Kidney iron deposition by R2* is associated with hemolysis and urinary iron

Abstract

Kidney iron deposition measured by R2* MRI is posited to result from tubular reabsorption of filtered hemoglobin due to intravascular hemolysis. In chronically transfused sickle cell disease (SCD), R2* is elevated and positively correlated with lactate dehydrogenase (LDH).(1) To account for contributions to renal iron from systemic iron overload, we evaluated kidney R2*, urinary iron and hemolysis markers in 62 non-transfused SCD patients. On multivariate analysis, kidney R2* was associated with urinary iron and LDH (R²=0.55, p<0.0001). Our study confirms that kidney R2* is associated with intravascular hemolysis and raises important questions regarding the role of iron in SCD nephropathy.

Introduction

Kidney iron deposition has been described in hemolytic disorders due to mechanical valves, paroxysmal nocturnal hematuria, and sickle cell disease (SCD). On MRI, this manifests as signal loss on gradient and spin echo sequences due to preferential hemosiderin deposition in the kidney.(2) The signal darkening is quantified by the parameter R2*, which has been shown to be directly proportional to tissue iron in the liver and heart. Kidney R2* has previously been demonstrated to rise proportionally to lactate dehydrogenase (LDH) in chronically transfused SCD patients.(1) LDH is not a specific marker of hemolysis, however, and chronically transfused patients could potentially deposit iron in the kidney through other mechanisms. To better understand the source of renal iron loading, we characterized the relationship between kidney R2*, urinary iron and markers of hemolysis in non-transfused SCD patients.

Methods

Sixty-two non-transfused SCD patients were recruited to the study, which was approved by the Institutional Review Board of Children’s Hospital Los Angeles. Following medical history and physical exam, subjects completed blood and urine testing, and then abdominal MRI for assessment of somatic iron stores. We used a 1.5 Tesla General Electric Cvi scanner (Philips Medical Systems, Best, The Netherlands) using a five element torso coil. Liver R2* was measured using a 16-echo gradient echo sequence with equally spaced echoes from 1 to 14.3 ms; other parameters were 36 x 36 cm field of view (FOV), 96 x 96 matrix, 10 mm slice thickness, 50 ms repetition time (TR), 20° flip angle, and 2083 Hz/px bandwidth. Kidney R2* was measured using the same sequence with echo times equally spaced from 1.1 to 17.3 ms; other parameters were 40 x 30 cm FOV, 136 x 198 matrix, 5 mm slice thickness, 18 ms TR, 20° flip angle, and 1485 Hz/px bandwidth. The pancreas R2* used 16 echoes equally spaced from 1.2 to 19 ms; other parameters were 40 x 30 cm FOV, 156 x 156 matrix, 5 mm slice thickness, 20.3 ms TR, 20° flip angle, and 1282 Hz/px bandwidth. Statistical analysis was performed using JMP® Pro, Version 14.0.0 (SAS Institute Inc., Cary, NC, 2018).

Results

Subjects were generally young adults with median age of 28 [12-70] years (Table 1). Nearly three quarters of subjects had homozygous SCD, while the remaining had SC disease or S-Beta thalassemia. Most subjects were anemic with median hemoglobin 9.1 g/dL, and all subjects had elevated markers of hemolysis. Ninety percent of subjects had mild or no systemic iron overload by LIC. Fifty-four percent had an elevated kidney R2* level (≥34 Hz). On univariate analyses, kidney R2* was associated with urinary iron, LDH, plasma hemoglobin, and hemoglobin (Figure 1), but it was not associated with absolute reticulocyte count (Table 1). LDH was associated with plasma hemoglobin (R²=0.4958, p<0.0001) and reticulocyte count (R²=0.0913, p=0.0161), and plasma hemoglobin was associated with reticulocyte count (R²=0.1634, p=0.0011). On multivariate analysis, kidney R2* was associated with urinary iron and LDH (R²=0.49, p<0.0001). Kidney R2* was not associated with liver or pancreas R2*.

Table 1.

Subject characteristics and their univariate associations with kidney R2* (n=62). Variables log-transformed to better fit normality as necessary, including kidney R2*. Significance determined at α=0.05 with Bonferroni correction as appropriate.

Parameter (x)	Summary		Kidney R2* vs. x R2 (p-value)
Age (years), median [IQR]	27	[20-39]	0.0021 (0.7218)
Sex, n [%]
Male	22	[35]	0.0129 (0.3790)
Ethnicity, n [%]
Black	56	[90]	0.0010 (0.8036)
Hispanic	6	[9]
Genotype, n [%]
SS	46	[74]	0.0897 (0.0180)
SC	13	[21]
S-Beta thalassemia	3	[5]
Vitals, median [IQR]
Height (cm)	168	[164-175]	0.0059 (0.5530)
Weight (kg)	66	[57-75]	0.0176 (0.3045)
SBP (mm Hg)	115	[105-128]	<0.0001 (0.9623)
DBP (mm Hg)	64	[58-73]	0.0037 (0.6404)
HR (bpm)	74	[66-80]	0.0505 (0.0790)
RR (bpm)	19	[18-20]	0.0038 (0.6826)
O2 Sat (%)	98	[96-99]	0.0305 (0.1901)
Laboratory values, median [IQR]
Hemoglobin (g/dL)	9.1	[8.0-10.5]	0.1236 (0.0051)
WBC (K cells/μL)	7.9	[6.1-10.0]	0.0394 (0.1219)
Platelets (K cells/μL)	348	[256-426]	0.0147 (0.3517)
Reticulocyte (K cells/μL)	196	[129-342]	0.0020 (0.7354)
Plasma hemoglobin (mg/dL)	15	[9-25]	0.2635 (<0.0001)
LDH (units/L)	905	[734-1264]	0.4195 (<0.0001)
HS-CRP (mg/L)	3.6	[1.7-7.3]	0.0275 (0.1974)
Ferritin (ng/mL)	122	[63-453]	0.0071 (0.5152)
Urinary iron (μg/L)	106	[8-257]	0.5087 (<0.0001)
Iron by MRI, median [IQR]
Liver R2* in LIC (mg/g DLW)	1.35	[1.15-2.07]	0.0026 (0.6921)
Pancreas R2* (Hz)	27	[21-30]	0.0740 (0.0339)
Kidney R2* (Hz)	38	[21-48]

transformed x-variables: log[SBP], log[DBP], log[HR], log[Hgb], log[wbc], log[absolute retic], log[plasma Hgb], log[LDH], log[CRP], log10[ferritin], log[urinary iron], log[R2* LIC], log[pancreas R2*]

Figure 1.

Univariate analyses of kidney R2* vs. (A) urinary iron, (B) LDH, (C) cell-free plasma hemoglobin, and (D) hemoglobin on log-transformed x- and y-axes. Blue line is log-log fit and red line indicates upper limit of normal kidney R2* of 34 Hz.

Discussion

Urinary iron appears when renal iron filtration is increased or tubular iron reabsorption is decreased.(3) Non-transferrin bound iron (NTBI), transferrin bound iron (TBI) and cell-free plasma hemoglobin can all appear in the urine in pathological situations. NTBI is freely filtered in the kidney, although virtually absent in plasma until transferrin binding capacity is saturated.(4) TBI, on the other hand, is minimally filtered except in the setting of glomerulopathy, then resorbed at the proximal and distal tubules, but it can appear in urine due to tubular damage.(5) Plasma hemoglobin results from intravascular hemolysis due to intrinsic red cell fragility or mechanical disruption. Most plasma hemoglobin is bound by haptoglobin, or metabolized to heme, which is bound by hemopexin, preventing renal filtration in both cases. Thus, hemolysis only leads to renally filtered iron if the hemolysis rate and duration swamp intrinsic buffering mechanisms.(6) In chronically transfused SCD patients, all three forms of renal iron loading can potentially co-exist.

The present work is important because it documents significant relationships between kidney R2*, urinary iron concentration, LDH and plasma hemoglobin in SCD patients without transfusional iron overload. In fact, the lack of association between kidney R2* and liver or pancreas R2* supports a different mechanism of iron loading. Transferrin saturation, a surrogate for NTBI, is not high in non-transfused SCD patients,(7) therefore NTBI is unlikely to contribute to either urinary iron or kidney R2* values. While extravascular heme recycling and iron export are extremely high in SCD due to shortened RBC survival, effective erythropoiesis and relatively low hepcidin leads to avid bone marrow utilization of iron via holo-transferrin, preserving circulating apo-transferrin levels. NTBI is lower even in transfused SCD than in thalassemia major, and extrahepatic manifestations of iron overload in the heart and endocrine glands are lower in SCD despite similar levels of hepatic iron overload.(8) Thus, highly effective erythropoiesis protects the kidney from NTBI filtration and damage in the same manner that it protects the heart and endocrine glands. However, renal filtration of holo-transferrin could theoretically contribute to urinary iron and kidney R2* in SCD patients with proteinuria.

Transferrin is the same size as albumin, and in patients with renal tubular dysfunction, urinary iron parallels albuminuria.(5) While albuminuria is common in SCD patients, it was not measured in this study. Previous reports in young SCD patients on chronic transfusion show an inverse relationship between proteinuria and kidney R2*, suggesting factors other than transferrinuria dominate.(9) The dependence of kidney R2* on LDH and plasma hemoglobin supports the hypothesis that filtered hemoglobin and heme are the dominant source of urinary iron and kidney R2*. Haptoglobin and hemopexin were not measured in this study but are characteristically depleted in SCD patients, and hemopexin deficiency is associated with renal injury in SCD.(10) Reticulocyte count represents marrow activity, and plasma hemoglobin correlates with red cell destruction, which indirectly correlates with LDH. Since LDH and plasma hemoglobin are more representative of intravascular rather than extravascular hemolysis, while reticulocytosis occurs with both, the lack of association between kidney R2* and reticulocyte count suggests that the site of red cell destruction is important in determining renal iron exposure. The strong correlation between kidney R2* and both LDH and urinary iron on multivariate analysis also supports kidney R2* as a marker of filtered iron from intravascular hemolysis.

Functional consequences of kidney iron in SCD are unknown. In diabetic nephropathy, urinary iron also appears to exacerbate tubular damage through free radical production from NTBI as transferrinuria worsens.(11) SCD nephropathy is associated with upregulation of endothelin-1, which is known to be a pro-inflammatory, mitogenic, natriuretic, and nociceptive mediator.(12) Hemolysis and subsequent heme production lead to nitric oxide depletion and increased renal production of endothelin-1, which in turn promotes iron deposition and renal oxidative stress. Endothelin-1 is active in the pathogenesis of chronic kidney disease, and preliminary studies of endothelin receptor antagonists in SCD suggest therapeutic benefit.(13)

In conclusion, this report suggests that hemolysis is linked to kidney iron deposition in SCD in the absence of chronic transfusions or iron chelation therapy, providing a clearer picture of the source of kidney iron in SCD patients. Kidney R2* and urinary iron concentration are strongly determined by the severity of intravascular hemolysis, independent of systemic iron overload. Hemolysis is an important marker of disease severity, e.g. priapism and stroke,(14) and kidney R2* may represent a more informative long-term biomarker than single LDH or cell-free hemoglobin measurements. Future studies should evaluate the trend of kidney R2* over time; similar to the use of hemoglobin A1c for chronic hyperglycemia in diabetes, kidney R2* may serve as a marker of chronic hemolysis in SCD.