The cause of chronic kidney disease (CKD)- Anemia
Mar 15, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
PART Ⅰ：Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism
Rosi Bissinger, Travis Nemkov & et al.
The development of anemia is a typical complication of advanced chronic kidney disease (CKD) and is associated with impaired quality of life1 and increased risk for cardiovascular events2 and hospitalization,3 as well as cognitive decline.4 The severity of anemia has been viewed as an independent predictor of mortality in both dialysis- and non– dialysis-dependent CKD (chronic kidney disease) patients.5 The pathophysiology of kidney disease–associated anemia is complex and involves iron and erythropoietin (EPO) deficiency in the setting of low-grade inflammation, which, in turn, compromise normal erythropoiesis in CKD (chronic kidney disease) patients.6 In advanced CKD (chronic kidney disease), the EPO response is inadequately low in relation to the degree of anemia.7,8 The high prevalence of concomitant iron deficiency in CKD (chronic kidney disease) is a consequence of disturbed iron homeostasis.9 A neglected mechanism of iron loss in CKD (chronic kidney disease) is proteinuria, which can lead to urinary losses of transferrin-bound iron (up to 0.3 mg/d) when proteinuria reaches the nephrotic range.10 Another factor that is thought to contribute to anemia in CKD (chronic kidney disease) patients is the shortened lifespan of red blood cells (RBCs), first described >60 years ago.6,11,12
Benefit of Cistanche: treating kidney disease and improving kidney function
A recent study using a carbon monoxide breath test demonstrated that the RBC lifespan progressively decreased from 120 days in patients with stage 1 CKD (chronic kidney disease) to 60 days in patients with stage 5 CKD (chronic kidney disease).13 Notably, transfusion of allogenic RBCs from healthy donors to CKD (chronic kidney disease) patients was followed by rapid clearance of transfused RBCs without evidence of hemolysis.12 A plausible mechanism for this observation may be the stimulation of apoptosis-like cell death in anucleate RBCs, denoting an injury pattern in which the cell membrane integrity is not compromised and the cytoplasmic content remains intact.14 RBCs undergoing cell death exhibit various morphologic alterations resulting from cytoskeletal damage, such as surface bleb formation, loss of membrane elasticity, and/or cellular dehydration.15 On a molecular level, RBC death is associated with intracellular Ca2þ accumulation, altered cellular energy status, and breakdown of phospholipid asymmetry, ultimately leading to externalization of phosphatidylserine (PS) on the outer plasma membrane.15,16 As a consequence, macrophages and specialized dendritic cells swiftly recognize PS-externalized RBCs, leading to erythrose phagocytosis and their catabolism in the spleen and liver.17 Because of the confounding pathophysiology of kidney disease–associated anemia in humans, animal studies are warranted to pinpoint the contributing mechanisms. Doxorubicin-induced nephropathy (DIN) in 129S1/SvImJ mice18 and mice with inducible podocin deficiency (Nphs2Dipod) 19 are 2 models that are characterized by the induction of nephrotic-range proteinuria within days, progression to kidney failure after 3 weeks, and death in 6 to 7 weeks.19–21 Both mouse models effectively recapitulate all stages of human CKD (chronic kidney disease). In the present study, we tested whether progressive kidney failure in these mice with proteinuric kidney disease affects RBC lifespan and contributes to anemia. In parallel, we examined the RBC phenotype in blood drawn from CKD (chronic kidney disease) patients with nephrotic-range proteinuria.
CKD (chronic kidney disease) and anemia: CISTANCHE
METHODS
Detailed information about the materials and methods is provided in the Supplementary Materials and Methods.
Mouse studies
Experiments were performed on 8-week-old wild-type 129S1/SvImJ mice of both sexes (Charles River). DIN was induced by a single injection of doxorubicin (14.5 mg/g body weight), as described previously.18,22 To control for the myelotoxic effect of doxorubicin unrelated to the development of nephropathy, doxorubicin-resistant C57BL/6 mice were also subjected to the same treatment protocol.23 In addition, similar experiments were conducted on 8-week-old mice with inducible deletion of podocin (B6-Nphs2tm3.1Antc*Tg [Nphs1-rtTA*3G] 8Jhm*Tg[tetO-cre]1Jaw) or Nphs2Dipod mice, which were treated with doxycycline for 14 days.19 All animal experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the German Law for the Welfare of Animals, with approval from the local authorities (Regierungspräsidium Tübingen, approval numbers M12/17 and M17/19G). The experimental design of the mouse studies is outlined in Supplementary Figure S1.
Patients
The patient study was conducted in compliance with the Declaration of Helsinki and was approved by the local ethics committee of the University Hospital Tübingen (556/2018BO2). Lithium-heparin blood and urine samples were obtained from patients with nephrotic-range proteinuria and preserved glomerular filtration rate (GFR; stages 1–2; n ¼ 10) and patients with reduced GFR (CKD (chronic kidney disease) stage 3–5; n ¼ 15) at the University Hospital Tübingen. As a control group, blood from age- and sex-matched healthy volunteers (n ¼ 25) was provided by the blood bank of the University Hospital Tübingen. All human samples were collected after informed consent. Clinical characteristics of the patients are stated in Table 1.
Table 1 | Characteristics of the CKD (chronic kidney disease) patients and healthy blood donors
Flow cytometry analyses
Different parameters of RBC cell death were determined by fellow cytometry.14 To determine RBC lifespan in vivo, 25 ml of 5,6-carboxyfluorescein diacetate succinimidyl ester [5(6)-CFDA, SE] dye was injected at a concentration of 9.96 mM (solubilized in dimethylsulfoxide) into the retro-orbital plexus of wild-type 129S1/SvImJ and doxorubicin-injected mice, as described previously.24 At the indicated time points, blood was drawn from the retro-orbital plexus of the mice, and the percentage of 5(6)-CFDA, SEþ cells was detected by fellow cytometry analysis. Finally, data were analyzed using FlowJo software (FlowJo LLC).
RBC deformability and osmotic gradient ektacytometry
RBC deformability was measured using the Laser-Assisted Optical Rotational Cell Analyzer (LORCA Max Sis; RR Mechatronics), which has been described in detail elsewhere.25 The osmotic gradient ektacytometry (cosmos can) analyses were also performed using the LORCA Max Sis and measure deformability under various osmotic conditions.26
Histologic examination
For hematoxylin and eosin staining, spleens and femurs were stained with hematoxylin and eosin. All slides were stained with the primary antibody Ter119 (BD Pharmingen; dilution 1:500). For periodic acid–Schiff staining, 2.5-mm-thick slices of the kidneys were stained with periodic acid–Schiff reagent (Carl Roth) and hematoxylin (Abcam). May-Grünwald-Giemsa staining (Pappenheim method) was performed to determine RBC shape changes, as described previously.27 Glomeruli isolation was done by using a biotinylation approach and cell sorting.19 For protein detection of podocin, an antibody from Sigma was applied (P0372).19 Roti-Mount Fluor Care 4,6-diamidino-2-phenylindole (DAPI; Carl Roth) was used to stain nuclei.
Ultra-high-performance liquid chromatography-mass spectrometry metabolomics from mouse RBCs
Analyses were performed as previously published.28 Brieflfly, the analytical platform employs a Vanquish ultra-high-performance liquid chromatography system (Thermo Fisher Scientific) coupled online to a Q Exactive mass spectrometer (Thermo Fisher Scientific).
Cistanche treat kidney disease caused by anemia
Statistical analyses
Data are provided as arithmetic means SEM or as median with interquartile range (25th–75th percentile) with n representing the number of used animals or included patients, respectively. Data were tested for normality with the Kolmogorov-Smirnov test, the D’Agostino test, and the Shapiro-Wilk test. Variances were analyzed by Bartlett test for equal variances. Tukey or Dunn multiple-comparison posttest, unpaired Student t-test, or Mann-Whitney U test was performed by GraphPad Prism 8 (GraphPad Software). P < 0.05 with 2- tailed testing was considered statistically significant. Additional graphs were plotted through GraphPad Prism 8.
RESULTS
Experimental proteinuric kidney disease induces anemia in mice After induction, 129S1/SvImJ mice with DIN and Nphs2Dipod mice developed nephrotic-range proteinuria (Figure 1a and Supplementary Figure S2C) and progressive kidney failure characterized by high plasma urea levels from day 20 onwards (Figure 1b and Supplementary Figure S2D). During the first 10 days, mice experienced bodyweight gain with ascites (Figure 1c and Supplementary Figure S2E), reflecting sodium retention caused by the excretion of serine proteases or proteinuria.18 After spontaneous reversal of sodium retention, these mice steadily lost weight. In mice with DIN and in Nphs2Dipod mice, light microscopy images, captured after 10 days, revealed typical histomorphologic changes consistent with focal segmental glomerular sclerosis (Figure 1d and Supplementary Figure S2B). These were absent in doxorubicin-injected C57BL/6 mice (Figure 1d). Doxorubicin treatment induced a strong decline in hemoglobin, RBC count, and hematocrit (Figure 1e–g) from day 10 on in 129S1/SvImJ and C57BL/6 mice, which in the latter were normalized at days 20 and 30. In contrast, on days 20 and 30, doxorubicin-injected 129S1/SvImJ and podocin-deficient mice developed progressive anemia, characterized by reduced mean corpuscular volume (Figure 1h) and reduced hemoglobin (Supplementary Figure S2F), suggesting that anemia is associated with progressive kidney failure and not with doxorubicin treatment per see.
Figure 1 | Kidney function and overt anemia in doxorubicin-injected 129S1/SvImJ mice. (a–d) Doxorubicin-injected 129S1/SvImJ mice developed (a) high proteinuria, (b) progressive increase of plasma urea concentration, (c) transient body weight increase, and (d) typical histomorphologic changes indicative of focal segmental glomerular sclerosis on day 10 (periodic acid–Schiff staining; bar ¼ 10 mm). (e–h) In addition, these mice developed anemia reflflected by (e) a decreased hemoglobin level, (f) lower red blood cell (RBC) numbers, (g) diminished hematocrit levels, and (h) decreased mean corpuscular volume. (a–d) Doxorubicin-injected C57BL/6 mice did not show any sign of kidney injury, and (e–g) anemia on day 10 was normalized on days 20 and 30. Arithmetic means SEM are shown. *P < 0.05, **P < 0.01, ***P< 0.001,and ****P < 0.0001 indicate signifificant difference between healthy 129S1/SvImJ and doxorubicin-injected 129S1/SvImJ mice; #P <0.05, ##P < 0.01, ###P < 0.001, and ####P < 0.0001 indicate signifificant difference to the baseline of doxorubicin-injected 129S1/SvImJ mice; and §P < 0.05, §§P < 0.01, §§§P < 0.001, and §§§§P < 0.0001 indicate signifificant difference between doxorubicin-injected 129S1/SvImJ and doxorubicin-injected C57BL/6 mice. Crea, creatinine.
Anemia in experimental proteinuric kidney disease is not caused by compromised erythropoiesis
Both anemic mouse models displayed a significant increase in the percentage of circulating reticulocytes (Figure 2a and Supplementary Figure S3C). Plasma EPO concentrations were dramatically increased at day 10 in 129S1/SvImJ with DIN and healthy C57BL/6 mice but were normalized again on days 20 and 30 (Figure 2b). In podocin-deficient mice, plasma EPO concentrations spiked at day 10 and remained increased at days 20 and 30 (Supplementary Figure S2H). In histologic analyses from bone marrow and spleen, the number of erythroid precursor cells compared with myeloid precursors was increased at day 30 (Figure 2d–g), pointing to stimulated erythropoiesis in anemic 129S1/SvImJ mice with DIN.
Figure 2 | Erythropoiesis is stimulated in doxorubicin-injected 129S1/SvImJ mice with anemia. (a) Anemia in doxorubicin-injected 129S1/SvImJ mice occurred despite increased reticulocyte numbers at days 20 and 30. (b) Plasma erythropoietin concentration was highly increased at day 10 in doxorubicin-injected 129S1/SvImJ and C57BL/6 mice but was normalized again on days 20 and 30. (c–e) Quantification of the absolute numbers of (c) myeloid and (d) erythroid cells in the bone marrow. The (e) ratio of myeloid-to-erythroid cells in healthy and doxorubicin-injected 129S1/SvImJ mice in bone marrow showed a higher number of erythroid precursor cells in doxorubicin-injected 129S1/SvImJ mice on day 30. (f,g) The (f) spleen and (g) bone marrow histology showed an increase in erythroid precursor cells, observed even at lower magnifications, in doxorubicin-injected 129S1/SvImJ mice (right panels) compared with healthy mice (left panels) on day 30. (f,g) Ter119 immunohistochemistry (lower panels) supported the hematoxylin and eosin (H&E) findings. Ter119 is positive in the erythroid precursors (nucleated cells) and in the red blood cells (nonnucleated cells). Bar ¼ 100 mm; insets ¼ 25 mm. Arithmetic means SEM are shown. *P < 0.05, **P < 0.01, and ***P < 0.001 indicate significant difference between healthy 129S1/SvImJ and doxorubicin-injected 129S1/SvImJ mice; ##P < 0.01 and ####P < 0.0001 indicate significant difference to baseline of doxorubicin-injected 129S1/SvImJ mice; and §P < 0.05, §§P < 0.01, and §§§§P < 0.0001 indicate significant difference between doxorubicin-injected 129S1/SvImJ and doxorubicin-injected C57BL/6 mice.
Benefit of Cistanche: treating kidney disease and improving kidney function
Reduced RBC lifespan is the primary cause of anemia in experimental proteinuric kidney disease
Externalization of PS on the outer leaflet of the RBC plasma membrane is an indicator of cell death and a promoter of erythro phagocytosis.14 RBC cell death was quantified using fluorescence-activated cell sorting analyses of fluorescent annexin V–bound surface PS.14 In freshly drawn blood, the percentage of PS-exposing cells was >4-fold higher on day 20 in mice with DIN (4.16% & 0.86%) compared with healthy mice (1.00% & 0.11%) (Figure 3a). Similarly, Nphs2Dipod mice showed an approximate 2-fold increase in PS exposure (1.27% & 0.20%) compared with healthy mice (0.58% & 0.05%) on day 30 (Supplementary Figure S3A). It is known that RBCs are eliminated from circulation by macrophages residing in the spleen.17 This observation may, therefore, explain the higher spleen/body weight ratio of Nphs2Dipod mice (Supplementary Figure S2G), wherein twice as many RBCs are degraded compared with healthy C57BL/6 mice. As nephrotic-range proteinuria leads to dysproteinemia,29 we further investigated whether enhanced RBC cell death may be stimulated by a component in the plasma of mice with DIN. As depicted in Figure 3b, PS exposure at days 10 and 20 were twice as high following incubation (30 minutes at 37OC) of healthy RBCs in plasma of doxorubicin-injected 129S1/ SvImJ mice compared with incubation in plasma of healthy mice. Ca2þ influx into RBCs, mediated by voltage-gated and voltage-independent nonselective cation channels,30,31 is one of the key regulators of RBC cell death. In RBCs collected at days 20 and 30 from 129S1/SvImJ mice with DIN, intracellular Ca2þ concentrations were increased (Figure 3c); this phenomenon was recapitulated in Nphs2Dipod mice on day 20 (Supplementary Figure S3B). In both mouse models, there was a significant negative correlation of the percentage of PS-positive RBCs, with the severity of anemia reflected by hemoglobin levels (Figure 3d and Supplementary Figure S3D). Moreover, there was a significant correlation with kidney damage reflected by plasma urea concentration (Figure 3e and Supplementary Figure S3E) and to a lesser degree with proteinuria (Figure 3f and Supplementary Figure S3F). To compensate for RBC loss in anemia, the formation of new RBCs was stimulated in both mice, as indicated by increased percentage of circulating reticulocytes, and was significantly correlated with the magnitude of PS exposing RBCs (Figure 3g and Supplementary Figure S3G).
Figure 3 | Mice with doxorubicin-induced nephropathy develop enhanced red blood cell (RBC) death mediated by increased intracellular calcium levels. (a) Externalization of phosphatidylserine (PS), reflecting RBC death, was enhanced on days 20 and 30 after induction of doxorubicin-induced nephropathy. (b) Incubation of healthy RBCs in plasma taken on days 10 and 20 from these mice led to PS externalization. (c) PS externalization was accompanied by enhanced intracellular calcium levels of RBCs taken on days 20 and 30 after induction. (d–g) The (d) percentage of PS-exposing RBCs was correlated with hemoglobin levels, kidney damage indicated by (e) plasma urea concentration and (f) proteinuria, as well as with (g) reticulocyte formation. (d–g) Data include each time point (0, 10, 20, and 30 days) of each healthy 129S1/SvImJ and 129S1/SvImJ mouse with doxorubicin-induced nephropathy. Arithmetic means SEM are shown. **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate significant difference between healthy 129S1/SvImJ and doxorubicin-injected (in.) 129S1/SvImJ mice; #P < 0.05, ##P < 0.01, and ####P < 0.0001 indicate significant difference to baseline of doxorubicin-injected 129S1/SvImJ mice; and §§§§P < 0.0001 indicate significant difference between doxorubicin-injected 129S1/SvImJ and doxorubicin-injected C57BL/6 mice. Crea, creatinine; MFI, mean fluorescence intensity.
Benefit of Cistanche: treating kidney disease and improving kidney function
