Clonal hematopoiesis of indeterminate potential is associated with acute kidney injury

Abstract

Age is a predominant risk factor for acute kidney injury (AKI), yet the biological mechanisms underlying this risk are largely unknown. Clonal hematopoiesis of indeterminate potential (CHIP) confers increased risk for several chronic diseases associated with aging. Here we sought to test whether CHIP increases the risk of AKI. In three population-based epidemiology cohorts, we found that CHIP was associated with a greater risk of incident AKI, which was more pronounced in patients with AKI requiring dialysis and in individuals with somatic mutations in genes other than DNMT3A, including mutations in TET2 and JAK2. Mendelian randomization analyses supported a causal role for CHIP in promoting AKI. Non-DNMT3A-CHIP was also associated with a nonresolving pattern of injury in patients with AKI. To gain mechanistic insight, we evaluated the role of Tet2-CHIP and Jak2^V617F-CHIP in two mouse models of AKI. In both models, CHIP was associated with more severe AKI, greater renal proinflammatory macrophage infiltration and greater post-AKI kidney fibrosis. In summary, this work establishes CHIP as a genetic mechanism conferring impaired kidney function recovery after AKI via an aberrant inflammatory response mediated by renal macrophages.

Fig. 1. CHIP is associated with a greater risk of incident AKI in three population-based cohorts.

a, Random effects meta-analysis of the association between CHIP and risk of incident AKI in the UKB, ARIC and CHS cohorts. b, Random effects meta-analysis of the association between CHIP driven by mutations in genes other than DNMT3A (non-DNMT3A-CHIP) and risk of incident AKI in the UKB, ARIC and CHS cohorts. c, Random effects meta-analysis of the risk of incident AKI across major CHIP driver genes. For the analyses in a–c, Cox proportional hazards analyses were conducted, adjusting for age, age², sex, baseline eGFR, baseline smoking status, diabetes and hypertension, as well as either ten principal components of genetic ancestry (UKB) or self-reported ethnicity (ARIC and CHS). These results are presented as HRs with 95% CIs. d, MR examining the association between CHIP and AKI risk. The results shown represent a random effects meta-analysis of conventional multiplicative random effects inverse variance weighted (IVW) estimators. These results are presented as odds ratios (ORs) with 95% CIs.

Fig. 2. CHIP is associated with impaired recovery from AKI in the ASSESS-AKI and BioVU cohorts.

a, Prevalence of CHIP among individuals with a resolving AKI pattern in ASSESS-AKI (n = 191) and BioVU (n = 88) compared to a nonresolving AKI pattern (n = 130 for ASSESS-AKI and n = 366 for BioVU), as defined by Bhatraju et al.. b, Odds of nonresolving AKI pattern according to CHIP status, adjusted for age, sex, baseline creatinine, AKI stage, smoking status, ethnicity and history of diabetes, hypertension and cardiovascular disease. c, Risk of significant kidney function impairment (primary study composite outcome of ESKD or eGFR decline by ≥50%) over 5 years of follow-up among ASSESS-AKI participants with baseline AKI according to CHIP status, adjusted for age, sex, baseline creatinine, AKI stage, smoking status, ethnicity and history of diabetes, hypertension and cardiovascular disease. *P < 0.05, ***P < 0.001.

Fig. 3. Early response of hematopoietic deletion of Tet2 to ischemic kidney injury in mice.

a, BUN and serum creatinine in WT and Tet2^−/− mice subjected to ischemic kidney injury (n = 6 mice each). b, mRNA of the tubule injury markers Kim-1 and Ngal (n = 6 mice each) and quantification of immunoreactive protein kidney levels of the kidney injury markers Kim-1 and Ngal, as well as the inflammatory markers Nlrp3 and Il-1β in the kidneys of WT and Tet2^−/− mice 8 days after injury (n = 4 mice each). c, Representative image of kidney histology with Periodic Acid–Schiff stain (left) and quantitation of the tubule injury score (n = 6 mice each, right). d, Kidney mRNA expression of the proinflammatory cytokines Tnf, Il1b, Ccl2, Ccl3 and Il6 in Tet2^−/− and WT mice (n = 6 mice each). e, Representative image of macrophage infiltration (F4/80 immunostaining). Data were analyzed using a two-tailed Student’s t-test or two-way analysis of variance (ANOVA) followed by Tukey’s or Bonferroni’s post hoc tests and presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 µm. Source data

Fig. 4. Tet2-deficient macrophages are hyperinflammatory in early ischemic kidney injury.

a, Kidney macrophage mRNA expression of the proinflammatory cytokines and chemokines Tnf, Il1b, Ccl2, Ccl3 and Il6 8 days after ischemic injury in Tet2^−/− and WT mice (n = 6 mice each). b, Kidney Cd68 mRNA expression in Tet2^−/− and WT mice (n = 6 mice each) and representative images and quantification of immunoreactive CD68 and its colocalization with immunoreactive NLRP3 and IL-β (n = 5 mice each). c, Representative uniform manifold approximation and projection (UMAP) plot of cell types examined in the scRNA-seq analyses of Tet2^−/− and WT mouse kidney cell samples enriched for CD45⁺ (hematopoietic) cells. d, Gene set enrichment analysis (GSEA) analysis of WT and Tet2^−/− macrophages after scRNA-seq. e, CellChat analyses of cell–cell communication pathways between macrophages and proximal tubules epithelial cells isolated from Tet2^−/− and WT mouse kidneys. Data were analyzed using a two-tailed Student’s t-test or two-way ANOVA followed by Tukey’s or Bonferroni’s post hoc tests and are presented as the mean ± s.e.m. **P < 0.01, ***P < 0.001; scale bar, 50 µm.

Fig. 5. Increased kidney interstitial fibrosis after ischemic kidney injury with hematopoietic deletion of Tet2.

a, Kidney mRNA of the profibrotic markers Tgfb1, Ccn2, Acta2, Col1a, Col3a1, Col4a1, Fn and Vim 8 days after injury in Tet2^−/− and WT mice (n = 6 mice each). b, Kidney Ngal1 mRNA (n = 6 mice each) and protein expression (n = 4 mice each) 28 days after injury in Tet2^−/− and WT mice. c, Kidney mRNA for the inflammatory cytokines Tnf, Il1b, Ccl2, Il23a and Il6 in kidneys 28 days after injury in Tet2^−/− and WT mice (n = 6 mice each). d, Kidney Emr1/F480 mRNA quantification and representative F4/80 macrophage immunohistochemistry (IHC) 28 days after injury in Tet2^−/− and WT mice (n = 6 mice each). e, Kidney macrophage mRNA expression of the proinflammatory cytokines Il1b, Ccl2, Ccl3 and Il6 and anti-inflammatory, pro-reparative cytokines Cd206, Cd290a, Cd163 and Il10 in kidney macrophages isolated from WT and Tet2^−/− mice 28 days after injury (n = 6 mice each). f, Kidney mRNA for the fibrotic markers Acta2, Col1a, Col3a1, Fn and Vim 28 days after injury in Tet2^−/− and WT mice (n = 6 mice each). g, Representative images of kidney histology stained with Masson trichrome blue and Picrosirius red, and quantification of interstitial collagen expression in the kidneys of WT and Tet2^−/− mice 28 days after injury (n = 6 mice each). Data were analyzed using a two-tailed Student’s t-test or two-way ANOVA followed by Tukey’s or Bonferroni’s post hoc tests and are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Scale bar, 50 µm. Source data

Fig. 6. The response of hematopoietic-specific Jak2^V617F mice to acute and chronic kidney injury.

a, Time course of BUN in response to ischemic kidney injury in WT and Jak2^V617F mice (n = 7 mice each). b, Quantification of kidney macrophages using flow cytometry in WT and Jak2^V617F mouse kidneys at baseline (n = 7 mice each) and 7 days after ischemic kidney injury (n = 7 in WT and 6 in Jak2^V617F mice). c, Kidney mRNA expression of the proinflammatory cytokines Tnf, Il1a, Il1b, Il6, Ccl2, Ccl3 and Il23 7 days after ischemic injury in WT and Jak2^V617F mice (n = 5 mice each). d, KIM-1 and NGAL protein level 7 days after ischemic kidney injury in WT and Jak2^V617F mice (n = 4 mice each). e, Kidney mRNA of the proinflammatory cytokines Il1a, Il6 and Ccl2 28 days after injury in WT and Jak2^V617F mice (n = 6 mice each). f, Kidney mRNA of the profibrotic factors Col1a1, Acta2 and Ctgf 28 days after injury in WT and Jak2^V617F mice (n = 6 mice each). g, Quantification of kidney immunoreactive protein expression of KIM-1, NLRP3 and α-SMA 28 days after injury in WT and Jak2^V617F mice (n = 5 mice each). h, Representative images of kidney histology stained with Picrosirius red and quantification of kidney interstitial collagen expression 28 days after injury in WT and Jak2^V617F mice (n = 4 mice each). i, Macrophage mRNA of the proinflammatory cytokines Tnf, I11b and Ccl2 in WT and Jak2^V617F mice 7 days after UUO (n = 4 mice each). j, Quantification of kidney α-SMA protein expression in the kidneys of WT and Jak2^V617F mice 7 days after UUO (n = 4 mice each). k, Representative images of kidney histology stained with Picrosirius red and quantification of kidney interstitial fibrosis in WT and Jak2^V617F mice 7 days after UUO (n = 4 mice each). Data were analyzed using a two-tailed Student’s t-test or two-way ANOVA followed by Tukey’s or Bonferroni’s post hoc tests and are presented as the mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001. Source data

Extended Data Fig. 1. Distribution of CHIP mutations and AKI rates by age.

A) Distribution of CHIP prevalence by age group across the cohorts included in the study. B) Proportion of individuals in each age group across the cohorts. C) Prevalence of major CHIP gene subtypes by age at baseline in the UKB. D) Rate of AKI across ages observed during follow-up.

Extended Data Fig. 2. Associations of individual CHIP genes with AKI risk in three prospective cohorts.

A) Risk of AKI by binary CHIP status. B) Risk of AKI among CHIP carriers per 10% increase in variant allele fraction (VAF). Analyses that did not converge due to small sample size or lack of events in the CHIP subgroup are left as blank rows. All analyses were adjusted for age, age, sex, baseline eGFR, baseline smoking status, diabetes, and hypertension, as well as either 10 PCs of genetic ancestry (UKB) or self-reported race/ethnicity (ARIC & CHS).

Extended Data Fig. 3. Kaplan-Meier plots illustrating cumulative AKI events as a function of time in the UKB cohort.

Cumulative AKI events are shown for (A) the whole cohort, (B) those with baseline CKD and (C) those without baseline CKD.

Extended Data Fig. 4. Creation of Tet2^-/–CHIP mice and WT controls.

A) Schema for production of wild type and Tet2^-/- mice. B) Representative images of flow cytometry of clonal hematopoietic expansion in Tet2^-/- mice. C&D) Tet2^-/- macrophages and neutrophils in kidneys without injury (n = 4 mice) and 8 days after ischemic injury (n = 10 mice). E) Kidney macrophage mRNA of proinflammatory cytokines Tnf, Il1b, Ccl2 and Ccl3 in Tet2^-/- and WT mice without injury (n = 8 mice). Data were analyzed using 2-tailed Student’s t-test or two-way ANOVA followed by Tukey’s or Bonferroni’s post hoc tests and presented as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, scale bar = 50 µm.

Extended Data Fig. 5. Survival curve of wild type and Tet2^-/- mice following severe kidney injury.

Survival curve of WT and Tet2^-/- mice following 33.5 minutes of renal pedicle clamping (n = 10 mice each).

Extended Data Fig. 6. Increased colocalization of Tet2^-/- CD45.2 cells but not Tet2^+/+ CD45.2 cells nor Tet2^+/+ CD45.1 cells with immunoreactive NLRP3 and IL-β.

Representative of n = 4 colocalization studies of Tet2^-/- CD45.2 cells or Tet2^+/+ CD45.2 cells and Tet2^+/+ CD45.1 cells with immunoreactive NLRP3 and IL-β. scale bar = 50 µm.

Extended Data Fig. 7. 28 days after ischemic injury, kidneys of Tet2^-/- mice had increased markers for neutrophils, total T cells, and CD4 and CD8 T cells.

mRNA markers for neutrophils (Ly6g), total T cells (Cd3) and CD4 and CD8 T cells in kidneys of WT and Tet2^-/- mice 28 days after ischemic injury (n = 7 WT mice and n = 6 Tet2^-/- mice). Data were analyzed using 2 tailed Student’s t-tests and presented as mean ± SEM. *p < 0.05.

Extended Data Fig. 8. Increased inflammation, immune cell infiltration, and fibrosis is observed in Tet2^-/- mice after unilateral ureteral obstruction compared to WT control mice.

A) mRNA of proinflammatory cytokines Il1b, Tnf, Ccl3 and Ccl2 in kidneys of WT and Tet2^-/- mice 7 days after UUO (n = 8 for WT and n = 7 for Tet2^-/-) B) Representative figures and quantification of macrophage and neutrophil infiltration7 days after UUO (n = 8 for WT and n = 7 for Tet2^-/-). C) Representative figure and quantification of interstitial fibrosis 7 days after UUO. (n = 8 for WT and n = 7 for Tet2^-/-). Data were analyzed using 2-tailed Student’s t-tests and presented as mean ± SEM. *p < 0.05; **p < 0.01; scale bar = 50 µm.