Impairment of Renal and Hematopoietic Stem/Progenitor Cell Compartments in Frailty Syndrome: Link With Oxidative Stress, Plasma Cytokine Profiles, and Nuclear DNA Damage

Abstract

Frailty is an age-related syndrome that drives multiple physiological system impairments in some older adults, and its pathophysiological mechanisms remain unclear. We evaluated whether frailty-related biological processes could impair stem cell compartments, specifically the renal stem compartment, given that kidney dysfunctions are frequent in frailty. A well-characterized in vitro nephrosphere model of human adult renal stem/progenitor cells has been instrumental to and was appropriate for verifying this hypothesis in our current research. Evaluating the effects of plasma from older individuals with frailty (frail plasma) on allogeneic renal stem/progenitor cells, we showed significant functional impairment and nuclear DNA damage in the treated cells of the renal stem compartment. The analysis of the frail plasma revealed mitochondrial functional impairment associated with the activation of oxidative stress and a unique inflammatory mediator profile in frail individuals. In addition, the plasma of frail subjects also contained the highest percentage of DNA-damaged autologous circulating hematopoietic progenitor/stem cells. The integration of both molecular and functional data obtained allowed us to discern patterns associated with frailty status, irrespective of the comorbidities present in the frail individuals. The data obtained converged toward biological conditions that in frailty caused renal and hematopoietic impairment of stem cells, highlighting the possibility of concomitant exhaustion of several stem compartments.

Keywords: Aging; Cellular senescence; Comorbidity; Frailty pathophysiology; Mitochondrial impairment.

Figure 1.

A framework of the study. (A) Experimental strategy. Forty-one frail, 35 non-frail older adults, and 38 young subjects (young: mean age ± standard deviation, 29.4 ± 2.3 years; males 14, females 24) were recruited. Collection of peripheral blood samples and separation of plasma and peripheral blood mononuclear cells (PBMCs). Plasma is used to treat human allogeneic renal cells growing as nephrospheres (NSs) that harbor adult renal stem/progenitor cells. Evaluation of the sphere-forming efficiency (SFE) of renal stem/progenitor cells. Evaluation of the proliferation, viability, DNA damage, and ROS production of NS cells. Evaluation of DNA damage in autologous cHPSCs present in PBMCs of plasma donors. Evaluation of plasma oxidative status and plasma inflammatory cytokines. The difficulty to obtain renal tissue did not permit to include pre-frail subjects in this study. (B) Clinical characteristics of older adults. Data are summarized as the mean ± standard deviation, frequency and percentage, or median and interquartile range (IQR). Comparisons between groups were analyzed by *Student’s t test, ^the chi-square test, or #the Mann‒Whitney U test, as indicated. Significance was set for values of p < .05; NS = not significant; N/A = not applicable; MMSE = Mini-Mental State Examination; COPD = chronic obstructive pulmonary disease. (C) Summary of the experiments and analyses performed for data generation. (B, C) Analytical data are provided in Supplementary Table 1A and B.

Figure 2.

Effects of 10 days of treatment with plasma from frail, non-frail, and young subjects on NS cells. (A) Phase contrast images representative of renal cells grown as NS, untreated or treated with the indicated plasma. Scale bar: 100 μm. Eleven NS cultures were used (Supplementary Table 2), and the treatments were made with 17 frail, 16 non-frail, and 16 young plasma samples (Supplementary Table 3). (B) Sphere-forming efficiency (SFE %) of renal cells grown as NSs and treated with the indicated plasma; data were obtained with a contrast phase microscope. (C) Percentage of NS cells positive for Ki-67 after the indicated plasma treatment. Flow cytometry data are shown (gating strategy in Supplementary Figure 1). (D) Cumulative percentage of live, early, late apoptotic, and necrotic NS cells after the indicated plasma treatments, flow cytometry data of Annexin V/PI staining are shown (representative dot plots in Supplementary Figure 2); color-code: light gray, live; white, early apoptotic; dark gray, late apoptotic; black, necrotic. (E) Representative flow cytometry dot plots of γ-H2AX+ NS cells treated with the same plasma samples as in Figure 2A. FSC = Forward Scatter (gating strategy in Supplementary Figure 1). (F) Percentage of nuclear DNA-damaged (γ-H2AX+) NS cells after the indicated plasma treatments, flow cytometry data. (G) Percentage of DNA-damaged (γ-H2AX+) cHPSCs (Supplementary Table 4) circulating in the subjects donors of the plasma used in panel F, flow cytometry data. (H) Representative cytospin immunofluorescence analysis of nuclear γ-H2AX+ foci in 3 different independent NS cultures grown with 7 frail, 4 non-frail, and 4 young plasma samples. Ten different fields were evaluated for each specific plasma treatment. Scale bars: 50 μm; zoomed inserts: 10 μm. Nuclei stained by DAPI. (I) Mean ± SEM. of γ-H2AX+ foci per 100 nuclei of untreated or treated NS cells. (J) Cumulative % of γ-H2AX+ multiple-focus nuclei. Color-code: light gray, 1 focus; white, 2 foci; dark gray, 3 foci; black, 4 foci; lines upward right, 5–23 foci per nucleus. (K) γ-H2AX fluorescence intensity per nucleus of NS cells. p < .05 obtained with 1-way ANOVA with Tukey’s test for pairwise multiple comparison was considered significant.

Figure 3.

Plasma oxysterols and cholesterol precursors, intracellular ROS in NS cells, plasma cytokine concentrations. (A) Levels of 27-OHC, lathosterol, lanosterol, and 24-OHC in plasma samples from 17 frail, 18 non-frail, and 10 young individuals (Supplementary Table 5). (B) Left, representative production of intracellular ROS in NS cells with or without treatment with the indicated plasma during growth, flow cytometry analysis of 2ʹ,7ʹ-dichlorofluorescein (DCF) (gating strategy in Supplementary Figure 1). Right, median DCF fluorescence intensity peak in dissociated NS cells of 5 different independent NS cultures, each one treated during growth with individual plasma from a set of 5 frail, 5 non-frail, or 5 young individuals; there were 5 untreated NS cultures (Supplementary Table 6). The DCF median values of plasma-treated cells were normalized to the DCF of corresponding untreated NS cells considered equal to 1. (C) Cytokine concentrations (pg/mL) of IL-6, CX3CL1, CXCL9, IL-1α, and CD40 were significantly higher in frail plasma. Cytokines were evaluated in 38 frail, 34 non-frail, and 36 young subjects by the Human Magnetic Luminex Screening Assay (Supplementary Table 7A). A p value < .05 by 1-way ANOVA with Tukey’s test or Holm‒Sidak’s test for pairwise multiple comparison was considered significant.

Figure 4.

PCA results, distribution of subjects along the first 2 principal components (PC1 and PC2). (A) Distribution of 13 frail, 18 non-frail, and 9 young individuals based on the merged data of the oxidative status, cytokines, and γ-H2AXcHPSC variables of their own plasma. (B) Distribution of 9 frail, 9 non-frail, and 5 young individuals based on the merged data of the oxidative status, cytokines, and γ-H2AXcHPSC variables of their own plasma and the variables SFE and γ-H2AXNSC for the effects of the same plasma on NS cells. (C) Distribution of 17 frail, 18 non-frail, and 10 young individuals based on data on their own plasma oxidative status variables. (D) Distribution of 36 frail, 34 non-frail, and 36 young individuals based on data on their plasma cytokine variables. (E) Distribution of 13 frail, 18 non-frail, and 9 young individuals based on the merged data of their plasma oxidative status and cytokine variables. Each point represents a subject. Color-coded: red, frail; green, non-frail; blue, young. Point shape represents the number of comorbidities of the subject: triangles, more than 2; circles, 2 or less than 2. Supplementary Table 1B shows the individual subjects whose data were used for this integrated analysis.