Noninvasive evaluation of renal oxygenation in primary nephrotic syndrome with blood oxygen level dependent magnetic resonance imaging: Initial experience

Abstract

Objectives

To use blood oxygen level dependent (BOLD) magnetic resonance imaging (MRI) to evaluate renal oxygenation in patients with primary nephrotic syndrome (PNS), and test the hypothesis that renal tissue oxygenation correlates with renal function, tubulointerstitial alterations and treatment response.

Methods

Patients with untreated first-onset PNS and healthy control subjects underwent BOLD MRI. Blood and urine samples were obtained on the day of MRI, and patients underwent renal biopsy the day after MRI. Renal tubulointerstitial damage scores (TIDS) were determined using Katafuchi criteria. All patients received corticosteroids within 7 days after MRI and were followed up for 12 months.

Results

Medullary R2* values were significantly lower in patients with PNS (n = 20) than controls (n = 18). Medullary R2* values were negatively correlated with estimated glomerular filtration rates and positively correlated with TIDS in patients with PNS. There were no significant differences in medullary or cortical R2* values when patients were classified according to treatment response.

Conclusions

The medullary oxygen concentration was higher in patients with PNS than in control subjects. BOLD MRI was a useful noninvasive method for the evaluation of renal function and tubulointerstitial impairment.

Introduction

Primary nephrotic syndrome (PNS) is caused by several types of glomerular disease, with or without concomitant renal tubulointerstitial injury, and is characterized by massive proteinuria, hypoalbuminaemia, oedema and hyperlipidaemia. PNS is usually treated with corticosteroids, but some patients do not respond to treatment and eventually progress to end-stage renal failure. Glomerular pathology is a crucial prognostic factor in PNS, with concomitant tubulointerstitial damage recognized as a significant factor contributing to poor prognosis. ¹ Since oxygenation abnormalities are a primary cause of (and unifying mechanism for) progression to tubulointerstitial fibrosis, ² a suitable noninvasive and reproducible method for evaluating changes in intrarenal oxygenation may aid in assessing tubulointerstitial alterations and predicting prognosis. Blood oxygen level dependent (BOLD) magnetic resonance imaging (MRI) is such a technique.

The basic principle of BOLD MRI is that magnetic field perturbations (caused by paramagnetic molecules such as deoxyhaemoglobin) lead to a loss of phase coherence and signal attenuation in gradient echo T2* weighted sequences. The R2* (1/T2*) relaxivity rate of spin dephasing is proportional to tissue deoxyhaemoglobin levels,^3,4 and changes in R2* can be interpreted as changes in oxygen partial pressure (pO₂) in tissues.^5,6 BOLD MRI has been shown to give reproducible results in oxygenation monitoring in normal kidneys,^7,8 and has been used to detect renal oxygenation changes in hypertensive disease, ³ diabetes, ⁹ transplanted kidneys ¹⁰ and acute renal ischaemia. ¹¹

The aim of the present study was to use BOLD MRI to evaluate renal oxygenation in patients with PNS, in order to test the hypothesis that renal tissue oxygenation correlates with renal function, tubulointerstitial alterations and treatment response.

Patients and methods

Study population

The study recruited sequential patients with PNS, admitted to the Department of Nephrology, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China, between January 2012 and December 2013. Inclusion criteria were: (i) initial onset of PNS; (ii) no prior corticosteroid therapy; (iii) no history of other kidney disease, hypertension, diabetes or other vascular disease; (iv) availability of follow-up data. Healthy control subjects with no history of renal disease, hypertension, diabetes or other vascular disease were recruited from individuals undergoing health examination at the Medical Examination Centre, the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China. Participants with contraindications to MRI (such as claustrophobia) were excluded.

The study protocol was approved by the Medical Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang, China, and all participants provided written informed consent prior to enrolment.

MRI protocol and analysis

In order to standardize hydration status and sodium balance, all participants were nil by mouth and received no intravenous transfusion 4 h prior to MRI. In addition, no diuretic agents were taken 12 h prior to MRI. ¹² MRI was performed using a 3 T clinical system (Signa HDx®, GE Medical systems, Milwaukee, WI, USA) with an eight-channel body coil. After conventional coronal, axial T1-weighted and T2-weighted imaging, BOLD MRI was acquired with a gradient-recalled-echoes sequence. A total of five coronal sections centred at the renal hilum were scanned with section thickness of 5 mm and intersection gap of 1 mm. Each section corresponded to 16 different gradient echoes and was acquired during a 15-s breath hold (echo times 1.6, 6.66, 11.72, 16.78, 21.84, 26.9, 31.96, 37.02, 42.08, 47.14, 52.2, 57.26, 62.32, 67.38, 72.44 and 77.5 ms). Scan parameters were repetition time 150 ms, flip angle 30°, bandwidth ± 31.25 kHz, FOV 35 cm, and matrix 128 × 128.

Imaging analysis of BOLD data was performed interactively using research software (FuncTool® 2, GE Healthcare) at the workstation (GE ADW 4.4, Sun Microsystems, Santa Clara, CA, USA). R2* maps of the kidney were automatically generated, and each map was delineated by two experienced radiologists (R.Z. and Q-D.W.) who were blinded to clinical data. Radiologists worked together and with consensus. For the R2* map, three coronal sections centred at the hilum of each kidney were selected. A total of three regions of interest (ROIs; 0.45 ± 0.04 cm² ) at the upper, middle, and lower parts of the kidney were traced in the cortex and medulla of each section, carefully avoiding renal sinus and susceptibility artefacts. In total, R2* values of nine cortical and nine medullary ROIs were collected per kidney (Figure 1), and the mean of these 18 cortical R2* and medullary R2* values was calculated.

Figure 1.

Representative blood oxygen level dependent (BOLD) magnetic resonance (MR) images of the right kidney of a healthy volunteer. (A) Coronal gradient-recalled-echo (GRE) T1 weighted image. (B) Corresponding R2* (1/T2*) BOLD MRI map. Small ovals indicate the placement of regions of interest in the cortex and medulla. The colour version of this figure is available online at www.sagepub.com

Laboratory analyses

Peripheral blood and urine samples were obtained from each participant on the day of the MRI. Analyses included haemoglobin, total serum protein, serum albumin, serum creatinine, urinary protein and estimated glomerular filtration rate (eGFR; calculated by the Modification of Diet in Renal Disease equation). ¹³

Renal pathology

Patients underwent renal biopsy the day after MRI, and biopsy specimens were processed for light, immunofluorescent, and electron microscopies. Formalin-fixed specimens were stained with hematoxylin and eosin, periodic acid–Schiff stain, periodic acid silver methenamine and Masson’s trichrome. Consecutive frozen sections were stained with direct immunofluorescence using fluorescein isothiocyanate (FITC)-labelled murine monoclonal antibodies (Dako, Denmark) for Immunoglobulin (Ig)G, IgA, IgM, C3, C4 and C1q. Formalin-fixed and frozen sections were examined via light microscopy.

Electron microscopy of two glomeruli per patient was performed to observe epithelial foot process fusion, electron dense deposits, and thickness of the glomerular basement membrane and tubuloreticular structures. Pathological grading (Katafuchi criteria ¹⁴ ) of renal tubulointerstitial damage was performed independently by two experienced pathologists, and agreement was reached by consensus if necessary. The total score (0–9) includes three components: interstitial inflammatory cell infiltration (0–3), interstitial fibrosis (0–3), and tubular atrophy (0–3).

Follow-up

Within 7 days after MRI scan, all patients received the initial adequate dose (1mg/kg per day) of prednisone, orally. Prednisone was gradually withdrawn over the following 8 weeks according to the individual needs of the patient. Patients received immunosuppressant therapy (cyclophosphamide, tripterygium or tacrolimus) as required. All patients were followed up for 12 months. If proteinuria resolved during the initial 8 weeks of treatment and did not return during the remaining 12 months of follow-up, the patient was defined as a treatment responder. Treatment nonresponders were defined as those patients whose proteinuria remained during the initial 8 weeks of treatment. Treatment-dependent patients were defined as those whose proteinuria disappeared during the initial 8 weeks of treatment but relapsed during the remaining 12 months of follow-up.

Statistical analyses

The study sample size was determined to be 20 patients, according to the findings of an earlier study. ¹⁰ Data were mean ± SD. Paired samples t-test was used to compare coronal R2* and medullary R2* between the right and the left kidneys; independent samples t-test was used to compare coronal R2* and medullary R2* between patients and controls. Patients were stratified into subgroups according to PNS pathological type and treatment response, and between-subgroup differences in coronal R2* and medullary R2* were analysed using one-way analysis of variance (ANOVA). Spearman’s correlation coefficient analysis was used to evaluate relationships between clinical, laboratory and pathological variables and R2*. Statistical analyses were performed using SPSS® version 16.0 (SPSS Inc., Chicago, IL, USA) for Windows®. P-values <0.05 were considered statistically significant.

Results

The study included 20 patients with PNS (eleven male/nine female; mean age 39.15 ± 14.58 years; age range 18–66 years) and 18 control subjects (nine male/nine female; mean age 41.39 ± 17.61 years; age range 19–69 years). There were no significant between-group differences in age or sex. Renal pathology and tubulointerstitial damage scores (TIDS) were determined in 18 patients (two patients refused biopsy). Seven patients (38.9%) had membranous nephropathy; five (27.8%) had mesangial proliferative glomerulonephritis; three (16.7%) had minimal change disease; one (5.6%) had focal segmental glomerulosclerosis; and two (11.1%) had mesangiocapillary glomerulonephritis. TIDS scores were 0 in three patients (16.7%), 1–3 in 11 patients (61.1%), 4–6 in four patients (22.2%), and 7–9 in one patient (5.6%). A total of seven patients were classified as treatment responders, eight were nonresponders and five were treatment dependent.

There were no significant differences between the right and left kidney in cortical or medullary R2* in controls or patients (data not shown). There was no significant difference between patients and controls in cortical R2* values (20.41 ± 3.92 s⁻¹ and 19.71 ± 0.66 s⁻¹, respectively). Medullary R2* values were significantly lower in patients than controls, however (23.97 ± 5.89 s⁻¹ vs 36.54 ± 1.34 s⁻¹; P < 0.001).

There was a significant positive correlation between tubulointerstitial damage score and medullary R2* in patients with PNS (r = 0.809, P < 0.001; Figure 2A). As shown in Figure 2B, there was a significant negative correlation between eGFR and medullary R2* in patients with PNS (r = −0.462, P = 0.040). There were no significant correlations between any other laboratory parameters (haemoglobin, total protein, serum albumin, serum creatinine and urine protein) and either medullary or cortical R2*values.

Figure 2.

Spearman’s correlation coefficient analysis of the relationship between medullary R2* values (determined by blood oxygen level dependent magnetic resonance imaging) and tubulointerstitial damage score (TIDS) or estimated glomerular filtration rate (eGFR) in patients with primary nephrotic syndrome (n = 20). (A) Positive correlation between TIDS and medullary R2* (r = 0.809, P < 0.001); (B) negative correlation between eGFR and medullary R2* (r = −0.462, P = 0.040).

There were no significant between-group differences in medullary or cortical R2* values when patients were classified according to disease type or treatment response (data not shown).

Discussion

This study demonstrated that medullary R2* values were significantly lower in patients with PNS than in healthy control subjects, indicating an increased medullary oxygen concentration in PNS, which is consistent with reports on many chronic renal diseases.^{10,15–17,18} Oxygenation of the kidney was determined by the balance between oxygen supply and consumption. The increased medullary oxygen content in patients may suggest that the reduction of renal oxygen consumption (as a result of the known reduced tubular fractional reabsorption of sodium) may influence the change in oxygen concentration.^19,20

Consistent with the findings of others, ²¹ medullary R2* values were negatively associated with eGFR in patients with PNS in the present study, indicating that BOLD MRI reflects renal function. The absence of correlation between R2* values and serum creatinine in the present study may be explained by the fact that this parameter is affected by extrarenal factors including age, weight, diet and drug use. ²² Our finding of a significant positive correlation between medullary R2* and TIDS is compatible with the findings of others, ²¹ and suggests that BOLD MRI may reflect the degree of tubulointerstitial changes.

Although medullary R2* values increased with more advanced kidney damage (low eGRF/high TIDS) in patients with PNS in the present study, mean medullary R2* values were significantly lower than those in healthy controls. This may be due to the effect of reduced blood supply (as a result of tubulointerstitial fibrosis and the loss of peritubular capillaries) lowering medullary oxygenation in patients with serious tubulointerstitial lesions.

The present study found no statistically significant relationship between treatment response and R2* values. Prognostic factors in PNS are complex; with severe tubulointerstitial damage recognized as a major contributing factor for poor prognosis. ²³ Prognosis may also be affected by factors including glomerular pathological type, although we found no significant between-group differences in R2* values when patients were classified according to disease type.

This study has several limitations, including the small sample size. Future studies should include larger patient groups. In addition, we performed BOLD MRI only once for each patient. Renal oxygenation is a dynamic characteristic that is influenced by a number of local and systemic factors, and may vary at different disease stages. ²⁴ Dynamic monitoring of renal oxygenation changes with noninvasive BOLD MRI would be useful throughout treatment. BOLD MRI may provide useful information for assessing the effect of pharmacological interventions and adjusting medication regimens.

In conclusion, the medullary oxygen concentration was higher in patients with PNS than in control subjects. BOLD MRI was a useful noninvasive method for the evaluation of renal function and tubulointerstitial impairment.