice and HK2 cells. Conclusion: The increase in FGF-23 may be associated with the progression of AKI to CKD and may mediate renal fibrosis via TGF-β and Wnt/β-catenin activation.

Left ventricular hypertrophy (LVH) is a primary feature of cardiovascular complications in patients with chronic kidney disease (CKD). miRNA-30 is an important posttranscriptional regulator of LVH, but it is unknown whether miRNA-30 participates in the process of CKD-induced LVH. In the present study, we found that CKD not only resulted in LVH but also suppressed miRNA-30 expression in the myocardium. Rescue of cardiomyocyte-specific miRNA-30 attenuated LVH in CKD rats without altering CKD progression. Importantly, in vivo and in vitro knockdown of miRNA-30 in cardiomyocytes led to cardiomyocyte hypertrophy by upregulating the calcineurin signaling directly. Furthermore, CKD-related detrimental factors, such as fibroblast growth factor-23, uremic toxin, angiotensin II, and transforming growth factor–β, suppressed cardiac miRNA-30 expression, while miRNA-30 supplementation blunted cardiomyocyte hypertrophy induced by such factors. These results uncover a potentially novel mechanism of CKD-induced LVH and provide a potential therapeutic target for CKD patients with LVH.

Background: Fibroblast growth factor 23 (FGF23) has become increasingly important in chronic kidney diseases (CKDs), cardiovascular calcification, and metabolic bone diseases. Fresh or stored blood samples are widely used for the FGF23 assay. Clarifying the factors influencing the FGF23 assay can help to quantify FGF23 more accurately. This study explored the effects of low-temperature storage time and repeated freeze–thaw cycles on the measurement of serum intact FGF23 (iFGF23). Materials and Methods: We selected 60 serum samples from patients with CKD stages 3–5 and hemodialysis patients. An enzyme-linked immunosorbent assay was used to measure the changes in serum iFGF23 levels after 6 years of storage at −80°C. In total, 18 fresh serum samples were frozen and thawed for 0, 1, 3, and 5 cycles to explore the effects of repeated freeze–thaw cycles on serum iFGF23 levels. Results: Median serum iFGF23 concentrations were 252.17 (interquartile range [IQR] 113.82–592.38) pg/mL and 203.85 (IQR 64.76–545.39) pg/mL before and after 6 years. There were no significant differences between them. However, we found a downward trend of 48% in the samples close to the normal level of iFGF23 (<150.34 pg/mL) after 6 years of storage ( p = 0.160). In addition, the iFGF23 levels of samples frozen and thawed for 0, 1, 3, and 5 cycles were 278.41 ± 39.51 (mean ± standard deviation) pg/mL, 262.84 ± 38.42 pg/mL, 252.97 ± 34.65 pg/mL and 250.49 ± 37.12 pg/mL, respectively. A slight downward trend in iFGF23 levels was observed with increasing freeze–thaw times; however, no significant differences were found among different freeze–thaw cycles. Conclusion: Serum iFGF23 levels remained stable after storage at −80°C for 6 years. In addition, five freeze–thaw cycles had no significant effects on serum iFGF23 levels.

Background: Cryopreserved whole blood, all-cell pellets (ACPs), and buffy coats in biobanks are widely used to obtain DNA for genetic testing. However, there are few studies concerning the quality control of DNA extracted from them. Our research aimed to perform quality control of DNA extracted from ACPs after cryopreservation for >10 years. Materials and Methods: A total of 1377 ACP samples (separated from 3 mL of whole blood) were retrieved from our biobank, where they had been cryopreserved for 10–15 years. Chemagic STAR was used to extract the DNA. Absorbance at A260, A280, and A230 were measured by spectrophotometry, and integrity was analyzed by agarose gel electrophoresis. The quality thresholds for an Illumina Asian Screening Array (ASA) were yields greater than 0.5 μg, concentration of 25–150 ng/μL, A260/280 ratio of 1.6–2.1, and no degradation fragments in the electrophoresis gel. Results: The median yield of genomic DNA was 54.30 μg (interquartile range [IQR] 35.55–74.64). The median A260/280 and A260/230 ratios were 1.90 (IQR 1.87–1.94) and 1.98 (IQR 1.64–2.41), respectively. In total, 1377 samples (100%) had qualified yields, and 1366 samples (99.20%) had qualified integrity results. Finally, 1328 (96.44%) samples were used for ASA. Of the remaining samples, 34 needed to be repurified, 4 were obtained at an insufficient concentration, and 11 were unqualified for integrity. In addition, we analyzed the influence of hemolysis (90 samples) and clots (102 samples) on the quality of DNA samples. Hemolysis and clotting did not influence yield or integrity, but a significant difference was found in A260/230 compared to normal samples (p < 0.05). Furthermore, the samples (14 samples) with both hemolysis and clots had higher A260/280 (p < 0.05). Conclusion: ACP samples stored for >10 years at −80°C produced DNA with high quality for use in genetic analysis. Hemolysis and clots in the ACPs led to lower purity, but did not significantly affect yield or integrity.