Corneal nerve fiber loss in diabetes with chronic kidney disease

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The Ocular Surface journal homepage: www.elsevier.com/locate/jtos

Original Research

Corneal nerve fiber loss in diabetes with chronic kidney disease Shyam Sunder Tummanapallia,∗, Tushar Issarb, Aimy Yanb, Natalie Kwaib, Ann M. Poyntenc, Arun V. Krishnanb, Mark D.P. Willcoxa, Maria Markoullia a b c

School of Optometry & Vision Science, University of New South Wales, Australia Prince of Wales Clinical School, University of New South Wales, Australia Department of Endocrinology, Prince of Wales Hospital, Australia

A R T I C LE I N FO

A B S T R A C T

Keywords: Chronic kidney disease Type 2 diabetes Peripheral neuropathy In vivo corneal confocal microscopy Corneal nerve fiber

Aims: Patients with chronic kidney disease (CKD) in type 2 diabetes typically manifest with severe peripheral neuropathy. Corneal confocal microscopy is a novel technique that may serve as a marker of nerve injury in peripheral neuropathy. This study examines the changes that occur in corneal nerve morphology as a result of peripheral neuropathy due to renal dysfunction in people with type 2 diabetes. Methods: Sixty-two participants (mean age, 62 ± 12 years) with type 2 diabetes and 25 age-matched healthy controls underwent a comprehensive assessment of neuropathy using the total neuropathy score (TNS). The corneal sub-basal nerve plexus was imaged using corneal confocal microscopy. Corneal nerve fiber length, fiber density, branch density, total branch density, nerve fractal dimension, inferior whorl length and inferior whorl nerve fractal dimension were quantified. Based on the eGFR, participants were classified into those with diabetic CKD (eGFR < 60; n = 22) and those without CKD (eGFR ≥ 60; n = 40). Results: Participants with diabetic CKD had significantly lower corneal nerve fiber density (P = 0.037), length (P = 0.036) and nerve fractal dimension (P = 0.036) compared to those without CKD. Multiple linear regression analysis revealed that reduced corneal nerve fiber density (ß coefficient = 0.098, P = 0.017), length (ß coefficient = 0.006, P = 0.008) and nerve fractal dimension (ß coefficient = 0.001, P = 0.007) was associated with low eGFR levels when adjusted for age, duration of diabetes and severity of neuropathy. Conclusion: Corneal confocal microscopy detects corneal nerve loss in patients with diabetic CKD and reduction in corneal nerve parameters is associated with the decline of kidney function.

1. Introduction Diabetes is associated with microvascular damage that leads to severe chronic complications such as retinopathy, nephropathy, neuropathy, and cardiovascular disease. Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes affecting up to 50% of all patients and is associated with high morbidity, poor quality of life and a high risk of lower-extremity amputation [1,2]. This prevalence and severity of neurological complications increases when diabetes is associated with chronic kidney disease (CKD) [3,4]. CKD is one of the most frequent and debilitating microvascular complications of type 2 diabetes, affecting about 30–40% of people with diabetes [5]. Neurological complications in CKD manifest in both the central and peripheral nervous systems with approximately 90% of patients undergoing dialysis develop peripheral neuropathy [3,6]. CKD and diabetes together can amplify the prevalence and severity of peripheral neuropathy, as both conditions are major and independent risk

∗

factors for the development of peripheral neuropathy [4,7,8]. Recent work has suggested that hyperkalemia may underlie the development of neuropathy in CKD [9,10] and a recent clinical trial has demonstrated that potassium restriction is neuroprotective [11] in people with both diabetic and non-diabetic CKD. Given these results, early detection of nerve injury is critical in patients with impaired renal function. In vivo corneal confocal microscopy allows direct, non-invasive imaging of the morphological changes of A-delta and C small nerve fibers of the cornea and has been proposed as a surrogate marker for small nerve fiber neuropathy in diabetes [12–16]. While small fiber neuropathy may occur in patients with diabetic CKD [8], there have been no previous reports examining the specific impact of renal dysfunction on small nerve fibers of the cornea in patients with diabetes. The aim of this study, therefore, was to investigate the changes that occur in corneal nerve morphology as a result of peripheral neuropathy due to renal dysfunction in people with type 2 diabetes.

Corresponding author. School of Optometry and Vision Science, University of New South Wales, Sydney, NSW, 2052, Australia. E-mail address: [email protected] (S.S. Tummanapalli).

https://doi.org/10.1016/j.jtos.2019.11.010 Received 20 July 2019; Received in revised form 15 November 2019; Accepted 22 November 2019 1542-0124/ © 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Shyam Sunder Tummanapalli, et al., The Ocular Surface, https://doi.org/10.1016/j.jtos.2019.11.010

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2. Methods

and pinprick sensibility (Neurotip™, Owen Mumford, UK). All eight subcategories were graded from 0 to 4 (0, no dysfunction to 4, severe dysfunction), to provide a total score of 0–32 points. Participants with a score greater than or equal to 2 were considered to have DPN (TNS ≥ 2). CKD was defined as glomerular filtration rate (GFR) < 60 ml/min/ 1.73 m2 or structural/functional abnormalities of the kidney for ≥3 months [38]. CKD stages were defined as stage 1: kidney damage with normal eGFR ≥90, stage 2: kidney damage with mild decrease in eGFR 60–90, stage 3: kidney damage with moderate decrease in eGFR 30–59, stage 4: kidney damage with sever decrease in eGFR 15–29 and stage 5 (ESKD, kidney failure): < 15 mL/min/1.73 m2 [38]. Based on the estimated GFR (eGFR) values, the diabetes cohort was classified into two sub-groups: diabetic CKD group (< 60 mL/min/ 1.73 m2; n = 22) and those without CKD group (≥60 mL/min/1.73 m2; n = 40).

2.1. Study participants The protocol of this prospective cross-sectional study was approved by the South-East Sydney Area Health Service and the University of New South Wales Research Committee and was conducted in accordance with the Declaration of Helsinki (2013). Written informed consent was obtained from 62 participants with type 2 diabetes recruited from the Diabetes Centre at the Prince of Wales Hospital, Sydney, Australia. For comparison, 25 age- and gender-matched healthy controls were recruited from The University of New South Wales, Sydney. This sample size was calculated based on achieving 90% power at a significance level of 5% to detect a difference of 4.0 ± 1.0 mm/mm2 in corneal nerve fiber length measurement, as per the previously reported data [17]. Participants were excluded from the study if they had end stage kidney disease (ESKD) or a history of other medical illnesses known to be associated with neuropathy, including treatment with neuromodulatory medication or cytotoxic agents and vitamin B12 deficiency. Participants were also excluded from the study if they presented with current eye infections, corneal abrasions, or had a history of herpes simplex virus infection or refractive surgery or contact lens wear or anterior segment trauma. Participants with a history of cataract surgery in the last 12 months were excluded [18–21].

2.4. Data and statistical analysis All statistical analysis was performed using SPSS version 23 (IBM Corp: Armonk, NY), Graph Pad Prism 7.0 (Graph Pad Software Inc., San Diego, CA) and SAS software, version 9.4 (SAS Institute, Inc., Cary, NC, USA). Descriptive statistical data were displayed as the mean ± standard deviation (SD) for continuous data. Categorical variables were presented as count with percentage and were compared between groups using Chi-square test. Normal distribution of the data was verified with the D'Agostino-Pearson normality test and visualization of quantile-quantile plots. The independent sample t-test or one-way ANOVA with Bonferroni as post-hoc test was used to compare the difference in means between groups regarding the parametric data, while Mann–Whitney U test or Kruskal-Wallis with Dunn's post hoc test was used to analyze the nonparametric data. Age-and BMI-adjusted intergroup comparisons of clinical variables were also performed using a general linear model. A P value less than 0.05 was considered statistically significant. Correlations were carried out to determine the relationship between corneal nerve parameters, risk factors neuropathy and clinical markers of renal function using Pearson's R (correlation coefficient) or Spearman's rho as appropriate and P values were adjusted for multiple comparisons by controlling the false discovery rate via the Benjamini-Hochberg procedure [39] using SAS software. Hierarchical multiple regression analysis was performed to assess the independent association between corneal confocal microscopy parameters and clinical markers of renal function while controlling the covariates such as age, duration of diabetes and severity of neuropathy. The multicollinearity among the corneal nerve parameters was examined prior to the hierarchical multiple regression analysis using the variance inflation factor (VIF). If the VIF value of any corneal nerve parameter exceeded 10, this indicated the presence of multicollinearity [40].

2.2. Corneal confocal microscopy and image analysis Corneal staining was assessed prior to confocal microscopy to rule out pre-existing ocular surface disease such as dry eye disease [22,23]. All participants were scanned bilaterally with a corneal confocal microscope (Heidelberg Retinal Tomograph III Rostock Cornea Module; Heidelberg Engineering GmbH, Heidelberg, Germany) as described elsewhere [24–26]. The acquired images corresponded to a scanned area of 2.5 mm on the central and inferior mid-periphery of corneal surface. Eight central and three to four inferior whorl images not overlapping by more than 20%, from both eyes of each participant, were selected for quantification [24,27]. Images were analyzed using a validated and purpose-designed fully automated nerve analysis software (Corneal Nerve Fiber Analyzer V.2, ACCMetrics, University of Manchester, Manchester, United Kingdom) [28,29]. This software is designed to quantify corneal nerve fiber density (CNFD, the total number of main nerves per square millimeter, no./mm2), corneal nerve fiber length (CNFL, the total length of main nerves and nerve branches per square millimeter, mm/mm2), corneal nerve branch density (CNBD, the total number of main nerve branches per square millimeter, no./ mm2), corneal total branch density (CTBD, the total number of branch points per square millimeter, no./mm2) and nerve fractal dimension (CNFrD). The fractional number that describes how an object fills the space, and therefore how complex it is [30–32]. A low CNFrD indicates fewer distorted nerve fibers, potentially reflecting an abnormality and vice versa for a high CNFrD [32]. In this study nerve fiber length and nerve fractal dimension at the inferior whorl region of the cornea was also quantified (IWL & IWNFrD). The IWL has been reported to be a sensitive parameter for detecting DPN [24,33]. Participants’ overall corneal nerve parameters were calculated by averaging measures obtained in each eye.

3. Results 3.1. Inter-group comparisons The demographics of 62 participants with type 2 diabetes were compared to 25 age- (P = 0.109) and gender- (P = 0.606) matched healthy controls (Table 1). Body mass index (BMI) was significantly higher (P < 0.001) in participants with type 2 diabetes when compared to healthy controls. The demographics and metabolic profiles of the sub-groups (diabetic CKD & those without CKD) are presented in Table 2. Participants with diabetic CKD were significantly older (P < 0.0001) and had a longer diabetes duration (P = 0.028) with more severe neuropathy (TNS; P < 0.001) than those without CKD. However, there were no significant differences in BMI (P > 0.99), HbA1c (P = 0.521), serum potassium (K) (P = 0.101), serum triglycerides (P = 0.684) and serum

2.3. Neuropathy assessment and renal disease staging All participants underwent comprehensive neurologic assessment and DPN was defined as per the Toronto consensus criteria [34]. Neuropathy severity was graded according to the Total Neuropathy Score (TNS) [35–37]. The TNS is a composite score composed of the assessment of sural and tibial nerve conduction studies, severity of sensory and motor symptoms, assessment of deep-tendon reflexes, lower-limb muscle strength, vibration sensibility (128-Hz tuning fork) 2

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Table 1 Demographic and clinical characteristics of the participants. Variables

Controls (n = 25)

T2D (n = 62)

P Value

Gender, F (%) Age (years), median (IQR) BMI (kg/m2), median (IQR) TNS, median (IQR)

12 (48%) 59 (56–63) 25 (22–27) 0

26 (42%) 63 (54–71) 29 (26–34) 4 (2–8)

0.606ᴪ 0.109‡ < 0.001‡

P values derived from ‡Mann-Whitney U test and ᴪChi-square test; Abbreviations: BMI = body mass index; kg/m2 = kilograms/meters; T2D, type 2 diabetes; n, number; IQR, interquartile range.

cholesterol (LDL, P = 0.979; HDL, P = 0.690) between the CKD and non-CKD subgroups of diabetes (Table 2). Participants with diabetic CKD had significantly lower serum bicarbonate levels (P = 0.033) and serum albumin (P < 0.001). They also had higher levels of urea (P < 0.001), creatinine (P < 0.001) and urine albumin-creatinine ratio (P < 0.001) than those in the non-CKD group. Serum sodium (Na) levels were not significantly different between the groups (Table 2). As the age and BMI were significantly higher in the diabetic CKD group compared to the control group, inter-group comparisons were carried out by adjusting for age and BMI using a general linear model. Comparisons of mean corneal confocal microscopy parameters between the three groups and age- and BMI-adjusted P values are presented in Table 2. There was a progressive trend of reduction in corneal nerve

Fig. 1. Title: Graphs showing the reduction in corneal nerve fiber density. Legend: Corneal nerve fiber density in controls and type 2 diabetic non-CKD (T2DNCKD) and type 2 diabetic CKD (T2DCKD) groups. Bar graph showing mean and standard error; CKD, chronic kidney disease.

Table 2 Demographic and clinical characteristics of the participants in three groups, results are expressed as Mean ± SD or Median (IQR). Variable

Controls (A)

T2DNCKD (B)

T2DCKD (C)

Overall P

A vs B

A vs C

B vs C

n, number Age (years) Gender, Female (%) BMI (kg/m2) HBA1c (%) HBA1c (mmol/mol) Duration of diabetes (years) eGFR TNS Systolic BP (mmHg) Diastolic BP (mmHg) Serum K+, mmol/l Triglycerides, mmol/l LDL cholesterol, mmol/l HDL cholesterol, mmol/l Serum HCO3−, mmol/l Serum Urea Creatine Albumin Serum Na+ Urine ACR (mg/mmol)

25 59 (56–63) 12 (48%) 25 (22–27) 0 ± 0 -

40 58 (47–63) 19 (48%) 25 (21–29) 7.5 (6.9–9) 58.5 (51–76) 11 ± 7 90 (85–90) 4.23 ± 5.44 130 (121–142) 78 ± 10 4.52 ± 0.41 1.60 (1.12–2.42) 2.42 ± 0.90 1.26 ± 0.33 26 (24–28) 5.45 ± 1.43 71 ± 14 44 (41–45) 139 (138–141) 0.00 (0–1.57)

22 72 (67–76) 7 (32%) 27 (23–30) 7.8 (7–9.3) 61.75 (53–78) 16 ± 7 45 (33–53) 10 ± 8 133 (120–140) 74 ± 12 4.71 ± 0.42 1.85 (1–2.70) 2.40 ± 1.51 1.22 ± 0.25 24.50 (22–26) 10 ± 3.50 143 ± 41 36 (35–42) 140 (137–141) 6.20 (2.65–41.7)

< 0.001^ 0.431ᴪ < 0.001^ 0.521‡ 0.521‡ 0.028† < 0.001‡ < 0.001^ 0.685‡ 0.096† 0.101† 0.684‡ 0.979† 0.690† 0.033‡ < 0.001† < 0.001† < 0.001‡ 0.935‡ < 0.001‡

> 0.99 < 0.001 < 0.001

< 0.001 < 0.001 < 0.001

< 0.001 > 0.99 0.007

-

-

-

Variable

Controls (A)

T2DNCKD (B)

T2DCKD (C)

Age and BMI adjusted P values Overall P A vs B

A vs C

B vs C

< 0.001 0.003 < 0.001 0.011 < 0.001 < 0.001 0.066

< 0.001 0.010 < 0.001 0.031 < 0.001 < 0.001 0.069

0.037 > 0.99 0.036 > 0.99 0.057 0.036 0.166

CNFD CNBD CNFL CTBD IWL CNFrD IWNFrD

30.39 49.44 17.24 67.12 17.92 1.501 1.496

± ± ± ± ± ± ±

6.09 19.67 2.94 24.82 3.18 0.029 0.023

23.21 32.93 13.92 48.13 13.21 1.471 1.460

± ± ± ± ± ± ±

5.53 14.3 3.09 18.34 3.54 0.030 0.049

17.86 ± 6.48 24.1 ± 16.13 10.97 ± 3.32 37.11 ± 21.26 8.99 ± 5.33 1.430 ± 0.050 1.280 ± 0.420

0.001 0.007 0.006 0.022 < 0.001 0.032 > 0.99

P values derived from ¶One-way ANOVA/^Kruskal-Wallis (Post hoc: Bonferroni/Dunn's multiple comparisons test), †t-test, ‡Mann-Whitney U test and ᴪChi-square test after adjusting for age and BMI; Abbreviations: CKD, chronic kidney disease; T2DNCKD, type 2 diabetic non-CKD; T2DCKD, type 2 diabetes with CKD, n, number; SD, standard deviation; BMI, body mass index; kg/m2, kilograms/meters; HbA1c, glycosylated hemoglobin; mmol/mol, millimoles per mole; eGFR, estimated glomerular filtration rate; TNS, total neuropathy score; BP, blood pressure; mmHg = millimeter of mercury; mmol/l, millimoles per liter; K+, potassium; LDL, lowdensity-lipoprotein; HDL, high low-density-lipoprotein; Na+, sodium; ACR, Albumin-creatinine ratio; mg/mmol (mg albumin: mmol creatinine); CNFD, corneal nerve fiber density; CNFL, corneal nerve fiber length; CNBD, corneal nerve branch density; CNBD, corneal nerve branch density; CTBD, corneal total branch density; IWL, inferior whorl length; CNFrD, Corneal nerve fractal dimension; IWNFrD, inferior whorl nerve fractal dimension; HCO3 -, bicarbonate ion; mm, millimeter; ms, n, number; IQR, interquartile range; Significant P-values are highlighted in bold. 3

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There were significant inverse correlations noted between corneal nerve parameters and TNS (CNFD, rho = −0.453, P = 0.001; CNBD, rho = −0.317, P = 0.024; CNFL, rho = −0.447, P = 0.002; CTBD, rho = −0.298, P = 0.035; IWL, rho = −0.387, P = 0.006; CNFrD: rho = −0.421, P = 0.003, IWNFrD, rho = −0.418, P = 0.003) (Table 4). All the corneal nerve parameters showed a significant positive correlation with eGFR values (CNFD, rho = 0.378, P = 0.007; CNBD, rho = 0.316, P = 0.024; CNFL, rho = 0.404, P = 0.004; CTBD, rho = 0.343, P = 0.016; IWL, rho = 0.482, P = 0.001; CNFrD: rho = 0.398, P = 0.004, IWNFrD, rho = 0.469, P = 0.001) (Table 4). All corneal nerve parameters showed a significant negative correlation with eGFR stages (CNFD, rho = −0.400, P = 0.004; CNBD, rho = −0.321, P = 0.024; CNFL, rho = −0.425, P = 0.003; CTBD, rho = −0.347, P = 0.015; IWL, rho = −0.514, P < 0.001; CNFrD: rho = −0.423, P = 0.003, IWNFrD, rho = −0.486, P = 0.001) (Fig. 3a; Fig. 3b; Table 4). There was were no correlation between corneal nerve parameters and serum K levels. Corneal nerve parameters such as CNFD (rho = −0.356, P = 0.0204) and CNFrD (rho = −0.331, P = 0.035) showed a significant negative correlation with urine albumin-creatinine ratio in cohort of type 2 diabetes with and without CKD (Table 4).

Fig. 2. Title: Graphs showing the reduction in corneal nerve fiber length. Legend: Corneal nerve fiber length in controls and type 2 diabetic non-CKD (T2DNCKD) and type 2 diabetic CKD (T2DCKD) groups. Bar graph showing mean and standard error; CKD, chronic kidney disease.

parameters between healthy controls and diabetic non-CKD and diabetic CKD groups (Fig. 1 & Fig. 2). In participants with diabetic CKD, there was a significant reduction in CNFD (P < 0.001), CNBD (P = 0.010), CNFL (P < 0.001), CTBD (P < 0.031), IWL (P < 0.001) and CNFrD (P < 0.001) compared to healthy controls. Similarly, participants with non-CKD had a significant reduction in CNFD (P = 0.001), CNBD (P = 0.007), CNFL (P = 0.006), CTBD (P = 0.022), IWL (P < 0.001) and CNFrD (P = 0.032) compared to healthy controls. A significant reduction was found in CNFD (P = 0.037), CNFL (P = 0.036) and CNFrD (P = 0.036) in the diabetic CKD group compared to those without CKD group (Table 2). IWNFrD showed no significant difference between the groups. Furthermore, corneal nerve parameters were compared according to the CKD stage (Table 3). All the corneal nerve parameters, except CNBD (P = 0.725) and CTBD (P = 0.278) were significantly reduced in the CKD stage 4 group compared with the CKD stage 1 group (P < 0.05). In addition, there were reductions in CNFD, CNFL, CNFrD and IWNFrD in participants with CKD stage 4 compared to CKD stage 2 participants (P = 0.039, 0.016, 0.001, 0.035, respectively). Participants with CKD stage 3 had significantly reduced CNFL and IWL compared to CKD stage 1 participants (P = 0.048, 0.002, respectively).

3.3. Hierarchical multiple regression (HMR) analysis HMR analysis was carried out with variables that were significantly correlated with eGFR values to analyze the relationship between corneal nerve parameters and renal function while controlling covariates such as age, duration of the diabetes and severity of the neuropathy. In the multicollinearity analysis, among all the corneal nerve parameters, CNFL had the highest VIF value of 28.93 compared to CTBD (24.60), CNBD (19.59) and CNFD (12.34). Parameters such as CNFrD (8.0), IWL (2.90) and IWNFrD (2.20) had VIF values less than 10, indicating no collinearity between these variables. Therefore, HMR analysis was performed separately for each corneal nerve parameter rather than in a single model. Individual corneal nerve parameters were included separately as dependent variables and age, duration of the diabetes and severity of the peripheral neuropathy (TNS) were entered as independent variables in the model 1 and eGFR values were added in model 2. Similarly, serum K levels and serum urea levels were used instead of eGFR in a separate HMR analysis. Corneal nerve parameters had a statistically significant association with eGFR stages: CNFD (ß coefficient = −2.684, 95% CI, −4.825 to −0.542, R2 = 0.087, P = 0.015), CNFL (ß coefficient = −1.666, 95% CI, −2.807 to −0.525, R2 = 0.114, P = 0.005), IWL (ß coefficient = −1.889, 95% CI, −3.351 to −0.427, R2 = 0.08, P = 0.012) and CNFrD (ß coefficient = −0.021, 95% CI, −0.034 to −0.007, R2 = 0.117, P = 0.004). Corneal nerve parameters also demonstrated a statistically significant association with eGFR levels: CNFD (ß coefficient = 0.098, 95% CI,

3.2. Clinical correlations Associations between corneal nerve structural changes, severity of neuropathy, renal function, serum potassium, serum urea levels and urine albumin-creatinine ratio in type 2 diabetes cohort are illustrated in Table 4.

Table 3 Comparisons of corneal nerve parameters according to the CKD stages in type 2 diabetes cohort.

CNFD CNBD CNFL CTBD IWL CNFrD IWNFrD

CKD stage 1

CKD stage 2

CKD stage 3

CKD stage 4

23.71 ± 5.75 34.62 ± 16.37 14.29 ± 3.05 50.62 ± 19.30 14.39 ± 2.60 1.47 ± 0.03 1.47 ± 0.03

22.46 ± 5.28 30.41 ± 10.45 13.35 ± 3.18 44.41 ± 16.69 11.42 ± 4.06 1.47 ± 0.03 1.44 ± 0.07

18.87 ± 5.01 24.61 ± 14.88 11.65 ± 2.77¶* 38.86 ± 21.14 9.64 ± 5.32¶† 1.44 ± 0.04 1.34 ± 0.34

13.29 ± 10.87¶†, §* 21.77 ± 23.62 7.95 ± 4.33¶†, §* 29.24 ± 23.02 6.09 ± 5.03¶† 1.38 ± 0.08¶‡, §†, #* 1.05 ± 0.7¶†, §*

Results are expressed as mean ± SD. Statistically significant differences using ANOVA Bonferroni post hoc test. *P < 0.05. †P < 0.01. ‡P < 0.001. ¶Significantly different from stage 1 group; §differ significantly from stage 2 group; #differ significantly from the stage 3 group. CNFD, corneal nerve fiber density; CNFL, corneal nerve fiber length; CNBD, corneal nerve branch density; CNBD, corneal nerve branch density; CTBD, corneal total branch density; IWL, inferior whorl length; CNFrD, corneal nerve fractal dimension; IWNFrD, inferior whorl nerve fractal dimension; CKD, chronic kidney disease. 4

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Table 4 Correlations (r and rho values) between corneal nerve parameters, nerve excitability measures and other variables in participants with type 2 diabetes.

eGFR stages^ eGFR^ TNS^ Serum K Serum Urea^ Urine ACR^

TNS^

CNFD

CNBD

CNFL

CTBD

IWL

CNFrD^

IWNFrD^

0.566** −0.519** 0.247 0.369* 0.310

−0.399** 0.378** −0.453** −0.191 −0.167 −0.356*

−0.321* 0.316* −0.317* −0.052 −0.191 −0.263

−0.425** 0.404** −0.447** −0.186 −0.161 −0.251

−0.347* 0.343* −0.298* −0.042 −0.182 −0.130

−0.514** 0.482** −0.387** −0.229 −0.324* −0.191

−0.423** 0.398** −0.421** −0.119 −0.185 −0.331*

−0.486** 0.469** −0.418** −0.166 −0.218 −0.140

*Correlation is significant at the 0.05 level. ** Correlation is significant at the 0.01 level; P values reported are adjusted for multiple comparisons by controlling the false discovery rate via the Benjamini-Hochberg procedure. Pearson's r correlation was unless otherwise specified, ^Spearman's rho test; Abbreviations; eGFR, estimated glomerular filtration rate; ACR, Albumin-creatinine ratio; TNS, total neuropathy score; CNFD, corneal nerve fiber density; CNFL, corneal nerve fiber length; CNBD, corneal nerve branch density; CNBD, corneal nerve branch density; CTBD, corneal total branch density; IWL, inferior whorl length; CNFrD, corneal nerve fractal dimension; IWNFrD, inferior whorl nerve fractal dimension; Significant correlations are shown in bold type.

Fig. 3. Title: Relationship between corneal nerve fiber measurements and CKD severity in participants with Type 2 diabetes. Legend: (a) Mean (SEM) corneal nerve fiber density (CNFD) declined with increasing CKD severity. (b) Mean (SEM) corneal nerve fiber length (CNFL) declined with increasing CKD severity.

greater reduction of nerve fiber length (11%) and fractal dimension (9.6%) at the inferior whorl region compared to the central cornea in patients with CKD associated with diabetes. This suggests that nerves at the inferior whorl, being the distal ends of the corneal nerves, are more sensitive to renal impairment in participants with diabetes and may further explain the length-dependent progression of axonal loss in this cohort. Further investigation is warranted to validate these findings. When the inter-correlations between the corneal nerve parameters were investigated, the only parameters shown to be truly independent were CNFL, CNFrD, IWL and IWNFrD. While the other corneal nerve parameters each provide specific information about the corneal nerve properties [42], the independence of CNFL, CNFrD, IWL and IWNFrD parameters highlights their utility in future analysis. Moreover, these data indicate that visualization of corneal nerves allows the detection of these neuronal changes. Given the increased severity of neuropathy when both diabetes and CKD are present, these findings suggest that renal function should be explored when managing patients with diabetic peripheral neuropathy in order to establish whether the renal dysfunction is a contributing factor. The severity of CKD was classified into five stages based on the eGFR [38]. Participants with stage 5 CKD and those receiving dialysis were excluded. Each corneal nerve parameter was grouped according to the severity of CKD and a dramatic stepwise reduction in all the corneal nerve parameters was noted with increasing severity of CKD, suggesting that axonal loss of peripheral nerve fibers is associated with a progressive decline of renal function (Fig. 3). Post hoc analysis revealed that there is an overlap of corneal nerve parameters especially between the CKD stages of 1 and 2 (Table 3). This could be due to the continuum of CKD stages, where there may not be much difference in corneal nerve parameters from CKD stage 1–2 because in those patients the eGFR may, in fact, be normal or borderline

0.019 to 0.178, R2 = 0.084, P = 0.017), CNFL (ß coefficient = 0.0058, 95% CI, 0.016 to 0.101, R2 = 0.101, P = 0.008) and CNFrD (ß coefficient = 0.001, 95% CI, 0 to 0.001, R2 = 0.104, P = 0.007). There was a significant association between CNFrD and serum urea levels (ß coefficient = −0.004, 95% CI, −0.008 to −0.001, R2 = 0.087, P = 0.027, Table 5). HMR analysis was also performed to determine the relationship between the corneal nerve parameters and severity of peripheral neuropathy while controlling factors such as age, duration of diabetes and renal function. When this was done, there were no statistically significant relationships between corneal nerve parameters and severity of peripheral neuropathy (P > 0.05). 4. Discussion This study demonstrated a significant loss of corneal nerve fibers in participants with diabetes-related CKD, above and beyond that experienced in diabetes without CKD. An association between corneal nerve morphology and severity of renal dysfunction and peripheral neuropathy was also found. After controlling for baseline differences in age and BMI, there was a significant reduction in all corneal nerve parameters in participants with type 2 diabetes without CKD compared to healthy controls (P < 0.05). These findings are consistent with the results of previously reported data [15,17,41]. There was a further significant reduction (P < 0.05) in corneal nerve fiber density (23%), length (21%) and nerve fractal dimension (2.7%) in the central region, and nerve fiber length (32%) and nerve fractal dimension (12.3%) in the inferior whorl region in participants with CKD associated with diabetes compared to those with diabetes alone (Table 2). This is an important finding as it suggests that the co-existence of renal dysfunction with diabetes may have an added detrimental effect on small nerve fibers. There was a 5

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and 0.021 in CNFrD (Table 5). In accordance with previous reports [13,17,44–46], a relationship between corneal nerve parameters and neuropathy severity was noted in this study (Table 4). However, the regression analysis demonstrated that this relationship was lost after controlling for the effects of eGFR, age and diabetes duration. The exact reasons for this are unclear. It is possible that in these patients, the presence of CKD is a more dominant contributor to neuropathy severity. This has been shown in a recent paper from our group [47], in which patients with diabetic kidney disease manifested more severe neuropathy and had changes in axonal function that mirrored those of non-diabetic CKD, not diabetes. The overall findings of regression analysis demonstrated that the abnormal eGFR is a major contributor to corneal nerve changes. It should be noted that all participants in the diabetic CKD group were neuropathic (TNS ≥2), explaining the high prevalence of nerve damage when diabetes was superimposed with the renal dysfunction. This prevalence (100%) is higher than the previous studies as the method of neuropathy assessment was different [48,49]. This study did not examine patients with CKD without diabetes to assess the true effect of renal impairment on corneal nerves as the study aimed to determine the impact of the combined effect of diabetes and its complications on corneal nerve parameters. There are multiple etiologies [50–52] associated with non-diabetic CKD. Some of these, including, hypertension and inflammatory kidney disease (i.e. glomerulonephritis) may cause corneal structural changes that are independent of the development of neuropathy [53,54]. We would envisage that a future study of non-diabetic CKD should be powered to assess the effects of each etiology on corneal parameters. Naturally, this will need to be a large undertaking and would have to be conducted independent of the present manuscript. A limitation of the present study is its cross-sectional design which limits longitudinal assessment of corneal nerve morphology and renal function in patients with CKD. A further limitation is that the study only included participants with type 2 diabetes. Therefore, the results of this study may not be generalized to CKD without diabetes or CKD with type 1 diabetes. In the current study, IWL was reduced in patients with type 2 diabetes and was associated with the severity of peripheral neuropathy. These findings are consistent with previous studies [17,33,55], and contrary to another study which used depth-corrected wide area mosaic images, rather than individual images for nerve analysis [56]. Studies have shown that selecting individual images for nerve analysis can overestimate measurements compared to composing a wider mosaic of the corneal nerve plexus using automated techniques [57–59]. Therefore, future studies should aim to employ these advanced imaging techniques to improve objectivity and reproducibility of analyses.

Table 5 Hierarchical multiple regression analysis showing the association between corneal nerve fiber, nerve excitability parameters and eGFR, eGFR stages, serum K and serum urea levels controlling for age, severity of peripheral neuropathy and duration of diabetes. This analysis was performed separately for each individual corneal nerve parameter because of the multicollinearity among them. Parameters

ß coefficient

Dependent Variable CNFD EGFR 0.098 eGFR Stages −2.684 K −2.131 Urea −0.51 Dependent Variable CNBD EGFR 0.112 eGFR Stages −2.869 K −0.203 Urea −0.591 Dependent Variable CNFL EGFR 0.058 eGFR Stages −1.666 K −1.139 Urea −0.313 Dependent Variable CTBD EGFR 0.181 eGFR Stages −5.304 K −0.486 Urea −0.877 Dependent Variable IWL EGFR 0.046 eGFR Stages −1.889 K −1.847 Urea −0.297 Dependent Variable CNFrD EGFR 0.001 eGFR Stages −0.021 K −0.021 Urea −0.004 Dependent Variable IWNFrD EGFR 0.002 eGFR Stages −0.072 K −0.15 Urea −0.011

SE

P*

95% CI (Lower)

95% CI (Upper)

R2 Value

0.04 1.069 2.07 0.302

0.017 0.015 0.308 0.098

0.019 −4.825 −6.291 −1.117

0.178 −0.542 2.029 0.098

0.084 0.087 0.018 0.051

0.103 2.765 5.182 0.766

0.279 0.304 0.969 0.444

−0.094 −8.405 −10.616 −2.132

0.318 2.667 10.211 0.95

0.019 0.017 0.001 0.012

0.021 0.57 1.123 0.162

0.008 0.005 0.316 0.06

0.016 −2.807 −3.396 −0.64

0.101 −0.525 1.119 0.014

0.101 0.114 0.018 0.066

0.134 3.577 6.769 0.998

0.18 0.144 0.943 0.384

−0.086 −12.467 −14.09 −2.887

0.449 1.86 13.118 1.132

0.029 0.035 0.001 0.015

0.028 0.73 1.409 0.209

0.108 0.012 0.196 0.161

−0.01 −3.351 −4.677 −0.717

0.102 −0.427 0.984 0.123

0.034 0.08 0.026 0.032

0 0.007 0.013 0.002

0.007 0.004 0.118 0.027

0 −0.034 −0.048 −0.008

0.001 −0.007 0.006 −0.001

0.104 0.117 0.042 0.087

0.002 0.046 0.085 0.013

0.263 0.125 0.082 0.386

−0.002 −0.164 −0.32 −0.037

0.005 0.02 0.02 0.015

0.019 0.036 0.053 0.014

Hierarchical multiple regression analysis showing the predictor capacity of dependent variables to predict the independent variables. P* value adjusted for age, severity of peripheral neuropathy and duration of diabetes. Abbreviations: CNFD, corneal nerve fiber density; CNFL, corneal nerve fiber length; CNBD, corneal nerve branch density; IWL, inferior whorl length; CNFrD, Corneal nerve fractal dimension; IWNFrD, inferior whorl nerve fractal dimension; STDC, stimulus time duration constant; RRP, relative refractory period; eGFR, estimated glomerular filtration rate; K, potassium; ms, milli seconds; R2 = coefficient of determination. CI = confidence interval.

5. Conclusions

normal (stage 1: ≥90, stage 2: 60–90). However, there was an overall decline of all the corneal nerve parameters in both stage 1 and 2 when compared to healthy controls (Table 2). Further, signs of neuropathy (TNS) were clinically apparent in patients with stage 3 CKD or above (eGFR < 60 ml/min) [37,43], which is also reflected in our study cohort as a greater decline of corneal nerve parameters in stage 3 and 4 as represented in Table 2. Similarly, there was a global reduction in all corneal nerve parameters, which correlated significantly with the severity of neuropathy (TNS) (Tables 3 and 4). After controlling for potential confounders including, age, severity of neuropathy and duration of diabetes, these association remained for CNFD, CNFL, CNFrD and IWL (Table 5). Of all corneal nerve parameters measured, CNFrD and CNFL exhibited the strongest independent associations and were the best predictors of severity of CKD. Regression analysis revealed that in participants with type 2 diabetes, every 1-unit (stage) increase in severity of CKD was associated with a decrease of approximately 1.16 mm/mm2 in CNFL

In conclusion, patients with type 2 diabetes-related CKD results in significant corneal nerve loss compared to patients with type 2 diabetes in the absence of CKD. This evidence suggests that corneal nerve assessment appears to be a valuable, non-invasive ophthalmic marker for identifying the peripheral nerve damage in participants with diabeticrelated CKD. Author contributions Mr Shyam Sunder Tummanapalli and Dr Maria Markoulli had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Shyam Sunder Tummanapalli wrote the manuscript and researched data. Tushar Issar, Aimy Yan and. Mark D. P. Willcox, Maria Markoulli, Arun V. Krishnan, Natalie Kwai and Ann M. Poynten reviewed/edited the manuscript. 6

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Financial disclosures

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