Leveraging Quantitative Systems Pharmacology and Artificial Intelligence to advance treatment of Chronic Kidney Disease Mineral Bone Disorder.

Chronic kidney disease mineral bone disorder (CKD-MBD) is a complex clinical syndrome responsible for the accelerated cardiovascular mortality seen in individuals afflicted with CKD. Current approaches to therapy have failed to improve clinical outcomes adequately, likely due to targeting surrogate biochemical parameters as articulated by the guideline developer, KDIGO (Kidney Disease: Improving Global Outcomes). We hypothesized that using a Systems Biology Approach combining machine learning with mathematical modeling, we could test a novel approach to therapy targeting the abnormal movement of mineral out of bone and into soft tissue that is characteristic of CKD-MBD. The mathematical model describes the movement of calcium and phosphate between body compartments in response to standard therapeutic agents. The machine learning technique we applied is Reinforcement Learning (RL). We compared calcium, phosphate, PTH, and mineral movement out of bone and into soft tissue under four scenarios: standard approach (KDIGO), achievement of KDIGO guidelines using RL (RLKDIGO), targeting abnormal mineral flux (RLFLUX), and combining achievement of KDIGO guidelines with minimization of abnormal mineral flux (RLKDIGOFLUX). We demonstrate through simulations that explicitly targeting abnormal mineral flux significantly decreases abnormal mineral movement compared to standard approach while achieving acceptable biochemical outcomes. These investigations highlight the limitations of current therapeutic targets, primarily secondary hyperparathyroidism, and emphasize the central role of deranged phosphate homeostasis in the genesis of the CKD-MBD syndrome.

Phosphorus: Chronicles of the epistemology of a vital element.

It was in the philosopher's stone quest that the alchemist Hennig Brand isolated chemiluminescent white phosphorus (P), Greek for "light bearer", from urine in 1669. By 1771 phosphorus was isolated from bone, and in 1777 it was identified by Antoine Lavoisier as a highly reactive element that exists predominantly in nature as ionic phosphate (PO43-) and in solution as phosphoric acid (H3PO4). Early 20th century studies revealed phosphorylated biomolecules as essential components of replicative nuclear material (RNA, DNA), a metabolic source of energy (ATP), and structural components of cellular membrane (phospholipid bilayer). Life on earth began as organophosphates of a self-replicating RNA that evolved into DNA and acquired a membrane to form the original eukaryotes, which eventually joined to form multicellular organisms of the deep sea. Tissue mineralization during transition from the ocean to land generated the endoskeleton, the largest phosphorus stores of evolving vertebrates. Subsequent studies of phosphate homeostasis elucidated its complex regulatory system based on the interaction of the kidney, small intestine, bone, and parathyroid glands, orchestrated by hormones (PTH, calcitriol, FGF23, Klotho), and carried out by phosphate-specific transporters (SLC34 and SLC20 families) all to ensure adequate phosphate for survival and health. Paradoxically, kidney replacement therapy in the 1970s, by prolonging the lives of millions of individuals with kidney failure, revealed the hazards of phosphorus excess. "Phosphorus the light bearer" has become in the eyes of many nephrologists "Phosphorus the cardiovascular toxin".

Application of artificial intelligence to chronic kidney disease mineral bone disorder.

The global derangement of mineral metabolism that accompanies chronic kidney disease (CKD-MBD) is a major driver of the accelerated mortality for individuals with kidney disease. Advances in the delivery of dialysis, in the composition of phosphate binders, and in the therapies directed towards secondary hyperparathyroidism have failed to improve the cardiovascular event profile in this population. Many obstacles have prevented progress in this field including the incomplete understanding of pathophysiology, the lack of clinical targets for early stages of chronic kidney disease, and the remarkably wide diversity in clinical manifestations. We describe in this review a novel approach to CKD-MBD combining mathematical modelling of biologic processes with machine learning artificial intelligence techniques as a tool for the generation of new hypotheses and for the development of innovative therapeutic approaches to this syndrome. Clinicians need alternative targets of therapy, tools for risk profile assessment, and new therapies to address complications early in the course of disease and to personalize therapy to each individual. The complexity of CKD-MBD suggests that incorporating artificial intelligence techniques into the diagnostic, therapeutic, and research armamentarium could accelerate the achievement of these goals.

Kidney Stone Pathophysiology, Evaluation and Management: Core Curriculum 2023.

Kidney stone disease, also known as nephrolithiasis or urolithiasis, is a disorder in which urinary solutes precipitate to form aggregates of crystalline material in the urinary space. The incidence of nephrolithiasis has been increasing, and the demographics have been evolving. Once viewed as a limited disease with intermittent exacerbations that are simply managed by urologists, nephrolithiasis is now recognized as a complex condition requiring thorough evaluation and multifaceted care. Kidney stones are frequently manifestations of underlying systemic medical conditions such as the metabolic syndrome, genetic disorders, or endocrinopathies. Analysis of urine chemistries and stone composition provide a window into pathogenesis and direct ancillary studies to uncover underlying diseases. These studies allow providers to devise individualized strategies to limit future stone events. Given its complexity, kidney stone disease is best addressed by a team led by nephrologists and urologists with input from multiple other health professionals including dietitians, endocrinologists, interventional radiologists, and endocrine surgeons. In this installment of AJKD's Core Curriculum in Nephrology, we provide a case-based overview of nephrolithiasis, divided by the individual stone types. The reader will gain a pragmatic understanding of the pathophysiology, evaluation, and management of this condition.

Understanding renal phosphate handling: unfinished business.

PURPOSE OF REVIEW: The purpose of this review is to highlight the publications from the prior 12-18 months that have contributed significant advances in the field of renal phosphate handling. RECENT FINDINGS: The discoveries include new mechanisms for the trafficking and expression of the sodium phosphate cotransporters; direct link between phosphate uptake and intracellular metabolic pathways; interdependence between proximal tubule transporters; and the persistent renal expression of phosphate transporters in chronic kidney disease. SUMMARY: Discovery of new mechanisms for trafficking and regulation of expression of phosphate transporters suggest new targets for the therapy of disorders of phosphate homeostasis. Demonstration of stimulation of glycolysis by phosphate transported into a proximal tubule cell expands the scope of function for the type IIa sodium phosphate transporter from merely a mechanism to reclaim filtered phosphate to a regulator of cell metabolism. This observation opens the door to new therapies for preserving kidney function through alteration in transport. The evidence for persistence of active renal phosphate transport even with chronic kidney disease upends our assumptions of how expression of these transporters is regulated, suggests the possibility of alternative functions for the transporters, and raises the possibility of new therapies for phosphate retention.

Preclinical and Clinical Evidence of Effect of Acid on Bone Health.

Acid can have ill effect on bone health in the absence of frank clinical acidosis but affecting the bone mioneral matrix and bone cells via complex pathways botyh ascute;y and chronically. While the reaction of bone to an acid load is conserved in evolution and is adaptive, the capacity can be overwhelmed resulting in dire consequences. The preclinical an clincl evidence of the acdi effect on bone is very convincing and the clinical evidence in both association and interventiopn studies are also quite credible, The adverse effects of acid on bone is underappreoicated, under-investigated, and the potential benefits of alkali therapy is not generrally known.

Artificial intelligence-guided precision treatment of chronic kidney disease-mineral bone disorder.

Chronic kidney disease (CKD)-mineral bone disorder (MBD) is a complex clinical syndrome that begins early during CKD and evolves into one of the deadliest complications of CKD through its effects on the cardiovascular and skeletal systems. Achievement of treatment goals to decrease the risk of accelerated cardiovascular events and fractures has been challenging. We hypothesized that application of quantitative systems pharmacology (QSP) modeling combined with artificial intelligence techniques could improve the management of CKD-MBD with the goal of improving outcomes for patients with CKD. We present the implementation of a reinforcement learning (RL) approach to achieve the prescribed goals for serum calcium, phosphorus, and parathyroid hormone through concurrent dosing of phosphate binders, vitamin D analogs, and calcimimetics by simulation in 80 subjects in Matlab. In silico simulation results demonstrate that the application of a QSP model coupled with RL more effectively and quickly achieves treatment goals even in the setting of inferior simulated subject compliance with medical therapy and identifies key decision variables for therapeutic recommendations.

Use of Artificial Intelligence to Identify New Mechanisms and Approaches to Therapy of Bone Disorders Associated With Chronic Kidney Disease.

Chronic kidney disease (CKD) leads to clinically severe bone loss, resulting from the deranged mineral metabolism that accompanies CKD. Each individual patient presents a unique combination of risk factors, pathologies, and complications of bone disease. The complexity of the disorder coupled with our incomplete understanding of the pathophysiology has significantly hampered the ability of nephrologists to prevent fractures, a leading comorbidity of CKD. Much has been learned from animal models; however, we propose in this review that application of multiple techniques of mathematical modeling and artificial intelligence can accelerate our ability to develop relevant and impactful clinical trials and can lead to better understanding of the osteoporosis of CKD. We highlight the foundational work that informed our current model development and discuss the potential applications of our approach combining principles of quantitative systems pharmacology, model predictive control, and reinforcement learning to deliver individualized precision medical therapy of this highly complex disorder.

Na/H Exchange Regulatory Factor 1 Deficient Mice Show Evidence of Oxidative Stress and Altered Cisplatin Pharmacokinetics.

(1) Background: One third of patients who receive cisplatin develop an acute kidney injury. We previously demonstrated the Na/H Exchange Regulatory Factor 1 (NHERF1) loss resulted in increased kidney enzyme activity of the pentose phosphate pathway and was associated with more severe cisplatin nephrotoxicity. We hypothesized that changes in proximal tubule biochemical pathways associated with NHERF1 loss alters renal metabolism of cisplatin or response to cisplatin, resulting in exacerbated nephrotoxicity. (2) Methods: 2-4 month-old male wild-type and NHERF1 knock out littermate mice were treated with either vehicle or cisplatin (20 mg/kg dose IP), with samples taken at either 4, 24, or 72 h. Kidney injury was determined by urinary neutrophil gelatinase-associated lipocalin and histology. Glutathione metabolites were measured by HPLC and genes involved in glutathione synthesis were measured by qPCR. Kidney handling of cisplatin was assessed by a kidney cortex measurement of γ-glutamyl transferase activity, Western blot for γ-glutamyl transferase and cysteine S-conjugate beta lyase, and ICP-MS for platinum content. (3) Results: At 24 h knock out kidneys show evidence of greater tubular injury after cisplatin and exhibit a decreased reduced/oxidized glutathione ratio under baseline conditions in comparison to wild-type. KO kidneys fail to show an increase in γ-glutamyl transferase activity and experience a more rapid decline in tissue platinum when compared to wild-type. (4) Conclusions: Knock out kidneys show evidence of greater oxidative stress than wild-type accompanied by a greater degree of early injury in response to cisplatin. NHERF1 loss has no effect on the initial accumulation of cisplatin in the kidney cortex but is associated with an altered redox status which may alter the activity of enzymes involved in cisplatin metabolism.

Phosphate Transport in Epithelial and Nonepithelial Tissue.

Phosphate is an essential nutrient for life and is a critical component of bone formation, a major signaling molecule, and structural component of cell walls. Phosphate is also a component of high-energy compounds (i.e., AMP, ADP, and ATP) and essential for nucleic acid helical structure (i.e., RNA and DNA). Phosphate plays a central role in the process of mineralization, normal serum levels being associated with appropriate bone mineralization, while high and low serum levels are associated with soft tissue calcification. The serum concentration of phosphate and the total body content of phosphate are highly regulated, a process that is accomplished by the coordinated effort of two families of sodium-dependent transporter proteins. The three isoforms of the SLC34 family (SLC34A1-A3) show very restricted tissue expression and regulate intestinal absorption and renal excretion of phosphate. SLC34A2 also regulates the phosphate concentration in multiple lumen fluids including milk, saliva, pancreatic fluid, and surfactant. Both isoforms of the SLC20 family exhibit ubiquitous expression (with some variation as to which one or both are expressed), are regulated by ambient phosphate, and likely serve the phosphate needs of the individual cell. These proteins exhibit similarities to phosphate transporters in nonmammalian organisms. The proteins are nonredundant as mutations in each yield unique clinical presentations. Further research is essential to understand the function, regulation, and coordination of the various phosphate transporters, both the ones described in this review and the phosphate transporters involved in intracellular transport.

Development of a quantitative systems pharmacology model of chronic kidney disease: metabolic bone disorder.

Chronic kidney disease mineral bone disorder (CKD-MBD) is a virtually universal complication of kidney diseases, starting early in the course of disease and resulting in devastating clinical consequences ranging from bone fragility to accelerated atherosclerosis and early cardiovascular death. Guidelines for therapeutic goals for CKD-MBD have been published, and achievement of these guidelines is associated with improved survival. However, the incomplete understanding of CKD-MBD and the individual variability in the manifestations of CKD-MBD have made it difficult to achieve these guidelines. We hypothesized that the progression of MBD through all stages of CKD, including end-stage kidney disease, could be represented by a quantitative systems pharmacology/systems biology (QSP) model. To address this hypothesis, we constructed a QSP model of CKD-MBD, building on an open-source model of calcium and phosphorus metabolism. Specifically, we estimated and validated the model using data from 5,496 patients with CKD enrolled in the Chronic Renal Insufficiency Cohort study. Our model accurately predicted changes in markers of mineral metabolism related to progressing CKD. We demonstrated that the incorporation of fibroblast growth factor 23 and the soft tissue compartment is essential for accurate modeling of the changes in calcium, phosphorus, intact parathyroid hormone, and calcitriol in CKD-MBD. We conclude that our systems biology model accurately represents CKD-MBD disease progression and can be used as a test bench for improving therapeutic interventions.

Transcriptomes of Major Proximal Tubule Cell Culture Models.

BACKGROUND: Cultured cell lines are widely used for research in the physiology, pathophysiology, toxicology, and pharmacology of the renal proximal tubule. The lines that are most appropriate for a given use depend upon the genes expressed. New tools for transcriptomic profiling using RNA sequencing (RNA-Seq) make it possible to catalog expressed genes in each cell line. METHODS: Fourteen different proximal tubule cell lines, representing six species, were grown on permeable supports under conditions specific for the respective lines. RNA-Seq followed standard procedures. RESULTS: Transcripts expressed in cell lines variably matched transcripts selectively expressed in native proximal tubule. Opossum kidney (OK) cells displayed the highest percentage match (45% of proximal marker genes [TPM threshold =15]), with pig kidney cells (LLC-PK1) close behind (39%). Lower-percentage matches were seen for various human lines, including HK-2 (26%), and lines from rodent kidneys, such as NRK-52E (23%). Nominally, identical OK cells from different sources differed substantially in expression of proximal tubule markers. Mapping cell line transcriptomes to gene sets for various proximal tubule functions (sodium and water transport, protein transport, metabolic functions, endocrine functions) showed that different lines may be optimal for experimentally modeling each function. An online resource (https://esbl.nhlbi.nih.gov/JBrowse/KCT/) has been created to interrogate cell line transcriptome data. Proteomic analysis of NRK-52E cells confirmed low expression of many proximal tubule marker proteins. CONCLUSIONS: No cell line fully matched the transcriptome of native proximal tubule cells. However, some of the lines tested are suitable for the study of particular metabolic and transport processes seen in the proximal tubule.

Current State of the Workforce in Nephrology.

The number of individuals with CKD and end-stage kidney disease continues to rise as the interest in nephrology as a career choice is declining among internal medicine residents. Simultaneously, the emergence of integrated healthcare delivery models encompassing multiple levels of nonphysician healthcare workers plus advanced technological capabilities offer innovative mechanisms for the delivery of optimal care for patients at risk for and suffering from CKD. Critical to the success of these models is the identification of aspects of nephrology care specific to and appropriate for each type of kidney care professional and the development of organizational structures that both define and facilitate the flow of patient care. However, several factors in addition to the declining interest in nephrology pose significant obstacles to the development of the optimal nephrology work force including gender imbalance in leadership and nonleadership positions, gender disparity in compensation, inadequate diversity in ethnicity of nephrologists, and perceptions of inadequate compensation and a poor work life balance. Recent studies suggest that some, but not all, of these challenges are being addressed, though full resolution will require creative and concerted efforts.

NHERF1 Loss Upregulates Enzymes of the Pentose Phosphate Pathway in Kidney Cortex.

(1) Background: We previously showed Na/H exchange regulatory factor 1 (NHERF1) loss resulted in increased susceptibility to cisplatin nephrotoxicity. NHERF1-deficient cultured proximal tubule cells and proximal tubules from NHERF1 knockout (KO) mice exhibit altered mitochondrial protein expression and poor survival. We hypothesized that NHERF1 loss results in changes in metabolic pathways and/or mitochondrial dysfunction, leading to increased sensitivity to cisplatin nephrotoxicity. (2) Methods: Two to 4-month-old male wildtype (WT) and KO mice were treated with vehicle or cisplatin (20 mg/kg dose IP). After 72 h, kidney cortex homogenates were utilized for metabolic enzyme activities. Non-treated kidneys were used to isolate mitochondria for mitochondrial respiration via the Seahorse XF24 analyzer. Non-treated kidneys were also used for LC-MS analysis to evaluate kidney ATP abundance, and electron microscopy (EM) was utilized to evaluate mitochondrial morphology and number. (3) Results: KO mouse kidneys exhibit significant increases in malic enzyme and glucose-6 phosphate dehydrogenase activity under baseline conditions but in no other gluconeogenic or glycolytic enzymes. NHERF1 loss does not decrease kidney ATP content. Mitochondrial morphology, number, and area appeared normal. Isolated mitochondria function was similar between WT and KO. Conclusions: KO kidneys experience a shift in metabolism to the pentose phosphate pathway, which may sensitize them to the oxidative stress imposed by cisplatin.

New roles of the Na+/H+ exchange regulatory factor 1 scaffolding protein: a review.

Na+/H+ exchange regulatory factor 1 (NHERF1), a member of a PDZ scaffolding protein family, was first identified as an organizer of membrane-bound protein complexes composed of hormone receptors, signal transduction pathways, and electrolyte and mineral transporters and channels. NHERF1 is involved in the regulation of Na+/H+ exchanger 3, Na+-dependent phosphate transporter 2a, and Na+-K+-ATPase through its ability to scaffold these transporters to the plasma membrane, allowing regulation of these protein complexes with their associated hormone receptors. Recently, NHERF1 has received increased interest in its involvement in a variety of functions, including cell structure and trafficking, tumorigenesis and tumor behavior, inflammatory responses, and tissue injury. In this review, we highlight the evidence for the expansive role of NHERF1 in cell biology and speculate on the implications for renal physiology and pathophysiology.

Sodium-hydrogen exchanger regulatory factor-1 (NHERF1) confers salt sensitivity in both male and female models of hypertension in aging.

Hypertension is a risk factor for premature death and roughly 50% of hypertensive patients are salt-sensitive. The incidence of salt-sensitive hypertension increases with age. However, the mechanisms of salt-sensitive hypertension are not well understood. We had demonstrated decreased renal sodium­hydrogen exchanger regulatory factor 1 (NHERF1) expression in old salt-resistant F344 rats. Based on those studies we hypothesized that NHERF1 expression is required for the development of some forms of salt-sensitive hypertension. To address this hypothesis, we measured blood pressure in NHERF1 expressing salt-sensitive 4-mo and 24-mo-old male and female Fischer Brown Norway (FBN) rats male and female 18-mo-old NHERF1 knock-out (NHERF1-/-) mice and wild-type (WT) littermates on C57BL/6J background after feeding high salt (8% NaCl) diet for 7 days. Our data demonstrate that 8% salt diet increased blood pressure in both male and female 24-mo-old FBN rats but not in 4-mo-old FBN rats and in 18-mo-old male and female WT mice but not in NHERF1-/- mice. Renal dopamine 1 receptor (D1R) expression was decreased in 24-mo-old rats, compared with 4-mo-old FBN rats. However, sodium chloride cotransporter (NCC) expression increased in 24-mo-old FBN rats. In FBN rats, age had no effect on NaK ATPase α1 and NKCC2 expression. By contrast, high salt diet increased the renal expressions of NKCC2, and NCC in 24-mo-old FBN rats. High salt diet also increased NKCC2 and NCC expression in WT mice but not NHERF1-/- mice. Our data suggest that renal NHERF1 expression confers salt sensitivity with aging, associated with increased expression of sodium transporters.

Loss of the Na+/H+ Exchange Regulatory Factor 1 Increases Susceptibility to Cisplatin-Induced Acute Kidney Injury.

Na+/H+ exchange regulatory cofactor (NHERF)-1, a scaffolding protein, anchors multiple membrane proteins in renal proximal tubules. Cultured proximal tubule cells deficient in Nherf1 and proximal tubules from Nherf1-deficient mice exhibit aberrant trafficking. Nherf1-deficient cells also exhibit an altered transcription pattern and worse survival. These observations suggest that NHERF1 loss increases susceptibility to acute kidney injury (AKI). Male and female wild-type C57BL/6J and Nherf1 knockout mice were treated with saline or cisplatin (20 mg/kg dose i.p.) to induce AKI and were euthanized after 72 hours. Blood and urine were collected for assessments of blood urea nitrogen and neutrophil gelatinase-associated lipocalin, respectively. Kidneys were harvested for histology (hematoxylin and eosin, periodic acid-Schiff) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay, Kim1 mRNA assessment, and Western blot analysis for cleaved caspase 3. Cisplatin treatment was associated with significantly greater severity of AKI in knockout compared with wild-type mice, as demonstrated by semiquantitative injury score (2.8 versus 1.89, P < 0.001), blood urea nitrogen (151.8 ± 17.2 mg/dL versus 97.8 ± 10.1 mg/dL, P < 0.05), and neutrophil gelatinase-associated lipocalin urine protein (55.6 ± 21.3 µg/mL versus 2.7 ± 0.53 µg/mL, P < 0.05). Apoptosis markers were significantly increased in cisplatin-treated Nherf1 knockout and wild-type mice compared to respective controls. These data suggest that NHERF1 loss increases susceptibility to AKI.

Clinical aspects of the phosphate transporters NaPi-IIa and NaPi-IIb: mutations and disease associations.

The Na+-dependent phosphate transporter NaPi-IIa (SLC34A1) is mostly expressed in kidney, whereas NaPi-IIb (SLC34A2) has a wider tissue distribution with prominent expression in the lung and small intestine. NaPi-IIa is involved in renal reabsorption of inorganic phosphate (Pi) from urine, and patients with biallelic inactivating mutations in SLC34A1 develop hypophosphatemia, hypercalcemia, hypercalciuria and nephrocalcinosis, and nephrolithiasis in early childhood. Monoallelic mutations are frequent in the general population and may impact on the risk to develop kidney stones in adulthood. SNPs in close vicinity to the SLC34A1 locus associate with the risk to develop CKD. NaPi-IIb mediates high-affinity transport of Pi from the diet and appears to be mostly important during low Pi availability. Biallelic inactivating SLC34A2 mutations are found in patients with pulmonary alveolar microlithiasis, a lung disease characterized by the deposition of microcrystals. In contrast, no evidence for disturbed systemic Pi homeostasis has been reported in these patients to date. Nevertheless, NaPi-IIb-mediated intestinal Pi absorption may be a target for pharmaceutical interventions in patients with chronic kidney disease and Pi overload.