Disruptions of the body’s internal clock in long-term diseases that cause organ scarring

A healthy liver is critical for maintaining metabolic homeostasis, and the liver CC plays a pivotal role in this process. Studies in mouse livers revealed rhythmic mRNA accumulation for approximately 10%–15% of the genome (,,,,). This rhythmicity in hepatic gene expression largely arises from circadian phase–specific DNA binding of CC genes and clock-regulated transcription factors (,–). Hepatic circadian transcription involves changes in three-dimensional genome and has implicated a role for REV-ERBα (–). Hepatic gene expression is also controlled by zonation, or spatial positioning relative to the liver's central vein and peripheral portal tracts (). A combination of single-cell RNA sequencing with FISH revealed that several key metabolic genes are controlled by both zonation and the liver CC (). Importantly, in mice with hepatocyte-specific knockout of the CC genesand, single-nucleus RNA sequencing revealed that the hepatocytic clock dictates cellular communication in liver by regulating gene expression in non-parenchymal cells such as liver endothelial cells and Kupffer cells (). Interestingly, HSCs have also been shown to control zonation and liver function (). Lipidomic, proteomic, and metabolomic studies have confirmed the widespread role of the CC oscillator and meal timing in dictating liver physiology (,–). ^{13 21 28 29 64 13 64 69 70 72 73 74 75 76 32 77 81} Rev-erba Rev-erbb

Since the role of the CC system in regulating liver metabolism has been reviewed (,,,–), here we summarize key features that, when deregulated, contribute to chronic metabolic liver disease resulting in fibrosis (). The liver CC controls blood glucose levels by regulating both preprandial gluconeogenesis and postprandial glycogen synthesis (,,,). The liver CC controls glucose metabolism by regulating expression of key genes, e.g., glucokinase (), phosphoenolpyruvate carboxykinase (), and glucose transporter 2 () (,,,). The CC also influences the glucoregulatory transcriptional activity of CREB, CHREBP, and GR (,–). Regarding lipid metabolism, plasma triglycerides (TGs), free fatty acids (FFAs), and cholesterol display circadian variations (,,,,) and are disrupted following knockout of CC genes such as(),, and(,). The liver CC regulates hepatic TG levels by controlling the expression of enzymes such as,, and, while REV-ERBα repressesandlevels to regulate SREBP1c, the driver of hepatic de novo lipogenesis (,,,). The metabolism of FFAs, bile acids, and xenobiotics is also controlled by the CC (,,). The liver CC also controls cellular processes such as endoplasmic reticulum (ER) stress, unfolded protein response (UPR), autophagy, and response to reactive oxygen species (ROS), all of which are indispensable for metabolic homeostasis (,,,–) (). ^{4 5 23 33 34} Table 1 ^{4 5 23 82 4 5 23 82 23 82 84 4 5 23 33 34 85 86 87 4 5 23 88 4 5 23 4 5 82 89 91} Figure 1 Gck Pck1 Glut2 Clock Rev-Erba Rev-Erbb Gpat2 Lipin1/2 Dgat2 Insig2 miR122

Recent studies also investigated the CC in myofibroblasts. Using enriched murine HSCs and human myofibroblasts, the existence of CC genes and their circadian expression were demonstrated (). Furthermore, this study identified nearly 2,000 rhythmically expressed genes involved in metabolism, stress response, collagen/ECM synthesis, and cell cycle in HSCs (). Among the rhythmically expressed HSC genes were members of the TGF-β/BMP/activin pathways, including,,, receptors (,,), and ligands (,,) (). Future studies (e.g., applying fibroblast-CC mutant mouse models) will be essential to decipher the importance of dynamic circadian communication between different liver cell types in health and disease. ^{92 92 92} Smad3 Smad7 Smad6 Acvr1 Bmpr1a Tgfbr1 Bmp2 Bmp3 Tgfb1

Although the circadian biology of the mouse liver is well studied, the identity of rhythmic genes in human hepatocytes is mostly unknown owing to challenges associated with collecting multiple biopsies over 24 hours. Recent studies (,) performed 24-hour transcriptomic analyses using humanized liver chimeric mice (HLCM) as a surrogate for human liver (,). These investigations unraveled the human hepatocytic circadian transcriptome, including common and distinct rhythmic genes and biochemical processes. These studies also revealed rhythmic pathways shared and discordant between human and mouse hepatocytes (,). Notably, genes metabolizing carbohydrates, FFAs, TGs, and bile acids were shown to display rhythmicity in both human and mouse hepatocytes (). ^{93 94 95 96 93 94 94}

Lifestyle changes and effective antiviral therapies have largely shifted the major causes of liver disease from viral to metabolic diseases. Owing to the global epidemic of obesity, metabolic dysfunction–associated steatotic liver disease (MASLD; formerly NAFLD) is currently emerging as the most prevalent chronic liver disease (CLD), affecting 20%–25% of the world population (–). MASLD is the liver manifestation of metabolic syndrome. MASLD encompasses a wide range of conditions, from benign metabolic dysfunction–associated steatotic liver (MASL) to metabolic dysfunction–associated steatohepatitis (MASH), a risk factor for developing hepatocellular carcinoma (HCC) (–). Modern diets characterized by overconsumption of energy-dense foods and fructose-containing drinks are key for driving metabolic syndrome. Both systemic and tissue-specific metabolic deregulation leads to hepatic stress and cell death, thereby creating an inflammatory milieu that eventually initiates fibrotic responses (–) (). ^{97 100 97 100 97 100} Figure 2

Fibrosis is the major determinant of clinical outcomes in MASH and is associated with increased risk of cirrhosis and HCC (,). In the healthy liver, HSCs are largely non-proliferative. However, liver injury leads to activation of HSCs, driving their differentiation to myofibroblasts, which are proliferative and contractile and upregulate expression of α-smooth muscle actin (αSMA) and multiple collagens. HSC activation results from several signals, such as proinflammatory cytokines, apoptotic hepatocytes, and increased ROS production (–,,). Activated HSCs increase the secretion of profibrotic molecules (TGF-β and PDGF-β), which enable further recruitment of immune cells (–,,). Thus, CLD-induced HSC activation eventually disrupts liver architecture and functions. ^{98 99 48 50 57 58 48 50 57 58}

Interestingly, the perturbation of the crosstalk between metabolism and CC drives different liver pathologies, including MASLD. MASLD arises due to energy surpluses created by (a) alterations in glucose and FFA metabolism, (b) increased de novo lipogenesis, and (c) compromised β-oxidation or reduced hepatic TG exports (). TG accumulation in hepatocytes increases ER stress, UPR, and ROS levels, which together act as triggers for MASLD and its sequela, fibrosis, by recruiting immune cells (,). Importantly, metabolic perturbations can alter the functioning of both the hepatic and myofibroblast CC oscillators (,,,). Circadian misalignment driven by daytime-restricted feeding or jet lag drives liver CC perturbation (,). Meal timing also affects liver proteome, phosphoproteome, and lipidome (). MASLD features, including increased TG levels and fibrosis, have been noted in mice fed with different versions of high-fat diet as well as with changes in meal timing (,,,,). MASLD breaks down the HSC CC oscillator, and this correlates with elevated expression of profibrotic collagens and αSMA () (). Furthermore, disruption of CC functioning induced by genetic mutations ofands leads to liver steatosis (–), while absence ofin hepatocytes is known to predispose to HCC (). Finally, not only metabolic but also infectious diseases perturb the rhythmic transcriptome in human hepatocytes, and predispose to CLD, fibrosis, and HCC, as shown for chronic hepatitis C virus infection (). ^{100 100 101 5 33 34 92 102 103 32 23 27 32 34 35 92} Table 1 ^{85 87 104 94} Clock Rev-Erb Bmal1

Kidneys filter blood and maintain fluid and electrolyte balance, which contributes to sustaining blood pressure (BP) (–). Circadian variation in renal functions has been observed in several species (–), including the circadian rhythmicity of urinary excretion of sodium, potassium, chloride, and phosphate (,). In humans, urinary volume and pH also display 24-hour rhythmicity under relatively constant conditions of eating, drinking, and sleeping (). Circadian rhythms also exist for the glomerular filtration rate (GFR), corticomedullary osmotic changes, blood flow, and transport of water and electrolytes, which are believed to be driven in part by the kidney CC (–). Global disruption ofin rats revealed a sex-dependent dissociation between circadian BP variation and control of sodium excretion, as only femalerats had significantly greater sodium excretion during the active phase compared with controls (). Additionally, murine kidney-specific cadherin-Cre–mediated (-Cre–mediated)knockout in distal tubules suggests its role in BP control and Nahandling in response to a K-depleted diet, but only in male mice (). ^{105 111 112 116 117 118 119 105 110 120 121} Bmal1 Bmal1 –/– Ksp Bmal1 + +

Kidneys and their functional units, nephrons, comprise over 30 different cell types that contribute to renal physiology. The existence of a "kidney clock" has been noted in rats and mice during embryonic development (–). Analyses from mouse fetal kidney (E18–E20) detected thousands of rhythmic transcripts, which included cell cycle and DNA repair genes, drivers of nephron development (and), epithelial sodium channel α subunit (, encoding αENaC), and sodium/hydrogen exchanger () (–). Investigations using global-KO and ureteric bud–specific (Cre--KO) mutant mice have revealed the role of CC in controlling nephron development. RNA sequencing from kidneys of adult mice indicated that approximately 13% of the genome (second only to what is observed in liver) is expressed in a circadian manner, suggesting a crucial role in driving renal physiology. Transcriptomic studies from different kidney parts, e.g., distal convoluted tubule, connecting tubule, and cortical collecting duct, not only revealed cell-intrinsic rhythmic expression of key CC genes but also identified numerous genes (involved in water and electrolyte balance, BP, and metabolic processes) that display circadian expression and were disrupted in-KO mice (–,,). Aldosterone controls expression of, which plays a major role in sodium handling and BP regulation (–). The CC geneparticipates in the aldosterone-mediated expression of, which further links renal CC machinery and a mediator of sodium balance (). ^{122 124 105 110 105 110 115 125 106 109 126} Hoxb7 Pax2 Scnn1a Slc9a3 Bmal1- Hoxb7 Bmal1 Clock Scnn1a Per1 Scnn1a

Posttranscriptional mechanisms also contribute to the rhythmic gene expression in the kidney (). Ribosome profiling from mouse kidney revealed a circadian translation pattern for several genes with known roles in renal functions, including aquaporins (,,), podocin (), the enzyme, transporters (,), adenosine receptor (), and(). The circadian rhythm of GFR (independent of cardiac function and autonomic nervous system) is critical for maintaining BP. Consistently, podocyte-specific (Cre-driven)knockout led to perturbed diurnal GFR and BP (,), raising novel pathophysiological questions about the relationship between podocytes and glomerular hemodynamics. Several renal-derived hormones (angiotensin, endothelins, and aldosterone) display rhythmic production/degradation, and the CC genesandare known to participate in this regulation (–). Collectively, these investigations confirm that key features of renal physiology (such as BP and electrolyte control and hormone production) display circadian rhythmicity in both humans and mice () (). ^{127 127 128 129 129 131 132} Figure 1 Aqp2 Aqp4 Aqp8 Nphs2 Cyp24a1 Glut9 Pept1 Adora1 Ppara Nephrin Bmal1 Cry1/2 Per1

Chronic kidney disease (CKD) is a global health problem affecting approximately 10% of the population that increases the risks of morbidity and mortality (–). CKD may originate from several heterogeneous pathological conditions that damage the cellular structures of kidney, leading to permanent loss of function. The pathogenesis of CKD and renal fibrosis has been extensively reviewed (,,,). Briefly, sustained pathological insults stemming from multiple conditions (metabolic syndrome/diabetes, drug toxicity, cardiovascular diseases, autoimmunity) lead to immune cell infiltration in the interstitial and glomerular regions (,,). This inflammatory response is largely driven through the activation of NF-κB and MAPKs (p38 and JNK), leading to increased production of several pathogenic cytokines, chemokines, and growth factors (,,). Persistence of this proinflammatory environment drives activation of pericytes as well as differentiation of myofibroblasts and phenotypic changes characterizing the epithelial-mesenchymal transition, which, along with TGF-β–induced deposition of collagens and fibronectin in the ECM, results in renal fibrosis (,,,–). In contrast to liver and lungs, the functional role of the kidney fibroblast–specific CC in health and disease remains unknown (). ^{133 136 51 52 133 134 46 51 52 46 51 52 46 51 52 133 136} Table 1

CKD is associated with perturbed diurnal rhythmicity of BP, i.e., a non-dipping pattern, which is independently correlated with a higher mortality rate or predisposition toward end-stage renal disease (–,). Polymorphisms in the humangene are associated with hypertension and type 2 diabetes (), both of which predispose to CKD. CKD disrupts sleep patterns (,), which are also documented in animal models of kidney disease (partial nephrectomy, adenine-induced) (–). The renin-angiotensin-aldosterone system is altered in CKD, and-KO mice display perturbations in renin production, GFR, and BP (). Thus, excess renin production may contribute to high BP and cardiorenal fibrosis. Another potent profibrotic mediator, the hormone endothelin-1 (ET-1), is atarget gene expressed in collecting duct cells and in the renal cortex (–). ^{105 110 135 137 138 139 105 110 140 105 108} BMAL1 Bmal1 Per1

Animal models have indicated that CC perturbation predisposes toward renal fibrosis. Notably, genomic--KO mice show aggravated unilateral ureteral obstruction–induced (UUO-induced) interstitial fibrosis (). Moreover, owing to increased TGF-β activity andexpression,-KO mice develop more severe renal fibrosis upon ureteral obstruction compared with control mice (). Whether kidney fibroblast–restricted CC gene mutations aggravate renal fibrosis remains unknown. Deoxycorticosterone acetate–treated (DOCA-treated) mice are a clinically relevant model for developing renal inflammation and fibrosis (). DOCA alters the renal expression of CC genes, suggesting a link between behavior-induced (diet-induced) alteration of clock and CKD (). Notably, BMAL1 regulates the circadian expression of, a master regulator of antioxidant responses protecting the kidney (). The NRF2 target genesandmediate glomerular protection in experimental diabetic glomerulopathy () and immune-mediated crescentic glomerulonephritis, respectively (). Collectively, these investigations suggest that disruption of renal CC function, which normally controls renal physiology, may drive CKD and renal fibrosis. Future studies will be required to unravel the role of kidney cell type–specific (epithelial, fibroblast, and immune) CC mutants in CKD and renal fibrosis (). Bmal1 Cox2 Clock Nrf2 Hmox1 Pparg ^{140 141 142 142 143 144 145} Table 1

Clock disruptions predispose to chronic respiratory disease and lung fibrosis.

Various aspects of respiratory activity are known to display a 24-hour variation in various species (–). Sleep-wake cycle influences the daily rhythmicity of pulmonary ventilation (). The metabolic rate drops during sleep and accompanies a decrease in. In healthy humans, several aspects of lung function, e.g., forced expiratory volume in 1 second (FEV1), peak expiratory flow (PEF), and airway resistance, display diurnal behavior (–). In controlled settings, it was observed that healthy human lungs attain their peak functional capacity (determined by FEV1 and FEV1/FVC [forced vital capacity]) around noon (mid-active phase), which gradually decreases and reaches its lowest level at midnight/early morning (late-rest phase) (,). Recordings of gaseous metabolism and body temperature in rats under controlled conditions have also confirmed the circadian rhythmicity in, which closely matches the pattern displayed by theand(measurements of oxygen consumed and carbon dioxide exhaled, respectively, in milliliters per minute). Rhythmicity inwas also noted in other species, suggesting an evolutionarily conserved phenotype (). Additionally, in mouse lungs diurnal variations exist for recruitment of immune cells (B cell, granulocytes, and macrophages) (). These studies indicate that the circadian system contributes to the daily rhythmicity observed for essential pulmonary functions in both humans and mice. ^{146 150 146 151 151 152 146 150} V E V E V E VO 2 VCO 2 V E

Molecular drivers and successive stages of lung development are considerably similar in mice and humans (). In utero investigations in mice and rats first detected expression of CC genes at a time frame roughly correlating with the pseudoglandular stage of lung development (E12–E17 in mice, corresponding to 5–17 weeks after conception for humans) (,). A histological study of human and rabbit bronchioles led to the identification of Clara cells (now termed club cells), which play essential roles in maintaining lung homeostasis (). The transcriptional activity of NKX2.1 (TTF1) is key for lung development and production of surfactant protein A from the Clara cells (–). Importantly,expression correlates with the transcription ofandduring lung development in mice. In prenatal murine lung,is presumed to regulate oxidative and inflammatory stress (). Investigations using-luciferase mice provided the initial evidence for suprachiasmatic nucleus–entrained rhythmic clock in the mature lungs (). CC genes are expressed in mouse larynx, trachea, bronchus, and lungs (). Importantly, circadian expression of CC genes and several of the muscarinic acetylcholine receptor genes (,, and) in respiratory tissues of mice was lost following double knockout ofand() (). Importantly, circadian disruptions modeling shiftwork and jet lag are known to disrupt CC gene expression and lung function (). Functional CC oscillators are known to be present in different lung cell types, e.g., alveolar and bronchial epithelium (club cells), lung fibroblasts, and macrophages (–) (). Critically, club cells are known to maintain CC coherence among various cell types of lungs, as specific ablation of these cells leads to altered rhythmicity in the remaining tissue (). Transcriptomic analyses from mouse lungs identified approximately 1,000 genes that are expressed in a diurnal manner, and pathway analyses indicated that the majority are immune related (). The role of the CC oscillator in pulmonary airway epithelial cells (AECs) was addressed by selectiveknockout in club cells. RNA sequencing showed that the AEC CC controls expression of genes involved in metabolism of lipids and xenobiotics, ECM remodeling, and chemokine/cytokine signaling (). Microarray-based study in rats identified numerous genes displaying circadian expression patterns, of which nearly 60% were expressed in the rest (inactive) phase (). Among the cycling transcripts in rat lungs are genes implicated in maintenance and repair of lung parenchyma, and vasculature (). Collectively, these investigations have established the "clock" as a major regulator of respiratory functions. ^{153 153 154 153 153 160 149 154 161 161 162 163 166} Table 1 ^{166 166 166 167 167} TTF1 Cry2 Clock Rev-Erba Per2 Chm2 Chm3 Chm4 Cry1 Cry2 Cry1Cry2 –/– –/– Bmal1

Chronic respiratory disease is exemplified by chronic obstructive pulmonary disease (COPD) and asthma, which both represent global health concerns affecting quality of life and mortality. Circadian biology influences various aspects of lung disease and fibrosis (,,–,). COPD and asthma symptoms worsen in the early morning. A study involving more than 7,000 asthmatic patients reported that around 60% suffered from nighttime symptoms more than three times each week, while 40% suffered every night (). Multiple factors, including oxidative stress, mucus production/levels, lung inflammation, and cortisol levels, influence the nighttime severity of asthma (–). Allergen-driven hyperactivity of eosinophils and mast cells drives bronchospasm. In humans and mice, mast cells and eosinophils are known to harbor functional CCs that regulate the expression of several disease-related genes (,). Notably, IgE-mediated activation of mast cells and consequent release of histamines and leukotrienes were found to exhibit circadian rhythmicity, andandtranscripts were reduced in bronchioles of asthmatic mice (). In allergic asthma models, macrophage-specific-KO mice show increased lung inflammation associated with increased IL-5 levels (). Furthermore, club cell–specificknockout is known to drive hyperinflammatory responses upon LPS challenge and bacterial infection as a result of perturbed diurnal expression of(). Critically, inflammation can disrupt the expression of CC genes in lungs and reprogram the transcriptome and metabolome (,). Collectively, these results suggest that CC perturbation in various cell types of lungs predisposes to inflammatory airway diseases like asthma. ^{53 54 146 150 168 168 168 170 171 172 173 173 164 146 147} Bmal1 Rev-Erba Bmal1 Bmal1 Cxcl5

COPD is a chronic disease resulting in reduction of respiratory function. The severity of COPD symptoms in humans shows marked diurnal variation (worsening during late night/early morning) (,). COPD results in oxygen desaturation, thus worsening sleep quality and heightening the risk of mortality due to cardiovascular pathologies (,). A cross-sectional study noted abnormal sleep patterns in approximately 70% of COPD patients, which suggests a feedforward cycle in which sleep disturbance and COPD worsen each other. Chronic exposure to cigarette smoke (CS) is a major cause of COPD (,). In mouse models of COPD, CS altered the expression ofand. Importantly, reduction of REV-ERBα protein levels was observed in COPD patients (). Genetic studies support a role for REV-ERBα activity in reducing overall inflammation (decreased neutrophil levels and cytokine expression) in COPD (,). Additionally, in mouse lungs, CS-induced COPD reduced levels of(), a key regulator of CC. Decreases in SIRT1 activity were found in COPD patients (). Alteration of molecular CC functioning has been linked to pulmonary fibrosis (–). Investigations using themodel showed that the mutants spontaneously develop a fibrotic phenotype with an increase in ECM remodeling gene expression, correlating with higher collagen deposition around bronchioles (). Mechanistically, CLOCK DNA binding was found to regulate the expression of, a regulator of ROS signaling (). Andmice presented with reducedand increased oxidative damage (). REV-ERBα activation could prevent progression of pulmonary fibrosis by limiting TGF-β activity (,). Finally, age-associated lung fibrosis is increased in-KO mice (). Collectively, these investigations reveal that perturbation of the CC is causally linked to chronic respiratory disease and lung fibrosis. ^{174 175 174 175 176 177 146 178 179 179 180 181 184 181 181 181 182 183 184} Rev-Erba Per2 Sirt1 Clock Δ19 Nrf2 Clock Δ19 Nrf2 Bmal1