Complex Disease Research Articles

The role of PD-1/PD-L1 signaling pathway in cancer pathogenesis and treatment: a systematic review

Aim: Cancer as a complex disease poses significant challenges for both diagnosis and treatment. Researchers have been exploring various avenues to find effective therapeutic strategies, with a particular emphasis on cellular signaling pathways and immunotherapy. One such pathway that has recently been suggested is the PD-1/PD-L1 pathway, which is an immune checkpoint signaling system that plays an important role in regulating the immune system and maintaining tissue homeostasis. Cancer cells exploit this pathway by producing PD-L1, which attaches to PD-1 on T cells, thus inhibiting immune responses and enabling the cancer cells to escape detection by the immune system. This study aimed to evaluate the role of the PD-1/PD-L1 pathway in cancer pathogenesis and treatment. Method: This study was performed based on the principles of Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA). All in vitro , in vivo , and clinical studies that were published in English have been considered during a thorough search of the Scopus, Web of Science, and PubMed databases without date restriction until March 2024. Results: According to the studies reviewed, the PD-1/PD-L1 signaling axis suggests promising therapeutic effects on various types of cancers such as non-small cell lung cancer, melanoma, breast cancer, hepatocellular carcinoma, squamous cell carcinoma, and colorectal cancer, among others. Additionally, research suggests that immune checkpoint inhibitors that block PD1/PD-L1, such as pembrolizumab, atezolizumab, nivolumab, durvalumab, cemiplimab, avelumab, etc. , can effectively prevent tumor cells from escaping the immune system. Moreover, there might be a possible interaction between microbiome, obesity, etc. on immune mechanisms and on the immune checkpoint inhibitors (ICIs). Conclusion: Although we have gained considerable knowledge about ICIs, we are still facing challenges in effectively prescribing the appropriate ICIs for individual patients. This is largely due to the complex interactions between different intracellular pathways, which need to be thoroughly studied. To resolve this issue, it is necessary to conduct more reliable clinical trials that can produce a scientific consensus.

The most frequent febrile syndromes and autoinflammatory diseases in adulthood

Autoinflammatory diseases are characterized by inflammatory manifestations in various organ systems, whereby recurrent febrile episodes, musculoskeletal complaints, gastrointestinal and cutaneous symptoms frequently occur accompanied by serological signs of inflammation. Autoinflammatory diseases include rare monogenic entities and multifactorial or polygenic diseases, which can manifest as a variety of symptoms in the course of time. Examples of monogenic autoinflammatory diseases are familial Mediterranean fever (FMF), cryopyrin-associated periodic syndrome (CAPS), tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) and the recently described VEXAS (vacuoles, E1 enzyme, X‑linked, autoinflammatory and somatic) syndrome. For non-monogenically determined autoinflammatory diseases, the most important representatives in adulthood are adult-onset Still's disease (AOSD) and the Schnitzler syndrome, in which a polygenic susceptibility and epigenetic factors are more likely to play a role.

Beneficial Effects of Capybara Oil Supplementation on Steatosis and Liver Apoptosis in Obese Mice.

Obesity is a complex chronic disease characterized by excess body fat (adipose) that is harmful to health and has been a major global health problem. It may be associated with several diseases, such as nonalcoholic fatty liver disease (NAFLD). Polyunsaturated fatty acids (PUFA) are lipid mediators that have anti-inflammatory characteristics and can be found in animals and plants, with capybara oil (CO) being a promising source. So, we intend to evaluate the hepatic pathophysiological alterations in C57Bl/6 mice with NAFLD, caused by obesity, and the possible beneficial effects of OC in the treatment of this disease. Eighteen 3-month-old male C57Bl/6 mice received a control or high-fat diet for 18 weeks. From the 15th to the 18th week, the animals received treatment-through orogastric gavage-with placebo or free capybara oil (5 g/kg). Parameters inherent to body mass, glucose tolerance, evaluation of liver enzymes, percentage of hepatic steatosis, oxidative stress, the process of cell death with the apoptotic biomarkers (Bax, Bcl2, and Cytochrome C), and the ultrastructure of hepatocytes were analyzed. Even though the treatment with CO was not able to disassemble the effects on the physiological parameters, it proved to be beneficial in reversing the morphological and ultrastructural damage present in the hepatocytes. Thus, demonstrating that CO has beneficial effects in reducing steatosis and the apoptotic pathway, it is a promising treatment for NAFLD.

Association of right aortic arch and agenesis of ductus arteriosus in prenatal tetralogy of Fallot spectrum and its clinical implications.

In our center, we observed an increased frequency of right aortic arch (RAA) with an agenesis of the ductus arteriosus (ADA) in prenatally diagnosed tetralogy of Fallot (ToF) and its variations. This study aimed to determine whether there is an association of RAA and ADA in fetuses with ToF. Distribution of genetic anomalies and impact on postnatal outcome were further evaluated. Single-center retrospective observational study including pregnancies with prenatal diagnosis of ToF from 2010 to 2023. All cases were subdivided into ToF with pulmonary stenosis (PS) and pulmonary atresia (PA). Clinical and echocardiographic databases were reviewed for pregnancy outcome, genetic anomalies, and postnatal course. The cohort included 169 cases, 124 (73.4%) with ToF/PS and 45(26.6%) with ToF/PA. Agenesis of the ductus arteriosus was significantly associated with RAA in both subtypes of ToF (p=0.001) compared to left aortic arch and found in 82.5% (33/40) versus 10.7% (9/84) of fetuses with ToF/PS and in 57.1% (8/14) versus 12.9% (4/31) of fetuses with ToF/PA. In both ToF/PS and ToF/PA, RAA/ADA versus RAA/patent DA revealed a significantly higher risk for the presence of genetic abnormalities, especially microdeletion 22q11.2, major aorto-pulmonary collateral arteries and a shorter time to complete surgical repair. We demonstrated a significantly increased frequency of RAA/ADA in patients with prenatally diagnosed ToF. Although this association revealed no significant impact on overall survival, the prenatal detection of RAA/ADA has implications for counseling, genetic evaluation and postnatal management.

BET Inhibitor JQ1 Selectively Reduce Tregs by Upregulating STAT3 and Suppressing PD-1 Expression in Patients with Multiple Myeloma.

Multiple myeloma (MM) stands as a prevalent hematological malignancy, primarily incurable, originating from plasma cell clones. MM's progression encompasses genetic abnormalities and disruptions in the bone marrow microenvironment, leading to tumor proliferation, immune dysfunction, and compromised treatment outcomes. Emerging evidence highlights the critical role of regulatory T cells (Tregs) in MM progression, suggesting that targeting Tregs could enhance immune functionality and treatment efficacy. In this study, a notable increase in Treg proportions within MM patients' bone marrow (BM) compared to healthy individuals is observed. Additionally, it is found that the bromodomain and extraterminal domain (BET) inhibitor JQ1 selectively diminishes Treg percentages in MM patients' BM and reduces TGF-β1-induced Tregs. This reduction occurs via inhibiting cell viability and promoting apoptosis. RNA sequencing further indicates that JQ1's inhibitory impact on Tregs likely involves upregulating STAT3 and suppressing PD-1 expression. Collectively, these findings suggest JQ1's potential to modulate Tregs, bolstering the immune response in MM and introducing a promising avenue for MM immunotherapy.

Elucidating the impact of Y chromosome microdeletions and altered gene expression on male fertility in assisted reproduction.

Genetic abnormalities like Y chromosome microdeletions are implicated in male infertility. This study investigated the association of azoospermia factor (AZF) region microdeletions with unsuccessful assisted reproductive techniques (ART), including in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). This cross-sectional analysis study examined 80 Iranian oligospermic men (mean age 34years) with prior failed ICSI and IVF cycles (IR.IAU.TNB.REC.1401.041). Semen analysis evaluated quantity/quality parameters based on World Health Organization guidelines. Participants were stratified by sperm DNA fragmentation (SDF) levels into: control (SDF < 15%, n = 20), mild elevation (15% ≤ SDF ≤ 30%, n = 60), and high (SDF > 30%, n = 20). Multiplex PCR mapped AZF microdeletions in the high SDF group. The AZF-associated genes were selected by RNA Seq analysis, and the candidate genes were checked for expression level by real-time PCR. High SDF individuals exhibited poorer semen metrics, including 69% lower sperm concentration (P = 0.04) than those without SDF. Of this subset, 45% (9/20 men) harboured predominately AZF microdeletions. Men with AZF microdeletions showed higher SDF (32% vs 21%, P = 0.02) and altered AZF-associated genes expression. As USP9Y 3-fold, UTY 1.3-fold, and BPY2 1-fold revealed up-regulation, while IQCF1 8-fold, CDY 6.5-fold, DAZ 6-fold, and DDX3Y 1-fold underwent down-regulation. The PAWP gene was also down-regulated (5.7-fold, P = 0.029) in the IVF/ICSI failure group. AZF microdeletions significantly impact male infertility and ART outcomes. High SDF individuals exhibited poorer semen metrics, with 45% AZF microdeletions. These microdeletions altered AZF-associated genes expression, affecting fertility mediator PAWP independently. Dual AZF and SDF screening enables personalized management in severe male infertility, potentially explaining IVF/ICSI failures.

Multi-omics research progress in early-onset colorectal cancer

Globally, the incidence of early-onset colorectal cancer (EOCRC) among individuals younger than 50 is escalating. Compared to late-onset colorectal cancer, EOCRC exhibits distinct clinical, pathological, and molecular features, with a higher prevalence in the left colon and rectum. However, the occurrence and development of EOCRC is a multi-factor and multi-stage evolution process, which is the result of the mutual effect of environmental, genetic and biological factors, and involves the multi-level regulation mechanism of other organisms. With the development and improvement of high-throughput sequencing technology, the application of multi-omics analysis has become an important development direction to resolve the pathogenesis of complex diseases and individualized treatment plans. This article aims to review the research progress of EOCRC at the multi-omics level, providing a theoretical foundation for earlier diagnosis and more precise treatment of this diseases.

P.062 Normal NCS in 42-year-old man with PMP22 duplication

Background: Charcot Marie Tooth disease is a polygenic disorder with cannonical features of distal amyotrophy, acrohypesthesia, and tight tendoachilles of either axonal (type 2) or demeylinating (type 1) varieties. Type 1 CMT patients are required to possess conduction slowing of a sufficient degree to qualify as demyelinating. Presented is a middle-aged man with an unremarkable neurologic exam and normal electrophysiology. Methods: Standard electrophysiological techniques were employed to obtain the nerve conduction data (Natus Nicolet EDX AT2; Middleton, WI, USA). Repeated next generation sequencing and deletion/duplication analyses were performed. Results: The nerve conduction studies showed no evidence of demyelination in the upper or lower extremeties. The duplication error was confirmed with repeat testing. The heterozygous PMP22 gene duplication encompassed the entire coding sequence involving exons 1-5. Conclusions: CMT1A accounts for the vast majority of dysmyelinating hereditary neuropathies. Phenotypic variability is well described. Presentations include (a) classic conduction slowing, (b) intermediate slowing, (c) conduction block, (d) HNPP-like, (e) absent CMAPs, and (f) normal NCSs in young infants. This is the first case of a neurologically intact adult with CMT1A. Cryptogenic genetic modifier-effect(s) are posited as a possible explanation of the lack of penetrance. Identifying the nature of this modification may prove instructive for future therapies.

DeepFace: Deep-learning-based framework to contextualize orofacial-cleft-related variants during human embryonic craniofacial development

Orofacial clefts (OFC) are among the most common human congenital birth defects. Previous multiethnic studies have identified dozens of associated loci for both cleft lip with or without cleft palate (CL/P) and cleft palate alone (CP). Although several nearby genes have been highlighted, the ‘casual’ variants are largely unknown. Here, we developed DeepFace, a convolutional neural network model, to assess the functional impact of variants by SNP activity difference (SAD) scores. The DeepFace model is trained with 204 epigenomic assays from crucial human embryonic craniofacial developmental stages of post-conception week (pcw) 4 to pcw 10. The Pearson Correlation Coefficient between the predicted and actual values for 12 epigenetic features achieved a median range of 0.50 to 0.83. Specifically, our model revealed that SNPs significantly associated with OFC tended to exhibit higher SAD scores across various variant categories compared to less-related groups, indicating a context-specific impact of OFC-related SNPs. Notably, we identified six SNPs with a significant linear relationship to SAD scores throughout developmental progression, suggesting that these SNPs could play a temporal regulatory role. Furthermore, our cell-type specificity analysis pinpointed the trophoblast cell as having the highest enrichment of risk signals associated with OFC. Overall, DeepFace can harness distal regulatory signals from extensive epigenomic assays, offering new perspectives for prioritizing OFC variants using contextualized functional genomic features. We expect DeepFace to be instrumental in accessing and predicting the regulatory roles of variants associated with OFC, and the model that can be extended to study other complex diseases or traits.

Pediatrics, Pediatricians and Child Care : Based on 16th Dr. K. C. Chaudhuri and Dr. Amala Chaudhuri Oration, on 15th October 2023.

Medical problems of children and their differences from adults have been mentioned in the ancient texts. Important contributions to medicine, including treatment of diseases of children were made by Greek and many scholars from middle East countries in 10th century. Pediatrics became widely recognized in Europe and USA during early 19th century and a number of children's hospitals were established in major cities. With technological advances, pediatric subspecialties also developed. In India, pediatrics was recognized around 1950s and thereafter, gradually progressed. Pediatric specialties came up in 1970s and became well established during 2020s.Pediatricians are regarded as doctors treating sick children. Pediatric specialists have the responsibility of providing tertiary care to patients with complex systemic diseases and critical care. In our country having a huge underprivileged population, pediatricians need to play a wider role and aim to provide comprehensive care that would lead to optimum development for every child. They should be aware of child rights, widely prevalent child abuse and exploitation and legal protective mechanisms, and attempt to tackle these issues in association with other agencies and organizations working for child welfare.

Neratinib impairs function of m6A recognition on AML1-ETO pre-mRNA and induces differentiation of t (8;21) AML cells by targeting HNRNPA3

Acute myeloid leukemia (AML) is frequently linked to genetic abnormalities, with the t (8; 21) translocation, resulting in the production of a fusion oncoprotein AML1-ETO (AE), being a prevalent occurrence. This protein plays a pivotal role in t (8; 21) AML's onset, advancement, and recurrence, making it a therapeutic target. However, the development of drug molecules targeting AML1-ETO are markedly insufficient, especially used in clinical treatment. In this study, it was uncovered that Neratinib could significantly downregulate AML1-ETO protein level, subsequently promoting differentiation of t (8; 21) AML cells. Based on “differentiated active” probes, Neratinib was identified as a functional inhibitor against HNRNPA3 through covalent binding. The further studies demonstrated that HNRNPA3 function as a putative m6A reader responsible for recognizing and regulating the alternative splicing of AML-ETO pre-mRNA. These findings not only contribute to a novel insight to the mechanism governing post-transcriptional modification of AML1-ETO transcript, but also suggest that Neratinib would be promising therapeutic potential for t (8; 21) AML treatment.

#817 Genetic analysis in a group of patients with persistent isolated microscopic hematuria

Abstract Background and Aims Autosomal dominant Alport syndrome (ADAS) is a genetic disease caused by pathogenic variants in COL4A3 and COL4A4 genes. ADAS is the main cause of isolated persistent microscopic hematuria (PMH). However, other diseases like IgA nephropathy (IgAN) can present with PMH. Establishing a precise genetic diagnosis in this type of patients would avoid invasive studies like kidney biopsies. In this study we performed a comprehensive genetic test in a cohort of patients with isolated PMH to evaluate the frequency of ADAS or other genetic abnormalities and compare the clinical characteristics of patients with or without ADAS Method Fifty-two patients with PMH were included in the study. PMH was defined by the presence of 3 or more dysmorphic red blood cells per field in at least 5 consecutive urine sediments and a time-averaged proteinuria &amp;lt;1 gr/day. Average proteinuria was determined for every 6 months of follow-up and time-averaged (TA) proteinuria represented an average of the mean proteinuria measurements in each 6-month period. A comprehensive genetic study using whole exome next-generation sequencing was performed. One hundred and one genes related to familial hematuria, focal segmental glomerulosclerosis and cystic kidney diseases were analyzed. Patients with known secondary causes of hematuria were excluded. The baseline clinical characteristics of patients with and without ADAS were compared. Patients with pathogenic variants in COL4A5 gene were excluded of this analysis. Results Thirty-one (60%) patients were diagnosed with ADAS (16 patients with pathogenic variants in COL4A3, 15 in COL4A4), 3 (6%) patients (2 women, 1 man) presented pathogenic variant in COL4A5 gene, and the genetic study was negative in the remaining 18 patients (variants of unknown significance in COL4A3 or COL4A4 were found in 3 of the latter). No other genetic abnormalities were detected in the panel of 101 genes. Table 1 shows the baseline clinical characteristics of patients with and without ADAS. Patients with ADAS presented a higher TA proteinuria [0.23 (0.11-0.6) vs 0.08 (0.05-0.16), p = 0.01], and a more frequent familial history of isolated microscopic hematuria (76% vs 40%, p = 0.01) chronic kidney disease (CKD) (44% vs 0%, p = 0.01) or end stage kidney disease (32% vs 0%, p = 0.02). In the multivariate analysis, only a family history of microscopic hematuria was associated with ADAS: OR 5.62 (1.25-25.49, p = 0.02). Conclusion ADAS is the most common cause of PMH. A genetic study should be the first diagnostic step in order to obtain a precise cause of the disease and avoid invasive procedures. A family history of microscopic hematuria and of CKD increases the likelihood of ADAS prior to the performance of genetic test.

Anatomic parameters of omphaloceles and their association with anatomic, genetic, or syndromic malformations: a retrospective study

PurposeThis retrospective study aims to describe anatomical parameters of omphaloceles and to analyze their association with anatomical, genetic, or syndromic malformations.MethodsCases were selected from digital records of two university centers, a certified regional registry and personal records. Patients from 1998 to 2018 with omphalocele and live birth (LB), termination of pregnancy due to fetal anomaly (TOPFA) and fetal death (FD) were included. Cases born outside Western Switzerland and/or with upper or lower coelosomy were excluded.ResultsWe analyzed 162 cases with the following distribution: 57 (35%) LB, 91 (56%) TOPFA and 14 (9%) FD. TOPFA was significantly more frequently performed in cases with non-isolated omphalocele, i.e., omphaloceles with associated major malformations (especially cardiovascular and genitourinary), genetic/chromosomal anomalies, or syndromes. For LB, associated anatomical malformations, genetic or chromosomal anomalies were not significantly associated with the size of the omphalocele or the liver involvement.ConclusionsThe proportion of cases resulting in TOPFA was higher among fetuses with major malformations, genetic or chromosomal anomalies. Despite the large size of this cohort, and in contrary to previous publications, the size of the omphalocele and/or liver involvement does not allow for conclusions regarding the presence or number of associated malformations, genetic or chromosomal anomalies.

#1428 Rnd3, a novel gene involved in ADPKD

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is an adult-onset, multi-systemic disorder, that affects ∼12.5 million people worldwide. ADPKD is characterised by progressive kidney enlargement caused by uncontrollable growth of cysts, alongside extra-renal phenotypes such as cysts in the liver, pancreas, and hypertension. Truncated mutations in PKD1 and PKD2 that encode for PC1 and PC2, respectively, are responsible for 85% and 15% of reported cases. Mutations in PKD1 are associated with the more severe form of ADPKD and earlier mean age of onset. The phenotypic variability of ADPKD can be attributed to genic, allelic and gene modifier effects. Modifier genes influence the molecular and/or phenotypic expression of other genes, altering the range of symptoms and the severity of the disease. In mice with a single gene mutation, modifier genes reduce the penetrance, dominance modification, expressivity, and phenotypic pleiotropy. To understand the involvement of modifier genes in the ADPKD kidney phenotype, a novel mouse model Rnd3tm1b(EUCOMM)Hmgu was generated, and in adult and aged Rnd3tm1b/+ mice, a kidney phenotype characteristic of ADPKD was evident. Rnd3 is an atypical member of the Rnd subgroup of the Rho family of GTPases and has a role in actin cytoskeleton reorganisation, cell cycle progression, and cell migration. Method 5 and 9-month-old kidneys from Rnd3tm1b/+ and age-matched wildtype mice were studied. In the Rnd3tm1b (EUCOMM)Hmgu mouse model, the TB1B mutation causes an inactivation of Rnd3 via the deletion of Exon 2 in C57BL/6N mice. RT-qPCR and SDS-Page Westerns were performed as well as staining for markers associated with Polycystic Kidney Disease. Results The onset of cyst formation in the cortex is apparent in 5-month-old Rnd3tm1b/+ kidneys compared to age-matched wildtype kidneys (Fig. 1a). A statistically significant 10% (p &amp;lt; 0.0465) increase in the proliferative index was observed in 5-month-old Rnd3tm1b/+ mice (Fig. 1e,f). Vimentin expression was also increased in podocytes and confirmed by a statistically significant 25% (p &amp;lt; 0.0461) increase in Rnd3tm1b/+ kidneys (Fig. 1g,h). Aquaporin-2 (Aqp2) expression was decreased in collecting ducts and verified by a statistically significant 0.4-fold-change (p &amp;lt; 0.0451) decrease in protein level in kidneys of 5-month-old Rnd3tm1b/+ mice (Fig. 1i-k). A statistically significant 0.75-fold (p &amp;lt; 0.0006) and 1.5-fold-increase (p &amp;lt; 0.02) in Pkd1 and Pkd2 expression was observed in 5-month-old Rnd3tm1b/+ kidneys, respectively (Fig. 1c). Kidneys of 9-month-old Rnd3tm1b/+mice presented with multiple cysts in the cortex, mimicking the ADPKD kidney phenotype (Fig. 1b). Vimentin staining in podocytes was increased and confirmed by a statistically significant 14% (p &amp;lt; 0.0419) increase in Rnd3tm1b/+mice (Fig. 1l,m). Expression of Pkd1 and Pkd2 was downregulated by a statistically significant 0.39-fold (p &amp;lt; 0.0001) and 0.2-fold-change (p &amp;lt; 0.004) in Rnd3tm1b/+mice (Fig. 1d). Conclusion Together, these findings highlight the importance of Rnd3 as a potential modifier in the onset of ADPKD by mediating the expression of Pkd1 and Pkd2. Interestingly, there is a critical window where Pkd1 and Pkd2 expression is altered, exacerbating the phenotype between 5 and 9-month-old Rnd3tm1b/+ mice. In addition to this, by understanding how modifiers are involved in triggering the onset of phenotypes in ADPKD, potential diagnostic and therapeutic measures can be discovered to ultimately aid in diagnosing ADPKD at a much earlier stage.

#623 One-year outcome of kidney transplant recipients harbouring complement regulatory gene mutation

Abstract Background and Aims Thrombotic Microangiopathy (TMA) is associated with genetic abnormalities in complement regulatory pathways. The impact of complement regulatory gene (CRG) mutation on post-transplant TMA in Indian kidney transplant recipients (KTR) is unknown. This study was conducted to ascertain an association between post-transplant renal limited TMA and the risk of graft failure in KTRs harbouring mutations in genes encoding complement-regulatory proteins. Method A Single Centre, Prospective Cohort Study was undertaken to assess the incidence of post-transplant renal limited TMA in KTRs with mutations in genes encoding complement regulatory proteins. Patients enrolled over one year were followed up for one-year post-transplantation. Genetic testing was performed with MLPA and Clinical exome sequencing. Results 138 KTRs with a mean age of 36.39 ± 12.39 years were enrolled. CRG mutation was seen in 96/138 (69.6%) KTRs. Duplication of the CFHR gene (n = 72) was most prevalent followed by mutations in CFH (n = 6), CFI (n = 2), MCP (n = 2), and DGKE (n = 1). Mutations in CFB, C3, and THBD genes were not detected. The Incidence of Post transplant renal limited TMA in KTRs with CRG mutations is 9.37% (9/96). Mutation related to aHUS was seen in 9 out of 11 patients who developed TMA. There was no significant difference in eGFR of patients with or without CRG mutation at the end of 1 year. Conclusion The prevalence of CRG mutation in KTRs is high and the majority of patients with post-transplant de novo TMA had CRG mutations. The graft functions at the end of 1 year were similar in patients with or without CRG gene mutation. This study suggests that the presence of CRG mutation should not preclude the option of Kidney transplantation in a patient with ESRD.

Congenital hand difference associated with 12q13.13 microdeletion.

We report the case of a 15-year-old boy with a de novo chromosomal deletion in the 12q13.13 region, presenting with congenital hand difference. This case emphasizes the clinical significance of recognizing such genetic anomalies and their implications.

#1699 The role of interleukin 12 cytokine family in autosomal dominant polycystic kidney disease physiopathology

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is an inherited, complex disease, characterized by cysts formation and phenotypic changes. ADPKD physiopathology is under research, the role of proinflammatory chemokines responsible for perpetuating the cycle of chronic inflammation leading to kidney damage being intensively studied. We aimed to explore the individualized contribution of IL-12 interleukin family members in ADPKD physiopathology. Method We developed a prospective, case control study, in Carol Davila Clinical Hospital of Nephrology, including a group of 66 ADPKD subjects (56.1 years old, men:women 35:32), and a group of 40 healthy subjects, similar as sex, mean age. ADPKD diagnosis was based on familial history, clinical exam and CT or MRI scan. We excluded from the study subjects that presented cysts in other organs except the kidney, with eGFR&amp;lt;60 mL/min/1.73 mp, with history of hematuria, cysts infection, urinary tract infection, renal lithiasis, with unstable blood pressure in the last 6 months. Laboratory tests: serum levels of monomers IL-12 p40 and IL-20 p35 and heterodimers IL-12 p70, IL-23, IL 35, assessed by ELISA method. Statistical analysis. All the results were analyzed using IBM SPSS Statistics 2015 and were presented as average value ± standard deviation. p &amp;lt; 0.05 was considered with statistical significance. Results In ADPKD patients, we detected altered production of IL-12 cytokine, with low production of IL-12 p35 and IL-35, respectively high production of IL-12 p70, IL-23 and IL-12 p40 versus control. IL-12 p35 and IL-35 had statistically significant lower levels in ADPKD patients with eGFR&amp;lt;90 ml/min/1.73 mp, when compared with ADPKD patients with eGFR&amp;gt;90 ml/min/1.73 mp (Table 1). IL-12 p70, IL-23 and IL-12 p40 had statistically significant higher levels in ADPKD patients with eGFR&amp;lt;90 ml/min/1.73 mp, when compared with ADPKD patients with eGFR&amp;gt;90 ml/min/1.73 mp (Table 1). IL-12p70, IL-23 and IL-12 p40 had a negative significant correlation with eGFR, while IL-12p35 and IL-35 had a positive significant correlation with eGFR. Though, IL-12p35 and IL-35 serum levels correlated negatively with IL-12p70, IL-23 and IL-12 p40 serum levels in ADPKD patients. Conclusion The results of the present study suggest that ADPKD is a cellular source of IL-12 family, cytokines produced by antigenic stimulation. IL-12 family programming is abnormal in ADPKD patients. We detected synergic and divergent action of IL-12 cytokine family members, relevant for ADPKD physiopathology.

#2869 Arterial hypertension: do self-care and knowledge of the disease matter?

Abstract Background and Aims Arterial hypertension (HTN) is the main risk factor for cardiovascular disease, which is in turn the leading cause of mortality worldwide. An adequate blood pressure (BP) control depends not only on biological factors, but also on individual behaviour and health literacy. In this area, there is scarce evidence on the potential impact of the level of knowledge and self-care related to the disease. This study aims to measure and characterise these variables in a population of hypertensive patients. Method We developed a retrospective, multi-centre study among patients with HTN, with follow-up at the hospital (at the Nephrology Department) or in primary care. The patients were randomly selected by convenience sampling, between November 2022 and December 2023. With their informed consent, we collected demographical and clinical variables, and applied the Portuguese version of the Self-Care of Hypertension Inventory (SC-HI) and Hypertension Knowledge Test (HKT) questionnaires. The statistical analysis was performed using Microsoft Excel and SPSS. The study is still ongoing; we hereby present a preliminary analysis. Results To date, we have collected data from 75 patients; 48% were female, with a median age of 69 (IQR 23). The follow-up was carried out at the hospital in 63% (n = 47) of the patients. 54.6% (n = 41) were retired and 66.7% (n = 50) had finished high school or higher education. Most patients (94.7%) had at least one comorbidity; the most common were dyslipidaemia (57.3%), obesity (33.3%), chronic kidney disease (20%) and diabetes (16%). 25 patients (33.3%) met the criteria for polypharmacy and 69.3% had been diagnosed for over 10 years. The median score at HKT was 16 (IQR 3), out of a maximum of 21, and the median score at SC-HI was 65 (IQR 16), out of a maximum of 92. There were 4.3% of invalid answers, which were scored as zero for the final calculations. Using the Spearman correlation test, we found no significant association between the scores (p = 0.061). When analysing subgroups (Figs 1 and 2), the polymedicated patients scored higher on SC-HI but lower on HKT. Those with controlled BP scored lower on both scores. Patients with Hypertension-Mediated Organ Damage (HMOD) scored higher on SC-HI but lower on HKT, while the contrary happened in those with a more long-standing diagnosis. Conclusion We must start by recognising that this is only a preliminary and mainly descriptive analysis of the sample, whose size is not yet representative. Moreover, the complexity of the questionnaires might have influenced the results, as seen by the non-negligible percentage of invalid responses. Taking these limitations into account, some considerations should still be made. Firstly, the median scores show that we still have a long way to go to increase health literacy, especially if we consider the relatively high level of education of the sample. The percentage of patients with comorbidities and polymedicated is also worthy of mention. The results suggest that these complex patients are forced to take more attitudes towards self-care, but do not necessarily know more about their disease; the same happens in patients with HMOD. On the other hand, those with a more long-standing diagnosis seem to know more but not necessarily be more self-cautious. These results, together with the absence of association between the scores, suggest that we must educate patients both on the appropriate self-care and the rationale behind it (and not solely on one of them), in order to maintain lasting positive behaviours. Surprisingly, patients whose BP was controlled were actually less self-conscious and informed—they could possibly have less complex disease, as there are many confounding factors that influence BP control for which the results were not corrected. In conclusion, this study reinforces the complexity of this topic and the need to invest in its understanding; the completion of this investigation, as well as the development of further studies and better tools to evaluate self-care and knowledge, are undoubtedly necessary.

#966 Targeting cAMP in ADPKD by small molecule activation of long form PDE4 enzymes

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most common hereditary kidney disorder, affecting over 12 million people worldwide. ADPKD is characterized by the uncontrolled growth of fluid-filled cysts in the kidney that can lead to kidney enlargement, loss of function, and associated complications. Currently, there are limited treatment options for ADPKD patients. By activating long form PDE4 enzymes and increasing cAMP hydrolysis, Mironid®’s first-in-class small molecule LoAc® compounds [1] directly target the increased kidney cAMP levels that are known to drive cyst formation in ADPKD, resulting in decreased cyst growth in vitro and in vivo. Here we present translational data demonstrating that LoAc® compound MR-L22 lowers renal cAMP after single or multiple doses and delivers efficacy in the Pkd1RC/RC mouse model of ADPKD, alongside favourable differentiation from tolvaptan, the only therapy approved for ADPKD patients. Method To demonstrate cAMP lowering in urine, male Han Wistar rats (n = 6) were dosed p.o. with 100 mg/kg MR-L22 or vehicle (1% methylcellulose, 0.2% Tween 80) either once, or on 7 consecutive days. Urine was collected on Day 1 or Day 7. To assess the ability of MR-L22 to ameliorate disease progression alone, or in combination with tolvaptan, doses of MR-L22 (60 mg/kg) and tolvaptan (0.1% in chow), both expected to be individually sub-maximally efficacious, were administered singly, or in combination in F1 hybrid progeny Pkd1RC/RC mice (8 m and 8f/group), from 4-16 weeks of age. Urine measurements were taken after 8 weeks of dosing. Total kidney volume (TKV) was measured by MRI prior to randomisation and termination. cAMP in urine and kidney tissue were measured by ELISA. Results A 100 mg/kg oral dose of MR-L22 in rat resulted in a &amp;gt;40% decrease in cAMP in urine compared to vehicle. This effect was maintained over multiple days of dosing demonstrating that urinary cAMP can be used as a biomarker of LoAc® target engagement. Consistent with the mechanism of action, a small increase in urine production and commensurate decrease in urine osmolality was observed. To assess the ability of MR-L22 to ameliorate disease progression in an in vivo model of ADPKD, and to determine whether additive or synergistic effects might be seen in combination with tolvaptan, MR-L22 (60 mg/kg) and tolvaptan (0.1% in chow) were dosed individually and in combination in the Pkd1RC/RC mouse model of ADPKD. Alongside a reduction in urinary cAMP, MR-L22 delivered reductions in TKV/Bw (Fig. 1), urinary and tissue cAMP, cystic index, terminal blood urea nitrogen (BUN) and creatinine, as well as trending reductions in urinary NGAL. Conclusion We have shown that in rat, acute or multiple dosing, of LoAc® compound MR-L22 results in a significant reduction of urinary cAMP and a small, but measurable, increase in urine production and commensurate decrease in urine osmolality. Furthermore, we have demonstrated that significantly reduced urinary and kidney cAMP levels correlate with reductions in TKV, cystic index, Kw/Bw, BUN and urinary NGAL in the Pkd1RC/RC model of ADPKD. In these studies, we have demonstrated beneficial differentiation vs tolvaptan on limiting aquaretic effects but been unable to conclusively demonstrate additive or synergistic effects of MR-L22 combined with tolvaptan due to experimental limitations; Further experiments will be needed to answer this question. Looking forwards, measurements of urine cAMP and urine osmolality will be measured as supportive of target engagement in phase 1 studies. Positive effects on TKV, seen in vivo in the Pkd1RC/RC model, will be assessed in a phase 2 clinical trial. By directly targeting cAMP, LoAc® compounds have the potential to slow renal disease progression, and to delay or prevent the need for dialysis, offering a potential new treatment option for ADPKD patients.

#788 Mitigation of tubular apoptosis and cystogenesis in ADPKD: the impact of TRPM8 through restriction of mitochondrial function

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD), characterized by numerous fluid-filled cysts and a progressive decline in renal function, is a prevalent hereditary kidney disorder. Approximately 70% of individuals affected by ADPKD may develop end-stage renal disease, typically manifesting around the median age of 58 years. Despite its prevalence, effective clinical treatments for ADPKD remain elusive. Transient receptor potential melastatin member 8 (TRPM8), serving as a sensor for cold temperatures and activated by the cooling agent menthol, orchestrates cellular Ca2+ signaling by facilitating Ca2+ entry into the cytosol. However, the role of TRPM8 in ADPKD remains undefined. Method The WT 9-12, an epithelial cell line isolated from the proximal tubule of a patient with ADPKD, was utilized for in vitro experiments. Eight-week-old wild-type (WT), Pkd1 knockdown (pkd1kd), and Trpm8, Pkd1 double mutant (T-/-P) mice were used for experiments. Renal Trpm8 was activated by administration of a 0.5% menthol diet for 7 months in mice. The cyst index was measured by H&amp;E stain and calculated using Image J. Apoptosis was evaluated by TUNEL assay (In Situ Cell Death Detection Kit, TMR red). Protein and mRNA expression of mice kidneys was detected by Western blotting and qRT-PCR, respectively. Blood urea nitrogen (BUN) was measured by HITACHI 7600 analyzer. Results The activation of TRPM8 by menthol treatment resulted in an elevation of intracellular Ca2+ levels, subsequently impeding proliferation in WT9-12 cells. Consistently, Trpm8 activation suppressed kidney weight, kidney weight-to-body weight ratio, BUN, fibrosis, and cystogenesis in Pkd1kd mice, while no such effects were observed in WT and T-/-P mice. This suggests a protective role of Trpm8 in ADPKD. Mechanistically, Trpm8 activation mitigated tubular apoptosis in the kidneys of Pkd1kd mice, as evidenced by reduced TUNEL-positive cells and downregulated Bax and cleaved caspase-3, with no such effects observed in WT and T-/-P mice. Intriguingly, the proliferation-associated signaling pathway, ERK, was also suppressed by Trpm8. Proteomic analysis of mice kidneys revealed that Trpm8 regulates mitochondrial dysfunction, EIF2 and Sirtuin signaling pathways. The result was verified in WT9-12 cells showing reduced mitochondrial respiration, particularly impacting oxidative phosphorylation by TRPM8. Conclusion Trpm8 inhibits tubular apoptosis and cyst formation through the restriction of mitochondrial function, subsequently retarding ADPKD development in mice. These findings illuminate a novel and promising strategy for intervening in ADPKD.