Targeting immune cell glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1) to prevent fibrosis after tubulointerstitial nephritis.

Glutamyl-prolyl-transfer RNA synthetase 1 is an enzyme that connects glutamic acid and proline to transfer RNA during protein synthesis. In this issue, a study by Kang et al. examined the role of the immune cell glutamyl-prolyl-transfer RNA synthetase 1 in toxin-induced tubulointerstitial nephritis mice. The study demonstrated that blocking glutamyl-prolyl-transfer RNA synthetase 1 may be a therapeutic target to attenuate fibrosis after toxin-induced tubulointerstitial nephritis.

Autosomal dominant chronic tubulointerstitial nephropathy: do not forget amyloidosis.

Amyloidosis is a rare cause of inherited kidney disease, with most variants responsible for prominent glomerular involvement. In this issue, Kmochová et al. reported the first description of autosomal dominant medullary amyloidosis due to apolipoprotein A4 variants, resulting in slowly progressive chronic kidney disease with minimal proteinuria. Combining next-generation sequencing with histopathological studies incorporating Congo red staining and mass spectrometry should be considered in the diagnostic workup of hereditary tubulointerstitial disorders not identified after routine genetic testing.

Glutamyl-prolyl-tRNA synthetase (EPRS1) drives tubulointerstitial nephritis-induced fibrosis by enhancing T cell proliferation and activity.

Toxin- and drug-induced tubulointerstitial nephritis (TIN), characterized by interstitial infiltration of immune cells, frequently necessitates dialysis for patients due to irreversible fibrosis. However, agents modulating interstitial immune cells are lacking. Here, we addressed whether the housekeeping enzyme glutamyl-prolyl-transfer RNA synthetase 1 (EPRS1), responsible for attaching glutamic acid and proline to transfer RNA, modulates immune cell activity during TIN and whether its pharmacological inhibition abrogates fibrotic transformation. The immunological feature following TIN induction by means of an adenine-mixed diet was infiltration of EPRS1high T cells, particularly proliferating T and γδ T cells. The proliferation capacity of both CD4+ and CD8+ T cells, along with interleukin-17 production of γδ T cells, was higher in the kidneys of TIN-induced Eprs1+/+ mice than in the kidneys of TIN-induced Eprs1+/- mice. This discrepancy contributed to the fibrotic amelioration observed in kidneys of Eprs1+/- mice. TIN-induced fibrosis was also reduced in Rag1-/- mice adoptively transferred with Eprs1+/- T cells compared to the Rag1-/- mice transferred with Eprs1+/+ T cells. The use of an EPRS1-targeting small molecule inhibitor (bersiporocin) under clinical trials to evaluate its therapeutic potential against idiopathic pulmonary fibrosis alleviated immunofibrotic aggravation in TIN. EPRS1 expression was also observed in human kidney tissues and blood-derived T cells, and high expression was associated with worse patient outcomes. Thus, EPRS1 may emerge as a therapeutic target in toxin- and drug-induced TIN, modulating the proliferation and activity of infiltrated T cells.

Autosomal dominant ApoA4 mutations present as tubulointerstitial kidney disease with medullary amyloidosis.

Sporadic cases of apolipoprotein A-IV medullary amyloidosis have been reported. Here we describe five families found to have autosomal dominant medullary amyloidosis due to two different pathogenic APOA4 variants. A large family with autosomal dominant chronic kidney disease (CKD) and bland urinary sediment underwent whole genome sequencing with identification of a chr11:116692578 G>C (hg19) variant encoding the missense mutation p.L66V of the ApoA4 protein. We identified two other distantly related families from our registry with the same variant and two other distantly related families with a chr11:116693454 C>T (hg19) variant encoding the missense mutation p.D33N. Both mutations are unique to affected families, evolutionarily conserved and predicted to expand the amyloidogenic hotspot in the ApoA4 structure. Clinically affected individuals suffered from CKD with a bland urinary sediment and a mean age for kidney failure of 64.5 years. Genotyping identified 48 genetically affected individuals; 44 individuals had an estimated glomerular filtration rate (eGFR) under 60 ml/min/1.73 m2, including all 25 individuals with kidney failure. Significantly, 11 of 14 genetically unaffected individuals had an eGFR over 60 ml/min/1.73 m2. Fifteen genetically affected individuals presented with higher plasma ApoA4 concentrations. Kidney pathologic specimens from four individuals revealed amyloid deposits limited to the medulla, with the mutated ApoA4 identified by mass-spectrometry as the predominant amyloid constituent in all three available biopsies. Thus, ApoA4 mutations can cause autosomal dominant medullary amyloidosis, with marked amyloid deposition limited to the kidney medulla and presenting with autosomal dominant CKD with a bland urinary sediment. Diagnosis relies on a careful family history, APOA4 sequencing and pathologic studies.

Cellular senescence in kidney biopsies is associated with tubular dysfunction and predicts CKD progression in childhood cancer patients with karyomegalic interstitial nephropathy.

Karyomegalic interstitial nephropathy (KIN) has been reported as an incidental finding in patients with childhood cancer treated with ifosfamide. It is defined by the presence of tubular epithelial cells (TECs) with enlarged, irregular, and hyperchromatic nuclei. Cellular senescence has been proposed to be involved in kidney fibrosis in hereditary KIN patients. We report that KIN could be diagnosed 7-32 months after childhood cancer diagnosis in 6/6 consecutive patients biopsied for progressive chronic kidney disease (CKD) of unknown cause between 2018 and 2021. The morphometry of nuclear size distribution and markers for DNA damage (γH2AX), cell-cycle arrest (p21+, Ki67-), and nuclear lamina decay (loss of lamin B1), identified karyomegaly and senescence features in TECs. Polyploidy was assessed by chromosome fluorescence in situ hybridization (FISH). In all six patients the number of p21-positive TECs far exceeded the typically small numbers of truly karyomegalic cells, and p21-positive TECs contained less lysozyme, testifying to defective resorption, which explains the consistently observed low-molecular-weight (LMW) proteinuria. In addition, polyploidy of TEC was observed to correlate with loss of lysozyme staining. Importantly, in the five patients with the largest nuclei, the percentage of p21-positive TECs tightly correlated with estimated glomerular filtration rate loss between biopsy and last follow-up (R2 = 0.93, p < 0.01). We conclude that cellular senescence is associated with tubular dysfunction and predicts CKD progression in childhood cancer patients with KIN and appears to be a prevalent cause of otherwise unexplained CKD and LMW proteinuria in children treated with DNA-damaging and cell stress-inducing therapy including ifosfamide. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Genome-Wide Association Study in Acute Tubulointerstitial Nephritis.

SIGNIFICANCE STATEMENT: Polymorphisms of HLA genes may confer susceptibility to acute tubulointerstitial nephritis (ATIN), but small sample sizes and candidate gene design have hindered their investigation. The first genome-wide association study of ATIN identified two significant loci, risk haplotype DRB1*14-DQA1*0101-DQB1*0503 (DR14 serotype) and protective haplotype DRB1*1501-DQA1*0102-DQB1*0602 (DR15 serotype), with amino acid position 60 in the peptide-binding groove P10 of HLA-DR ß 1 key. Risk alleles were shared among different causes of ATIN and HLA genotypes associated with kidney injury and immune therapy response. HLA alleles showed the strongest association. The findings suggest that a genetically conferred risk of immune dysregulation is part of the pathogenesis of ATIN. BACKGROUND: Acute tubulointerstitial nephritis (ATIN) is a rare immune-related disease, accounting for approximately 10% of patients with unexplained AKI. Previous elucidation of the relationship between genetic factors that contribute to its pathogenesis was hampered because of small sample sizes and candidate gene design. METHODS: We undertook the first two-stage genome-wide association study and meta-analysis involving 544 kidney biopsy-defined patients with ATIN and 2346 controls of Chinese ancestry. We conducted statistical fine-mapping analysis, provided functional annotations of significant variants, estimated single nucleotide polymorphism (SNP)-based heritability, and checked genotype and subphenotype correlations. RESULTS: Two genome-wide significant loci, rs35087390 of HLA-DQA1 ( P =3.01×10 -39 ) on 6p21.32 and rs2417771 of PLEKHA5 on 12p12.3 ( P =2.14×10 -8 ), emerged from the analysis. HLA imputation using two reference panels suggested that HLA-DRB1*14 mainly drives the HLA risk association . HLA-DRB1 residue 60 belonging to pocket P10 was the key amino acid position. The SNP-based heritability estimates with and without the HLA locus were 20.43% and 10.35%, respectively. Different clinical subphenotypes (drug-related or tubulointerstitial nephritis and uveitis syndrome) seemed to share the same risk alleles. However, the HLA risk genotype was associated with disease severity and response rate to immunosuppressive therapy. CONCLUSIONS: We identified two candidate genome regions associated with susceptibility to ATIN. The findings suggest that a genetically conferred risk of immune dysregulation is involved in the pathogenesis of ATIN.

Heterozygous Variants in a Patient with Karyomegalic Interstitial Nephritis.

Karyomegalic interstitial nephritis (KIN) is a rare kidney disease marked by large tubular nuclei, interstitial inflammation, tubular atrophy, and interstitial fibrosis. The current study presented the case of a 39-year-old man with deteriorating kidney function and a serum creatinine level of 2.08 mg/dL. The renal biopsy revealed that the main pathological features of renal tubular epithelial cells were obvious enlargement, irregular shape, and hyperchromatic nuclei. The genetic analysis of the patient revealed two heterozygous variants in the FAN1 gene, c.2485c>T, and c.2928dupT, located in exons 10 and 13, respectively. A diagnosis of KIN was rendered. The two variations of the proband are identified in separate alleles from the father and mother, respectively, according to his family's sequencing. This phenotype is consistent with an autosomal recessive pattern of inheritance. The patient was treated with the Chinese patent medicine Niaoduqing Particles. After 38 months of follow-up, renal function was barely changed with a serum creatinine of 1.73 mg/dL.

Granulomatous interstitial nephritis with CTLA-4 haploinsufficiency: a case report.

BACKGROUND: Cytotoxic T lymphocyte antigen-4 (CTLA-4) is an essential inhibitory regulator of immune activation. CTLA-4 haploinsufficiency is known to be associated with dysregulation of FOXP3+ regulatory T cells, hyperactivation of effector T cells, and lymphocytic infiltration of multiple organs. However, there have only been a few reports of renal involvement with CTLA-4. Herein, we present a case of acute granulomatous tubulointerstitial nephritis (TIN) in a patient with CTLA-4 haploinsufficiency. CASE PRESENTATION: A 44-year-old man presented with a 3-week history of fever and malaise, and subsequently developed acute kidney injury (AKI) a few days after treatment with levofloxacin (LVFX). A kidney biopsy and immunohistochemical staining revealed granulomatous TIN with dominantly infiltrating CD4+ T cells. General symptoms and renal impairment showed improvement after discontinuation of LVFX and initiation of oral steroids. However, they worsened following steroid tapering. Further, a colon biopsy analysis showed similar findings to the renal tissue analysis. We suspected that granulomatous TIN was possibly associated with CTLA-4 haploinsufficiency. Therefore, the patient was transferred to another hospital for further treatment of CTLA-4 haploinsufficiency using immunosuppressive agents. CONCLUSIONS: There have been few reports regarding renal involvement of CTLA-4 haploinsufficiency. In the present case, granulomatous TIN could have arisen due to instability of immune regulatory functions, such as CTLA-4 haploinsufficiency, and treatment with LVFX could have triggered immunologic activation and severe inflammation as well as renal dysfunction.

Polyploid tubular cells and chronic kidney disease.

Defective DNA repair drives chronic kidney disease (CKD), but mechanisms are unclear. Airik and colleagues use a genetic model of defective DNA repair mimicking karyomegalic nephritis, a form of CKD characterized by tubular epithelial cells (TEC) with large nuclei and tubulointerstitial nephritis. They show that DNA damage in TEC triggers endoreplication leading to polyploid TEC and CKD. Blocking endoreplication preserved kidney function, suggesting that DNA damage triggers CKD via TEC polyploidization, questioning the concept of G2/M-arrest.

Prevalence of hereditary tubulointerstitial kidney diseases in the German Chronic Kidney Disease study.

Hereditary chronic kidney disease (CKD) appears to be more frequent than the clinical perception. Exome sequencing (ES) studies in CKD cohorts could identify pathogenic variants in ~10% of individuals. Tubulointerstitial kidney diseases, showing no typical clinical/histologic finding but tubulointerstitial fibrosis, are particularly difficult to diagnose. We used a targeted panel (29 genes) and MUC1-SNaPshot to sequence 271 DNAs, selected in defined disease entities and age cutoffs from 5217 individuals in the German Chronic Kidney Disease cohort. We identified 33 pathogenic variants. Of these 27 (81.8%) were in COL4A3/4/5, the largest group being 15 COL4A5 variants with nine unrelated individuals carrying c.1871G>A, p.(Gly624Asp). We found three cysteine variants in UMOD, a novel missense and a novel splice variant in HNF1B and the homoplastic MTTF variant m.616T>C. Copy-number analysis identified a heterozygous COL4A5 deletion, and a HNF1B duplication/deletion, respectively. Overall, pathogenic variants were present in 12.5% (34/271) and variants of unknown significance in 9.6% (26/271) of selected individuals. Bioinformatic predictions paired with gold standard diagnostics for MUC1 (SNaPshot) could not identify the typical cytosine duplication ("c.428dupC") in any individual, implying that ADTKD-MUC1 is rare. Our study shows that >10% of selected individuals carry disease-causing variants in genes partly associated with tubulointerstitial kidney diseases. COL4A3/4/5 genes constitute the largest fraction, implying they are regularly overlooked using clinical Alport syndrome criteria and displaying the existence of phenocopies. We identified variants easily missed by some ES pipelines. The clinical filtering criteria applied enriched for an underlying genetic disorder.

Upregulation of C/EBP Homologous Protein induced by ER Stress Mediates Epithelial to Myofibroblast Transformation in ADTKD-UMOD.

Autosomal dominant tubulointerstitial kidney disease due to UMOD mutations (ADTKD-UMOD) results in chronic interstitial nephritis, which gradually develops into end-stage renal disease. It is believed that the accumulation of mutant uromodulin causes the endoplasmic reticulum (ER) stress, then leads to the kidney damage. But the underlying mechanism remains unclear. To find the ADTKD-UMOD patients, UMOD gene screening was performed in 26 patients with unexplained chronic interstitial nephritis, during the past 10 years in our department, and among them three ADTKD-UMOD cases were discovered. Routine pathological staining and electron microscopy sections were reviewed again to confirm their kidney lesions. Immunostaining of UMOD and ER stress marker GRP78, as well as CHOP have all been done. The strong colocalization of UMOD with GRP78 and CHOP in ADTKD-UMOD patients but not in other chronic interstitial nephritis patients had been found. Moreover in vitro experiments, ER stress induced by tunicamycin (TM) not only significantly increased the expression of GRP78 and CHOP, but also caused the epithelial to myofibroblast transformation (EMT) of renal tubular epithelial cells, evidenced by decreased expression of E-cadherin and increased expression of vimentin, and extracellular matrix (ECM) deposition, evidenced by increased expression of fibronectin (FN). CHOP knockdown could restore the upregulation of vimentin and FN induced by TM. Thus, specific activation of CHOP in renal tubular epithelial cells induced by UMOD protein might be the key reason of renal interstitial fibrosis in ADTKD-UMOD patients.

Pirfenidone Attenuates Renal Tubulointerstitial Fibrosis through Inhibiting miR-21.

BACKGROUND: Our previous studies had shown pirfenidone (PFD) not only improved tubulointerstitial fibrosis (TIF) but also inhibited the expression of microRNA-21 (miR-21) in the renal tissue of unilateral urethral obstruction (UUO) rats. This study aims to investigate whether PFD can attenuate TIF through inhibiting miR-21 in UUO rats. METHODS: Sprague Dawley rats were divided randomly into sham-operated group, UUO group, and PFD and olmesartan (Olm) treatment groups. Samples were collected on day 14. Expression of miR-21, TGF-ß1, Smad3, and Smad7 mRNA in the renal tissue was detected using real-time quantitative PCR. Immunohistochemistry was performed to assess the protein expressions of collagen III, E-cadherin, and α-SMA. Automated capillary Western blotting was used to detect the quantitative expression of TGF-ß1, Smad3, p-Smad3, Smad7, collagen III, E-cadherin, and α-SMA in renal tissues. The expression of miR-21 and Smad7 mRNA and the protein levels of collagen III and α-SMA were examined in the miR-21-overexpressing cell line, NRK-52E. RESULTS: Compared with the UUO group, both PFD and Olm inhibited renal tubular dilation, diffused epithelial cell degeneration and necrosis, and reduced renal interstitial edema, inflammatory cell infiltration, and collagen fiber deposition, while no significant difference between PFD group and Olm group. Informatics-based approaches identified Smad7 as a likely candidate for regulation by miR-21. Compared with the sham group, miR-21 expression was upregulated in the UUO group resulting in the downregulation of Smad7 expression due to degradation. The overexpression of miR-21 in the in vitro model downregulated Smad7 and promoted EMT and ECM accumulation. Protein levels of TGF-ß1, Smad3, p-Smad3, collagen III, and α-SMA were upregulated, while E-cadherin protein was downregulated in the UUO group than in the sham group. PFD rather than Olm decreased the expression of miR-21 and increased the expression level of Smad7 mRNA and then inhibited the TGF-ß1/Smad3 signaling pathway. Olm only downregulated the TGF-ß1/Smad3 signaling pathway. CONCLUSIONS: PFD improves TIF by downregulating the expression of miR-21, then elevating Smad7, and finally inhibiting the activation of the TGF-ß1/Smad3 signaling pathway in UUO rats.

Aristolochic Acid Induces Renal Fibrosis and Senescence in Mice.

The kidney is one of the most susceptible organs to age-related impairments. Generally, renal aging is accompanied by renal fibrosis, which is the final common pathway of chronic kidney diseases. Aristolochic acid (AA), a nephrotoxic agent, causes AA nephropathy (AAN), which is characterized by progressive renal fibrosis and functional decline. Although renal fibrosis is associated with renal aging, whether AA induces renal aging remains unclear. The aim of the present study is to investigate the potential use of AAN as a model of renal aging. Here, we examined senescence-related factors in AAN models by chronically administering AA to C57BL/6 mice. Compared with controls, the AA group demonstrated aging kidney phenotypes, such as renal atrophy, renal functional decline, and tubulointerstitial fibrosis. Additionally, AA promoted cellular senescence specifically in the kidneys, and increased renal p16 mRNA expression and senescence-associated ß-galactosidase activity. Furthermore, AA-treated mice exhibited proximal tubular mitochondrial abnormalities, as well as reactive oxygen species accumulation. Klotho, an antiaging gene, was also significantly decreased in the kidneys of AA-treated mice. Collectively, the results of the present study indicate that AA alters senescence-related factors, and that renal fibrosis is closely related to renal aging.

NCTD Prevents Renal Interstitial Fibrosis via Targeting Sp1/lncRNA Gm26669 Axis.

In our previous study, we demonstrated that norcantharidin (NCTD) is a potential therapeutic agent for renal interstitial fibrosis (RIF). Recently, we found that lncRNA Gm26669 (Gm26669) contributed to the development of RIF and could be regulated by NCTD. However, the upstream mechanisms of Gm26669 and whether the anti-RIF effects of NCTD are related to its regulatory action on Gm26669 remain unclear. Our bioinformatics analysis indicated that special protein1 (Sp1), a transcription factor, may bind to the promoter of Gm26669. In the present study, we observed a significant increase in the nuclear translocation of Sp1 using both in vivo and in vitro models of RIF. Furthermore, the knockdown of Sp1 inhibited the expression of collagen type I (CoL-I) and fibronectin (Fn). Mechanistically, Sp1 promoted the expression levels of CoL-I and Fn by directly binding to the promoter of Gm26669 to elevate its expression level. Moreover, we found that NCTD alleviated RIF by inhibiting Gm26669 and the nuclear translocation of Sp1. Collectively, above results suggested that NCTD might prevent RIF via targeting the Sp1/Gm26669 axis, thus providing a new theoretical basis for the clinical application of NCTD in the treatment of RIF.