The authors have declared that no competing interests exist.
Conceived and designed the experiments: M. Pruijm BV LH MS MB. Performed the experiments: M. Pruijm M. Piskunowicz CZ MM IB. Analyzed the data: M. Pruijm M. Piskunowicz LH IB MM BV MB. Wrote the paper: M. Pruijm MB.
Experimentally renal tissue hypoxia appears to play an important role in the pathogenesis of chronic kidney disease (CKD) and arterial hypertension (AHT). In this study we measured renal tissue oxygenation and its determinants in humans using blood oxygenation level-dependent magnetic resonance imaging (BOLD-MRI) under standardized hydration conditions. Four coronal slices were selected, and a multi gradient echo sequence was used to acquire T2* weighted images. The mean cortical and medullary R2* values ( = 1/T2*) were calculated before and after administration of IV furosemide, a low R2* indicating a high tissue oxygenation. We studied 195 subjects (95 CKD, 58 treated AHT, and 42 healthy controls). Mean cortical R2 and medullary R2* were not significantly different between the groups at baseline. In stimulated conditions (furosemide injection), the decrease in R2* was significantly blunted in patients with CKD and AHT. In multivariate linear regression analyses, neither cortical nor medullary R2* were associated with eGFR or blood pressure, but cortical R2* correlated positively with male gender, blood glucose and uric acid levels. In conclusion, our data show that kidney oxygenation is tightly regulated in CKD and hypertensive patients at rest. However, the metabolic response to acute changes in sodium transport is altered in CKD and in AHT, despite preserved renal function in the latter group. This suggests the presence of early renal metabolic alterations in hypertension. The correlations between cortical R2* values, male gender, glycemia and uric acid levels suggest that these factors interfere with the regulation of renal tissue oxygenation.
Numerous experimental studies have suggested that disturbed oxygenation plays a role in the development and progression of kidney disease including hypertensive nephropathy
Until recently, human data were largely lacking mainly due to the lack of non-invasive methods to estimate renal tissue oxygenation. Over the last decade, blood oxygenation level- dependent magnetic resonance imaging (BOLD-MRI) has become a powerful tool to estimate renal tissue oxygenation non-invasively in humans. The basic principle of BOLD-MRI is that changes in renal tissue desoxyhemoglobin concentrations involve generation of phase incoherence of magnetic spins, leading to an increase in apparent relaxation rate R2* (expressed in sec−1). Under the assumption that blood pO2 is in equilibrium with tissue pO2, R2* values provide estimates of tissue oxygenation, a low R2* indicating a high tissue oxygenation
Several studies have used BOLD-MRI in humans to investigate renal oxygenation in different forms of chronic kidney disease (CKD)
Changes in renal tissue oxygenation may also contribute to the development of ischemic and hypertensive nephropathies
The aim of the present study was therefore to assess renal tissue oxygenation at baseline and after an acute administration of furosemide in patients with various levels of renal function as well as in patients with essential hypertension and a normal renal function and in control subjects. Moreover, we analyzed the potential determinants of renal cortical and medullary tissue oxygenation in these patient groups.
This research project was approved by the local institutional review committee (Ethical Committee of the Canton de Vaud, Switzerland) and conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from each participant.
Patients with CKD stage 1–5, or with hypertension without CKD were eligible for this study. CKD was defined as an estimated glomerular filtration rate (eGFR) ≤60 ml/min/1.73 m2, or the presence of structural or functional abnormalities for at least three months
Patients were recruited at the outpatient nephrology and hypertension clinic of the university hospital in Lausanne (CHUV). Controls were recruited by local advertisement. Participants were maintained on their regular diet. Salt intake, proteinuria, and creatinine clearance were measured before BOLD-MRI by a 24 h urine collection. On the day of each BOLD-MRI measurement, an identical oral hydration protocol was followed by each participant at home (loading dose of 5 ml/kg of water at 8am, followed by 3 ml/kg every hour till 12am), see extended methods for further details and justification of this hydration protocol. Subjects joined our research unit at 11.30 am. BP was measured three times by an experienced research nurse using an automated Omron 705IT oscillometric device according to the recommendations of the European Society of Hypertension
BOLD-MRI was performed between 1 and 2 pm in the radiology department. Magnetic resonance measurements were carried out on a 3T whole-body MR system (MAGNETOM Trio, Siemens Medical Systems, Erlangen, Germany), as described previously
Anatomical templates are shown on the left, R2* maps in the middle, and color maps on the right (low R2* levels corresponding to higher tissue oxygenation in red, high R2* levels in yellow). Two regions of interest (ROIs) are traced in the form of circles (20 voxels each) in the cortex and medulla of each kidney; this procedure is repeated on four different slices.
Clinical data were analyzed using STATA 11.0 (StataCorp, College Station, Texas, USA). Quantitative variables were expressed as mean ± standard deviation, or as median (25th–75th percentile range), as appropriate. Qualitative variables were expressed as number of patients and percentage. Comparisons between baseline characteristics of study groups were analyzed with ANOVA. Distribution of variables was also expressed using the probability density function according to Kernel. In case of non-normal distribution, variables were log-transformed. Multivariable logistic regression, adjusting for the predefined variables age, sex, current smoking, body mass index, diabetes, hemoglobin, and 24 h urinary sodium excretion, was used to determine the independent association of cortical and medullary R2* values with CKD status. These variables were selected based on their known association with CKD (body mass index, diabetes, age), or because of a theoretical association with renal tissue oxygenation (hemoglobin, 24 h urinary sodium excretion, smoking).
Similarly, in order to determine the independent association of each independent variable of interest with the dependent variables cortical and medullary R2* values, multivariate linear regression was performed, adjusted for the same predefined covariates.
Associations between the eGFR slope and R2* values were examined with Spearman's rank correlation and multivariable linear regression analysis. Results of the logistic multivariate analysis are presented as Odds-ratio (OR) and 95% confidence interval (95% CI). Results of all linear multivariate analyses are presented as beta-coefficients (β) and their 95% confidence intervals.
Details of the screening procedure are provided in the 'extended
Control (n = 42) | CKD (n = 95) | AHT (n = 58) | |
Age (years) | 46±13 |
56±15 | 57±11 |
Sex (% female) | 52 |
30 | 32 |
Currently smoking (%) | 7 |
27 | 35 |
Body Mass Index (kg/m2) | 26±5 | 27±5 | 29±5 |
Systolic BP (mmHg) | 122±13 |
135±19 | 142±16 |
Diastolic BP (mmHg) | 73±10 | 76±12 | 82±10 |
eGFR (CKD-EPI, ml/min/1.73 m2) | 97±14 |
57±31 | 91±15 |
eGFR (MDRD, ml/min/1.73 m2) | 94±15 |
57±29 | 91±16 |
Hemoglobin (g/dl) | 136±11 | 130±18 | 138±13 |
Blood glucose (mmol/l) | 5.6±0.9 |
6.5±2.1 | 6.1±1.2 |
Diabetes (%) | 0 |
23 | 17 |
Blood potassium (mmol/l) | 3.9±0.2 |
4.2±0.6 | 3.8±0.3 |
Venous bicarbonate (mmol/l) | 27(20;30) |
25 (14;32) | 27 (23;32) |
Blood uric acid (μmol/l) | 289 (130;450) |
391 (168;662) | 342 (163;548) |
Oxygen saturation (%) | 97±2.0 | 96±1.9 | 96±1.6 |
24 h Urinary volume (ml) | 1731 (694;5008) | 2068 (585;4356) | 1812 (780;3945) |
24 h Urinary sodium excretion (mmol) | 156±72 | 173±92 | 174±87 |
24 h Urinary protein excretion (g) | 0.06 (0;0.12) | 0.3 (0;9.4) | 0.07 (0;0.17) |
24 h Urinary albumin excretion (mg) | 4.7 (0;23) | 100 (1;6131) | 10 (0;29) |
24 h Urinary creatinine clearance (ml/min) | 125 (83;213) | 68 (14; 170) | 115 (73;249) |
Values are expressed as mean±SD, or as median (min; max) as appropriate. CKD = chronic kidney disease; AHT = arterial hypertension.
* p<0.05: control group versus CKD;
** p<0.05 AHT versus CKD.
CKD (n = 95) | AHT (n = 58) | Controls (n = 42) | |
Beta blocker | 34.9 | 39.6 | 0 |
Alpha blocker | 2.3 | 0 | 0 |
ACE-inhibitor | 17.4 | 11.5 | 0 |
AT-II type 1 receptor blocker | 52.3 | 26.4 | 0 |
Calcium channel blockers | 32.6 | 35.9 | 0 |
Thiazide diuretic | 27.9 | 20.8 | 0 |
Loop diuretic | 17.4 | 3.9 | 0 |
Statine | 57 | 33 | 5 |
Fibrate | 1.2 | 0 | 0 |
Ezetimibe | 5.8 | 9.6 | 0 |
Aspirin | 31.4 | 41.5 | 2.4 |
Allopurinol | 17.4 | 3.9 | 0 |
23.3 | 3.9 | 0 | |
Oral antidiabetics | 8.1 | 11.5 | 0 |
Insuline | 17.4 | 3.8 | 0 |
All values are expressed as the percentage of patients of each group treated with the drug in question; CKD: chronic kidney disease (CKD), AHT: arterial hypertension AHT.
There were no differences in mean or median cortical and medullary R2* levels between the right and left kidney in all groups (cortex (median (range)) respectively 17.4 (16.1; 18.6) and 17.6 (16.3; 18.8) sec−1, p = 0.29; and medulla (mean±SD): 28.7±4.2 vs 28.7±2.5 sec−1, p = 0.94). Therefore, only the mean cortical and medullary R2* values of both kidneys are shown and used for statistical analysis. The distribution of medullary R2* was comparable in all groups, but that of cortical R2* values differed markedly between groups (
A/medullary R2* values and B/cortical R2* values.
The number of subjects was n = 10 (for the eGFR>125 ml/min/1.73 m2 category), 64 (eGFR 90–125), 65 (60–89), 36 (30–59), 15 (15–29) and 5 (<15), respectively.
Control | CKD | AHT | p (ANOVA) | |
men | 29.5±2.7 | 29.1±2.6 | 28.7±2.1 | 0.53 |
women | 29.0±1.7 | 28.3±2.7 | 28.4±2.2 | 0.50 |
men | −6.8±3.3 | −3.7±2.5 | −4.9±2.1 | 0.001 |
women | −5.4±2.2 | −3.7±2.1 | −4.2±2.1 | 0.02 |
men | 17.8 (16.9;19.0) | 17.8 (17.0;18.5) | 17.4 (16.4;18.2) | 0.36 |
women | 16.2 (15.3;17.8)* | 17.5 (16.1;18.7) | 17.4 (16.0;18.6) | 0.18 |
men | −1.0 (−0.88;−2.2) | −1.2 (−0.55;−1.8) | −1.3 (−1.0;−1.78) | 0.81 |
women | −1.2 (−0.52;−1.5) | −1.5 (−0.59;−2.0) | −1.9 (−0.9;−2.4) | 0.08 |
men | 1.64±0.2 | 1.62±0.2 | 1.66±0.1 | 0.71 |
women | 1.76±0.1 | 1.63±0.16 | 1.64±0.13 | 0.01 |
men | 1.35±0.2 | 1.51±0.2 | 1.48±0.11 | 0.06 |
women | 1.54±0.2* | 1.52±0.1 | 1.55±0.16 | 0.98 |
All values are shown before and after the administration of intravenous furosemide. Subjects on loop diuretics (n = 2 in the AHT group and n = 15 in the CKD group) are not included in the furosemide-induced changes.
Values expressed in sec−1, as mean±SD or as median (25th–75th percentile), as appropriate. CKD = chronic kidney disease; AHT = arterial hypertension. * P<0.05 concerning within-group differences between men and women.
In addition, within the CKD group no significant difference was observed according to the renal diagnosis (
Values shown as boxplots; No (no CKD, n = 100), DM (diabetic nephropathy, n = 20), AHT (hypertensive nephropathy, n = 31), GN(glomerulonephritis, n = 17), reflux (reflux nephropathy, n = 6), one (solitary kidney, n = 7), IF (interstitial nephritis, n = 6), other (other cause of kidney disease, n = 8). There were no differences between cortical and medullary R2* levels between the groups (ANOVA p = 0.10 and p = 0.99, respectively)
The situation was different in acute conditions, since the response to furosemide differed significantly between groups. Medullary R2* but not cortical values decreased significantly (suggesting an increase in medulla oxygenation) in the three groups, both in men and women (
The difference in furosemide-induced change between the AHT and control group was not explained by a difference in eGFR. In multivariate logistic regression adjusted for age, sex, and eGFRmdrd, the furosemide-induced change in medullary change in R2* level remained significantly smaller in the presence of AHT (OR 0.81; 0.64–0.98, p = 0.047).
In multivariate logistic regression, adjusted for age, sex, body mass index, hemoglobin, current smoking and urinary sodium excretion, with CKD status as dichotomized dependent variable (CKD compared with healthy controls and hypertensive subjects pooled together), medullary R2* levels were not associated with CKD status (OR(95% CI): 1.01 (0.86;1.18), p = 0.92). Medullary R2* levels were also not associated with CKD status when comparing the CKD group separately with healthy controls (CKD vs controls, OR: 0.95 (0.79; 1.15), p = 0.59), or with the hypertensive group (CKD vs. AHT, OR: 1.1 (0.91;1.35), p = 0.3).
Although cortical R2* levels were higher in CKD patients, this difference was not statistically significant in adjusted models (CKD versus non-CKD, OR: 1.20 (0.95; 1.51, p = 0.14)). The same trend was seen when comparing the CKD group separately with healthy controls (OR: 1.10 (0.85; 1.41), p = 0.49).
The determinants of renal cortical and medullary oxygenation were also analyzed in the entire population using linear models. There was no association between medullary R2* levels and eGFR or any of the other predefined variables in the adjusted multivariable linear regression analysis (
Medullary R2* | Cortical R2* | |||
Sex (female vs. male) | −0.43 | 0.49 | −1.8 | |
Age (per year) | −0.01 | 0.72 | −0.01 | 0.7 |
BMI (per kg/m2) | −0.03 | 0.61 | −0.04 | 0.58 |
eGFR (MDRD) | −0.003 | 0.75 | −0.01 | 0.29 |
Smoking (yes vs. no) | 0.28 | 0.77 | 1.04 | 0.16 |
Urinary 24 h sodium excretion (mmol) | 0.002 | 0.52 | 0.004 | 0.13 |
Diabetes (yes vs. no) | −1.01 | 0.41 | 1.67 | 0.07 |
Mean Arterial BP (per mmHg) | −0.04 | 0.036 | 0.003 | 0.91 |
Urinary 24 h protein excretion (per g) | −0.18 | 0.58 | −0.39 | 0.32 |
RAAS-blocker (yes vs.no) | −0.86 | 0.11 | 0.34 | 0.6 |
Serum glycemia (per mmol/l) | −0.11 | 0.54 | 0.91 | <0.001 |
Serum uric acid (per μmol/l) | −0.0001 | 0.76 | 0.014 | <0.001 |
Correlations are expressed as regression coefficient β. Correlations between R2* levels and predefined factors are shown under (A). The analysis including the additional variables glycemia, serum uric acid level and 24 h urinary proteinuria in the model is shown under (B).
adjusted for gender, age, BMI, eGFR, smoking, urinary sodium excretion, Hemoglobin, and diabetes.
Results for cortical R2* levels and its associations with predefined variables were also largely negative (
Since only a small proportion of the variability in R2* levels was explained by the predefined variables, some additional analyses were performed on the entire population. Thus, a negative correlation was found between medullary R2* levels and mean arterial blood pressure (
The positive correlation found between uric acid and cortical R2* levels did not change after the inclusion of allopurinol and diuretic treatments, or mean arterial blood pressure in the model. Results for cortical R2* levels stratified by sex are shown in
Cortical R2* | Men | Women | ||
β° | β° | |||
Age (per year) | −0.05 | 0.17 | 0.049 | 0.02 |
BMI (per kg/m2) | −0.07 | 0.57 | −0.03 | 0.54 |
eGFR (MDRD) | −0.03 | 0.11 | −0.002 | 0.81 |
Smoking (yes vs. no) | 1.69 | 0.1 | −1.06 | 0.16 |
Urinary 24 h sodium excretion (mmol) | −0.007 | 0.11 | 0.001 | 0.65 |
Diabetes (yes vs. no) | 2.47 | 0.06 | −0.63 | 0.46 |
Glycemia (per mmol/l) | 1.13 | <0.001 | 0.31 | 0.15 |
Uric acid (per mmol/l) | 0.015 | 0.004 | 0.01 | 0.001 |
°adjusted for age, BMI, eGFR, smoking, urinary sodium excretion, Hb, and diabetes.
In order to assess whether some of the results were linked to an over-representation of young women in the control group, sensitivity analyses were performed in a subgroup excluding healthy women <40 years (n = 13) and men aged >75 years suffering from CKD (n = 5). The main results of the study remained unchanged (see
The main findings of this study are that: 1) mean cortical and medullary R2* values as a proxy for renal tissue oxygenation are similar in hypertensive patients, CKD patients and healthy controls; however, the distribution of cortical R2* values differs markedly between groups, 2) the medullary R2* response to furosemide is blunted in hypertensive patients and markedly reduced in CKD patients, 3) baseline renal tissue oxygenation appears to be remarkably stable over different degrees of kidney dysfunction, independently of the cause of kidney disease and 4) cortical R2* levels are positively associated with male gender, glycemia and uric acid levels.
The first interesting observation of this paper is that although mean cortical and medullary R2* look identical in hypertensive and CKD patients and controls, the distribution of cortical R2* differs between groups with a clear bimodal distribution in controls and AHT and a unimodal shape of the distribution in CKD. As found in our population, part of the bimodal distribution is linked to the male/female ratio. However, this ratio is similar in hypertensives and CKD patients suggesting other mechanisms explaining the differences in distribution, which should be the subject of further study.
The second finding of our study is that renal cortical and medullary oxygenation as measured by BOLD-MRI appears to be extremely well maintained in patients with CKD. Indeed, in contrast to what has been observed experimentally we did not find any decrease in cortical or medullary oxygenation even in advanced CKD; the nature of the underlying renal disease does not appear to play a major role. In this respect, our data are in accordance with the study by Michaely et al
The discrepancy between animal studies and BOLD-studies in humans regarding oxygenation can be interpreted in different ways. First of all, it might be that BOLD-MRI is not sensitive enough or simply not as good a tool to assess renal tissue oxygenation in CKD-patients as is direct invasive measurements using microelectrodes. Nonetheless, early animal studies performed to validate the BOLD-MRI technique have reported linear relationships between directly measured renal pO2 values and the BOLD signal
A role of renal handling of sodium in mediating oxygen consumption is supported by our observation that the medullary R2* response to furosemide differs between controls and hypertensives with a blunted response in hypertension and an even more marked reduction in CKD patients. In CKD, the markedly reduced response to furosemide can be explained by the reduced renal function leading to lower concentrations of furosemide within the kidney. However, this cannot be the explanation for hypertensive patients who had a comparable renal function as controls. Pratt et al have previously demonstrated ethnic differences in the response to furosemide
Our multivariate analysis enabled us to identify several new factors associated with renal tissue oxygenation. Thus, cortical R2* levels was positively and strongly associated with male gender. The relationship with male gender was robust and persisted in sensitivity analyses, and suggests that cortical oxygenation might be regulated differently in men and women. It may also provide some clues why renal function declines faster in men. However, our data do not offer any explanation for the higher R2* values in men, and studies measuring simultaneously renal perfusion and tubular sodium handling are necessary to clarify this issue.
Renal tissue hypoxia has been a consistent finding in mouse and rat models of diabetic nephropathy
Our observation that circulating uric acid levels correlate positively with cortical R2* levels adds information to the ongoing debate about the role of uric acid in cardiovascular disease. Hyperuricemia-induced cortical vasoconstriction could be partly responsible for the herein described correlation between uric acid and cortical hypoxia
This study has some limitations. Firstly, no information was acquired on renal perfusion. We therefore cannot determine whether changes in oxygenation were the result of alterations in oxygen delivery. Nevertheless, Textor and colleagues have shown previously that renal tissue oxygenation is largely independent of renal blood flow, and that cortical (but not medullary) oxygenation only falls in case of severe renal artery stenosis occluding more than 90% of the lumen
Our data suggest that renal tissue oxygenation at rest is comparable in controls, treated hypertensives and CKD patients. However, the response to furosemide differs between groups providing some insights on the mechanisms linking renal tubular function, oxygen consumption and renal function in hypertension and/or chronic kidney diseases. Our data do not support the concept that chronic kidney disease is associated with decreased renal tissue oxygenation in humans, as repeated observed in animal models. However, acute changes in oxygenation most likely occur in humans, as illustrated by the changes in R2* in response to furosemide. Future interventional studies should clarify the role of renal sodium handling, blood glucose and serum uric acid in the regulation of renal tissue oxygenation as newly described in this study. The differences in cortical R2* levels between men and women suggest that renal oxygenation is possibly regulated differently in men and women; this point also needs further study. Although no correlation was found between R2* values and (previous) kidney function, it is still possible that renal tissue R2* values predict kidney function decline but this is to be demonstrated in prospective studies.
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