Abstract
Introduction: Sickle cell disease (SCD) is associated with damage to all of the segments of the kidney's nephron, including the glomeruli, tubules, and collecting duct. Due to procedural risks, renal biopsies are rarely performed, limiting our understanding of the molecular changes occurring in the kidney. In diabetic chronic kidney disease (CKD), urine mRNA expression of segment-specific biomarkers reflect underlying nephron injury and predict progression of CKD (PMID: 33077837, PMID: 36787638). Urinary gene expression profiling in SCD remains unexplored and may clarify the pathophysiology, timing, and location of nephron injury. We hypothesized that expression of nephron biomarkers would correlate with clinical indicators of kidney dysfunction in adults with SCD.
Methods: Urine samples (≥10mL) were collected from 50 adults with SCD and processed within 2 hours of collection to isolate mRNA. Samples were first centrifuged for 10 minutes at 1000rpm at room temperature. Supernatant was discarded and pellet was washed with PBS. The pellets were stored in -80°C in RLT buffer (RNeasy Plus Mini Kit; Qiagen) with β-mercaptoethanol. RNA was extracted from the urine pellets and converted to cDNA (High Capacity cDNA Reverse Transcription Kit; ThermoFisher Scientific) according to the manufacturer's protocol. Real time PCR was performed to determine gene expression of glomerular (NPHS1; nephrin), proximal tubular (HAVCR1; kidney-injury molecule-1), and collecting duct (AQP2, aquaporin-2) segments and values were normalized to GAPDH. Clinical variables and laboratory data were obtained at the time of urine sample collection. Statistical associations between mRNA expression and clinical variables were assessed using univariate linear regression or one-way ANOVA, as appropriate, using Systat.
Results: The median age of the cohort was 37 (interquartile range [IQR], 28–42) years, and 52% were female. 94% were HbSS or Sβ⁰-thalassemia genotype, 48% were on hydroxyurea and 20% were on renin-angiotensin-aldosterone inhibitors at the time of sample collection. The median systolic and diastolic blood pressures were 118 (IQR, 110–129) mmHg and 70 (IQR, 64–78) mmHg, respectively. Older age was associated with increased expression of proximal tubular (HAVCR1; p=0.012) and collecting duct (AQP2; p=0.024) stress biomarkers. Higher systolic blood pressure was associated with increased expression of stress biomarkers in all three segments of the nephron (AQP2; p=0.001; HAVCR1; p=0.042; NPHS1; p=0.07) while higher diastolic blood pressure was associated with increased AQP2 expression (p=0.02). We did not observe an association between mRNA expression and sex, hydroxyurea or RAASi use.
Next, we evaluated the association of the mRNA stress biomarkers with kidney function. The median eGFR, calculated using the CKD-EPI creatinine-cystatin C 2021 equation, was 97 (IQR, 76–113) mL/min/1.73 m² and median urine albumin-to-creatinine ratio (UACR) was 33 (IQR, 2–195) mg/g. CKD was present in 54% of participants (27/50); among those, 22 were classified as CKD Stage I and 5 as Stage II or higher. Increased expression of the glomerular stress biomarker, NPHS1, was significantly associated with higher UACR (log-transformed β 0.76±0.26; p=0.007) and a trend for lower eGFR (β -6.6±3.7; p=0.089) and worsening CKD stage (β 0.90±0.47; p=0.068). We did not observe significant associations between mRNA expression of proximal tubular (HACVR1) or collecting tubule (AQP2) stress biomarkers with kidney function (p≥0.1).
Discussion: In conclusion, we demonstrate that increased urine mRNA expression of AQP2, a biomarker of collecting duct stress, was associated with systolic and diastolic blood pressure while increased NPHS1 expression, a biomarker of glomerular stress, was associated with kidney dysfunction. AQP2 expression is regulated by vasopressin and implicated in the development and progression of hypertension, a risk factor for cardiac, renal, and stroke complications in SCD. NPHS1 encodes nephrin, an important component of the glomerular slit diaphragm, and increased NPHS1 expression reflects increased glomerular stress in diabetic CKD. Future studies evaluating the urine expression of NPHS1 and AQP2 to predict the risk of CKD and hypertension may facilitate the development of noninvasive tools to better understand the pathophysiology of kidney damage and guide closer monitoring and treatment strategies.
