Diabetic nephropathy (DN) is one of the most frequent microvascular complications of diabetes mellitus, affecting 25 to 40% of patients with type 1 diabetes (T1D) (1). It is the single most common cause of end stage renal disease (ESRD) in adults in the Western world (1, 2). Diabetic nephropathy is marked by pathological changes occurring in the renal glomeruli that lead to the development of albuminuria, hypertension, and progressive decline in renal function (3). Hypertension is both a feature of, and a risk factor for the progression of diabetic nephropathy (4). Studies using ambulatory blood pressure monitoring (ABPM) have examined early patterns of blood pressure abnormalities in individuals with diabetes and the link to renal disease progression. Lurbe et al. found that abnormal nocturnal dipping of systolic blood pressure (SBP) was linked to progression of microalbuminuria (5) while Marcovecchio et al. found that daytime diastolic blood pressure was most predictive of future microalbuminuria (6).

Traditionally, measurement of microalbuminuria (urine microalbumin/Cr ratio: 30 to 300 mg/g) has been used in the clinical setting to screen for diabetic nephropathy. However, evidence suggests that renal pathological changes such as nephromegaly and glomerular basement membrane thickening develop soon after diabetes diagnosis and much earlier than microalbuminuria can be detected (7–9). Several serum and urine markers are increased in children with T1D compared to healthy controls (10, 11) signaling the different aspects of pathophysiology involved in this complication. Of interest to our study, urinary neutrophil gelatinase-associated lipocalin (uNGAL) has been studied extensively as a marker of tubular damage in both acute (12) and chronic kidney disease of various etiologies (13–17). Urinary pentosidine is an advanced glycation end product (AGE) that marks oxidative stress caused by tissue exposure to hyperglycemia, and levels of urine pentosidine are elevated in patients with T1D (10). Changes in the levels of these markers in the urine has been documented in patients with T1D. However, correlation with clinical features of diabetic nephropathy such as blood pressure patterns and with glycemic risk factors has not been fully established.

While the role of overall hyperglycemia, as measured by glycated hemoglobin (HbA1c), in the pathogenesis of diabetic kidney disease is well established (18), the role of glycemic variability (GV) is an area of debate. In-vitro studies have demonstrated that glycemic excursions are more deleterious to human endothelial cells than sustained hyperglycemia (19). GV has been linked clinically to the development of diabetic retinopathy and neuropathy in both T1 and T2 diabetes (20, 21). Using self-measured blood glucose levels, earlier studies concluded that GV was not associated independently with diabetes complications (22). However, more recent studies using continuous glucose monitoring (CGM) data demonstrated a role for GV in diabetic nephropathy (23). The specific contribution of GV to the disease process in various stages of DN is yet to be elucidated.

The current study was designed to evaluate the use of NGAL and Pentosidine as urinary markers of DN in children with T1D and their relationship with ABPM patterns. Secondarily, we studied the association of glycemic variability measures as well as measures of glycemic control (HbA1c) with the levels of urinary markers of nephropathy.

This is a cross-sectional study. Subjects between the ages of 10 and 21 years with T1D for more than 1 year were recruited from the Diabetes and Nephrology pediatric outpatient clinics. Given the known effects of puberty on both glycemic control and microvascular complications, the age range of cases was chosen to avoid prepubertal population. Patients were excluded if they had type 2 or monogenic diabetes, obesity (BMI ≥95th percentile or ≥30 kg/m2 for patient ≥ 18 years of age), secondary hypertension unrelated to diabetes, chronic kidney disease from another etiology, chronic inflammatory diseases, acute infection, or a history of malignancy. Consents and assents were obtained from all participates. Controls (n=10) were enrolled from Nephrology- Urology clinics.

The study entailed a single visit for each case. Weight (kg), height (cm) and manual blood pressure in the seated position were obtained. Pubertal staging was assessed by a pediatric endocrinologist or a pediatric endocrinology fellow. Random urine samples were collected at the time of the visit. ABPM was performed for 24 hours and CGM for 3 to 7 days. Electronic medical records were reviewed for the most recent HbA1c (within 1 month of enrollment) and for lipid panel. Records were also reviewed for a recent serum Creatinine (Cr) value to estimate the renal function; most cases did not have a Cr value within one year of enrollment and thus was not included in the analysis. During the monitoring period, cases continued their usual diabetes care as recommended by their pediatric endocrinologist.

For the controls, they only provided a urine sample and did not get ABPM or CGM.

The study enrolled 21 cases. Table 1 shows the subject characteristics. More females participated in the study (62%). Three cases (14%) met the ADA recommended glycemic targets of 2019 ADA standards (26). Controls (N= 10) were similar in characteristics of sex, race, and BMI, though tended to be slightly younger at enrollment ( Table 1 ). Controls had no significant renal disease (idiopathic microscopic hematuria 6, elevated BP 2, stones 1, joint hypermobility 1). Most cases (81%) used continuous subcutaneous insulin infusion for diabetes management at the time of study enrollment. Eleven cases (52%) had LDL cholesterol levels ≥100 mg/dl; none of the study cases had levels exceeding 130 mg/dl ( Table 2 ). Median HbA1C of study cases was 8.4% (IQR: 7.5, 9.3). Retinal exam results were available for 18 (86%) study cases and none had evidence of diabetic retinopathy.

Diabetic nephropathy is a multifactorial, insidious disease process and represents a major complication for individuals with T1D. This study in a pediatric T1D cohort aims to explore the characteristics of BP profile and glucose variability and their correlation with urinary markers of glomerular and tubular damage as well as markers of oxidative stress. We found that subjects with T1D have high urinary NGAL/Cr (marker of tubular injury) and pentosidine/Cr (marker of oxidative stress) than controls, when microalbuminuria was negative. We also found a high prevalence of abnormal blood pressure dipping in our cohort, which is consistent with the results of other studies, including normotensive and normoalbuminuric pediatric T1D patients (28, 29). Abnormal BP dipping has been shown to be a predictor for micro- and macro-vascular complications in individuals with diabetes (28, 30, 31). Urinary NGAL has been shown to be of value in the evaluation and progression of both acute and chronic renal damage of different etiologies (12, 13, 15, 32). T1D patients with microalbuminuria have higher urine NGAL and NGAL/Cr levels compared to those with normal urine microalbumin (33, 34). Our study further demonstrates that even with normal urine microalbumin/Cr, NGAL/Cr correlates with albumin excretion. Furthermore, we demonstrate that urine NGAL correlates with loss of nocturnal dipping of SBP, one of the earliest blood pressure profile abnormalities detected for children with T1D. This highlights the potential role of this urine inflammatory biomarker in the early detection of diabetic nephropathpy prior to the microalbuminuric phase.

Oxidative stress is another prominent contributor to the pathophysiology of DN. Urinary pentosidine is a major component of advanced glycation end (AGEs) products resulting from tissue exposure to hyperglycemia causing oxidative stress. It has been shown to be elevated in children with T1D starting at disease onset. Pentosidine levels are higher in children with microalbuminuria compared to those with normal urine albumin excretion (UAE) and correlate with traditional measures of glycemic control (HbA1c) (11, 35). Our results indicate that urinary pentosidine levels correlate with UAE (even within the normal urinary albumin excretion) as well as with mean arterial blood pressure. These findings add to evidence promoting the use of AGEs as early markers of diabetes complications and potential targets for monitoring and intervention in the early stages of disease progression. The small number of subjects in our study is a limiting factor that might have precluded the elucidation of relationships between urinary pentosidine levels and blood pressure measures.

Since its discovery, HbA1c has been used as the primary measure of metabolic control in people with diabetes, representing the mean average glucose over the preceding 2-3 month period. Apart from that “average”, not a lot is known about how the frequency and/or amplitude of day-to-day and hour-to-hour blood glucose fluctuations might affect complications. Glycemic variability is recognized as a potential risk factor for diabetes complications. The increasing availability of glucose data from CGM systems provides the potential to understand the role of blood glucose excursions in relationship to diabetes comorbidities. In addition, recent advances in insulin pump technologies (Hybrid closed-loop systems) target the reduction of such variability and improve overall diabetes control. Interestingly in our cohort, the higher glycemic variability (higher CV) group tended to have lower HbA1c and would be viewed in “better” glycemic control using this traditional measure of metabolic control. This observation makes our finding of a negative correlation between uNGAL and HbA1C, HBGI, and mean blood glucose levels highly provocative and points towards a potential greater contribution of glycemic excursions in the pathophysiology of nephropathy than the exposure to significant but stable hyperglycemia. Further clarification of relationships between measures of GV and markers of DN as well as blood pressure patterns might be possible with a larger cohort.

Our study had limitations, such as the small number of cases and the lack of long term follow up. However, this study was designed as a pilot in order to explore the feasibility of larger, prospective and multicenter studies looking into the development of DN and mitigation of its risk factors.

In summary, diabetic nephropathy is a multifactorial complication that starts early after diagnosis and eventually has a large impact on patient’s quality of life and prognosis. Our study highlights the high prevalence of abnormal BP pattern as well as identifies early urinary biomarkers of renal injury before the development of microalbuminuria. Our results suggest that higher blood glucose values are not necessarily associated with higher markers of tubular injury, raising the question of whether glycemic variability rather than mean blood glucose has a stronger impact on development of DN. Future research is needed to identify the disease process earlier to allow for intensified intervention and new therapeutic targets.

The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.

The studies involving human participants were reviewed and approved by University at Buffalo/Oishei Children’s Hospital of Buffalo, and Nationwide Children’s Hospital. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

All authors contributed to the study conception and design. Material preparation, data collection and analysis were performed by all authors. The first draft of the manuscript was written by LeM and all authors commented on previous versions of the manuscript. All authors contributed to the article and approved the submitted version.

Study was funded by the fellow’s division funding at University at Buffalo, NY and the Research Institute, Nationwide Children’s Hospital, Columbus, OH, USA.

Author LuM has received research grants from Novo Nordisk, Astrazeneca, Sanofi Aventis, and from Juvenile Diabetes Research Foundation.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.