Do you know anemia may cause kidney disease?
Mar 15, 2022
Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com
PART Ⅱ：Proteinuric chronic kidney disease is associated with altered red blood cell lifespan, deformability and metabolism
Rosi Bissinger, Travis Nemkov & et al.
Anemia is a common complication of chronic kidney disease, affecting the quality of life of patients. Among various factors, such as iron and erythropoietin deficiency, reduced red blood cell (RBC) lifespan has been implicated in the pathogenesis of anemia. However, mechanistic data on in vivo RBC dysfunction in kidney disease are lacking. Herein, we describe the development of chronic kidney disease-associated anemia in mice with proteinuric kidney disease resulting from either administration of doxorubicin or an inducible podocin deficiency. In both experimental models, anemia manifested at day 10 and progressed at day 30 despite increased circulating erythropoietin levels and erythropoiesis in the bone marrow and spleen. Circulating RBCs in both mouse models displayed altered morphology and diminished osmotic-sensitive deformability together with increased phosphatidylserine externalization on the outer plasma membrane, a hallmark of RBC death. Fluorescence-labelling of RBCs at day 20 of mice with doxorubicin-induced kidney disease revealed premature clearance from the circulation. Metabolomic analyses of RBCs from both mouse models demonstrated temporal changes in redox recycling pathways and Lands'cycle, a membrane lipid remodeling process. Anemic patients with proteinuric kidney disease had an increased proportion of circulating phosphatidylserine-positive RBCs.
Thus, our observations suggest that reduced RBC lifespan, mediated by altered RBC metabolism, reduced RBC deformability, and enhanced cell death contribute to the development of anemia in proteinuric kidney disease.
Cistache can treat CKD (chronic kidney disease) and anemia
Proteinuric chronic kidney disease (CKD) patients with anemia display enhanced RBC death To confirm that PS-exposing RBCs occur also in human CKD (chronic kidney disease), as described earlier,37 we analyzed blood samples from 25 patients treated by our outpatient clinic. To match the mouse models that represent nephrotic syndrome with preserved GFR during the first 10 days, and then advanced CKD with reduced GFR from day 20 onwards (Figure 1 and Supplementary Figure S2), we analyzed 10 patients with primary nephrotic syndrome representing proteinuric CKD (chronic kidney disease) with preserved GFR (>60 ml/min per 1.73 m2 ) and 15 patients with CKD (chronic kidney disease) with nephrotic-range proteinuria and GFR <60 ml/min per 1.73 m2. The patient characteristics are shown in Table 1. Kidney disease–associated anemia, as defined by a hemoglobin concentration <13.5 g/dl in men and <12 g/dl in women, was observed in 4 of the 10 primary nephrotic patients (red triangles in Figure 7), whereas 14 of 15 CKD (chronic kidney disease) patients with nephrotic-range proteinuria and reduced GFR were anemic (Figure 7a). In the latter group, plasma EPO concentrations and reticulocyte production index were not increased (Figure 7b and c), consistent with reduced erythropoiesis. In fluorescence-activated cell sorting analysis, primary nephrotic patients and patients with advanced CKD had a higher rate of PS-exposing cells (mean, 1.0% ± 0.3% and 1.4% ± 0.7%, respectively) compared with healthy subjects (mean, 0.6% ± 0.1%; Figure 7d). RBC cell death in patients with primary nephrotic syndrome and advanced CKD (chronic kidney disease) was triggered by higher levels of reactive oxygen species (Figure 7e) and increased ceramide levels (Figure 7f). Augmented intracellular calcium concentration was found in patients with advanced CKD (chronic kidney disease) (Figure 7g).
Human RBCs from patients with primary nephrotic syndrome and advanced CKD (chronic kidney disease) showed morphologic alterations, as observed in the mouse models (Figures 4c and 7j–l and Supplementary Figure S4A). Although RBC morphology was normal in controls, anemic patients with primary nephrotic syndrome and advanced CKD (chronic kidney disease) patients had an increased number of teardrop cells (black triangles) and schistocytes (black crosses) (Figure 7k and l). In addition, target cells occurred in primary nephrotic patients with anemia and in patients with advanced CKD (chronic kidney disease) (red crosses; Figure 7k and l). All patient groups, including primary nephrotic patients without anemia, had an increased proportion of spherocytes (blue arrows; Figure 7j–l).
To analyze the deformability of human RBCs, ektacytometry was performed. In comparison to healthy controls, maximum deformability (EImax) was reduced in patients with advanced CKD (chronic kidney disease) (Figure 7h); EImax tended to be lower in patients with a primary nephrotic syndrome without reaching statistical significance (Figure 7h). The parameters SS1/2, Omin, Ohyper, and EImax at isotonicity were not significantly different between healthy controls, primary nephrotic patients, and patients with advanced CKD (chronic kidney disease) (Supplementary Figure S8A–D).
Figure 7 | Red blood cell (RBC) death in proteinuric chronic kidney disease (CKD) patients with anemia. (a–c) The (a) hemoglobin, (b) plasma erythropoietin concentration, and (c) reticulocyte production index in healthy, primary nephrotic patients and patients with advanced CKD (chronic kidney disease). (d–f) Percentages of (d) phosphatidylserine (PS)–exposing RBCs, (e) dichlorodihydrofluorescein diacetate (DCFDA) fluorescence, and (f) ceramide-dependent fluorescence as factors associated with RBC death was augmented in primary nephrotic patients and in patients with advanced CKD. (g) Intracellular calcium concentration was enhanced in advanced CKD (chronic kidney disease) patients. (h) Ektacytometry measurements revealed that RBC deformability of patients with advanced CKD (chronic kidney disease) was significantly impaired, as indicated by a diminished maximum elongation index (Imax). (i–l) May-Grünwald-Giemsa staining (Pappenheim method) revealed morphologic alterations in primary nephrotic syndrome patients (j) without anemia and (k) with anemia, and in (l) patients with advanced CKD (chronic kidney disease) compared with RBCs obtained from (i) healthy donors. Arithmetic means SEM are shown. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 indicate significant difference between groups.Dep., dependent; GFR, glomerular filtration rate; MFI, mean fluorescence intensity; NS, not significant.
Cistache can treat kidney disease
DISCUSSION
The present study reveals novel pathophysiological mechanisms leading to kidney disease–associated anemia in 2 murine models of proteinuric kidney disease with severely impaired kidney function. Our study demonstrates that in these models, anemia is the result of a reduced RBC lifespan triggered by exposure of PS and accelerated phagocytic clearance. Intriguingly, anemia in these mice developed despite stimulated erythropoiesis, suggesting that reduced RBC lifespan, through increased RBC cell death, might be an alternative explanation for these findings. Contrary to CKD (chronic kidney disease) patients with anemia (Figure 7 ), both mouse models were characterized by increased plasma EPO concentration. This can be surmised by the preservation of EPO-secreting ability in these models that probably spares the EPO-secreting cells located in the interstitium of the kidney. The increased EPO secretion in these models, however, does not invalidate the conclusion that RBC cell death is a major player in the pathogenesis of kidney disease–associated anemia. On the contrary, stimulation of erythropoiesis by increased EPO secretion can be considered as a compensatory mechanism to increased RBC death induced by kidney failure in these models. Along these lines, increased extramedullary erythropoiesis with increased spleen volume was recently observed in another proteinuric mouse model with anemia.38
In patients with proteinuric CKD (chronic kidney disease) and concomitant anemia, we also observed an increased percentage of PS-exposing RBCs along with higher levels of reactive oxygen species and ceramide. This suggests that accelerated RBC death might be involved in the pathogenesis of kidney disease–associated anemia in human CKD (chronic kidney disease). Plasma EPO concentrations and reticulocyte production index were not increased in anemic CKD patients, pointing to reduced erythropoiesis, which in concert with RBC death is expected to aggravate kidney disease–associated anemia. The reasons for the loss of EPO secretion of the kidney in human CKD (chronic kidney disease) remain unclear. Remarkably, although not all patients with normal GFR had anemia, those with reduced GFR were all anemic, pointing to an effect of long-standing and advanced CKD (chronic kidney disease). Notably, the relative EPO deficit in CKD (chronic kidney disease) can be overcome by using the new class of prolyl hydroxylase inhibitors,39 suggesting perturbed oxygen sensing as a possible cause for EPO hyposecretion.
Our data demonstrate diminished RBC deformability in both mouse models of proteinuric nephropathy, which may be directly related to elevated cytoplasmic Ca2þ levels.40 Together, these mechanisms could act in concert to facilitate the induction of RBC cell death and removal of senescent and injured RBCs from the blood circulation.15 Furthermore, we observed metabolic reprogramming in these cells, indicative of oxidative stress and membrane lipid remodeling. Although CoA and acyl-CoA were not directly measured in these samples, they are actively converted in RBCs to acylcarnitines by carnitine palmitoyltransferase.36 Accumulating levels of the latter compound class indicate activation of these mechanisms in nephropathy, as these metabolites are not readily transported across RBC membranes.41 In further support, we observed accumulation in both models of CoA precursors, including pantothenate, which is taken up42 and metabolized43 by RBCs, in parallel to increasing free fatty acids and decreasing free carnitine. Interestingly, we previously found that these alterations occur in association with supraphysiologic levels of intracellular Ca2þ. 16 Although those results were generated ex vivo, we report herein similar responses in vivo. Furthermore, acylcarnitines are capable of directly modulating membrane properties44 and correlate with RBC deformability,45 as well as osmotic and oxidative hemolysis.46 Unconjugated free carnitine promotes membrane deformability through the mediation of interactions between membrane proteins.47 Our observations of significantly decreased levels of carnitine in RBCs from mice with nephropathy, presumably due to increased consumption for the generation of acylcarnitines, may contribute to the impaired rheological parameters we observed in parallel.
Cistache can treat chronic kidney disease
Our findings suggest common mechanisms leading to RBC death in mice with both DIN and podocin deficiency, which may be related to both nephrotic-range proteinuria and, more important, the development of severe kidney failure in the mouse models observed from day 20 on. In humans, advanced CKD (chronic kidney disease) with reduced GFR is a strong predictor of anemia,48 and stimulation of RBC death could be related to the uremic milieu. One has to acknowledge that in advanced CKD (chronic kidney disease), many factors and derangements might come into play and promote kidney disease–associated anemia. The contribution of heavy proteinuria to the stimulation of RBC death remains unclear, but, although not proven, might involve factors that are lost in the urine, such as transferrin or others regulating RBC metabolism.49 So far, the current treatment of kidney disease–associated anemia focuses on increasing erythropoiesis by iron or EPO substitution,50 by application of oral hypoxia-inducible factor protein stabilizers,51 or by oral or i.v. iron administration.52 However, these treatments do not consider increased RBC death. In a previous cross-sectional study in hemodialysis and peritoneal dialysis patients, we found that patients with a higher percentage of PS-exposing RBCs were treated with higher EPO doses.14 Therefore, amelioration of RBC cell death promises to be a possible therapeutic approach in treating kidney disease–associated anemia. In this context, the inhibitory effect of various pharmacologic agents on RBC cell death53 requires further human and animal studies.
In conclusion, altered cellular metabolism contributes to RBC dysfunction, enhanced RBC death, and hence anemia in mouse models of proteinuric CKD (chronic kidney disease), despite increased serum EPO levels. The findings of this study may partly explain the mechanisms of anemia associated with CKD (chronic kidney disease) in humans. DISCLOSURE Although unrelated to the contents of the articles, ADA and TN are founders of Omix Technologies, Inc. All the other authors declared no competing interests. DATA STATEMENT Data will be made available on reasonable request.
Treatment for kidney disease and anemia:cistanche
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