Background The risk of chronic diseases and multimorbidity increases with age, yet, individuals of the same age can strongly differ in healthspan, ranging from early manifestation of age-related disease to robust health into very old age. Plasma biomarkers, including metabolites and proteins, can capture intrinsic health status, thereby providing insights into the nature of this variation. These biomarkers have been widely explored to understand chronic and early disease risk but less so for disease resilience in mid- and late-life survival (i.e. after 90 years), or for multigenerational longevity. Methods We quantified 326 plasma proteins using data-independent mass spectrometry in two generations of the Leiden Longevity Study cohort: F1 nonagenarian siblings (late-life; age ≥89; N=852) and F2 offspring and their partners (mid-life; age 30-80; N=2,282). Baseline plasma protein levels were tested for association with mid- and late-life survival, with up to 22 years of follow-up, and cardiometabolic healthspan, with up to 16 years of follow-up. By comparing F2 offspring and partners, we tested for plasma proteins associating with familial longevity. Findings Four proteins: GSN, F2, CRTAC1, and HP, consistently associated with increased mid- and late-life survival, prolonged cardiometabolic healthspan, and familial longevity; representing overall resilience. Moreover, six proteins: APCS, C7, FCN2, HPR, GSN, and PIGR, associated with mortality independent of MetaboHealth, a well-established metabolomics-based mortality score. Interpretation We identified GSN, F2, CRTAC1, and HP as promising candidate indicators of healthy aging and resilience, meriting further study. Funding ZonMw, LUF, BBMRI-NL, VOILA, Joerg Bernards-Stiftung, Koeln Fortune, and CECAD ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement The research leading to these results has received funding from Netherlands Organization for Scientific Research, domain Health Research and Medical Sciences (09120012010052), and Leiden University Fonds (LUF; Elise Mathilde foundation). The Leiden Longevity Study has received funding from the Innovation-Oriented Research Program on Genomics (SenterNovem IGE05007), the European Union Seventh Framework Programme (FP7/2007 2011: grant Agreement Nr 259679), BBMRINL, a Research Infrastructure financed by the Dutch government (NWO 184.021.007 and 184.033.111), and the VOILA consortium (ZonMw; 457001001). PA and RUM received support from the Joerg Bernards-Stiftung as well as Koeln Fortune and CECAD (funded by the Deutsche Forschungsgemeinschaft DFG under Excellence Strategy of Germany EXC 2030 390661388) ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The Leiden Longevity Study protocol was approved by the Medical Ethical Committee of the Leiden University Medical Center before the start of the study (P01.113) at 16th of August 2002. The first participant was enrolled at 5th of September 2002. In accordance with the Declaration of Helsinki, the LLS obtained informed consent from all participants prior to their entering the study. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The datasets analyzed in the current study are available following a data access procedure (https://leidenlangleven.nl/data-access/). For each request it will be tested whether the research is in compliance with the informed consent that has been signed by the LLS participants. This study does not report original code. All data were analysed and processed using published software packages, the details of which are provided and cited in the Methods section.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) syndrome is caused by heterozygous germline variants in the fumarate hydratase (FH) gene. Inheritance follows an autosomal dominant pattern. Loss of FH confers a predisposition for various benign and malignant neoplasms, including cutaneous leiomyomas, uterine fibroids and FH-deficient renal cell carcinoma. While benign, cutaneous and uterine manifestations have a relevant impact on quality of life and risk for complications, the vast majority of FH-deficient RCCs exhibit an aggressive behaviour with invasive growth and the potential for early metastatic spread. Additionally, pathogenic germline FH variants have been associated with other neoplasms, such as adrenal gland and Leydig cell tumours. The aggressive behaviour of FH-deficient RCC challenges nephron-sparing resection strategies, as a wide margin is recommended. Even after early nephrectomy for surgical removal of FH-deficient renal cell carcinomas, there is a relevant risk for distant metastasis as well as the remaining predisposition for de novo primary renal tumours in the other kidney. Active screening is central to HLRCC care since no preventative HLRCC-specific treatment exists. Vascular endothelial growth factor/epidermal growth factor receptor-directed treatment regimes, such as erlotinib/bevacizumab, demonstrate efficacy against HLRCC-associated RCC. This emphasizes the importance of establishing the correct diagnosis in HLRCC early on to guide therapeutic decisions. Morphologic criteria as well as specific immunohistochemical staining and molecular genetics allow the identification of FH-deficient RCC. Changes made in the recent 2022 World Health Organization classification impact the diagnosis of HLRCC in multiple ways. This commentary aims to discuss this impact and raise awareness among pathologists as well as clinicians involved in the care of patients with HLRCC.

Patients with ADPKD exhibit an increased prevalence of kidney stones with a significant overrepresentation of uric acid (UA) stones which has led to the previous suggestion that UA may be associated with disease progression in ADPKD. However, available results are controversial and the exact mechanism remains unknown. Since we have previously reported that calcium-based microcrystals worsen ADPKD disease progression we hypothesized that renal UA crystallization could affect PKD disease progression and aimed to investigate this in animal models of PKD and the AD(H)PKD registry at the University Hospital Cologne. WT and Pkd1+/− rats were supplemented with UA and the uricase inhibitor oxonic acid from 8 to 10 weeks of age to induce moderate hyperuricemia causing renal UA crystal deposition. This was followed by washout from 10 to 18 weeks to analyze crystal clearance. For additional treatment with the ketone beta-hydroxybutyrate (BHB) and alkaline Citrate, adult rats received a solution containing 2.1% BHB and 13.75 mM tripotassium citrate/16.75 mM citric acid in the drinking water from 14 to 18 weeks of age. Human data including eGFR, UA levels in serum, spot urine and 24-hour urine and urine pH were used from the AD(H)PKD study (Clinical Trials ID: NCT02497521) and analyzed using multivariable linear models. UA crystal deposition in Pkd1+/− rats led to significantly increased kidney growth, tubule dilation and UA crystal burden compared to UA-treated WT controls. Dilated tubules exhibited strong TLR-4 expression, NLRP3 activation, recruitment of peritubular macrophages and intraluminal neutrophils undergoing NETosis. Strikingly—although untreated heterozygous Pkd1+/− rats fail to develop a polycystic phenotype—UA-treated Pkd1+/− rats developed polycystic kidney disease after 8 weeks washout period with persistent activation of TLR4, NLRP3, pericystic macrophages and intracystic neutrophils. UA-treated WT rats displayed a normalization of renal tubules. Previously, we demonstrated that treatment with BHB and Citrate can ameliorate PKD disease progression. BHB has been reported to inhibit TLR4-NLRP3 signaling and citrate can prevent UA deposition due to urine alkalinization. Therefore, we induced UA crystal deposition in Pkd1+/− followed by 4 weeks of normal diet to allow crystal clearance, followed by treatment with BHB/Citrate in the drinking water. Remarkably, BHB/Citrate potently inhibited the progression to PKD in Pkd1+/− rats by ameliorating cyst growth, TLR4-NLRP3 activation, and macrophage and neutrophil recruitment. Translational analysis of UA stone forming parameters in the AD(H)PKD patient registry showed a correlation of serum and urine UA concentration with eGFR slope. However, significance of this association was lost after adjusting for eGFR, age and sex. In contrast, analysis of the Marshal lithogenic risk score which combines urine UA with urine pH to predict the risk for UA crystal/stone formation showed a significant and independent impact on eGFR slope. Our results suggest that UA exposure is a sufficient environmental hit to induce cystogenesis in a heterozygous Pkd1 rat model. A heterozygous Pkd1 gene mutation as observed in ADPKD patients creates a hyperresponsive renal phenotype upon UA crystal formation leading to exaggerated tubule dilation, persistent inflammation, fibrosis and progression to PKD. Treatment with BHB/Citrate significantly reduces TLR4-NLRP3 activation and macrophage/ neutrophil recruitment leading to a significant amelioration of UA-induced cystogenesis. We conclude that UA crystal deposition could accelerate PKD disease progression by NLRP3-mediated and macrophage/neutrophil-based hyperinflammation. BHB/Citrate could not only prevent UA crystallization but ameliorate UA-associated kidney injury and ADPKD disease progression. Finally, the translational analysis in the AD(H)PKD patient registry suggest that, although not showing serum uric acid as independent risk factor, the lithogenic risk- largely driven by the combination of urine uric acid and urine pH- could be a driver of PKD disease progression.

BACKGROUND:RNA-binding proteins (RBPs) are fundamental regulators of cellular biology that affect all steps in the generation and processing of RNA molecules. Recent evidence suggests that regulation of RBPs that modulate both RNA stability and translation may have a profound effect on the proteome. However, regulation of RBPs in clinically relevant experimental conditions has not been studied systematically.METHODS:We used RNA interactome capture, a method for the global identification of RBPs to characterize the global RNA-binding proteome (RBPome) associated with polyA-tailed RNA species in murine ciliated epithelial cells of the inner medullary collecting duct. To study regulation of RBPs in a clinically relevant condition, we analyzed hypoxia-associated changes of the RBPome.RESULTS:We identified >1000 RBPs that had been previously found using other systems. In addition, we found a number of novel RBPs not identified by previous screens using mouse or human cells, suggesting that these proteins may be specific RBPs in differentiated kidney epithelial cells. We also found quantitative differences in RBP-binding to mRNA that were associated with hypoxia versus normoxia.CONCLUSIONS:These findings demonstrate the regulation of RBPs through environmental stimuli and provide insight into the biology of hypoxia-response signaling in epithelial cells in the kidney. A repository of the RBPome and proteome in kidney tubular epithelial cells, derived from our findings, is freely accessible online, and may contribute to a better understanding of the role of RNA-protein interactions in kidney tubular epithelial cells, including the response of these cells to hypoxia.

Autosomal Dominant Polycystic Kidney Disease is the most common genetic cause of kidney failure. Outcome prediction is essential to guide therapeutic decisions. However, currently available models are of limited accuracy. We aimed to examine the potential of serum proteomics for improved risk stratification. Here we show that 29 proteins are significantly associated with yearly kidney function decline. Functional enrichment on these 29 proteins reveals GO:BP terms related to immune response, lipoproteins and metabolic processes. A comparison to an Immunoglobulin A nephropathy cohort provides information regarding the eGFR-dependency and disease specificity of these proteins. The final outcome prediction model (adjusted R² 0.31) contains six proteins, namely Endothelial Plasminogen Activator Inhibitor (SERPINF1), Glutathione Peroxidase 3 (GPX3), Afamin (AFM), FERM Domain Containing Kindlin-3 (FERMT3), Complement Factor H Related 1 (CFHR1), and Retinoic Acid Receptor Responder 2 (RARRES2), the predictive value of which is independent from the clinical and imaging parameters currently used in clinical care. The validation of these models in different cohorts indicates the accuracy of the models. It will now be important to move towards targeted validation in a prospective study.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic cause of kidney failure with very limited therapeutic options. Data from animal models show a large potential of ketogenic diets (KD) to ameliorate disease progression. The phase-2-like randomized controlled trial “KETO-ADPKD” [1] revealed first insights into safety and efficacy of this approach in humans, showing significantly improved development of kidney function. Building upon these findings, these in-depth analyses highlight the factors and mechanisms of ketosis in ADPKD, aiming to advance the clinical application of this promising intervention. KETO-ADPKD is a randomized and controlled clinical trial (NCT04680780) with 66 patients over the course of three months. Here, we examine the suitability of three different ketosis readout parameters and thoroughly investigate the correlations and moderating effects between biochemical markers of ketosis and disease progression. We identified both capillary beta-hydroxybutyrate and breath acetone levels as reliable and appropriate markers for assessing ketosis, while urine ketone measurements demonstrated a poor correlation. Patients with higher baseline kidney volume and better baseline kidney function exhibited a more substantial reduction in kidney volume. Those who achieved higher biochemical ketosis thresholds experienced a significant reduction in height-adjusted total kidney volume (htTKV), with a decrease of up to 17.6 mL/m in the ketogenic diet group (KDG), compared to a 14.8 mL/m increase in the control group (P = 0.02). Additionally, the degree of ketosis was positively correlated with improvements in estimated glomerular filtration rate (eGFR). We show that ketosis moderates ameliorating effects on disease progression in ADPKD. We identify patient groups that might profit most from the intervention and evaluate adequate biochemical readout parameters. Taken together, these data provides important insights to implement ketogenic dietary interventions to routine clinical care in the future, which addresses a strong clinical need.

Degenerative diseases are marked by the progressive accumulation of cellular damage, leading to impaired cellular function and tissue degeneration. Despite advances in single-cell technologies, capturing the gradual decline of individual cells in vivo remains challenging. Here, we present a novel, universal, cross-model framework for quantifying cellular damage at single-cell resolution, to uncover conserved molecular trajectories of cellular degeneration. This method uses single-cell RNA sequencing data and enables the detection of progressive damage within distinct cell populations under physiological and pathological conditions. We developed the Podocyte Damage Score (PDS) and Hepatocyte Damage Score (HDS) to monitor cellular deterioration in murine models of kidney glomerulosclerosis and liver steatosis, respectively. The application of these scores to both murine and human datasets accurately quantified cellular damage across diverse disease models and distinguished varying degrees of damage even in unperturbed samples. Notably, the PDS revealed circadian gene expression dysregulation as a hallmark of podocyte injury, while the HDS identified a critical threshold of hepatocyte damage leading to cellular senescence and metabolic dysfunction. The approach provides a scalable tool for decoding disease progression and identifying therapeutic targets across degenerative disorders. ### Competing Interest Statement The authors have declared no competing interest.

Acute kidney injury (AKI) is a common complication among hospitalized patients and is associated with significant mortality and morbidity. Although being a frequent and substantial threat, therapeutic and preventive strategies for AKI are still lacking. Importantly, a reduction of cellular stress resistance contributes to the development of AKI in rodents. Protective dietary strategies activate evolutionarily conserved metabolic pathways leading to an increase in cellular stress resistance and may prevent from AKI. However, the successful transfer to the clinic is still lagging behind since the optimal diet for humans and the underlying molecular mechanisms remain unknown. Five established dietary preconditioning protocols—caloric restriction (CR), a fasting-mimicking diet (FMD), a ketogenic diet (KD) and the dietary restriction of either branched-chained amino acids (BCAA) or sulfur-containing amino acids (SAA) - were systematically examined in a. rodent model of renal ischemia-reperfusion injury (IRI) to quantify diet-induced kidney protection. Based on this effort, the most beneficial dietary interventions were determined and are now examined in a prospective, randomized pilot trial (NCT05709600, DILKID trial) during living kidney donation in humans to elucidate feasibility and safety in humans. In addition, this trial allows to obtain human tissue and biofluids in the context of the beneficial dietary interventions. An integrative multi-layered omics approach including single nucleus RNA sequencing (snRNAseq) is used to delineate the underlying mechanisms of diet-induced kidney protection in rodents and man. FMD, a low-SAA diet and CR efficiently protect from IRI-induced kidney failure as quantified by survival rates, kidney function and tissue damage in mice. Of note, these beneficial dietary interventions show overlapping changes in cysteine catabolism as a potential underlying mechanism of diet-induced kidney protection. Preconditioning with KD leads to moderate benefits after IRI, whereas BCAA fails to protect from ischemic kidney damage. With regard to humans, more than 50% of participants have been enrolled in the DILKID-Trial to date without any indication towards relevant safety issues. This points towards an overall good feasibility and tolerability of the diets at hand. Dietary preconditioning with FMD, CR, KD and low-SAA diets effectively protect against renal IRI in rodents. As these diets are feasible in humans, our findings provide an important outlook towards novel protective strategies in the clinic. Based on the identification of an underlying mechanism mediating diet-induced organ protection, our molecular analyses will provide additional information on potential future pharmacological approaches to protect from organ injury in the kidney and beyond.

Background. Primary hyperoxaluria (PH) is a rare disorder with significant morbidity and mortality if left untreated. Given the rarity, global inequities in diagnostics and treatment are expected. Recently introduced RNA interference therapeutics (RNAi) have dramatically changed the outcome for PH patients, potentially disproportionately affecting low-resource regions. Understanding these disparities is crucial for implementing measures to ensure equitable healthcare access for PH patients worldwide. This study aims to evaluate the current global health situation for PH patients upon the introduction of targeted therapeutics. Methods. An international cross-sectional questionnaire study was conducted among healthcare providers involved in PH care. Responses were gathered between March 2023 and April 2024 and distributed by e-mail via various international nephrology networks. Meta-analysis (mixed random effects model with inverse-variance weighting) was used to analyze data and adjust for subgroup differences. Results. We gathered 136 responses from 57 countries, representing all World Bank regions. Overall access to genetic analysis diagnostics was 82% (confidence interval 77%-91%) and to urinary oxalate measurement 97% (93%-100%). Significant differences (P < .05) between low- and high-income countries were found for most diagnostics including genetic testing, plasma oxalate, plasma and urinary glycolate. Conservative therapies (e.g. pyridoxine and alkalinizing agents) were highly available globally (98% and 95%), but significant differences in access to peritoneal dialysis, and kidney and liver transplantation were reported (P < .05). Access to the RNAi therapeutic lumasiran was limited to high- and middle-income countries, with 53% (40%-66%) of all countries having access (78% high-income versus 56% middle-income). Even in high-income countries, RNAi was not always accessible. Conclusions. We found global disparities in access to optimal management of PH patients, disproportionately affecting low-income countries, but even existing between high-income countries. These results may provide support for initiatives to improve the outcome of PH patients worldwide in an era of new targeted therapeutic treatments.

The cellular response to hypoxia is crucial to organismal survival, and hypoxia-inducible factors (HIF) are the key mediators of this response. HIF-signaling is central to many human diseases and mediates longevity in the nematode. Despite the rapidly increasing knowledge on RNA-binding proteins (RBPs), little is known about their contribution to hypoxia-induced cellular adaptation. We used RNA interactome capture (RIC) in wild-type Caenorhabditis elegans and vhl-1 loss-of-function mutants to fill this gap. This approach identifies more than 1,300 nematode RBPs, 270 of which can be considered novel RBPs. Interestingly, loss of vhl-1 modulates the RBPome. This difference is not primarily explained by protein abundance suggesting differential RNA-binding. Taken together, our study provides a global view on the nematode RBPome and proteome as well as their modulation by HIF-signaling. The resulting RBP atlas is also provided as an interactive online data mining tool (http://shiny.cecad.uni-koeln.de:3838/celegans_rbpome).

A substantial number of patients with autosomal dominant polycystic kidney disease (ADPKD) undergo a nephrectomy, especially in workup for a kidney transplantation. Currently, there is no evidence-based algorithm to guide clinicians about which patients should undergo nephrectomy, the optimal timing of this procedure, or the preferred surgical technique. This systematic review-based consensus statement aimed to answer important questions regarding nephrectomy in ADPKD. A literature review was performed and extended to a meta-analysis when possible. For this purpose, PubMed and EMBASE were searched up to May 2024. Fifty-four publications, describing a total of 2391 procedures, were included. In addition, an exploratory questionnaire was sent to urologists, nephrologists, and transplant surgeons. These sources were used to develop practice points about indications, complications, mortality, and timing and technique of nephrectomy. In addition, data on renal embolization as a potential alternative to nephrectomy were explored and summarized. To reach consensus, practice points were defined and improved in three Delphi survey rounds by experts of the European Renal Association Working Group Genes & Kidney and the European Association of Urology Section of Transplantation Urology. A total of 23 practice points/statements were developed, all of which reached consensus. Among others, it was deemed that nephrectomy can be performed successfully for various indications and is an intermediate risk procedure with acceptable mortality and minimal impact on kidney graft function when performed before, in the same session or after transplantation. The complication rate seems to increase when the procedure is performed as an emergency. During the workup for transplantation, patient complaints should be assessed routinely by questionnaires to indicate symptom burden. Deciding on the need for nephrectomy and exploring potential alternatives such as kidney embolization should be a process of shared decision-making, preferably after multidisciplinary consultation.

Background Autosomal dominant polycystic kidney disease (ADPKD) leads to progressive renal cyst formation and loss of kidney function in most patients. Vasopressin 2 receptor antagonists (V2RA) like tolvaptan are currently the only available renoprotective agents for rapidly progressive ADPKD. However, aquaretic side effects substantially limit their tolerability and therapeutic potential. In a preliminary clinical study, the addition of hydrochlorothiazide (HCT) to tolvaptan decreased 24-h urinary volume and appeared to increase renoprotective efficacy. The HYDRO-PROTECT study will investigate the long-term effect of co-treatment with HCT on tolvaptan efficacy (rate of kidney function decline) and tolerability (aquaresis and quality of life) in patients with ADPKD. Methods The HYDRO-PROTECT study is an investigator-initiated, multicenter, double-blind, placebo-controlled, randomized clinical trial. The study is powered to enroll 300 rapidly progressive patients with ADPKD aged ≥ 18 years, with an eGFR of > 25 mL/min/1.73 m 2 , and on stable treatment with the highest tolerated dose of tolvaptan in routine clinical care. Patients will be randomly assigned (1:1) to daily oral HCT 25 mg or matching placebo treatment for 156 weeks, in addition to standard care. Outcomes The primary study outcome is the rate of kidney function decline (expressed as eGFR slope, in mL/min/1.73 m 2 per year) in HCT versus placebo-treated patients, calculated by linear mixed model analysis using all available creatinine values from week 12 until the end of treatment. Secondary outcomes include changes in quality-of-life questionnaire scores (TIPS, ADPKD-UIS, EQ-5D-5L, SF-12) and changes in 24-h urine volume. Conclusion The HYDRO-PROTECT study will demonstrate whether co-treatment with HCT can improve the renoprotective efficacy and tolerability of tolvaptan in patients with ADPKD.