Evidence of retinal arteriolar narrowing in patients with autosomal-dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease, with a prevalence of 1:400 to 1:1000 in Caucasians. It is caused by mutations in the PKD1 gene located on chromosome 16p13.3 [1] (in approximately 85% cases) as well as in the PKD2 gene on chromosome 4q13-23 [2]. These genes encode the polycystin-1 (PC-1) and polycystin-2 (PC-2) proteins [3]. PC-1 and PC-2 assemble in the plasma membrane to regulate the calcium (Ca²⁺) entry mechanism. The other biological functions of polycystins (PCs) are still under investigation [4].

The presence of kidney cysts is a characteristic feature in ADPKD patients and is necessary to establish the diagnosis [5]. Cystic pathology in the course of ADPKD is believed to be the result of abnormal cell proliferation and deregulated apoptosis [6]. Cysts may cause abdominal pain, hematuria and infection [7]. The number and diameter of cysts increases with age [8], which leads to progressive renal enlargement and subsequent loss of kidney function [9]. By the age of 60 years, half of ADPKD patients develop chronic end-stage kidney disease (ESRD), requiring kidney replacement therapy [10]. In developed countries, patients with ADPKD constitute 4–10% of all patients undergoing dialysis. In ADPKD, cyst generation and enlargement are associated with transformed vascular architecture: microvasculature tends to develop the regression of larger capillaries with flattened arterioles. It can be the result of reduced expression of the polycystins in the vasculature, where they are important for mechanosensation, fluid-shear stress sensing, signaling, and preserving structural integrity [11, 12, 13]. Vascular endothelial growth factor (VEGF) is an angiogenic cytokine playing pivotal roles in the maintenance of vascular networks; the VEGF family is chronically downregulated in ADPKD. Moreover, many cells in ADPKD aberrantly express VEGF, which in turn stimulates interstitial fibroblasts and endothelial cells, contributing to disease progression [14]. Abnormal kidney function due to polycystic kidney disease may result in systemic osmotic imbalances resulting from hyponatremia, which causes an osmotic gradient across the vascular interface. Interestingly, VEGF acting via VEGF receptor-2 can induce a release of glutamate from Müller cells, a type of retinal glial cell, which serve as support cells for retinal neurons, preventing cellular swelling of Müller glia in hypoosmotic stress [15]. Under prolonged osmotic stress conditions, the local production of reactive oxygen species (ROS) is induced, which is associated with the development of different chronic inflammatory retinal diseases, such as exudative age-related macular degeneration, diabetic retinopathy, uveitis, or atherosclerotic vascular retinal disorders [16, 17, 18].

In ADPKD patients, cardiovascular abnormalities are significantly more common than in the general population, including hypertension (HT), higher left ventricular mass or cardiac valvular defects, and intracranial and extracranial aneurysms [19]. Intracranial aneurysms occur in 4–8% of patients with ADPKD and in 25–50% may cause intracranial bleeding [20]. In ADPKD patients, especially 40–50-year-olds, subarachnoid bleeding occurs 5 times more often than in the general population and causes 5% of deaths among those patients [21]. In the general population, analysis of retinal vessels according to the Keith-Wagener classification [22] can serve as a predictive marker for the occurrence, clinical course, and prognosis of cerebrovascular and cardiovascular diseases. Retinal vessel analysis (RVA) is a modern methodological approach that is used to analyze retinal vessel caliber and vascular function in the retinal microcirculation [23]. The association of retinal vessel caliber abnormalities, such as arteriolar narrowing (AN) and a lower arteriovenous ratio (AVR), with HT and chronic kidney disease has been shown [24, 25, 26]. The width of the arterial vessels at the level of the retina depends on the age and gender, and as a person ages, the arterial vessels gradually narrow [27]. To date, no studies on the advancement of vascular changes have been performed in the retinal microcirculation of ADPKD patients.

The aim of this study was to analyze in detail the retinal vessel quality in ADPKD patients with normal kidney function and without concurrent diabetes mellitus (DM), using standard ophthalmological examination and RVA testing, and to compare it to a non-ADPKD control group. We also wanted to verify the hypothesis that retinal vessels in ADPKD patients may contain microaneurysms, as is are typical in the other vascular beds of ADPKD subjects, and to determine whether ADPKD may be a risk factor for retinal vascular abnormalities, independent of HT and kidney function.

The study group included 39 adult individuals with ADPKD (15 males, 24 females), while the control group comprised 45 gender- and age-matched individuals (20 males, 25 females).

The following inclusion criteria were applied: the presence of cysts in both kidneys according to Ravine et al. criteria of the ADPKD phenotype [5], ADPKD in family history, serum creatinine concentration ≤1.35 mg/dL, and lack of developed DM. Individuals with a negative family history of ADPKD, an absence of cysts in the kidneys (Ravine's criteria not fulfilled), serum creatinine concentration ≤1.35 mg/dL, and no prior diagnosis of diabetes were enrolled for the control group.

Initially, both cohorts of patients qualified for prospective observation, according to the established inclusion criteria [28], as mentioned above. Since all examinations, including thorough ophthalmological examinations, were performed after 6 years of observation in both groups, at this time some of these initial inclusion criteria had already not been fulfilled. DM was not diagnosed in any patient from the ADPKD group, while it developed in five patients from the control group (DM type 2: four cases, DM type 1: one case; one patient treated exclusively with an anti-diabetic diet, two patients with oral anti-diabetic drugs, one with insulin therapy). The duration of diabetes at the time of the ophthalmological examination was approximately 2 years. Patients who developed DM were not excluded from the study due to its prospective, cohort nature, which requires monitoring of parameter changes in all patients in the study and control groups who met the specified inclusion criteria for the study at the beginning of the observation period. Additionally, the short duration of DM in these patients and its good control (HbA1C; 5.34 ± 0.56%) determine the low probability of developing diabetic retinopathy, the features of which were not found in the ophthalmological examination.

The study adhered to the tenets of the Declaration of Helsinki, and the study protocol was approved by the Local Research Bioethics Committee of the Pomeranian Medical University in Szczecin (decision BN-001/135/06). Each patient provided written informed consent for his/her involvement.

A full medical history review was obtained from the participants. The waist-hip ratio (WHR) was calculated as the proportion of the waist and hip circumferences, and the body mass index (BMI) was calculated as weight/height squared (kg/m²). The fasting venous blood sample of each participant was tested for glucose (the enzymatic-amperometric method (Cobas GLUC 800: 04,404,483,190 with a Super GL system, Diagnostic Systems, Germany)), insulin (microparticle enzyme immunoassay (Ax-Sym MEIA, Abbott Laboratories, Abbot Park, USA)), C-peptide (electrochemiluminescent method (Cobas 6000 system, Roche, Mannheim, Germany)), HbA1c (immunoturbidimetric method (Cobas HbA1c 150: 20753521322)). The measurements of serum creatinine, urea, uric acid, and lipid levels (total cholesterol (TC), LDL-cholesterol, HDL-cholesterol, triacylglycerol (TG)) were performed with a Bio-Autoanalyzer Cobas Integra 800 (Roche, Mannheim, Germany). Homeostatic model assessment (HOMA) indices of insulin action were analyzed. Insulin resistance in patients was expressed with the HOMA-% sensitivity (HOMA%S) formula, and pancreatic beta-cell function was expressed with the HOMA-% beta (HOMA%B) index [29]. The e-GFR was calculated from a single serum creatinine measurement with the chronic kidney disease epidemiology collaboration (CKD-EPI) equation [30].

Since most of the analyzed quantitative parameters presented distributions significantly different from a normal distribution (Shapiro-Wilk test), the non-parametric Mann-Whitney U test was applied to compare values between groups, and the Spearman rank correlation coefficient (Rs) was used to measure the strength of the association between variables. Qualitative parameters were compared between groups with Fisher's exact test. Multivariable analysis of independent factors associated with retinal vessel parameters was performed with the general linear model (GLM). P < 0.05 was considered statistically significant. Statistical analysis was performed with Statistica 13 software.

Both the ADPKD and control groups were comparable in terms of mean age (43.58 ± 11.6 vs 43.71 ± 9.1 years), gender distribution (36.8% vs 44.4% of males), and BMI. HT was observed more frequently in ADPKD patients, with both systolic blood pressure (SBP) and diastolic blood pressure (DBP) values significantly higher. The serum concentrations of creatinine, urea, and uric acid were significantly higher and the e-GFR was significantly lower in the ADPKD group than in the controls (Table 1). Fasting levels of the metabolic parameters (glucose, insulin, C-peptide, total cholesterol, HDL-cholesterol, LDL-cholesterol and triacylglycerol) were not significantly different between groups (Table 1).

Outstandingly, the severity of hypertensive retinopathy appeared to be more advanced in the ADPKD group than in the control group. According to Keith-Wegener classification, retinal arteriolar narrowing (Grade I) and arteriovenous crossing (Grade II) were significantly more common in the ADPKD group in comparison to controls (Table 2). Importantly, in both study groups, we did not find vascular abnormalities such as retinal hemorrhages, exudates, cotton wool spots, sclerosis, spastic lesions of the retinal arterioles, or papilledema, or microaneurysms, which are very characteristic of ADPKD patients in other vascular beds.

Table 3 provides the values of ocular together with macro-and micro-retinal vessel parameters obtained in both investigated groups of patients. We observed that the AVR values were considerably lower in ADPKD patients than in controls (p < 0.0001). No significant difference in arterial and venous responses to flicker stimulation was identified between the eyes from the ADPKD and control groups. There was no difference in the values of the IOP and OPA between the investigated groups. We observed that the MOPP values were significantly higher (p < 0.0001) in the ADPKD group than in the controls and correlated positively with SBP and DBP. Interestingly, the OPA measurements were strongly related to the MOPP values. An increase in the MOPP was associated with lower OPA values (Rs = −0.39, p = 0.004 in the ADPKD group and Rs = −0.26, p = 0.02 in the control group).

Significantly, the AVR measurements were dependent on the arterial blood pressure values. An increase in systolic blood pressure was associated with a lower AVR index (Rs = −0.25, p = 0.04 in the ADPKD group and Rs = −0.12, p = 0.29 in the control group). Similarly, diastolic blood pressure results correlated negatively with AVR values (Rs = −0.16, p = 0.20 in the ADPKD group and Rs = −0.25, p = 0.03 in the control group). Accordingly, the severity of hypertensive retinopathy according to the Keith-Wagener classification correlated positively with SBP values (Rs = +0.27, p = 0.03 in the ADPKD group and Rs = +0.27, p = 0.02 in the control group). Remarkably, the severity of hypertensive retinopathy positively correlated with participant age (Rs = +0,27; p = 0.02 in the ADPKD group and Rs = +0.28, p = 0.006 in the control group). A similar correlation was found in the ADPKD group between the arterial and venous responses to flicker stimulation and age (Rs = −0.35, p = 0.003 and Rs = −0.24, p = 0.045, respectively). This finding indicates that older patients have more advanced vascular changes than their younger counterparts.

Importantly, we also observed in univariate analysis that AVR values were lower in men than in women among controls (0.853 ± 0.051 vs 0.892 ± 0.054, p = 0.007) but not among ADPKD patients (0.804 ± 0.068 vs 0.808 ± 0.062, p = 0.84). The multivariate analysis of ADPKD patients and controls revealed that male gender was an independent factor significantly associated with lower AVR values (β = −0.171, p = 0.027). Finally, the multivariable analysis of ADPKD patients and controls (Table 4), adjusted for age, gender, e-GFR and the presence of HT, revealed that ADPKD was an independent factor associated with lower AVR values (by 0.069 on average, β = −0.50, p < 0.0001).

Hypertension, arterial vasospasm, and extensive remodeling of small renal arteries and arterioles at early stages of cystic kidney disease result in cardiovascular complications that form the leading cause of death in ADPKD [19]. Therefore, verifiable detection of vascular changes in retinal vessels seems to be an important diagnostic task in subjects suffering from ADPKD. The early detection of vascular retinopathy signs is an important step in the risk stratification of patients with this chronic incurable illness for medical purposes and suitable treatment onset. In the present work, our effort was devoted to analyzing in detail the retinal vessel quality in non-diabetic ADPKD patients using selected, highly objective ophthalmological tests and comparing it to that of non-ADPKD subjects.

The quantitative measurement of different retinal vessel metrics, including the AVR, permits the objective detection of retinal microvascular changes. The AVR quantifies the change in diameter of retinal blood vessels, and it is basically the ratio of arteriole to venule diameter [33, 34]. To our knowledge, this is the first study exploring retinal vessel caliber and function using the RVA method in ADPKD patients with preserved normal kidney function. Here, we showed that ADPKD was an independent factor associated with a lower AVR irrespective of age and the presence of HT. We found no retinal hemorrhages, cotton wool spots, exudates, or papilledema, or microaneurysms in ADPKD patients.

High blood pressure primarily affects the blood vessels in the retina, altering the vessel caliber. Several study groups documented low AVR values in the course of HT [25, 27]. Despite numerous studies quantifying retinal vessel caliber in HT and other cardiovascular diseases, only a few analyzed retinal vascular measurements and AVR in the course of chronic kidney disease (CKD). Accordingly, Liu et al. observed that the retinal arteriole diameter was narrower in patients with CKD and that a low retinal arterial caliber was significantly associated with a lower e-GFR [35]. Baumann et al. [24] documented lower AVR in CKD patients than in controls. The higher age of non-diabetic CKD patients and their higher SBP independently predicted a lower retinal arteriolar diameter [24]. Similarly, in our study, AVR measurements were lower in ADPKD and dependent on the SBP values. Interestingly, we observed that male sex was an independent factor associated with lower AVR values. Likewise, Ponto et al. also observed that men had a lower AVR than women, and the decrease in AVR values with increasing age was steeper in men than in women [27].

It was previously shown in Pkd2^+/− mice, which are the orthologous animal model of ADPKD, that the resistance arteries in their bodies present with enhanced vasoconstriction and increased arterial remodeling [36, 37]. Both of these processes may also be responsible for the increased risk of diverse vascular complications reported in ADPKD patients. Importantly, in the retinal microvasculature, we did not find microaneurysms, which are common in other vascular beds in ADPKD patients [20]. We hypothesize that mutations of either the PKD1 or PKD2 gene, encoding polycystins responsible for ADPKD, may participate in the mechanism leading to a decrease in AVR value through abnormalities of intracellular calcium ion (Ca²⁺) metabolism. Changes in the tone of the retinal arterioles are primarily achieved through changes in [Ca²⁺] within the smooth muscles of these vessels, which regulate muscle cell contraction and, subsequently, vascular constriction. Vascular smooth muscle cell (VSMC) contraction can be evoked by an increase in cytosolic [Ca²⁺] owing to transmembrane Ca²⁺ influx or sarcoplasmic reticulum (SR) Ca²⁺ release. Ca²⁺ influx across the plasma membrane can occur via a variety of Ca²⁺ channels, including voltage-gated, store-operated, and ligand-gated channels [38]. Ca²⁺ may also be released from intracellular stores via RyR- or IP3-R-gated channels in the SR membrane. Using high-speed confocal Ca²⁺ imaging, it has been demonstrated that the resulting signals are far from homogeneous, with spontaneous activity in retinal arterioles being characterized by both localized Ca²⁺ sparks and more global Ca²⁺ waves and oscillations [39, 40, 41]. PKD1 and PKD2 gene products (PC-1 and PC-2 proteins, respectively) form a subgroup of the transient receptor potential (TRP) superfamily, which consists of a large number of nonselective cation channels with variable degrees of Ca²⁺ permeability [42]. Recently, Yanda et al. [43] found that several key components of Ca²⁺ signaling are elevated in mouse PN (PC1 null) vs PH (PC1 containing) cells and proposed that elevated Ca²⁺ release from the SR is actually the key factor stimulating renal cyst growth. Qi Qian et al. [44], using an in vitro model, found decreased PC-2 expression in pkd2^+/− vessels, roughly half of the wild-type level. Consistent with these observations, freshly isolated pkd2^+/− VSMCs have significantly altered intracellular Ca²⁺ homeostasis. The resting [Ca²⁺]j was approximately 17% lower in pkd2^+/− cells than in wild-type cells (p=0.0003), and the total SR Ca²⁺ stores were significantly decreased (p<0.0001). Pkd2^+/− VSMCs have decreased resting [Ca²⁺] j and capacity of the SR Ca²⁺ store. PC-2 not only conducts Ca²⁺ from SR stores but is also intimately related to cytosolic Ca²⁺ levels, as they the open probability of PC-2. Low levels of Ca²⁺ (up to 1 μM) increase the open probability of PC-2, whereas high levels (>1 μM) are inhibitory, ultimately giving PC-2 a bell-shaped Ca²⁺ response [45]. The PC-2/PC-1 complex is important in maintaining proper signaling throughout the cell, thus explaining the requirement for both PCs in maintaining calcium homeostasis and how gene mutation in either polycystin can distinctly affect cell function. Interestingly, in our previous study [46], we found that ADPKD patients with preserved normal kidney function had higher Ca²⁺ concentrations in serum and erythrocytes than non-ADPKD controls, indicating the potential role of the polycystin complex as a putative membrane-based calcium channel and regulator of calcium homeostasis in different types of cells and in the retinal vasculature. This might also indicate that the natural course of ADPKD leads to disorders of intra- and extracellular calcium metabolism preceding the onset of renal failure. Based on our previous molecular observations in ADPKD patients, the results of our current study may indicate that dysfunction of the PC-1 and PC-2 in the SR membrane and in the cellular membrane, caused by mutation of PKD1 or PKD2 gene, interferes with Ca²⁺ flow through these membranes, leading to anomalous Ca²⁺ homeostasis, resulting in arteriole contraction, perhaps also in the human retina. In addition, Coban et al. showed that in patients with advanced stages of ADPKD, the arterial stiffness (AS) development, which contributes to endothelial dysfunction, was increased compared to patients at earlier stages or to healthy individuals [47]. Accordingly, results of Choi et al. suggest that the changes of AS in ADPKD patients may be caused by aberrant Ca²⁺ homeostasis [48].

On the other hand, inhibition of the endogenous glutamate receptor-dependent signal transduction cascade in the Müller cells of PKD21/703 transgenic rats with mutated polycystin-2 or pharmacological inhibition of purinergic P2Y1 and adenosine A1 receptors in Müller cells of wild-type retinas, results in swelling of these cells under hypo-osmotic conditions. Abrogation of osmotic-related adenosine-triphosphate (ATP) release through the glutamate signaling pathway might be glioprotective; that is, it may avoid cytotoxic Ca²⁺ overload due to overstimulation of P2Y1 receptors [49], prevent excessive release of different growth factors from the cells [50], or be neuroprotective through the upregulation of NTPDase1 in retinal Müller cells [15]. However, the loss of osmotic ATP release from Müller cells may also contribute to the development of cytotoxic retinal vasculature edema in PKD21/703 rats [15], affecting vessel caliber. Notwithstanding, further research is essential to determine the molecular mechanisms of osmotic ATP release from Müller cells and calcium metabolism in the retinal cells of transgenic animal models with polycystic kidney disease due to PC mutations.

Overall, we suggest that a lower AVR value found in the retinal microcirculation, independent of HT, could be a specific pathophysiological ocular manifestation of the systemic vasculopathy that develops in the course of ADPKD. Increased awareness of retinal vascular symptoms and signs in ADPKD patients may provide an earlier diagnosis of retinal dysfunction and subsequent treatment before irreversible vision defects develop in the eyes of ADPKD patients.