**1. Introduction**

Chronic kidney disease (CKD)-mineral and bone disorder (CKD-MBD) becomes an important issue in CKD management [1]. CKD patients show a bone and mineral disor‐ der as follows: (i) laboratory abnormal metabolisms of calcium, phosphate, and vitamin D; (ii) evidence of calcified tissue disturbance; (iii) and arterial calcification. Arterial calcifica‐ tion is known to be related to numerous worse symptomatic outcomes, such as ischemic cardiovascular attacks and death [2]. The pathogenesis of vascular calcification in CKD is complex, and instead of occurring by a simple process of calcium and phosphate precipita‐ tion, it is produced by an active process in which vascular smooth muscle cells (VSMCs) undergo apoptosis and vesicle formation and are transformed into osteoblast-like cells that induce matrix formation and attract local factors that are involved in the mineralization process [3] (Fig. 1).

The pathogenesis of the multifactorial interactions between aging and progression of vascular calcification remains uncertain. However, there is no doubt that end-stage renal disease (ESRD) patients are at high risk of and have a common finding of vascular calcification due to multiple confounders that promote the differentiation of VSMCs to osteoblast-like cells, which are able to enhance the tissue calcium deposition process [4]. Vascular calcification has recently reported to be associated with many traditional risk factors, aging, high blood pressure, diabetes, and hyperlipidemia, and with nontraditional risk factors, including elevated serum phosphate level, hyperparathyroidism, and high-dose prescription of calcium [5]. Vascular calcification causes a decrease in vascular elasticity, an increase in pulse wave velocity [6], an induction of cardiomyopathy [7], a decrease in coronary artery flow, and an ischemic change (Fig. 2).

© 2015 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Figure 1.** Relationship between calcium and phosphorus and vascular calcification.

**Figure 2.** Schematic representation of the clinical effects of arterial intimal and medial calcification.

Recent therapeutic regimens that are performed to suppress vascular calcification are concen‐ trated on the control of metabolic markers of skeletal disorder, including phosphate, and vitamin D. The antiosteoporosis drugs such as bisphosphonates [8] have shown therapeutic possibility, but more additional clinical trials are needed. The ADVANCE study has recently shown that cinacalcet and low-dose vitamin D reduce vascular and cardiac valve calcification in hemodialysis (HD) patients with secondary hyperparathyroidism (SHPT) [9]. This review article describes recent progress in terms of the pathogenic mechanisms and methods of assessing and managing vascular calcification in ESRD patients.


**Abstract**

Nanoparticles are small particles that range from 1 to 100 nm in size, exhibit several physical and chemical features. An understanding of nanoparticles would reveal great qualities and potential applications that would aid the diversification of thin film technologies. The synthesis methods employed like top-down, bottom-up, chemical, biological, and mechanical processes have great influence on the properties exhibited by such nanomaterials. This review covers an insight into the knowledge of nanoparticles, their classifications, parameters affecting their efficient performance, synthesis and characterization techniques of nanoparticles. Nanoparticles are also characterized to obtain their morphological, structural, optical, elemental, size, and physiochemical features. The potential applications of nanoparticles have not been left undiscussed.

**Keywords:** nanoparticles, thin film technologies, synthesis, characterizations, applications

## **1. Introduction**

Nanotechnology involves synthesizing and developing different nanomaterials. The field of nanotechnology allows different nanoparticles of unique features to be produced. Nanoparticles (NPs) are complex material particles that fall within the range of one to hundred nanometers. Their nanometer sizes drive the chemical, optical, physical, and electric features of the nanoparticles [1]. Naturally, nanoparticles can be sourced from geological, biological, meteorological, and cosmological means. However, nanoparticles can be created from liquid and solid materials by breaking down biopolymers, condensing gases, wet chemical process, implantation of ions, hydrothermal process, pyrolysis, radiolysis etc. Nanoparticles are usually viewed with the aid of electron microscopes, can penetrate filters, and have unique mechanical properties that distinguish them from the bulk materials. Nanoparticles exist in various shapes like nanorods, nanostars, nanofibers, nanospheres, nanoflowers, nanoboxes etc. [2].

Nanoparticles comprise a functionalized surface, a shell of different layered materials, and the core/main nanoparticle [3]. The features of materials in their bulk form are different from their nanoparticle forms because of the large area to volume ratio, interfacial layer, affinity to solvents, kind of coating, quantum mechanics effects, rate of diffusion, mechanical, and ferromagnetic features [1]. The large area to volume ratio makes the nanoparticles highly reactive and able to penetrate membranes. The chemical nature of nanoparticles should be studied to enhance their molecular attachment to surfaces.


**4. Conclusions**

Flood events (major and minor) are inevitable, regardless of the amount of existing protection. Flood protection is critical, yet the financial cost of maintaining high levels of protection can prove rather excessive. In major flood events, failures occur, often at different locations each time depending on the spatial distribution and evolution of the phenomenon. However, it is possible to take into account the parameters that highlight the weaknesses and the problematic or insufficient flood protection design so that rational and reliable alternative proposals and solutions emerge.

From the correlations and analyses of the data in this study, it was derived that an important element for the spatial determination of the new measures is the infrastructure already developed for flood protection. The study showed that, despite the existence of multiple structural measures/work, many of them show poor efficiency, as shown by the high rate of repeated maintenance and its cost.

The methodology followed in this study was based on the results of the performance of existing flood protection measures, their characteristics, and their spatial correlation. The evaluation of structural measures by assessing their vulnerability via their yearly maintenance implemented the economic loss and financial impact that flooding events might have on infrastructure. The new measures should be related to the cost of construction and maintenance each time, applying the rule of cost–benefit [10] and life cycle analysis, and infrastructure that costs more to maintain should be replaced.

**Author Contributions:** Conceptualization, E.T.; methodology, E.T., C.S. and A.C.; software, E.T., C.S. and A.C.; validation, E.T., C.S. and A.C.; formal analysis, E.T., C.S. and A.C.; investigation, E.T., C.S. and A.C.; resources, E.T., C.S. and A.C.; data curation, E.T., C.S. and A.C.; writing—original draft preparation, E.T.; writing—review and editing, E.T. and C.S.; visualization, E.T. and C.S.; supervision, E.T. and C.S.; project administration, E.T.; funding acquisition, E.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the INTERREG V-A European Territorial Cooperation Program "Greece-Bulgaria 2014–2020 Flood Protection—Cross Border Planning and Infrastructure Measures for Flood Protection".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** All data, models, and code that support the findings of this study are available from the corresponding author upon reasonable request.

**Conflicts of Interest:** The authors declare no conflict of interest.


*3.1. LC-MS*/*MS Analysis of DWP16001, Dapagliflozin, and Ipragliflozin*

To compare the pharmacokinetics and tissue distribution of DWP16001, dapagliflozin, ipragliflozin in mice, analyses of the three SGLT2 inhibitors using LC-MS/MS were applied. Figure 2 shows the selected precursor and product ions of DWP16001, dapagliflozin, ipragliflozin, and D4-DWP16001 (IS). The selected precursor and product ions of dapagliflozin and ipragliflozin were consistent with previously published findings [12,15].

Representative multiple reaction-monitoring (MRM) chromatograms of DWP16001, D4-DWP16001 (IS), dapagliflozin, and ipragliflozin (Figure 3) showed that all the analyte peaks obtained using the liquid-liquid extraction method using MTBE were well separated with no interfering peaks at their respective retention times.

**Figure 2.** Product ion spectra of (**A**) DWP16001, (**B**) D4-DWP16001 (IS), (**C**) dapagliflozin, and (**D**) ipragliflozin.

**Figure 3.** Representative multiple reaction-monitoring (MRM) chromatograms of (**A**) DWP16001, (**B**) D4-DWP16001 (IS), (**C**) dapagliflozin, and (**D**) ipragliflozin in mouse double-blank plasma, blank plasma spiked with DWP16001, dapagliflozin, ipragliflozin at the lower limit of quantification (LLOQ) (5 ng/mL), and plasma samples at 1 h following single oral administration of DWP16001, dapagliflozin, or ipragliflozin at a dose of 1 mg/kg.


**4. Ambient noise level**

Wenz curves are very helpful in the prediction of the level of ambient noise under given constraints. Wenz curves as shown in **Figure 1** represent the average ambient noise for different shipping traffic levels and wind speeds.

Ambient noise level variation w.r.t frequency can be well understood from Wenz curves, and this can be summarized as follows:

**157**

*Underwater Ambient Noise*

*DOI: http://dx.doi.org/10.5772/intechopen.93057*

60–90 dB with less-frequency dependence.

marine mammals or rain is present.

sources are relatively closer to the surface."

**5. Ambient noise in shallow water**

total ocean area.

the sea surface and the seafloor.

• 10–100 Hz—In this band noise level depends mostly on shipping density and activities related to industries. In such cases noise levels are in the range of

• 1–100 kHz—Sea surface agitiation is the dominant factor unless and otherwise

• >100 kHz—Noise in this range is dominated by electronic and thermal noise.

Shallow water ambient noise is highly random due to the waveguide nature of the environment and bottom reflection [12]. There are two definitions of shallow water: hypsometric and acoustic. The hypsometric definition is based on the fact that most continents have continental shelves bordered by the 200 m bathymetric contour, beyond which the bottom generally falls off rapidly into deep water. Therefore, shallow water is often taken to mean continental shelf waters shallower than 200 m. Using this definition, shallow water represents about 7.5 percent of the

Acoustically, shallow water conditions exist whenever the propagation is characterized by numerous encounters with both the sea surface and the seafloor. By this definition, some hypsometrically shallow water areas are acoustically deep. Alternatively, the deep ocean may be considered shallow when low-frequency and long-range propagation conditions are achieved through repeated interactions with

Shallow water regions are distinguished from deep water regions by the relatively greater role played in shallow water by the reflecting and scattering boundaries. Also differences from one shallow water region to another are primarily driven by differences in the structure and composition of the seafloor. Apart from water depth, the seafloor is perhaps the most important part of the marine environment that distinguishes shallow water propagation from deep water propagation. The most common shallow water bottom sediments are sand, silt, and mud, with

In shallow water, in the absence of local shipping and biological noise, wind noise dominates the noise of distant shipping over the entire frequency range [1]. The reason for this is that the deep favorable propagation paths traveled by distant

Shallow water is known for its variability. Waters close to shore and in busy harbors are dynamic locations, where rapid noise changes take place. Even though shallow water has the variability as a notable feature due to variable background of biological and shipping activities, at high frequencies and even at high wind speeds, this level is significantly constant from location to location at the same wind speed. Although noise field characterization is complex in shallow waters, it has to be car-

compressional sound speeds greater than that of the overlying water.

shipping noise in deep water are absent in shallow water.

ried out because of its greater applications in naval operations.

The main inference can be drawn out of Wenz curves as "noise level (NL) decreases as depth increases and also w.r.t change in frequency, because most noise

• 100–1000 Hz—Here shipping is the primary source of noise. Sea surface agitation is also a contributing factor to noise with this frequency range.

**Figure 1.** *Wenz curve [11].*

*Noise and Environment*

**4. Ambient noise level**

the absence of ships and tumid marine life.

curves, and this can be summarized as follows:

discovered that it correlated very well with wind speed [7].

ambient noise for different shipping traffic levels and wind speeds.

to 50 KHz. In this range of frequency, i.e., in KHz, hydrophone is used as an anemometer [1]. Wind speed is considered as the major constituent of ambient noise in

Wind noise can be treated as a typical case of random noise. Wind noise in the frequency range of 500 Hz to 20 KHz is called as Knudsen noise, because Knudsen

Wenz curves are very helpful in the prediction of the level of ambient noise under given constraints. Wenz curves as shown in **Figure 1** represent the average

Ambient noise level variation w.r.t frequency can be well understood from Wenz

**156**

**Figure 1.** *Wenz curve [11].*

The main inference can be drawn out of Wenz curves as "noise level (NL) decreases as depth increases and also w.r.t change in frequency, because most noise sources are relatively closer to the surface."


**8. Receptor promiscuity and biased signaling**

GPCR "promiscuity" is defined as the ability of a given receptor to activate several different effectors (for review: (Hermans, 2003). While most receptors can probably induce parallel signaling by the GαGTP and Gβγ subunits (see above), some are capable of activating different G proteins; and some use both G protein dependent and G protein independent signaling pathways.

"Biased signaling" refers to the observation that when receptors two or more signaling pathways, a few agonists preferentially use only one of the signaling pathways available to the other agonists – an observation that suggests that the activated receptor takes different conformations, depending on the agonist occupying its binding site.


*Review* **Current Therapeutic Approach to Atrial Fibrillation in Patients with Congenital Hemophilia**

**Minerva Codruta Badescu 1,2, Oana Viola Badulescu 3,4, Lacramioara Ionela Butnariu 5,\*, Mariana Floria 1,6,\*, Manuela Ciocoiu 3, Irina-Iuliana Costache 1,7, Diana Popescu 1, Ioana Bratoiu 8, Oana Nicoleta Buliga-Finis 1 and Ciprian Rezus 1,2**

	- 8 Department of Rheumatology and Physiotherapy, "Grigore T. Popa" University of Medicine and Pharmacy, 700115 Iasi, Romania<EMAIL_ADDRESS>
**Abstract:** Cardiovascular disease in hemophiliacs has an increasing prevalence due to the aging of this population. Hemophiliacs are perceived as having a high bleeding risk due to the coagulation factor VIII/IX deficiency, but it is currently acknowledged that they also have an important ischemic risk. The treatment of atrial fibrillation (AF) is particularly challenging since it usually requires anticoagulant treatment. The CHA2DS2-VASc score is used to estimate the risk of stroke and peripheral embolism, and along with the severity of hemophilia, guide the therapeutic strategy. Our work provides the most complete, structured, and updated analysis of the current therapeutic approach of AF in hemophiliacs, emphasizing that there is a growing interest in therapeutic strategies that allow for short-term anticoagulant therapy. Catheter ablation and left atrial appendage occlusion have proven to be efficient and safe procedures in hemophiliacs, if appropriate replacement therapy can be provided.

**Keywords:** atrial fibrillation; anticoagulant; catheter ablation; left atrial appendage occlusion; cardiovascular disease; hemophilia

#### **1. Introduction**

Hemophilia is a rare genetic disorder. It is caused by inherited X-linked mutations in the genes encoding coagulation factor VIII (hemophilia A) or factor IX (hemophilia B). It is characterized by a lifelong increased tendency to bleed, which is generally proportional to the degree of coagulation factor deficiency. Of the two types, hemophilia A (HA) is six times more prevalent than hemophilia B (HB) [1]. While women are carriers, men clinically express the disease.

Due to advances in treating hemophilia, the life expectancy of hemophiliacs has currently increased, approaching that of the general male population [2–6]. Once uncommon, age-related diseases have emerged in hemophiliacs and the number of cases is growing. Among the most frequent non-hemophilia-related medical comorbidities are cancer and cardiovascular disease, both associated with old age. Their diagnosis and treatment are extremely challenging because it often requires procedures and drugs that

**Citation:** Badescu, M.C.; Badulescu, O.V.; Butnariu, L.I.; Floria, M.; Ciocoiu, M.; Costache, I.-I.; Popescu, D.; Bratoiu, I.; Buliga-Finis, O.N.; Rezus, C. Current Therapeutic Approach to Atrial Fibrillation in Patients with Congenital Hemophilia. *J. Pers. Med.* **2022**, *12*, 519. https://doi.org/ 10.3390/jpm12040519

Academic Editor: José Miguel Rivera-Caravaca

Received: 14 February 2022 Accepted: 21 March 2022 Published: 23 March 2022

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

may worsen the already deficient hemostasis. The treatment of cardiovascular diseases in patients from the general population usually includes antithrombotic therapy, either anticoagulant, antiplatelet agents, or both. Evidence-based guidelines for the management of cardiovascular diseases in hemophiliacs are lacking and the antithrombotic treatment is guided by the consensus of experts based on the international guidelines dedicated to non-hemophilia patients [1,7–9].

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia in the general adult population. The prevalence is 2–4% and rises due to the increase in the number of elderly people worldwide and the intensification of the effort to diagnose AF [10–12]. Observational studies note similar rates of AF in patients with hemophilia compared to the general population [13,14]. Since its treatment usually involves anticoagulant therapy and interventional techniques, AF poses unique therapeutic challenges in hemophiliacs because these patients have a bleeding-prone hemostatic balance. Our work provides an in-depth analysis of the modern therapeutic approach to AF in hemophiliacs, taking into account the new advances in the field. In order to extract the necessary data, we performed an extensive search in the Web of Science. The keywords "atrial fibrillation" and ("hemophilia" or "haemophilia") were searched in the titles and abstracts of the articles. As this review aimed to provide information on the current treatment of atrial fibrillation, the search was restricted to the last 10 years. We manually screened the titles and abstracts of all 138 articles retrieved from the automatic search, removing duplicates and articles in languages other than English.

#### **2. AF Epidemiology and Risk Factors**

Age and many of the comorbidities associated with aging are important risk factors for AF [12,15–27]. Current evidence shows that hypertension, diabetes mellitus, dyslipidemia, obesity, and smoking are very common among hemophiliacs [28–32]. Moreover, of these cardiovascular risk factors, hypertension is more prevalent in hemophiliacs than in the general male population [29–33]. The clustering of cardiovascular risk factors is also present, with more than a third of hemophiliacs having metabolic syndrome [33]. The unfavorable cardiovascular risk profile sets in earlier in life in hemophiliacs than in the general population [29,31,34] and the cardiovascular risk factor burden is present, irrespective of hemophilia severity [32,35].

In men, the risk of AF increases steeply after the age of 50 [36]. Thus, with the prolongation of the lifespan of hemophiliacs and the accumulation of risk factors for AF, the number of patients with this arrhythmia is expected to increase. In the largest European cohort of hemophiliacs analyzed to date, the prevalence of AF was 0.84% [13]. A low value of 0.2% was found in patients younger than 60 years of age and seventeen times higher (3.4%) in those over 60 years of age, similar to that of the general population [9]. While there was a very important age-dependent upward trend, the prevalence of AF was inversely correlated with the severity of hemophilia. Other European studies reported a prevalence of 2.2–2.4% [37,38]. The lowest prevalence of AF was found in Asian hemophiliacs (0.28%) [30], and the highest in two North American cohorts, 5.4% in young hemophiliacs [39] and 7.5% in those aged between 54 and 73 [32]. This highlights the negative impact of aging on the prevalence of AF.

#### **3. Assessment of the Ischemic Risk**

Blood stasis in the left atrium and impaired contractility of the left atrial appendage are the main contributors to cardioembolism [40]. It is considered that 20–30% of all ischemic strokes and 10% of cryptogenic strokes are the consequence of AF [12]. Generally, AF multiplies the risk of stroke by five [41]. This risk is not homogeneous in the general population, but is dependent on comorbidities and ranges between 1% and 20% [42,43]. Moreover, strokes associated with AF are usually severe, highly recurrent, often fatal, or with permanent disability [12].

The risk of AF-related stroke is influenced by additional factors, therefore, risk scores have been developed to stratify patients and guide the anticoagulant treatment [44–46]. The CHADS2 score was originally used [47], but the CHA2DS2-VASc score outperformed the CHADS2 score and simplified the anticoagulation decision-making as it could identify individuals with a truly low thromboembolic risk that do not require anticoagulant treatment (0 in men and 1 in women) [48,49].

Data on the risk of ischemic stroke in hemophiliacs compared to men in the general population are scarce. Still, the rate of ischemic stroke is expected to be low [50]. A retrospective study showed that the prevalence of ischemic stroke was lower in hemophiliacs with severe disease (0%) than in the general male population (1.5%), but this difference was not statistically significant in hemophiliacs with mild-moderate disease [37]. Of four patients with ischemic stroke, the event was correlated with the presence of AF in one.

Few data are available on the risk of ischemic stroke and systemic embolism in hemophiliacs with AF. CHADS2 and subsequently CHA2DS2-VASc scores were used to estimate this risk, but none have been prospectively validated in a population of hemophiliacs thus far. The CHA2DS2-VASc score was calculated in 33 patients with AF from a large pan-European cohort of almost 4000 hemophiliacs [13]. The score ranged from 0 to 4, driven principally by age and hypertension. The score's mean value was 1.3. Of the 33 hemophiliacs with AF, 16 (48%) had hypertension, three (9%) peripheral vascular disease, three (9%) diabetes, and two (6%) had previous stroke. None had congestive heart failure. Eleven patients (33%) were between 65 and 74 years and three (9%) were 75 years or older. There were ten hemophiliacs with a CHA2DS2-VASc score of 0. Based on the CHA2DS2-VASc score, in hemophiliacs with AF in this cohort, the risk of stroke was low [13].

The French registry included 68 hemophiliacs requiring antithrombotic treatment for acute coronary syndromes (ACS)/coronary artery disease (CAD), AF, or both [51]. Seventeen patients only had AF and one patient had both AF and CAD. The CHA2DS2- VASc score ranged from one to seven. Sixteen patients had a CHA2DS2-VASc score ≥2. The score's mean value was three, much higher than previously reported [13]. This difference was not determined by the age of the enrolled patients, but by the load of comorbidities.

Data also came from a short series of cases and case reports. In seven HA patients, the CHA2DS2-VASc score ranged between one and six, with a mean value of three, where age and hypertension were the most important contributors [52]. The patient with severe hemophilia had the highest CHA2DS2-VASc score mainly due to advanced age, previous stroke, and myocardial infarction. Cheung et al. reported the case of a HA patient with mild disease who also had a score of six due to age, left ventricular dysfunction, hypertension, history of stroke, and atherosclerotic carotid stenosis [53]. A very high CHA2DS2-VASc score of seven was reported in a 79-year-old patient with mild HA, based on age, chronic heart failure, hypertension, transient ischemic attack, and coronary artery disease [54].

Although the value of the CHA2DS2-VASc score varies widely in this population, it has become evident that due to the multiple risk factors that accumulate with age, hemophiliacs with AF have an increased risk of ischemic stroke and systemic embolism, regardless of the severity of the disease. Therefore, stroke prevention measures should be discussed with the patient and implemented by a cardiologist–hematologist team.

The first attempt to incorporate a stroke risk score in anticoagulation decision-making was based on the CHADS2 score [55]. Currently, the CHA2DS2-VASc score is endorsed by the ADVANCE (age-related developments and comorbidities in hemophilia) Working Group [8]. The proposed algorithm incorporates both the CHA2DS2-VASc score and the severity of hemophilia. Still, caution is advised as the absolute risk of stroke may be overestimated because the CHA2DS2-VASc score includes parameters that are either irrelevant—female sex—or rarely relevant— peripheral arterial disease and very old age—for the hemophilia population. It has been proposed that only hemophiliacs with a CHA2DS2-VASc score ≥3 should be considered with a high risk of stroke [50]. Considering the severity of hemophilia and the risk of bleeding, other researchers have considered

that a CHA2DS2-VASc score ≥2 should be used to start anticoagulation in patients with factor level ≥20% and a higher threshold—CHA2DS2-VASc score ≥4—was suggested in hemophiliacs with a factor level <20% [8,9].

#### **4. Assessment of the Bleeding Risk**

The HAS-BLED score is used in the general AF population for estimating the basal bleeding risk before and during the anticoagulant treatment [56]. A HAS-BLED score ≥3 indicates a high risk of bleeding, but this value should not be used as an absolute parameter for withholding or withdrawing anticoagulation. This score must be seen as a tool to reduce the bleeding risk by identifying those risk factors that can be avoided or reversed.

The reliability of this score in hemophiliacs has long been questioned because two major determinants of the risk of bleeding in hemophiliacs—the severity of the disease and the presence of inhibitors—are not directly assessed by this score.

The frequency of bleedings depends on the severity of hemophilia. The highest risk is in patients with a factor level <1% of normal, who may bleed spontaneously or after minimal trauma [57,58]. Hemophiliacs have a particular hemorrhagic profile. Bleedings are usually recurrent and mainly affect the joints and muscles. Although it is a rare complication, intracranial hemorrhage (ICH) is the most feared because it is life-threatening. Its incidence is higher in hemophiliacs than in the general population, especially in those with severe disease [59,60]. The implementation of modern therapies has led to a major reduction in ICH incidence so that currently, the patients on prophylactic treatment no longer develop ICH [59–62]. Moreover, hypertension has long been associated with ICH [63]. Since hypertension is very prevalent in hemophiliacs, its diagnosis and intensive treatment should be promptly implemented to reduce the ICH risk in the entire hemophiliac population.

Hemophilia patients without inhibitors typically have a predictable response to clotting factor replacement, and bleeding can be reliably prevented or treated. Those with baseline factor levels >5% and those with severe hemophilia under clotting factor prophylaxis have the lowest risk of bleeding [7]. Patients with inhibitors have a much less predictable hemostatic response to bypassing agents. The problem with developing neutralizing anti-FVIII antibodies is of great importance as it occurs in 25–33% of HA patients [64,65]. Studies have found that 10–20% of bleedings in hemophiliacs with inhibitors are either unresponsive or only partially responsive to bypassing agents [66]. Therefore, the risk of uncontrolled bleeding almost always outweighs the benefit of anticoagulation.

When reported, often in a small series of cases and case reports, the HAS-BLED score was frequently three. In seven HA patients, the HAS-BLED score ranged between one and three, with a mean value of three. Higher values of the score such as five or six have also been reported [54,67].

Until 2021, when the results of the French registry were published, there was a general agreement that the HAS-BLED score underestimated the bleeding risk in hemophiliacs and was considered not suitable for use in this population [50,68]. The French registry provided evidence to the contrary. The HAS-BLED score of the 18 hemophiliacs with AF ranged from zero to four, with a mean value of two [51]. A HAS-BLED score ≥3 was associated with increased bleeding risk. In the two years of follow-up under antithrombotic treatment, five out of eight patients with a HAS-BLED score ≥3 reported bleeding episodes, while no hemorrhagic events were reported in any of the 10 patients with a HAS-BLED score <3. Of note, none of the hemophiliacs had inhibitors. The median coagulation factor level and the proportion of patients on prophylaxis were similar between the two groups. This was the first report to show that the HAS-BLED score is applicable to hemophiliacs.

The type, intensity, and duration of antithrombotic therapy are determined, on one hand, by the ischemic risk, and on the other hand, by the risk of bleeding and need for replacement therapy, which must ensure a level of coagulation factor that allows for safe antithrombotic treatment. Intensive replacement therapy as required by anticoagulants is unlikely to be sustainable in the long-term. In this context, finding alternative therapeutic solutions has become a priority. Of particular interest are the catheter ablation techniques and the devices for closing the left atrial appendage.

#### **5. Rhythm or Rate Control in AF**

When choosing between rhythm and rate control, it should be considered that over time, AF becomes less responsive to treatment or is irreversible [69] and that AF progression leads to an increased risk of ischemic stroke or systemic embolism, a high risk of hospitalization for heart failure [70] and a decrease in the quality of life [71]. The current guideline highlights the fact that the primary indication for rhythm control is to reduce the symptom burden related to the arrhythmia and to improve the quality of life, especially when factors favoring rhythm control are prevailing [12]. While no major differences were observed in cardiovascular mortality or stroke rate [72], the rhythm control strategy improved the left ventricular function and quality of life in patients with heart failure [73].

The drugs used for rate or rhythm control and the antiarrhythmic selection algorithm in hemophiliacs mirror the guidelines for AF management in the general population [12]. One option to achieve rhythm control is through cardioversion and the currently available data in hemophiliacs reflect a preference for the pharmacologic approach [74–78]. The anticoagulant therapy should be started as soon as possible before cardioversion, and concomitant coagulation factor replacement therapy should be administered when necessary. The algorithm that includes transesophageal echocardiography (TEE) should be considered first—when available—because TEE can rule out atrial thrombi and the 3-week period of anticoagulation prior to procedure is avoided [12]. Of note, successful cardioversion does not imply that long-term anticoagulant treatment is no longer necessary. The stroke occurrence in high-risk patients is the same, regardless of the rhythm or rate control strategy adopted [79], therefore, the decision regarding long-term oral anticoagulant treatment should be driven by the presence of the stroke risk factors [12]. Thus, other therapeutic options should be considered in hemophiliacs with a CHA2DS2-VASc score of one or higher in whom long-term anticoagulant therapy treatment is not feasible.

Patients with AF require both antiarrhythmic and anticoagulant treatment, therefore, the guidelines provide specific recommendations to help in the selection of the appropriate drug and dose [80]. Recent data on clinical implications of drug–drug interactions show that in patients over 66 years old on anticoagulant treatment with a NOAC —apixaban, dabigatran, rivaroxaban—the bleeding risk was not increased by the concomitant use of amiodarone, diltiazem, and verapamil [81]. If dronedarone is used, it should be noted that it increases the frequency of gastrointestinal bleedings when associated with dabigatran and rivaroxaban and the risk of overall bleeding in patients receiving rivaroxaban [82]. Thus, when selecting AF treatment for hemophiliacs, it should be considered that the underlying hemorrhagic risk will be increased by the administration of the antithrombotic therapy and that some antiarrhythmic drugs may further potentiate this risk.

Due to the increased bleeding risk of hemophiliacs, there is a permanent pursuit for the use of the minimum effective dose of anticoagulant. The increasing availability of thrombin generation assays could facilitate this approach. While conventional coagulation tests provide only partial information on hemostasis, the global coagulation tests evaluate the functionality of all its components. By assessing the dynamics of clot formation, clot resistance, and stability, global coagulation tests reflect the interaction between procoagulants, natural anticoagulants, platelets, and the fibrinolytic system. Guided by a thrombin generation assay, a reduced dose of low molecular weight heparin was safely administered to a HA patient before cardioversion [68].

#### **6. Surgical and Catheter Ablation of AF**

AF catheter ablation for pulmonary vein isolation (PVI) is an efficient and safe rhythm control strategy. It represents a first-line therapy in selected patients with symptomatic AF and it is also recommended after antiarrhythmic drug therapy failure [12,83,84]. The interest in catheter ablation is growing because this therapy is perceived as a way to avoid long-term treatment with oral anticoagulants. It was hypothesized that after successful ablation, the risk of thromboembolic events in patients with AF will be the same as in patients without AF [85–87], thus the anticoagulant treatment could be stopped. However, discontinuation of oral anticoagulant therapy is associated with a low risk of stroke only in patients with a low CHA2DS2-VASc score [84]. Patients with a previous stroke continue to have a high risk of thromboembolic events despite the successful procedure [88].

Anticoagulant treatment is necessary during the procedure to prevent thrombus formation on sheaths and catheters and at the sites of ablation. The standard protocol using unfractionated heparin is also implemented in hemophiliacs given that during the procedure, the coagulation is restored to normal by the factor replacement therapy [78,84]. Most centers adopt the 300–350 s interval [78], but a slightly lower activated clotting time (ACT) target has been used [77]. There is evidence that even an ACT target range of 225–250 s is safe and effective in specified settings [89,90].

AF ablation is followed by a period of high embolic risk due to atrial stunning, tissue damage caused by the procedure, and possible early recurrence of arrhythmia [77]. Therefore, the anticoagulant treatment is recommended for at least two months after the procedure [12]. Because the predictive value of the CHA2DS2-VASc score is maintained after AF ablation regardless of the arrhythmic outcome [88], it is recommended that the decision to stop or continue anticoagulation beyond two months be made based on the patient's risk profile and not on the result of the ablation procedure [12,84].

Cardiac ablation includes the catheter, surgical, and hybrid surgical–catheter ablation. Data on protocol and outcome of the catheter ablation procedure for AF treatment in hemophiliacs are very recent (Table 1). Cryoablation was successfully performed in a HA patient with symptomatic AF that could not be amended through multiple pharmacological attempts [77]. Anticoagulant and replacement factor therapy were used during the PVI procedure, but no long-term antithrombotic therapy was recommended due to the patient's CHA2DS2-VASc score of 0.

A total of seven PVI procedures for symptomatic AF—with an average duration of five years—were performed in five HA patients [78]. Three patients remained in sinus rhythm during follow-up, while in two cases, the procedure was repeated at nine and 16 months after the first PVI due to AF recurrence. The success rate of PVI was comparable to that reported in the general population [91] and the complications were only hematoma at the femoral vein puncture site. Procedures were performed under anticoagulant treatment and replacement factor therapy, aiming at a peak coagulation factor level of 80–100% during ablation and in the following 24 h. On the first day after the procedure, the trough level of the coagulation factor was kept above 50%. For at least four weeks after the procedure, patients received either dabigatran 110 mg bid or VKA and bridging with low-molecularweight heparin (LMWH) until INR >2. A coagulation factor level of at least 20% was considered safe during the anticoagulant treatment.

Surgical AF ablation performed concomitantly with cardiac surgery is an opportunity to add supplementary benefits [92]. A 50-year-old HA patient with mild disease underwent minimally invasive mitral valve repair for valve leaflets prolapse with severe mitral regurgitation. Exclusion of the left atrial appendage and left and right atrial cryoablation for concurrent AF was also performed [93]. His CHA2DS2-VASc score was one. At a 2-year follow-up, the patient had sinus rhythm without AF paroxysms. No antithrombotic was recommended after surgery.

Severe complications after catheter ablation occur in 5–7% of patients in the general population, of which 2–3% are life-threatening. Stroke (<1%) and pericardial tamponade (1–2%) are the most severe. Clinical relevant hematoma at the site of venous puncture occurs in 0.9% of patients in the general population. Periprocedural inguinal bleeding has been reported in hemophiliacs, in two cases with a significant decrease in the hemoglobin level [78]. No fatalities have been reported in hemophiliacs to date.

**Table 1.** Reports on the

non-pharmacological

 treatment of AF.

**Table 1.** *Cont.*

**Table 1.** *Cont.*

fraction,

HCV = hepatitis C virus, DES = drug-eluting

tion, AMI = acute myocardial

structive

wk = week, mo = month, NR = not reported.

 pulmonary

 disease,

 CABG = coronary

 stent, ACS = acute coronary

> infarction,

 HF = heart failure, LAAO = left atrial appendage

 artery bypass grafting,

 ICA = internal

 syndrome,

 PCI = percutaneous

 occlusion,

 carotid artery, GI=

 COPD = chronic ob-

 coronary

 intervengastrointestinal;

#### **7. Percutaneous Left Atrial Appendage Occlusion**

As more than 90% of emboli originate in the left atrial appendage (LAA) [99], percutaneous left atrial appendage occlusion (LAAO) arose from the need to provide protection from ischemic stroke and systemic embolism in AF patients for whom long-term anticoagulant treatment is contraindicated. Data provided by several studies confirmed that LAAO is a feasible, effective and safe alternative to oral anticoagulant treatment [100]. LAAO has comparable efficacy with VKA at a lower rate of major bleedings, particularly hemorrhagic stroke [101,102]. This strategy provides a 90% reduction in the rate of hemorrhagic stroke [103]. In high-risk AF patients, LAAO has comparable efficacy with NOAC in stroke prevention and a similar or better safety profile [104,105]. To prevent device-related thrombosis after implantation, antithrombotic treatment is required until the complete endothelialization of the device surface. In patients without contraindications to oral anticoagulation, the postprocedural antithrombotic therapy consists of 45 days of VKA/NOAC and aspirin, followed by six months of dual antiplatelet therapy (DAPT) with aspirin and clopidogrel and then lifelong single antiplatelet therapy (SAPT) with aspirin [100].

Patients who have had bleeding complications or who have contraindications to long-term oral anticoagulant treatment will receive DAPT with aspirin and clopidogrel for 1–6 months, followed by lifelong SAPT with aspirin [100]. In naïve patients, loading doses of aspirin and clopidogrel are indicated. Patients at extremely high risk of bleeding should receive SAPT for at least 2–4 weeks. If no antithrombotic drug can be administered, epicardial LAA occlusion or thoracoscopic LAA clipping should be considered instead of LAA occluder implantation [100]. Early data from an ongoing study suggest that reduced doses of rivaroxaban (10 mg od and 15 mg od) may be an alternative to postprocedural DAPT [106].

Watchman, Amplatzer Cardiac Plug, and Amulet devices are the most widely used (Table 1). The Amplatzer device family is less thrombogenic than the Watchman device [107], therefore, postprocedural anticoagulation is never necessary [12]. The patients receiving Amplatzer Amulet devices did not have a higher risk of device-related thrombosis while on antiplatelets, especially those on SAPT [108]. In patients at high risk of stroke and contraindication to oral anticoagulant treatment, the implantation of Watchman device followed by DAPT was also an effective strategy [109]. The first case of LAAO in a hemophiliac with a high risk of stroke due to AF was published ten years ago [53]. Since then, many successful implantations of Amplatzer Cardiac Plug and Amplatzer Amulet devices have been reported [53,67,95,96]. Patients had high ischemic and bleeding risks. Their CHA2DS2-VASc score ranged from three to six and the HAS-BLED score from three to six. The postprocedural antithrombotic treatment consisted of antiplatelet agents.

Successful use of the Watchman device was also reported in hemophiliacs with AF [54,98]. Postprocedural antithrombotic therapy varied widely, from anticoagulation to no treatment. In a 60-year-old HB patient with mild disease, AF catheter ablation and placement of the Watchman device were performed concomitantly in order to minimize the patient's exposure to the anticoagulant [94]. After the procedure, the patients received only one month of VKA treatment. Still, the outcome was favorable at a 6-month follow-up.

A combined therapy consisting of LAA closure and cardioversion was successfully performed in a 69-year-old patient with moderate HB while on apixaban 2.5 mg bid and prophylaxis with eftrenonacog alfa [97]. After the procedure, he received one-month apixaban, three months DAPT, and long-term clopidogrel with favorable outcomes.

The largest series of cases of percutaneous LAAO published to date included seven patients with HA [52]. Three patients received the Amplatzer Amulet device and the rest the Watchman device. Before the procedure, two patients were on NOAC, one on LMWH, and one on DAPT and LMWH. After the device implantation, only antiplatelets were recommended, mainly SAPT with aspirin. In two cases, short-term DAPT was used.

Procedural risks associated with LAAO are cardiac tamponade (2–4%), stroke (1–2%), and inguinal hematoma (1%). Device embolization is extremely rare (<1%), but may require emergency cardiac surgery [103,110]. Most complications in hemophiliacs were

minor or significant bleedings at the site of venous puncture. In one case, periprocedural arterial puncture occurred and in two cases, pericardial effusion without tamponade was diagnosed [52,67].

Although periprocedural therapy varies between treating centers, prophylactic replacement of the coagulation factor is always necessary, as a very high factor level of ≥80% must be achieved during the procedure [52–54]. This level of coagulation factor is the same as that recommended for major surgery [57]. The percutaneous LAAO procedure is minimally invasive and does not require such intense replacement therapy, but the high level is considered necessary due to the risk of intracardiac lesions and the possible conversion to thoracic surgery. Moreover, full heparinization is used during the procedure [52,54,94]. In the first three days after the procedure, a trough level of the coagulation factor of 80% was frequently used [52,53].

#### **8. Long-Term Antithrombotic Treatment**

Current guidelines do not support the use of antiplatelet therapy alone for stroke prevention in AF patients in the general population [12]. It was proven that aspirin alone has limited or no protective effect against ischemic stroke [111,112]. Adding clopidogrel to aspirin reduced the stroke rate compared to aspirin alone, but major bleedings significantly increased [113]. Moreover, DAPT with aspirin and clopidogrel did not offer better protection from stroke than VKA at a similar rate of major bleedings [114].

AF patients requiring antithrombotic treatment receive an anticoagulant. VKA is more effective than aspirin in preventing ischemic stroke, with similar rates of major bleedings even in those older than 75 years [115]. Four NOACs—apixaban, dabigatran, edoxaban, and rivaroxaban—are currently indicated for stroke prevention in AF patients. A meta-analysis of their pivotal trials showed that NOACs reduced by 19% the risk of stroke or systemic embolism, with 51% in the risk of hemorrhagic stroke, and with 10% all-cause mortality comparative to VKA [116]. There was also a 14% reduction in the risk of major bleedings with NOACs, most coming from a 52% reduction in intracranial hemorrhages. Still, gastrointestinal bleedings increased by 25% with NOACs compared to warfarin. Of note, fatal bleedings are halved by using a NOAC versus VKA [117]. Due to the more favorable benefit–risk ratio, NOACs are preferred over VKAs in patients with AF eligible for a NOAC—without prosthetic mechanical heart valves or moderate-severe mitral stenosis [12].

In men with AF in the general population, the anticoagulant treatment should be considered if the CHA2DS2-VASc score is ≥1 [12]. Since there is no evidence that hemophiliacs are protected from cardioembolism by their underlying coagulation defect [57], the antithrombotic treatment should be considered in the presence of AF. Table 2 provides a practical algorithm for the antithrombotic treatment.

The most recent therapeutic algorithm was proposed by Schutgens et al. and is based on both the CHA2DS2-VASc score and the severity of hemophilia [8]. Among hemophiliacs with baseline FVIII/IX level ≥20%, the anticoagulant treatment is recommended only if the CHA2DS2-VASc score is ≥2. All oral anticoagulants are allowed, but the use of NOACs over VKA is encouraged because of the superior safety profile of NOACs to that of VKA, particularly regarding intracranial bleeding [116]. NOACs are preferred, especially in patients with HB because VKA reduces plasma FIX levels and increases the severity of hemophilia and consequently the risk of intracranial hemorrhage. While on VKA, these patients will need even more intensive replacement therapy, which is burdensome and may increase the frequency of inhibitor development. The high frequency of intravenous coagulation factor infusion and the related costs make long-term VKA use prohibitive [94].

The plasma FVIII/IX level considered safe for oral anticoagulation is ≥30% [7,55]. Thus, only a few patients will benefit from anticoagulant treatment without requiring replacement therapy, namely the patients with very mild hemophilia and native factor activity level above this threshold. For hemophiliacs with more severe disease, this high threshold is difficult to maintain in the long-term because it requires very frequent administration of

replacement products, often every 1–2 days [51]. Several studies have reported the safe use of anticoagulant treatment in hemophiliacs with FVIII/IX levels ≥20% [118,119] including after catheter ablation [78]. Therefore, it has been proposed to lower the threshold from 30% to 20% [8].

**Table 2.** Practical guide for long-term antithrombotic treatment.

The patient should be informed of the advantages and disadvantages of the proposed treatments.

ACO = anticoagulant, DAPT = dual antiplatelet therapy, SAPT = single antiplatelet therapy, mo = month, LAAO = left atrial appendage occlusion, NOAC = non-vitamin K oral anticoagulant, VKA = vitamin K antagonist, HA = hemophilia A, HB = hemophilia B.

Dabigatran is a direct thrombin inhibitor. In its pivotal trial, the dose of 150 mg bid reduced the risk of stroke by 36% compared to VKA at a similar rate of major bleedings. The dose of 110 mg bid was associated with a similar risk of stroke and a lower rate of major bleedings [120]. Of note, both regimes were associated with lower rates of hemorrhagic stroke than VKA, but the 150 mg bid dose increased the incidence of gastrointestinal bleeding by 50%. Dabigatran was successfully administered in hemophiliacs with AF eligible for anticoagulant treatment [8,121]. The 110 mg bid regimen was used due to its similar efficacy to VKA at a lower risk of bleeding.

Apixaban is a direct inhibitor of FXa, more efficient and safe than VKA in preventing stroke or systemic embolism in patients with AF eligible for a NOAC [122]. A recent real-world data analysis evaluated the efficacy and safety of apixaban according to its dose [123]. The rates of stroke/systemic embolism were reduced by 30% with the 5 mg bid regimen and by 37% with the 2.5 mg bid regimen. The rates of major bleeding decreased by 41% for each of the two regimes. To date, the successful use of low-dose apixaban has been reported in hemophiliacs with AF [52,97].

Rivaroxaban is a direct inhibitor of FXa, non-inferior to VKA in preventing stroke and systemic embolism in patients with AF eligible for a NOAC and with similar overall safety [124]. In the pivotal trial, the intracranial bleedings were less frequent with rivaroxaban than VKA, but the number of gastrointestinal bleedings increased. A recent real-world dose-based data analysis of rivaroxaban showed that the 20 mg od regimen reduced the risk of stroke and systemic embolism and was associated with fewer major bleedings compared to VKA. The 15 mg od regimen was as effective and safer than VKA [125]. In Asians with AF, the efficacy and safety of the 10 mg dose were also assessed [126]. Data from studies conducted on the Asian population showed that rivaroxaban was associated with a lower risk of stroke or systemic embolism than warfarin, regardless of dose [127]. In patients with mild hemophilia, the 10 mg od regimen was used [52].

Edoxaban is a direct inhibitor of FXa non-inferior to VKA in preventing stroke or systemic embolism in patients with AF eligible for a NOAC, with significantly lower rates of major bleeding than VKA for both 60 mg od and 30 mg od regimen [128]. The 60 mg od regimen was slightly more efficient than VKA. The rates of life-threatening bleeding, intracranial bleeding, and major bleeding plus clinically relevant non-major bleeding were favorable to edoxaban for both regimes, except for gastrointestinal bleedings that were higher with 60 mg od edoxaban than with VKA and lower with 30 mg od edoxaban than with VKA.

Thus, the preference for NOACs in hemophiliacs with AF is justified as they provide protection against ischemic stroke at least as well as VKA, but with a lower hemorrhagic risk including reduction in intracranial bleeding.

Hemophiliacs with baseline FVIII/IX level 1–19% and patients with the severe disease under FVIII/IX prophylaxis are candidates for catheter ablation or antiplatelet therapy with aspirin only if the CHA2DS2-VASc score ≥4 [8]. As VKA doubles the risk of intracranial hemorrhage compared to aspirin [129], it is considered unsuitable in hemophiliacs with basal FVIII/IX level <20% [8]. The plasma FVIII/IX level considered safe for SAPT is ≥5–10% [1,7,55].

Of note, apixaban is the only NOAC that has been compared in a large randomized clinical trial with aspirin in AF patients considered unsuitable to receive a VKA [130]. The rate of stroke/systemic embolism was more than halved, while the risk of major bleeding or intracranial hemorrhage did not significantly increase. Thus, the administration of apixaban could be a solution for those hemophiliacs receiving aspirin instead of VKA because their risk of bleeding is considered too high or intensive prophylactic replacement therapy is not feasible [75].

The oral anticoagulant treatment is not recommended in hemophiliacs with severe disease without prophylaxis because the thrombin generation is comparable to that in patients with a therapeutic INR [131]. In hemophilia patients with inhibitors, the antithrombotic therapy is generally contraindicated due to the less predictable hemostatic response to bypassing agents [66].

#### **9. Discussion**

Modern replacement therapies of the deficient coagulation factor prolong the lifespan of hemophiliacs, enabling the occurrence of diseases associated with old age. Atherosclerotic cardiovascular disease challenges most physicians' ability to provide adequate care because it often requires antithrombotic therapy. Hemophilia is a rare disease, so there are no conditions for conducting randomized clinical trials. Therefore, the therapeutic

approach to cardiovascular diseases in hemophiliacs including AF is based on case reports, observational studies, and the consensus of experts [1,7–9,55], which is unlikely to change in the near future.

The management of patients with AF relies on three pillars: avoiding stroke, better symptom management, and cardiovascular and comorbidity optimization [12]. The CHA2DS2-VASc and HAS-BLED scores are recommended for the assessment of the risk of stroke and bleeding in the general population, respectively. Stroke prevention has evolved considerably in the last decade. Anticoagulant therapy with VKA or better with NOAC is the mainstream treatment. Patients with contraindication to oral anticoagulants or at risk of bleeding under anticoagulant therapy perceived as too high to be acceptable can now be candidates for left atrial appendage occlusion/exclusion [12]. Rhythm control to reduce the AF related symptoms and to improve the quality of life can be implemented through drugs or more recently, cardiac ablation. Pulmonary vein isolation is usually performed, but surgical or thoracoscopic ablation is also available in selected cases.

Although used to guide treatment in hemophiliacs with AF [7,8], the CHA2DS2-VASc score has not been prospectively validated in this population. Still, high values of this score have been found in hemophiliacs [52–54,67], suggesting that the risk of stroke and systemic embolism may be higher than anticipated [13]. The exact risk of stroke/systemic embolism in hemophiliacs with AF is still unknown, but it is certain that it is present even in patients with severe hemophilia. The HAS-BLED score was rarely used in hemophiliacs to estimate the bleeding risk because it was believed that the coagulation factor deficiency itself carries a high risk of bleeding. Moreover, it has been hypothesized that this score underestimates the bleeding risk in these patients [50,68]. However, recent evidence showed that a HAS-BLED score ≥3 was associated with increased bleeding risk even in hemophiliacs [51].

The antithrombotic treatment increases the risk of bleeding in all hemophiliacs, regardless of the severity of the disease. It was found that during the anticoagulant treatment, the bleeding risk of hemophiliacs with the mild disease increased eight times compared to the controls [51]. In hemophiliacs eligible for long-term oral anticoagulants, a preference for NOAC instead of VKA is highlighted, justified by their better efficacy and safety profile. Moreover, low doses of NOACs prevent stroke equal to or more than warfarin, with less major bleedings [120,122–128]. According to the available data, apixaban and 10 mg rivaroxaban seem to be the most suitable to recommend to hemophiliacs presenting AF [75,127,132]. Reports of successful use of NOACs in hemophiliacs with AF have already begun to appear [8,52,97].

HB patients benefit greatly from these recommendations. While a large fraction of factor VIII in blood originates from liver sinusoidal endothelial cells [133], FIX is a vitamin K-dependent coagulation factor produced in the liver. The treatment with VKA will further reduce plasma FIX levels, increasing the severity of hemophilia and the need for substitution therapy. Moreover, FIX replacement therapy may interfere with VKA and make INR value unreliable [55]. Since the coagulation factors II, VII, and X are also vitamin K-depending coagulation factors synthesized in the liver, VKA administration has the potential to further aggravate the imbalance of the hemostasis in hemophiliacs.

The experience gained so far suggests that the anticoagulant therapy is safe for hemophiliacs under appropriate replacement therapy, so working with a hematologist is vital for therapeutic success. In general, levels ≥80% and aiming at 100% are recommended intraprocedural and up to three days thereafter [52–54,78], ≥20% under anticoagulant treatment and DAPT [8,9,78], and ≥5–10% under SAPT [1,7,55].

The management of AF in patients with hemophilia can be quite complex, particularly for the reduction in the risk of stroke in the long-term and in the context of invasive procedures. Since the anticoagulant treatment is considered safe at FVIII/FIX level ≥20% [8], few hemophiliacs can receive it without coagulation factor replacement therapy. Many will require frequent administrations, possibly every 1–2 days, which is burdensome in the long run. It should be noted that this type of replacement therapy is also very expensive. Avoiding the need for long-term anticoagulant therapy should be a priority, therefore, the

optimal therapeutic solution must be sought with great care. Cardioversion helps to control AF-related symptoms, improves left ventricular function, and quality of life, but long-term anticoagulant therapy is often needed [12].

PVI with short-term replacement therapy during periprocedural treatment with VKA or NOAC seems to be a good option [8]. NOACs appear safer than bridging VKA with LMWH for PVI [78]. Catheter ablation proved to be effective for rhythm control and safe to perform in hemophiliacs [77,78]. Although the mandatory duration of the anticoagulant treatment is two months, it is recommended that the treatment be extended in patients with high ischemic risk [12]. Still, it must also be considered that by continuing the oral anticoagulant treatment beyond three months from the procedure, the thrombotic events will slightly decrease while the major bleedings will increase [134].

Percutaneous LAAO is another possibility for these patients with a high bleeding risk [135]. It attracts increasing interest due to its favorable antithrombotic therapy profile, namely, the possibility of using short-time DAPT or anticoagulant, then lifetime SAPT. While DAPT requires the same level of coagulation factor as the anticoagulant treatment [9], SAPT may be safely recommended in hemophiliacs with factor level ≥5–10% [1,7,55]. This is a feasible approach in hemophiliacs. Thus, advances in the treatment of hemophilia allow for the implementation of the modern therapeutic solutions of AF in hemophilic patients.

This study has several limitations. First, there are little data on the management of AF in hemophiliacs, especially on percutaneous cardiac ablation and LAA occlusion techniques, therefore the conclusions cannot be generalized. Second, studies that have reported the use of antithrombotic therapy in hemophiliacs have often included, in addition to patients with AF, those with acute coronary syndromes or elective coronary procedures (coronary artery bypass grafting and percutaneous coronary intervention with stent implantation), which made it difficult to extract the necessary information. Moreover, data on the management of AF in hemophiliacs have sometimes been reported along with those from patients with other hereditary bleeding disorders.

#### **10. Conclusions**

Patients with hemophilia are often perceived as having only an increased risk of bleeding. However, in the presence of AF and ischemic risk factors, they also have an increased risk of stroke and systemic embolism. The choice of appropriate therapy is greatly hampered by the lack of guidelines based on strong evidence. Although the physicians treat hemophiliacs with AF on a case-by-case basis, it should be highlighted that the treatment offered in the general population can be implemented in hemophiliacs if appropriate replacement therapy can be provided. As far as we know, our work provides the most complete, structured, and updated analysis of the current therapeutic approach of AF in hemophiliacs.

**Author Contributions:** Conceptualization, M.C.B., O.V.B., M.F., and C.R.; Methodology, D.P., I.B., and O.N.B.-F.; Writing—original draft preparation, M.C.B., L.I.B., and M.F.; Writing—review and editing, M.C.B., O.V.B., and L.I.B.; Supervision, M.C., I.-I.C., and C.R. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

With Atrial Fibrillation): Developed in collaboration with the European Heart Rhythm Association and the Heart Rhythm Society. *Circulation* **2006**, *114*, e257–e354. [CrossRef]


**3. Design Considerations**

The main features of the FD IA proposed are analysed and discussed in view of the fundamental performance, in order to facilitate the design procedure. In a FD implementation, the CM signal must be processed at least with the same accuracy and speed as the DM signal. Therefore, the CMFB network, in particular, and the CM feedback loop, in general, must be designed so that the open-loop gain (LG) and gain-bandwidth product (LGBW) of both components are similar [30]. This requirement can be analytically expressed as

$$LG\_{CM} \simeq LG\_{DM} \tag{7a}$$

$$LGBV\_{CM} \simeq LGBV\_{DM} \tag{7b}$$

Therefore, it is recommendable to provide similar paths to the DM and the CM signal in order to accomplish these requisites. In the case of the IA represented in Figures 3 and 4, the output branch of the core circuit (Figure 3) is common to both the DM and the CM section. Nevertheless, the differential input stage of each loop, and hence the corresponding effective transconductance, is different in every case. Indeed, for the DM signal, the input transconductance is given by (4), whereas for the CM component the transconductance is equal to the individual transconductances of transistors MCM1 to MCM4 in Figure 4. As the linearization carried out in *GmI* implies a reduction of the transconductance value, it is expected that effective input transconductance of the DM loop is lower as compared to the CM loop. This fact ensures that an appropriate treatment of the DM signal will result in an adequate processing of the CM signal.

Regarding the signal processing of the FD IA, only the DM component gives rise to an output current in the input and output *V*-to-*I* converters, being the CM signal rejected by the differential structure of these stages. However, a CM signal can also produce an output current, given that the presence of mismatches is unavoidable in a real implementation. In order to evaluate the impact of the join action of a CM signal and the mismatches on the output current produced, the residual transconductance of the input and the output transconductor in the IA, defined as Δ*Gm* ≡ *i vCM* , has been analytically calculated. With this purpose, each small signal parameter *gi* has been assumed to have values equal to *gi* + Δ*gi*/2 and *gi* − Δ*gi*/2 for a given pair of ideally matched transistors. In addition, the contributions to the residual transconductance, due to considering mismatches in every

pair of transistors, have been evaluated individually. The corresponding expressions were obtained by means of a hand analysis and the main terms were determined by simulations, resulting to be dominant the responses associated to mismatches in the transconductance (Δ*gm*) and output conductance (Δ*go*) of the input driver transistors, MDI and MDO. The corresponding expressions are:

$$\Delta G\_{m}|\_{\Delta \mathcal{G}\_{m,MD}} \approx \frac{2}{R} \cdot \frac{\Delta \mathcal{G}\_{m,MD} \mathcal{G}\_{o,MD}}{\mathcal{G}\_{m,MD}^2} \cdot \frac{1}{\left[1 + \frac{2}{R} \frac{1}{\mathcal{G}\_{m,MD}} \left(\frac{\mathcal{G}\_{o,MD} + \mathcal{G}\_{o,MD}}{\mathcal{G}\_{m,MD}}\right)\right]} \tag{8a}$$

$$
\Delta G\_{\rm m}|\_{\Delta \mathcal{G} \Rightarrow MD} \approx \frac{2}{R} \cdot \frac{\Delta \mathcal{G}\_{\rm o, MD}}{\mathcal{G}\_{\rm m, MF}} \cdot \frac{1}{\left[1 + \frac{2}{R} \frac{1}{\mathcal{G}\_{\rm m, MD}} \left(\frac{\mathcal{G}\_{\rm o, MD} + \mathcal{G}\_{\rm o, MD}}{\mathcal{G}\_{\rm m, MF}}\right)\right]} \tag{8b}
$$

where MD represents the driver transistors in *GmI* and *GmO*. The impact of the transconductance and output conductance mismatches of other transistors on Δ*Gm* is negligible and, hence, is not reported here for the sake of conciseness.

The use of the CM rejection ratio (CMRR) is a very widespread habit in order to compare the magnitude of the CM gain with respect to the DM gain. As the proposed IA has a single-stage structure, the voltage gain for DM and CM signals will be given by the product of the input transconductance and the output impedance. Assuming the same output impedance for both signal components, the CMRR of the IA can be expressed in terms of the ratio of the effective and the residual transconductance, given respectively by (4) and (8b), as:

$$\text{CMRR} \equiv \frac{G\_{mI}}{\Delta G\_{mI}} = \frac{1}{\left(\frac{\Delta\_{\text{S},nMII}\varrho\_{\text{s},MDI}}{\mathcal{S}\_{m,MDI}^2} + \frac{\Delta\varrho\_{\text{s},MDI}}{\mathcal{S}\_{m,MDI}}\right)} \cdot \frac{\left[1 + \frac{2}{\mathcal{K}\_I} \frac{1}{\mathcal{S}\_{m,MDI}} \left(\frac{\mathcal{G}\_{\text{s},MDI} + \mathcal{G}\_{\text{s},MDI}}{\mathcal{S}\_{m,MDI}}\right)\right]}{\left[1 + \left(1 + \frac{2}{\mathcal{K}\_I} \frac{1}{\mathcal{S}\_{m,MDI}}\right) \left(\frac{\mathcal{G}\_{\text{s},MDI} + \mathcal{G}\_{\text{s},MDI}}{\mathcal{S}\_{m,MDI}}\right)\right]}\tag{9}$$

The most-right term in (9) represents the ratio of the load regulation effects of resistor *R* for the CM and the DM signals, respectively. Thanks to the improved response of the SSF cell, the value of these terms is very close to unity, which allows the expression of the CMRR as a function of the different mismatches in the actual implementation of the circuit. At this point it is worth to mention that, as observed in Figure 2, the structure of the input section of both the PD IA and the FD IA is the same and, hence, both structures present a similar rejection to CM signals form the architecture point of view.

Another key parameter for an IA is the noise, as it indicates the minimum signal level that can be processed. In the case of an IA for bioimpedance spectroscopy, the signal bandwidth required is usually wide and, hence, thermal noise is dominant. The spectral density of the input referred thermal noise has been analytically determined, assuming that the main contributions are due to the input *V*-to-*I* converter, and can be expressed as:

$$\frac{\sigma\_{IN,th}^2}{\Delta f} = \left[1 + \left(1 + \frac{2}{R\_I} \frac{1}{g\_{m,MDI}}\right) \left(\frac{g\_{o,MDI} + g\_{o,MDI}}{g\_{m,MFI}}\right)\right]^2 \cdot 4kTR\_I \cdot \left[1 + \frac{4}{3}(g\_{m,MDI} + g\_{m,MSII})R\_I\right] \tag{10}$$

where *k* and *T* are Boltzmann's constant and the absolute temperature, respectively. The first term in (10) represents the conversion factor for referring the noise from the resistor to the input of the circuit, and is the inverse of the load regulation effect of resistor *RI* on the SSF cell (see Equation (4)), the second factor is the thermal noise of resistor *RI*, and the last term includes the main thermal noise contributions of the devices involved in the circuit implementation of the input *V*-to-*I* converter, *GmI*. It can be inferred from (10) that the noise of the IA can be decreased by reducing the value of the source degeneration resistor *RI*, which is possible until a certain limit thanks to the use of SSF sections.

The fact of linearizing the *V*-to-*I* converters in the IA by means of a resistor, requires a given level of biasing current to achieve a given input DM voltage range with a determined linearity. In each SSF cell in the input and output transconductors, the bias current 2*IB* is split into two branches corresponding to the input and feedback transistors. As the tail current of the driver devices is fixed to *IB* by the lower current sources, a current equal to *IB* is steered towards the feedback transistors. Consequently, the maximum input DM signal that can be processed by each *V*-to-*I* converter is that leading to a current equal to zero through one of the feedback transistors. This condition can be expressed for *GmI* as

$$
\upsilon\_{I,DM\_{max}} = \pm R\_I \cdot I\_B \tag{11}
$$

(**b**)

where the voltage gain of the SSF cells has been assumed to be equal to unity. Nevertheless, this is an extreme situation that leads to switching off one of the branches of the input transconductor. Instead, a specific criterion, such as considering a given total harmonic distortion (THD) level, is assumed in a practical case to determine the value of *vI*,*DMmax* in an objective way.


*2.1. Clinical Assessments*

Patients underwent annual clinical evaluations and cognitive assessments, including tests standardized for use in individuals with DS (Test of Severe Impairment [27], Verbal Fluency Test [28] and the Dementia Questionnaire for People with Learning Disabilities [26]). Dementia status was determined by a neurologist with experience in diagnosing AD in the DS population. Blood collected at the time of clinical diagnosis of AD included thyroid function tests (TSH and Free T4), Vitamin B12 levels, and APOE genotyping; in a subset of the cohort, blood was sent for the evaluation of thyroid autoantibodies including TPO antibodies and/or thyroglobulin (Tg).

Age of onset of AD was determined based on at least a one-year progressive decline in two or more cognitive domains. Early onset was defined as having an AD onset greater than one standard deviation (SD) below the mean age of AD onset for the cohort, and late onset was defined as greater than one SD above the mean age of AD onset for this group. In our cohort, the mean (SD) age of AD onset was 53 (6). Therefore, early AD onset refers to onset before the age 47, typical AD onset as occurring between ages 47 and 59, and late AD onset as occurring after the age of 59.

*Article*

**and Herminia Diana Rosas 1,2,3**

**and Herminia Diana Rosas 1,2,3**

*Article*

*Article*

**Citation:** Lai, F.; Mercaldo, N.D.; Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*

**Citation:** Lai, F.; Mercaldo, N.D.; Wang, C.M.; Hersch, M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*

**Citation:** Lai, F.; Mercaldo, N.D.; Wang, C.M.; M.S.; Hersch, G.G.; Rosas, H.D.; Association between Hypothyroidism Onset and Alzheimer Disease Onset in Adults with Down Syndrome. *Brain Sci.*

https://doi.org/10.3390/xxxxx

Academic Editor: Rebecca Sims

Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021

https://doi.org/10.3390/xxxxx

**2021**, *11*, x.

Academic Editor: Rebecca Sims

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institu-

tional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021

https://doi.org/10.3390/xxxxx

**2021**, *11*, x.

Academic Editor: Rebecca Sims

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institu-

tional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Received: 31 July 2021 Accepted: 8 September 2021 Published: 16 September 2021

tional affiliations.

**2021**, *11*, x.

Age of hypothyroidism onset, which was obtained from caregivers and available for 111 of the 152 patients (73%) with a reported history of hypothyroidism, was the age at which thyroid supplementation was first started based on abnormal thyroid function tests<EMAIL_ADDRESS>(H.D.R.) 2 Massachusetts General Hospital, Boston, MA 02114, USA 3 McLean Hospital, Belmont, MA 02478, USA 4 Harvard College, Harvard University, Cambridge, MA 02138, USA;

**Disease Onset in Adults with Down Syndrome**

<EMAIL_ADDRESS>
**Association between Hypothyroidism Onset and Alzheimer**

**Florence Lai 1,2,3,\*, Nathaniel D. Mercaldo 1,2, Cassandra M. Wang 4, Micaela S. Hersch 5, Giovi G. Hersch 6**

1 Harvard Medical School, Boston, MA 02115, USA<EMAIL_ADDRESS>(N.D.M.);

5 School of Nursing, Simmons University, Boston, MA 02115, USA<EMAIL_ADDRESS>


**2.8. Regulation of urinary acidification**

Hereditary forms of hypomagnesemia include rare, genetically determined disorders that may affect renal magnesium handling either primarily or secondarily. **Table 5** summarizes

**Gene Protein Disorder Mode of inheritance**

Bartter syndrome type 1 AR

Bartter syndrome type 2 AR

AR

AR

AR

AD

with hypercalciuria and nephrocalcinosis

with hypercalciuria and nephrocalcinosis with ocular

Hypercalciuric hypercalcemia AD

Hypomagnesemia 2, renal AD

Hypomagnesemia 4, renal AR

with secondary hypocalcemia

hypomagnesemia

impairment

SLC12A1 Sodium‐potassium‐

KCNJ1 Potassium channel

CASR Calcium‐sensing receptor (CasR)

ATPase

(Pro‐EGF)

KCNA1 Kv1.1 Potassium channel Myokymia 1 with

FXYD2 Gamma subunit Na/K/

EGF Epidermal growth factor

chloride transporter

CLCNKB Chloride channel ClC‐Kb Bartter syndrome type 3 AR BSND Barttin Bartter syndrome type 4 AR

14 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

CLDN16 Claudin‐16, tight junction Familial hypomagnesemia

CLDN19 Claudin‐19, tight junction Familial hypomagnesemia

TRPM6 TRPM6 cation channel Hypomagnesemia 1, intestinal

SLC12A3 NaCl cotrasporter (NCCT) Gitelman syndrome AR

KCNJ10 Kir4.1 potassium channel SESAME syndrome AR HNF1B HNF1β transcription factor HNF1B nephropathy AD

(NKCC2)

(ROMK1)

Pyrophosphate (PPi) is present in urine and can contribute 50% CaOx monohydrate (COM) crystal growth inhibition in the collecting duct and up to 80% in the urine [33, 34]. It has been postulated that hypopyrophosphaturia is a metabolic risk factor for recurrent stone formers [35]. That the concentration of inorganic PPi is higher in urine than in plasma cannot fully explain the origin of urinary PPi, but does suggest that it is somehow either secreted

the spectrum of underlying genetic defects [31].

*Note:* AD = autosomal dominant; AR = autosomal recessive.

**Table 5.** Inherited disorders of renal magnesium loss.

**2.7. Pyrophosphaturia**

One of the main functions of the kidney is to keep the systemic acid‐base chemistry constant. The kidney has evolved so that it can regulate blood acidity by means of three key func‐ tions: (1) by reabsorbing the HCO3− filtered through the glomeruli to prevent its excretion in the urine; (2) by generating a sufficient quantity of new HCO3− to compensate for the loss of HCO3− due to dietary metabolic H+ loads and loss of HCO3− in the urea cycle; and (3) by excreting HCO3− (or metabolizable organic anions) following a systemic base load [40]. For the kidney to be able to perform these functions, various types of cell throughout the nephron have to respond to changes in acid‐base chemistry by modulating specific ion transport and/or metabolic processes in a coordinated fashion, such that the urine and renal vein chemistry is adjusted appropriately. The kidney contributes to acid‐base homeostasis by recovering filtered bicarbonate in the proximal tubule. Distally, intercalated cells of the collecting duct generate new bicarbonate, which is consumed by the titration of non‐volatile acid [41].

The renal tubular acidosis (RTA) syndromes encompass a disparate group of tubular trans‐ port defects that share the inability to secrete hydrogen ions (H+ ). This inability results in fail‐ ure to excrete acid in the form of ammonium (NH4+ ) ions and titratable acids or to reabsorb some of the filtered bicarbonate (HCO3− ). Either situation coincides with a drop in plasma bicarbonate levels, leading to chronic metabolic acidosis. Much of the morbidity of RTA syn‐ dromes is attributable to the systemic consequences of chronic metabolic acidosis, including growth retardation, bone disease, and kidney stones [42].

Dysfunction of the proximal tubules, where approximately 90% of the bicarbonate is reab‐ sorbed, leads to proximal RTA [43], whereas malfunctioning of the intercalated cells in the collecting ducts accounts for all known genetic causes of distal RTA (dRTA).

Inherited proximal RTA is a rare disorder that may be inherited as an autosomal recessive or dominant trait [44]. The more common autosomal recessive form has been associated with mutations in the basolateral sodium bicarbonate cotransporter NBCe1, encoded by the SLC4A4 gene. Mutations in this transporter lead to a reduced activity and/or trafficking, thus, disrupting the normal bicarbonate reabsorption process in the proximal tubules [45]. As an isolated defect of bicarbonate transport, proximal RTA is rare. It is more often associated with Fanconi syndrome, which features urinary wastage of solutes such as phosphate, uric acid, glucose, amino acids, and low‐molecular‐weight proteins, as well as bicarbonate. The distal acidification mechanisms remain intact, however, and acid urine can still be produced. The clinical phenotype is of a metabolic acidosis with hypokalemia; metabolic bone disease is common, but nephrocalcinosis and nephrolithiasis are rare [46].

In contrast, 80% of cases of distal RTA (dRTA) are associated with medullary nephrocalcino‐ sis. The molecular basis underlying primary dRTA is a defective functioning of alpha inter‐ calated cells [41]. The molecular defects behind proximal and distal RTA are listed in **Table 6**.

The clinical signs and symptoms of dRTA can vary, depending on the underlying mutation: patients may reveal a mild metabolic acidosis after the incidental detection of kidney stones, or they may have severe health issues with failure to thrive and growth retardation in chil‐ dren, rickets, severe metabolic acidosis, and nephrocalcinosis. Kidney stones in dRTA consist of CaP due to the release of Ca and Pi from bone to buffer the acidosis, leading to hypercalci‐ uria and consequent CaP precipitation due to an alkaline pH [47].

#### **2.9. Macromoleculuria**

The formation of crystal aggregates involves interaction between crystals and urinary mac‐ romolecules (UMs) that serve as an adhesive. The number of UMs isolated in urine has been

**Table 6.** The inherited renal tubular acidoses.

steadily increasing, and they now form a large group of proteins and some glycosaminogly‐ cans [48, 49]. The main macromolecules involved in crystallization are summarized in **Table 7**.

Dysfunction of the proximal tubules, where approximately 90% of the bicarbonate is reab‐ sorbed, leads to proximal RTA [43], whereas malfunctioning of the intercalated cells in the

Inherited proximal RTA is a rare disorder that may be inherited as an autosomal recessive or dominant trait [44]. The more common autosomal recessive form has been associated with mutations in the basolateral sodium bicarbonate cotransporter NBCe1, encoded by the SLC4A4 gene. Mutations in this transporter lead to a reduced activity and/or trafficking, thus, disrupting the normal bicarbonate reabsorption process in the proximal tubules [45]. As an isolated defect of bicarbonate transport, proximal RTA is rare. It is more often associated with Fanconi syndrome, which features urinary wastage of solutes such as phosphate, uric acid, glucose, amino acids, and low‐molecular‐weight proteins, as well as bicarbonate. The distal acidification mechanisms remain intact, however, and acid urine can still be produced. The clinical phenotype is of a metabolic acidosis with hypokalemia; metabolic bone disease is

In contrast, 80% of cases of distal RTA (dRTA) are associated with medullary nephrocalcino‐ sis. The molecular basis underlying primary dRTA is a defective functioning of alpha inter‐ calated cells [41]. The molecular defects behind proximal and distal RTA are listed in **Table 6**. The clinical signs and symptoms of dRTA can vary, depending on the underlying mutation: patients may reveal a mild metabolic acidosis after the incidental detection of kidney stones, or they may have severe health issues with failure to thrive and growth retardation in chil‐ dren, rickets, severe metabolic acidosis, and nephrocalcinosis. Kidney stones in dRTA consist of CaP due to the release of Ca and Pi from bone to buffer the acidosis, leading to hypercalci‐

The formation of crystal aggregates involves interaction between crystals and urinary mac‐ romolecules (UMs) that serve as an adhesive. The number of UMs isolated in urine has been

SLC4A1 AE1 AD

cotransporter 1 (NBC1)

 ‐ ATPase AR with early onset hearing loss

loss

AR

AR

‐ ATPase AR with later onset hearing

**Disorder Gene Protein Mode of inheritance**

ATP6V1B1 β1 Subunit of H+

ATPV0A4 α4 Subunit of H+

Ca2 Carbonic anhydrase 2

(CAH2)

Distal RTA type1 SLC4A1 AE1 AR

Proximal RTA type2 SLC4A4 Sodium bicarbonate

collecting ducts accounts for all known genetic causes of distal RTA (dRTA).

16 Updates and Advances in Nephrolithiasis - Pathophysiology, Genetics, and Treatment Modalities

common, but nephrocalcinosis and nephrolithiasis are rare [46].

uria and consequent CaP precipitation due to an alkaline pH [47].

**2.9. Macromoleculuria**

Combined Proximal and Distal RTA type3

PH, primary hyperoxaluria; AR, autosomal recessive.

**Table 6.** The inherited renal tubular acidoses.

Although the role of these proteins in stone formation is still far from clear, coating of the crystals by the urinary macromolecules seems to prevent crystal aggregation or at least delay it for long enough for the urine to transit through the kidney.

An inhibitory role has repeatedly been confirmed for osteopontin (OPN) [50–54], which is synthesized in the kidney and excreted in the urine in concentrations that suffice to inhibit CaOx crystallization. No naturally occurring mutations in the SSP1 gene encoding OPN have ever been reported in human diseases, but SSP1 polymorphisms have been associated with the risk of nephrolithiasis [55–57].

Tamm‐Horsfall protein (THP), also called uromodulin, is a kidney‐specific protein synthe‐ sized by cells in the TAL of the loop of Henle. It is the most abundant protein in human urine. It is a potent inhibitor of crystal aggregation *in vitro*, and its ablation *in vivo* predisposes one of the two existing mouse models to spontaneous intrarenal calcium crystallization, but there are still some key issues to clarify regarding the role of THP in nephrolithiasis. By conduct‐ ing a long‐range follow‐up of more than 250 THP‐null mice and their wild‐type controls, Liu et al. [58] demonstrated that renal calcification was a highly consistent phenotype of the THP‐null mice. The crystals consisted primarily of CaP in the form of hydroxyapatite. They were located in the interstitial space of the renal papillae more frequently than in the tubules (particularly in older animals), and there was no accompanying inflammatory cell infiltration. The interstitial deposits of hydroxyapatite observed in THP‐null mice strongly resemble the renal crystals found in human kidneys with idiopathic CaOx stones. In humans, a number of naturally occurring THP mutations are reportedly linked to autosomal dominant medullary

**Table 7.** Crystallization‐modulating macromolecules (Modified from Khan SR and Canals BK 2009 [13]).

cystic disease and familial juvenile hyperuricemic nephropathy (Uromodulin‐related dis‐ eases). Mutations lead to a defective intracellular trafficking of THP, and to a reduced THP excretion and secretion. No renal stone disease has been described in patients with any of these mutations to date, however [13].

#### **2.10. Medullary sponge kidney**

Medullary nephrocalcinosis is a frequent finding in medullary sponge kidney (MSK), a renal malformation associated with renal stones, urinary acidification and concentration defects, cystic anomalies in the precalyceal ducts, a risk of urinary infections, and renal failure. In a large series of 375 patients with macroscopic nephrocalcinosis, it was found that the clinical diagnoses most frequently associated with MSK were hyperparathyroidism and dRTA [9]. The prevalence of MSK in the general population is not known because no systematic autopsy searches have been performed. In a large series of subjects undergoing iv urography for vari‐ ous reasons, pictures ranging from clearly evident MSK to faint radiological signs of the disease were seen in 0.5–1% of cases [59]. MSK is relatively well represented in renal stone patients, however, and has been found in up to 20% of recurrent renal calcium stone formers [60, 61].

Why this malformative condition may predispose to medullary nephrocalcinosis remains to be established. MSK is considered a rare and sporadic disorder, but a recent study showed that 50% of MSK stone formers had relatives with milder forms of MSK, suggesting that it is relatively common for MSK to be familial, and it may be inherited as an autosomal dominant trait [62]. It has also been reported that 12% of unrelated MSK patients carried in heterozygos‐ ity two very rare variants of the glial cell line‐derived neurotrophic factor (GDNF) gene, and these variants were inherited and co‐segregated with the MSK phenotype in some families [63].

Mezzabotta et al. [64] had the chance to conduct an *in vitro* analysis on the behavior of papil‐ lary renal cells coming from the healthy portion of a kidney resected due to renal cancer in a MSK patient with medullary nephrocalcinosis, who harbored one of these rare GDNF gene variants. They found an unexpected and previously never reported phenomenon involving the spontaneous formation of Ca2 PO4 nodules very similar to those of calcifying vascular cells. They demonstrated that silencing the GDNF gene in a human renal cell line and cultivating the silenced cells in osteogenic conditions triggered the deposition of Ca2 PO4 . These results demonstrate the functional role of GDNF gene mutation in determining the medullary neph‐ rocalcinosis associated with the MSK phenotype. They also provide the first experimental evidence of human renal tubular cells having a pivotal role in driving a calcification process. The role of renal cells in nephrocalcinosis is discussed in the subsequent paragraphs.