Kidney International

BackgroundFGF23 excess is associated with morbidity and mortality, but the role of excessive circulating FGF23 concentrations as a mere biomarker or causative factor of pathology is controversial. Here, we investigated the consequences of FGF23 excess in kidney disease. MethodsThis study used three independent disease models: i) anti-glomerular basement membrane (Anti-GBM) disease in male C57BL/6 mice, ii) Adriamycin (doxorubicin)-induced nephropathy in female BALB/c mice, and iii) male DBA/2J mice fed an adenine-containing diet. Anti-GBM and Adriamycin mice and matched control mice received intravenous injections of recombinant FGF23 1{micro}g or vehicle for six consecutive days (Anti-GBM) or once (Adriamycin model), with dissection 24h after the last injection. Adenine mice underwent organ harvesting after 15 weeks to establish ex vivo precision-cut kidney slices (PCKS) and 24h treatment with recombinant FGF23 or vehicle. In addition to histological and biochemical profiling, we assessed serum cytokines, biochemistry and renal transcriptomes and histology of mice and patients with IgA nephropathy. RNAseq data and published transcriptomes underwent gene set enrichment, bulk ligand-receptor interaction analysis and cell-type decomposition. ResultsMice with Anti-GBM disease showed decreased glomerular filtration rate, albuminuria and renal tubular casts. FGF23 treatment increased phosphaturia, but also circulating soluble TNF receptor-1. Renal transcriptomes revealed FGF23-driven proinflammatory transcriptional signatures in murine Anti-GBM and also adverse Vcam1, Pdgfrb and chemokine ligand-receptor signaling in Anti-GBM but not in healthy mice. FGF23 increased transcriptome-inferred renal macrophage content in Anti-GBM mice. Findings were confirmed by immunofluorescence. In Adriamycin-induced nephropathy and in PCKS from the adenine nephropathy model, a short-term FGF23 excess caused expression of proinflammatory transcripts. Finally, human data revealed associations between histopathological or transcriptome-inferred renal immune cell infiltration and circulating FGF23 concentrations. ConclusionFGF23-driven patterns of proinflammatory gene and protein expression or leukocyte overabundance in the kidney were observed in several different models or states of FGF23 excess. The present data provide evidence that excess FGF23 directly drives inflammation in kidney disease and may serve as a therapeutic target.

BackgroundPersistent kidney fibroblast activation and tubular epithelial cell (TEC) injury are key contributors to CKD. However, transcriptional and cellular identities of advanced kidney disease, along with renal fibroblast specific markers and molecular targets contributing to persistent tubular injury, remain elusive. MethodsWe performed single-cell RNA sequencing with two clinically relevant murine kidney fibrosis models. Day 28 post-injury was chosen to ensure advanced fibrotic disease. Identified gene expression signatures were validated using multiple quantitative molecular analyses. ResultsWe revealed comprehensive single cell transcriptomic profiles of two independent kidney fibrosis models compared to normal control. Both models exhibited key CKD characteristics including renal blood flow decline, inflammatory expansion and proximal tubular loss. We identified novel populations including "secretory", "migratory" and "contractile" activated fibroblasts, specifically labelled by newly identified fibroblast-specific Gucy1a3 expression. Fibrotic kidneys elicited elevated embryonic and pro-fibrotic signaling, including separate "Embryonic" and "Pro-fibrotic" TEC clusters. Also, fibrosis caused enhanced cell-to-cell crosstalk, particularly between activated fibroblasts and pro-fibrotic TECs. Analysis of factors mediating mesenchymal phenotype in the injured epithelium identified persistent elevation of Ahnak, previously reported in AKI, in both CKD models. AHNAK knockdown in primary human renal proximal tubular epithelial cells induced a pro-fibrotic phenotype and exacerbated TGF{beta} response via p38, p42/44, pAKT, BMP and MMP signaling. ConclusionsOur study comprehensively examined kidney fibrosis in two independent models at the singe-cell resolution, providing a valuable resource for the field. Moreover, we newly identified Gucy1a3 as a kidney activated fibroblast specific marker and validated AHNAK as a putative disease target. Significance StatementMechanistic understanding of kidney fibrosis is principal for mechanistic understanding and developing targeted strategies against CKD. However, specific markers and molecular targets of key effector cells - activated kidney fibroblasts and injured tubular epithelial cells - remain elusive. Here, we created comprehensive single cell transcriptomic profiles of two clinically relevant kidney fibrosis models. We revealed "secretory", "contractile" and "migratory" fibroblasts and identified Gucy1a3 as a novel marker selectively labelling all three populations. We revealed that kidney fibrosis elicited remarkable epithelial-to-stromal crosstalk and pro-fibrotic signaling in the tubular cells. Moreover, we mechanistically validated AHNAK as a putative novel kidney injury target in a primary human in vitro model of epithelial-to-mesenchymal transition. Our findings advance understanding of and targeted intervention in fibrotic kidney disease.

Lupus nephritis (LN), a severe manifestation of Systemic lupus erythematosus (SLE), is a heterogeneous disease driven by diverse immune and tissue cell types. Current treatments of LN are non-specific, increase risk of infection, and have high rates of relapse. To better understand LN, we obtained 538K single-cell and 143K single-nuclear profiles from kidney biopsies of 156 LN patients and 30 pre-implantation transplant biopsy controls, along with 327K single-cell blood profiles from many of these patients. We built an atlas of kidney tissue cell types in LN encompassing 55 immune and 36 tissue states. Integrating renal immune single cell data with blood data enabled the identification of tissue-specific immune cell states. For example, within the myeloid compartment, plasmacytoid dendritic cells (DC20) were shared across tissue and blood, while macrophage cell states were tissue-specific (M5-14). We identified LN pathological features associated with cell states. Specifically, we examined the chronicity index, which quantifies irreversible tissue damage, and the activity index, which measures active reversible inflammation. Increasing chronicity index tracked with the most dramatic changes in both tissue and immune cell states. The proportion of injured or degenerating proximal tubule cells expanded with chronicity, reflecting ongoing tissue damage. Increasing chronicity index was also associated with an expansion of tissue-specific CD56bright NK cells (NK1), GPNMBhighNUPR1high Macrophages (M5), and CLEC10Alow cDC2 (DC17). Importantly, most expanded immune populations associated with chronicity index were tissue-specific states. After adjusting for chronicity effects, we identified specific cell states in the myeloid compartment that associated with activity. Highly tissue-specific macrophage populations expressing known Scar Associated Macrophage (SAM) gene signatures were particularly expanded, including M5, GPNMBhigh LYVE1low (M11), C1Qlow SPP1high, SPP1highFABP5high (M7), and MERTKhighFABP5high (M9). We hypothesize that M5 may be a profibrotic pathogenic population that could be targeted for therapeutic benefit. We further demonstrate that blood only partially reflects renal tissue. Our results define key renal cell states in the tissue and immune compartments and argue that profound changes in both compartments are associated with increasing chronicity, while specific changes in the myeloid compartment track with activity. In aggregate, these observations strongly support that therapeutic targeting of myeloid populations, in addition to B cells, may offer an unproved strategy to prevent progression of renal inflammation and ongoing kidney damage in LN.

Patients suffering from distal renal tubular acidosis (dRTA) are sometimes diagnosed with proximal tubule dysfunction with leaks of phosphate, uric acid, amino acids, and low-molecular-weight proteins, also known as Fanconi-like syndrome. The underlying molecular basis is largely elusive. We previously reported on Atp6v0a4 knockout (KO) mice, which exhibit severe metabolic acidosis in combination with proximal tubule dysfunction as evidenced by phosphaturia and proteinuria. Here, we show that Rab7, a key regulator of endo-lysosomal trafficking and lysosomal biogenesis, is strongly diminished in proximal tubules of Atp6v0a4 KO mice, while the number of abnormal Ist1-labelled Lamp1-positive vesicles is increased. This is accompanied by the accumulation of autophagosomes, autolysosomes and autophagic substrates. Importantly, correction of metabolic acidosis with bicarbonate therapy resolves proximal tubule dysfunction and trafficking defects in Atp6v0a4 KO mice. Acid-challenged wildtype mice also show trafficking defects with Rab7-downregulation and an increase in Ist1-labeled Lamp1-positive vesicles and develop proximal tubule damage in the long-term. Similar acidosis-induced alterations also occur in human kidney organoids. Altogether, our data provide insights, why patients suffering from severe dRTA may develop a Fanconi-like syndrome, which may contribute to the progression of chronic kidney failure. Translational StatementPatients with renal acidosis caused by impaired proton secretion in the collecting duct (distal renal tubular acidosis - dRTA) sometimes show unexplained symptoms of proximal tubule dysfunction such as proteinuria and phosphaturia. Here, we show that proximal tubules are particularly sensitive to acidosis as evidenced by impaired trafficking, lysosomal damage and accumulation of autophagic substrates. We also show that early treatment of dRTA by alkali supplementation can prevent proximal tubule dysfunction. Because metabolic acidosis represents a well-known risk factor for the progression of chronic kidney disease (CKD), our findings highlight the potential clinical importance of early alkali supplementation to delay disease progression.

BackgroundClassic Fabry disease (FD) is caused by GLA mutations that result in enzymatic deficiency of alpha-galactosidase A (AGAL), lysosomal storage of globotriaosylceramide, and a resulting multisystemic disease. In non-classic later-onset FD, patients have some preserved AGAL activity and a milder disease course, though female carriers may also be affected. While FD pathogenesis has been mostly attributed to catalytic deficiency of mutated AGAL, lysosomal storage and impairment of lysosomal functions, other pathogenic factors may be important, especially in non-classic later-onset FD. MethodsWe characterized the clinical, biochemical, genetic, molecular, cellular and organ pathology correlates of the p.L394P AGAL variant that was identified in six individuals with end-stage kidney disease by the Czech national screening program for FD and by further screening of 25 family members. ResultsClinical findings revealed a milder clinical course with ~15% residual AGAL activity. Laboratory investigations documented intracellular retention of mutated AGAL with resulting ER stress and the unfolded protein response (UPR). Kidney biopsies did not show lysosomal storage. We observed similar findings of ER stress and UPR with several other classic and non-classic FD missense GLA variants. ConclusionsWe identified defective proteostasis of mutated AGAL resulting in chronic ER stress and UPR of AGAL expressing cells (hereafter referred to as AGALopathy) as an important contributor to FD pathogenesis. These findings provide insight into non-classic later-onset FD and may better explain clinical manifestations with implications for pathogenesis, clinical characterization and treatment of all FD forms. Significance statementCatalytic deficiency of mutated AGAL is responsible for classicFabry disease (FD) pathogenesis but does not fully explain the findings in non-classic later-onset FD, in which affected individuals and female carriers develop clinical manifestations despite some AGAL activity and variably mitigated lysosomal storage. In this investigation of individuals with the p.L394P AGAL variant, we identified defective proteostasis of mutated AGAL resulting in chronic endoplasmic reticulum stress and the unfolded protein response as significant contributors to pathogenesis of non-classic later-onset FD. Similar effects were documented also in other AGAL variants identified in classic and non-classicFD. Endoplasmic reticulum stress and the unfolded protein response therefore play an important role in FD.

BackgroundAcute kidney injury (AKI) occurs frequently in critically ill patients and is associated with adverse outcomes. Cellular mechanisms underlying AKI and kidney cell responses to injury remain incompletely understood. MethodsWe performed single-nuclei transcriptomics, bulk transcriptomics, molecular imaging studies, and conventional histology on kidney tissues from 8 individuals with severe AKI (stage 2 or 3 according to Kidney Disease: Improving Global Outcomes (KDIGO) criteria). Specimens were obtained within 1-2 hours after individuals had succumbed to critical illness associated with respiratory infections, with 4 of 8 individuals diagnosed with COVID-19. Control kidney tissues were obtained post-mortem or after nephrectomy from individuals without AKI. ResultsHigh-depth single cell-resolved gene expression data of human kidneys affected by AKI revealed enrichment of novel injury-associated cell states within the major cell types of the tubular epithelium, in particular in proximal tubules, thick ascending limbs and distal convoluted tubules. Four distinct, hierarchically interconnected injured cell states were distinguishable and characterized by transcriptome patterns associated with oxidative stress, hypoxia, interferon response, and epithelial-to-mesenchymal transition, respectively. Transcriptome differences between individuals with AKI were driven primarily by the cell type-specific abundance of these four injury subtypes rather than by private molecular responses. AKI-associated changes in gene expression between individuals with and without COVID-19 were similar. ConclusionThe study provides an extensive resource of the cell type-specific transcriptomic responses associated with critical illness-associated AKI in humans, highlighting recurrent disease-associated signatures and inter-individual heterogeneity. Personalized molecular disease assessment in human AKI may foster the development of tailored therapies.

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common genetic kidney disorder resulting in 10% of patients with renal failure. The molecular events responsible for the relentless growth of cysts are not defined. Thus, identification of novel drivers of ADPKD may lead to new therapies. Ankyrin Repeat and Single KH domain-1 (ANKHD1) controls cancer cell proliferation, yet its role in ADPKD is unexplored. Here, we present the first data that identify ANKHD1 as a driver of proliferative growth in cellular and mouse models of ADPKD. Using the first Ankhd1-deficient mice, we demonstrate that Ankhd1 heterozygosity potently reduces cystic growth and fibrosis, in a genetically orthologous mouse model of ADPKD. We performed transcriptome-wide profiling of patient-derived ADPKD cells with and without ANKHD1 siRNA silencing, revealing a major role for ANKHD1 in the control of cell proliferation and matrix remodelling. We validated the role of ANKHD1 in enhancing proliferation in patient-derived cells. Mechanistically ANKHD1 promotes STAT5 signalling in ADPKD mice. Hence, ANKHD1 is a novel driver of ADPKD, and its inhibition may be of therapeutic benefit.

BackgroundDistal renal tubular acidosis (dRTA) is a rare kidney disorder characterized by impaired urinary acidification due to defective proton secretion in type A intercalated cells of the collecting duct. Recently, pathogenic variants in the human gene encoding the WD Repeat Domain 72 protein (WDR72) have been reported in patients with dRTA, yet the physiological role of WDR72 in the kidney remains unknown. MethodsTo elucidate the renal function of Wdr72, we generated a kidney-specific knockout mouse model (Wdr72fl/fl;Pax8-Cre+) and assessed acid-base homeostasis under baseline, acute, and chronic acid loading. ResultsWdr72fl/fl;Pax8-Cre+ mice displayed persistently elevated urinary pH, reduced titratable acid and net acid excretion under basal and acid-loaded conditions, consistent with incomplete dRTA. While the systemic pH remained unchanged compared to controls under standard diet, chronic acid load led to mild hyperchloremic, hypokalemic metabolic acidosis. Notably, urinary NH excretion was increased upon acid loading accompanied by upregulation of key ammoniagenesis enzymes, which was detected even under basal conditions, consistent with a compensatory activation of proximal tubular acid excretion pathways. The total and membranous abundance of the V-ATPase B1 subunit decreased markedly within the kidney, despite unchanged transcript levels, suggesting a defect in V-ATPase trafficking or assembly. In addition, morphometric analyses revealed an increased proportion of type A intercalating cells that failed to expand upon acid loading, indicating defective adaptive plasticity. ConclusionsKidney-specific Wdr72 deletion impairs distal urinary acidification through reduced V-ATPase abundance and membranous targeting, altered intercalated cell morphology, and limited adaptive remodeling, resulting in incomplete dRTA. Upregulation of renal ammoniagenesis partially compensates the acidification defect. These findings highlight WDR72 as a key regulator of distal nephron acid-base homeostasis and offer mechanistic insight into WDR72-associated dRTA. Key PointsO_LIKidney-specific deletion of Wdr72 reduced Atp6v1b1 membranous localization in intercalated cells. C_LIO_LIKidney-specific Wdr72 knockout altered intercalated cell morphology, and limited their adaptive remodeling. C_LIO_LIThe lack of the renal Wdr72 resulted in incomplete dRTA, compensated partially by elevated ammoniagenesis. C_LI

Key PointsO_LIRenin deficiency in renin-lineage cells worsened crescentic injury and impaired cell migration, revealing a protective role for renin in crescentic glomerulonephritis. C_LIO_LILoss of renin shifted renin-lineage cells signaling toward interferon/STAT1-driven C_LIO_LIRenin-lineage cell ablation in crescentic glomerulonephritis induced a less inflammatory disease time-course. C_LI BackgroundThe adult juxtaglomerular renin-lineage cell (RLC) niche contributes to intraglomerular repair after injury, but their role in highly inflammatory crescentic glomerulonephritis (cGN) remains unclear. While angiotensin II-AT1R signaling promotes fibrosis and inflammation, the contribution of the RLCs, and of renin expression within RLCs, to cGN outcome has not been investigated. MethodsWe used tdTomato lineage-tracing to track RLCs in wild-type (WT) and renin-knockout (RenKO) mice following cGN induction. RLC migration and glomerular injury were quantified histologically. Single-cell RNA sequencing was performed on isolated tdTomato-positive cells at day 10 and 21 after injury to characterize transcriptional programs. Disease progression was additionally examined in mice with diphtheria toxin A-mediated (DTA) RLC ablation. ResultsRLCs were detected within injured glomeruli during cGN, with sporadic localization to crescentic lesions. Genetic renin deletion in RLCs worsened cGN outcomes, with RenKO mice developing increased albuminuria (by 306%), crescent formation (by 50%) and podocyte loss (by 15%) by day 21 compared to WT controls. Renin-deficient RLCs exhibited a reduced intraglomerular migratory response with decreased colocalization with mesangial and podocytes cell markers. Single-cell transcriptomic analysis supports an immunomodulatory reparative phenotype in WT RLCs. In contrast, RenKO RLCs displayed enrichment of interferon-stimulated genes and pathways suppressing cell migration. RLC ablation reduced macrophage infiltration, but did not alter disease progression, suggesting compensatory cellular mechanisms. ConclusionsRenin expression supports the plasticity and injury-associated responses of RLCs during cGN. Loss of renin shifts RLCs toward an interferon-driven inflammatory and antimigratory phenotype that aggravates glomerular injury, while ablation of the RLCs may be compensated without major outcome changes.

Inverted formin-2 (INF2) gene mutations are among the most common causes of genetic focal segmental glomerulosclerosis (FSGS) with or without Charcot-Marie-Tooth (CMT) disease. Recent studies suggest that INF2, through its effects on actin and microtubule arrangement, can regulate processes including vesicle trafficking, cell adhesion, mitochondrial calcium uptake, mitochondrial fission, and T-cell polarization. Despite roles for INF2 in multiple cellular processes, neither the human pathogenic R218Q INF2 point mutation nor the INF2 knock-out allele is sufficient to cause disease in mice. This discrepancy challenges our efforts to explain the disease mechanism, as the link between INF2-related processes, podocyte structure, disease inheritance pattern, and their clinical presentation remains enigmatic. Here, we compared the kidney responses to puromycin aminonucleoside (PAN) induced injury between R218Q INF2 point mutant knock-in and INF2 knock-out mouse models and show that R218Q INF2 mice are susceptible to developing proteinuria and FSGS. This contrasts with INF2 knock-out mice, which show only a minimal kidney phenotype. Co-localization and co-immunoprecipitation analysis of wild-type and mutant INF2 coupled with measurements of cellular actin content revealed that the R218Q INF2 point mutation confers a gain-of-function effect by altering the actin cytoskeleton, facilitated in part by alterations in INF2 localization. Differential analysis of RNA expression in PAN-stressed heterozygous R218Q INF2 point-mutant and heterozygous INF2 knock-out mouse glomeruli showed that the adhesion and mitochondria-related pathways were significantly enriched in the disease condition. Mouse podocytes with R218Q INF2, and an INF2-mutant human patients kidney organoid-derived podocytes with an S186P INF2 mutation, recapitulate the defective adhesion and mitochondria phenotypes. These results link INF2-regulated cellular processes to the onset and progression of glomerular disease. Thus, our data demonstrate that gain-of-function mechanisms drive INF2-related FSGS and explain the autosomal dominant inheritance pattern of this disease.

BackgroundMUC1 and UMOD pathogenic variants cause autosomal dominant tubulointerstitial kidney disease (ADTKD). MUC1 is expressed in kidney, nasal mucosa and respiratory tract, while UMOD is expressed only in kidney. Due to haplo-insufficiency ADTKD-MUC1 patients produce approximately 50% of normal mucin-1. MethodsTo determine whether decreased mucin-1 production was associated with an increased COVID-19 risk, we sent a survey to members of an ADTKD registry in September 2021, after the initial, severe wave of COVID-19. We linked results to previously obtained ADTKD genotype and plasma CA15-3 (mucin-1) levels and created a longitudinal registry of COVID-19 related deaths. ResultsSurveys were emailed to 637 individuals, with responses from 89 ADTKD-MUC1 and 132 ADTKD-UMOD individuals. 19/83 (23%) ADTKD-MUC1 survey respondents reported a prior COVID-19 infection vs. 14/125 (11%) ADTKD-UMOD respondents (odds ratio (OR) 2.35 (95%CI 1.60-3.11, P = 0.0260). Including additional familial cases reported from survey respondents, 10/41 (24%) ADTKD-MUC1 individuals died of COVID-19 vs. 1/30 (3%) with ADTKD-UMOD, with OR 9.21 (95%CI 1.22-69.32), P = 0.03. The mean plasma mucin-1 level prior to infection in 14 infected and 27 uninfected ADTKD-MUC1 individuals was 7.06{+/-}4.12 vs. 10.21{+/-}4.02 U/mL (P = 0.035). Over three years duration, our longitudinal registry identified 19 COVID-19 deaths in 360 ADTKD-MUC1 individuals (5%) vs. 3 deaths in 478 ADTKD-UMOD individuals (0.6%) (P = 0.0007). Multivariate logistic regression revealed the following odds ratios (95% confidence interval) for COVID-19 deaths: ADTKD-MUC1 8.4 (2.9-29.5), kidney transplant 5.5 (1.6-9.1), body mass index (kg/m2) 1.1 (1.0-1.2), age (y) 1.04 (1.0-1.1). ConclusionsIndividuals with ADTKD-MUC1 are at an eight-fold increased risk of COVID-19 mortality vs. ADTKD-UMOD individuals. Haplo-insufficient production of mucin-1 may be responsible.

IntroductionAutosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of fluid filled cysts, progressive fibrosis and chronic inflammation, often leading to kidney failure. Renal fibrosis in ADPKD is primarily driven by myofibroblast activation and excessive extracellular matrix (ECM) accumulation, which contribute to disease progression. Here we investigated the therapeutic potential of pirfenidone, an antifibrotic drug, on myofibroblast activity, ECM production, and ADPKD progression. MethodsPrimary cultures of myofibroblasts from human ADPKD kidneys were treated with pirfenidone in vitro, and cell proliferation, migration, contractility and changes in ECM production were measured. In vivo, the effect of pirfenidone on cyst growth, fibrosis and renal function were determined in the Pkd1RC/RC male mouse model of ADPKD and wild type controls. ResultsAnalysis of single-nucleus RNA sequencing data of human ADPKD kidneys revealed that fibroblasts are a primary source of fibrous and cell-adhesive ECM, with higher ECM gene expression compared to normal human kidneys. Treatment of human ADPKD renal myofibroblasts with pirfenidone led to reduced ECM gene expression, cell proliferation, migration and contractility. In vivo, pirfenidone treatment in Pkd1RC/RC mice reduced renal fibrosis, collagen deposition, myofibroblast accumulation, pro-fibrotic gene expression and decreased TGF-{beta}/SMAD3 and mTOR signaling. While kidney cyst number remained unchanged, kidney size and cyst area were reduced, leading to improved kidney morphology and improved renal function in RC/RC mice. ConclusionThese findings suggest that pirfenidone mitigates renal fibrosis and preserves renal architecture in ADPKD, supporting its potential as a therapeutic strategy to inhibit fibrosis in ADPKD. Translational StatementAutosomal dominant polycystic kidney disease (ADPKD) is characterized by cyst growth and progressive renal fibrosis, which contributes significantly to kidney function decline. Current therapies predominantly target cyst expansion but do not adequately address fibrosis. Our study demonstrates that myofibroblasts are the principal source of extracellular matrix (ECM) deposition in ADPKD kidneys and that pirfenidone, an FDA-approved antifibrotic drug for idiopathic pulmonary fibrosis, effectively reduces myofibroblast activation, ECM production, and key profibrotic signaling pathways both in human ADPKD cells and in a rodent model. Importantly, pirfenidone treatment also decreased kidney enlargement and cyst growth without nephrotoxicity. These findings support the repurposing of pirfenidone as a complementary therapeutic strategy in ADPKD to target fibrotic remodeling alongside cystic disease. Clinical trials investigating pirfenidones safety and efficacy in ADPKD patients could potentially slow disease progression and improve renal outcomes.

Hematopoietic stem cell transplant-associated thrombotic microangiopathy (HSCT-TMA), characterized by microvascular endothelial damage and severe renal injury, negatively affects HSCT survivorship with high mortality and long-term renal morbidity. After HSCT, innate immunity and inflammation are often dysregulated. The alternative complement pathway (AP) of the innate immune system is overactivated in HSCT-TMA, but the mechanisms of its initiation are poorly described. Complement component C3 of the AP can be cleaved by proteases outside of the AP. Because mRNA expression of tissue kallikrein 1 (KLK1), an inflammatory serine protease that produces kinins, has been found to be markedly elevated in the renal endothelium of inflamed mice, we hypothesized that increased KLK1 activity during inflammation contributes to AP overactivation and endothelial injury in HSCT-TMA. We assessed AP activation, KLK1 activity, endothelial injury, and renal function in HSCT-TMA experimental models and disease settings and investigated C3 cleavage and AP activation by KLK1. We found that patients with HSCT-TMA had significantly increased AP activation and decreased KLK1 inactivation at TMA diagnosis compared to pre-HSCT. Mice challenged with HSCT-TMA triggers cyclosporine A (CsA) and lipopolysaccharide (LPS) exhibited increased AP activation, renal endothelial injury, and impaired renal function in the setting of decreased KLK1 inactivation. We further demonstrated that KLK1 cleaved AP component C3 to C3b that functionally activated the AP. Our data indicate a noncanonical mechanism for AP activation by KLK1 in settings of HSCT-TMA. Key PointsO_LITissue kallikrein (KLK1) functionally cleaves complement factor C3. C_LIO_LIReduced KLK1 inhibition is associated with activation of the alternative pathway of complement. C_LI O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/674260v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@8f260aorg.highwire.dtl.DTLVardef@a6511org.highwire.dtl.DTLVardef@7160f6org.highwire.dtl.DTLVardef@194d36d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical Abstract.C_FLOATNO HSCT-TMA triggers (e.g. inflammation, immunosuppression) upregulate expression of the inflammatory serine protease KLK1, which cleaves C3 to initiate AP activation and amplification, leading to renal endothelial injury. Created with BioRender.com under its Academic License Terms with Baylor College of Medicine. C_FIG

Elevated galactose deficient IgA1 (Gd-IgA1) is known to be associated with IgA nephropathy (IgAN) and is often regarded as the first of four steps in the "four hit" hypothesis to explain disease pathogenesis. However, while the proposed downstream hits have support from unbiased genetic association studies, similar genetic evidence to support a causal role for Gd-IgA1 in the disease is lacking. Multiple previous genome-wide association studies have shown that common variation in the gene C1GALT1 is strongly associated with Gd-IgA1 levels. We used this established relationship first to calculate power to detect an association of C1GALT1 alleles with IgAN risk and second to conduct association and Mendelian randomisation analyses to detect and quantify evidence for a causal role of Gd-IgA1 in IgA nephropathy. Despite adequate power, we did not observe significant genetic evidence for a causal role of Gd-IgA1 in IgAN that would explain the well-established observational association, and infer that this may not be explained by causation. This raises the possibility that Gd-IgA1 might better be regarded as a biomarker than a cause of IgA nephropathy and suggests that caution is needed when inferring clinical efficacy of treatments based on their effects on Gd-IgA1 levels. Lay SummaryImmunoglobulin A (IgA) nephropathy is a kidney disease in which IgA builds up in the kidneys, causing inflammation there. People with the condition often have higher levels of a form of IgA that lacks a sugar called galactose (galactose-deficient IgA1, or Gd-IgA1) but it has been unclear whether this abnormal IgA actually causes the disease, or whether it is simply a marker of it. To address this, we used two genetic experiments: first, we tested whether people who inherit genetic variants that increase Gd-IgA1 levels are more likely to develop IgA nephropathy; and second, we used Mendelian randomization methods that compare the genetic effects on Gd-IgA1 levels with their effects on disease risk. Despite adequate power, neither approach showed that Gd-IgA1 causes IgA nephropathy, suggesting the raised levels seen in patients might be a consequence of other processes. This means treatments should not be judged solely on whether they lower Gd-IgA1.

Polycystic kidney diseases (PKD) are genetic disorders characterised by the formation of fluid-filled cysts, which disrupt kidney architecture and function. Autosomal recessive PKD (ARPKD) is a rare form of PKD, caused by mutations in PKHD1, and clinically more severe than the more common autosomal dominant PKD (ADPKD). Prior studies have implicated the ciliary-located Hedgehog (Hh) pathway in ADPKD, with increased levels of Hh components in experimental ADPKD models, and reduced cystogenesis following pharmacological Hh inhibition. In contrast, the role of the Hh pathway in ARPKD is poorly understood. We hypothesised that Hh pathway activity would be elevated during ARPKD pathogenesis, and its modulation may inhibit cystogenesis, akin to prior findings in ADPKD. To test this, we utilised Cpk mice, a model which replicates the pathophysiology of ARPKD, and generated a human cellular ARPKD 3-dimensional cystogenesis model by mutating PKHD1 in human collecting duct cells through CRISPR-Cas9 technology. We found significantly elevated levels of the Hh transcriptional effector Gli3 in the Cpk mouse, a finding replicated in our human cellular ARPKD model. In the Cpk mouse, we also observed an increase in total GLI3 and GLI3 repressor protein levels. However, reduction of increased Gli3 levels via genetic deletion in the Cpk mouse did not affect cyst formation. Similarly, lowering GLI3 transcript to wildtype levels, did not influence cyst size in our human cellular ARPKD model. Collectively, these data show that elevated Gli3 does not modulate cyst progression in the context of ARPKD, highlighting the complexity of the Hh pathway in PKD. New and NoteworthyThe role of the Hedgehog pathway in autosomal recessive polycystic kidney disease (ARPKD) is poorly understood. Here, we describe elevated levels of Gli3, the Hedgehog transcriptional effector, in murine and human ARPKD models. However, reversal of the increase in Gli3 did not significantly affect cystogenesis in a human cell model of ARPKD or disease progression in a mouse model which replicates ARPKD pathophysiology. Collectively, our data indicates that Gli3 does not modulate ARPKD progression.

BackgroundVisual scoring of tubular damage has limitations in capturing the full spectrum of structural changes and prognostic potential. We investigate if computationally quantified tubular features can enhance prognostication and reveal spatial relationships with interstitial fibrosis. MethodsDeep-learning and image-processing-based segmentations were employed in N=254/266 PAS-WSIs from the NEPTUNE/CureGN datasets (135/153 focal segmental glomerulosclerosis and 119/113 minimal change disease) for: cortex, tubular lumen (TL), epithelium (TE), nuclei (TN), and basement membrane (TBM). N=104 pathomic features were extracted from these segmented tubular substructures and summarized at the patient level using summary statistics. The tubular features were quantified across the biopsy and in manually segmented regions of mature interstitial fibrosis and tubular atrophy (IFTA), pre-IFTA and non-IFTA in the NEPTUNE dataset. Minimum Redundancy Maximum Relevance was used in the NEPTUNE dataset to select features most associated with disease progression and proteinuria remission. Ridge-penalized Cox models evaluated their predictive discrimination compared to clinical/demographic data and visual-assessment. Models were evaluated in the CureGN dataset. ResultsN=9 features were predictive of disease progression and/or proteinuria remission. Models with tubular features had high prognostic accuracy in both NEPTUNE and CureGN datasets and increased prognostic accuracy for both outcomes (5.6%-7.7% and 1.6%-4.6% increase for disease progression and proteinuria remission, respectively) compared to conventional parameters alone in the NEPTUNE dataset. TBM thickness/area and TE simplification progressively increased from non- to pre- and mature IFTA. ConclusionsPreviously under-recognized, quantifiable, and clinically relevant tubular features in the kidney parenchyma can enhance understanding of mechanisms of disease progression and risk stratification.

There is a wealth of indirect evidence that extracellular RNA (exRNA) signalling can regulate renal tubular epithelial cell function. However, the physiological importance of this signalling is uncertain. We sought to determine the extent of extracellular RNA transfer between cells in a healthy kidney. We tested the hypothesis that RNA travels from glomerular podocytes to renal tubular epithelial cells. We developed a method to track exRNA in the kidney using SLAMseq (SH-linked alkylation for the metabolic sequencing of RNA in tissue). We crossed podocin-Cre mice with floxed-stop-UPRT mice to express recombinant uracil phosphoribosyl transferase (UPRT) in podocytes. Mice were injected with the modified nucleobase 4-thiouracil, which is incorporated into nascent RNA with high efficiency only in UPRT-expressing cells. We harvested glomeruli or tubular cells, extracted RNA and prepared libraries for SLAMseq, in which sites of mRNA labelling with 4-thiouracil are detected as T>C conversions in 3UTRs. In glomeruli, we detected labelling of known podocyte genes but not of genes known to be restricted to endothelial, renal tubular or white blood cells. Setting a false-discovery rate of 1%, the proportion of genes deemed to be labelled with high confidence was 7.1% (95% confidence interval 6.8 - 7.4%) in 4TU-treated podocyte-UPRT mice, 2.5% (2.3 - 2.7%) in Cre-negative controls and 1.0% (0.9 - 1.1%) in 4TU-naive controls. In tubular cells, we detected a small but statistically significant increase in RNA labelling in podocyte-UPRT mice compared to Cre-negative controls (p = 7.4 x 10-16 in a zero-inflated Poisson regression model). We conclude that RNA is transferred from podocytes to renal tubular epithelial cells in vivo under physiological conditions. Our model provides the opportunity to explore the consequences of this novel signalling pathway in health and kidney disease.

BackgroundClass switching toward spike (S)-binding IgG4 antibodies after mRNA-based COVID-19 vaccination has been observed, an antibody subclass with strong neutralizing but limited effector activity. While this has been reported in healthy individuals, subclass dynamics in immunocompromised kidney patients are unclear. We assessed IgG subclass patterns and S-specific B-cell phenotypes up to 6 months after a two-dose mRNA-1273 vaccination schedule in kidney transplant recipients (KTRs), dialysis patients, and patients with chronic kidney disease (CKD). MethodsIn this exploratory study, KTRs (n=11), dialysis patients (n=5), CKD stage G4-5 patients (eGFR <30 ml/min/1.73m2, n=5), and controls without known kidney disease (eGFR >45 ml/min/1.73m2, n=8) received two mRNA-1273 doses 28 days apart. Blood was collected pre-vaccination (V1), and at 28 days (V3) and 6 months (V4) after the second dose. S1-specific IgG antibodies were measured by a validated fluorescent bead-based multiplex-immunoassay, and participants seronegative at V1 and seropositive at V3 were included. B cells were phenotyped by flow cytometry. ResultsFive of 11 KTRs had no detectable S-binding B cells, whereas all other groups mounted responses. Across responders, the frequency of S-binding B cells increased from V1 (median 0.08%) to 0.49% at V3 and to 0.84% at V4 (both p<0.0001). S-binding B cells mainly comprised IgG+ plasmablasts. The IgG4:IgG1 log-ratio increased significantly from V3 to V4 (p<0.001), indicating a relative shift toward IgG4; absolute frequencies were comparable across the groups. ConclusionsApproximately half of KTRs lacked detectable S-binding B cells after two mRNA-1273 doses, despite antibody formation. Among responders, S-binding B cells persisted up to 6 months after vaccination with a relative shift toward IgG4, a pattern also observed in dialysis patients, CKD patients and controls. The clinical significance of this subclass skewing requires confirmation in larger cohorts with functional antibody readouts.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease. Cysts develop through dedifferentiation of tubular epithelial cells, but the sequence of molecular events and their relative importance remain unclear. To address this gap in knowledge, 40 cysts from 4 ADPKD kidneys and 4 microcystic tissues were mapped on transcriptomic and histological level. Cyst were heterogenous and we identified 6 cystic subclusters with 2 deviations from the main trajectory, dependent on the rate of interstitial remodeling, inflammation and dedifferentiation. Loss of proximal tubular marker gene expression was more pronounced compared to those of other tubular segments. Altered expression of metabolic pathways was consistent among the cysts, which was further analyzed in human and mouse cell lines. Purine metabolism was similarly altered in all ADPKD cell lines, and its modulation with azathioprine suppressed cyst formation in vitro. In conclusion, by focusing on common altered pathways in cysts and cell models, we have identified purine metabolism as a novel potential target in ADPKD.

A major therapeutic obstacle in diabetes mellitus is the metabolic or hyperglycemic memory: the persistence of impaired organ function despite improvement of blood glucose. Therapies reversing the hyperglycemic memory and thus improving already established organ-dysfunction are lacking, but urgently needed considering the increasing prevalence of diabetes mellitus worldwide. Here we show that glucose-mediated changes in gene expression largely persist in diabetic kidney disease (DKD) despite reversing hyperglycemia. The senescence-associated cyclin-dependent kinase inhibitor p21 (Cdkn1a) was the top hit among genes persistently induced by hyperglycemia and was associated with sustained induction of the p53-p21 pathway. Persistent p21 induction was confirmed in various animal models, in several independent human samples and in in vitro models. Tubular p21 expression and urinary p21-levels were associated with DKD severity and remained elevated despite improved blood glucose levels in humans, suggesting that p21 may be a biomarker indicating persistent ("memorized") kidney damage. Glucose-mediated p21 induction and tubular senescence were enhanced in mice with reduced levels of the disease resolving protease activated protein C (aPC). Mechanistically, glucose-induced and sustained tubular p21 expression is linked with demethylation of its promoter and reduced DNMT1 expression. aPC reverses already established p21 expression independent of its anticoagulant function through receptor signaling. Accordingly, new pharmacological approaches specifically mimicking aPC signaling (3K3A-aPC, parmodulin-2) enabled the reversal of glucose-mediated sustained tubular p21 expression, tubular senescence, and DKD. Thus, p21-dependent tubular senescence contributes to the hyperglycemic memory but can be therapeutically targeted. Single sentence summaryaPC signaling targets persistent p21 expression and tubular senescence and reverses the hyperglycemic memory in diabetic kidney disease.