The Ob protein (leptin) and the kidney

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1 Kidney International, Vol. 53 (1998), pp The Ob protein (leptin) and the kidney KUMAR SHARMA and ROBERT V. CONSIDINE PERSPECTIVES IN CLINICAL MEDICINE Department of Medicine, Division of Nephrology, Thomas Jefferson University, Philadelphia, Pennsylvania, and Department of Medicine, Division of Endocrinology and Metabolism, Indiana University, Indianapolis, Indiana, USA The Ob protein (leptin) and the kidney. Mutation of the Ob gene, which encodes for leptin, or mutation of the leptin receptor leads to obesity in mice. Humans, for the most part, have a positive correlation of leptin with body fat mass suggesting possible defects in leptin effector mechanisms that may contribute to obesity. As patients on hemodialysis have difficulty with appetite, we investigated whether leptin is cleared by the kidney and is elevated in hemodialysis patients. In patients with intact renal function there was a net renal uptake of 12% of circulating leptin, whereas in patients with renal insufficiency there was no renal uptake of leptin. In a separate cohort of 36 patients with end-stage renal disease (ESRD), peripheral leptin levels factored for body mass index was increased by fourfold as compared to a group of healthy controls (N 338). The leptin receptor exists in a long and short form, with the long form primarily expressed in the hypothalamus but also in the lungs and kidneys of the mouse. Further studies are necessary to clarify the role of leptin in regulating appetite in patients with ESRD and the role of leptin in directly affecting kidney function via its receptors. The prevalence of obesity has increased dramatically in the United States during the past decade. The recent National Health and Nutrition Examination Survey (NHANES) reported that the prevalence of overweight Caucasian men and women increased from 20% to 30% in the past decade [1]. In African American men and women the prevalence of obesity has increased from 30 to 40% in men and 30% to 48% in women. Hispanic Americans had a similar increase in the prevalence of obesity. The estimated cost of treating obesity and its complications is estimated to be 68 billion dollars annually. The increased prevalence of obesity contributes to the development of NIDDM and hypertension and thus will promote increased prevalence and progression of kidney disease [2]. Several excellent reviews discuss the pathogenesis of obesity in humans [3] and the molecular genetics of obesity [4]. The present review will highlight the identification of two proteins, leptin and its receptor, that cause obesity in mice and then focus on the relationship of these two proteins with the kidney. IDENTIFICATION OF LEPTIN AND THE LEPTIN RECEPTOR The identification of the genes responsible for obesity in two strains of mice has recently been elucidated and allows for analysis of these genes in the human population. The identification has Key words: obesity, hemodialysis, end stage renal failure, progression of renal disease, leptin by the International Society of Nephrology also led to novel concepts in how the body deals with disturbances in energy balance. The ob/ob mouse is characterized by hyperphagia, decreased energy expenditure, reproductive deficiency, NIDDM with hyperinsulinemia and onset of obesity by three to four weeks of age. Parabiosis experiments performed in 1959 with normal mice sharing the circulation of ob/ob mice resulted in the ob/ob mouse losing weight and reducing its appetite, whereas there was no effect on the normal mouse [5]. This suggested that the ob/ob mouse was missing a circulating factor that controls appetite and weight. The ob/ob gene was identified in 1994 by Zhang et al by positional cloning [6]. The gene was found to encode a secreted 167 amino acid protein named leptin. Mutations were found in codon 105 leading to a defective leptin protein and in the promoter leading to absence of leptin mrna synthesis in two different strains of ob/ob mice, respectively. Administration of leptin resulted in weight loss, increased locomotor activity and improved insulin and glucose levels [7 9]. The db/db mouse has a phenotype identical to the ob/ob mouse. However, in parabiosis experiments between normal mice and db/db mice, the normal mouse stopped eating and lost weight whereas the db/db mouse was not affected [10]. This and other data were interpreted to suggest that the db/db mouse had a defective response to a circulating factor that controls appetite. Upon the identification of leptin, it was found that the db/db mouse was markedly hyperleptinemic and it was reasoned that this mouse had a defect in the leptin receptor [11]. This was indeed the case. Tartaglia et al isolated the db gene by assaying expression libraries for CNS receptors demonstrating strong affinity for leptin [12]. A loss of function mutation was identified in db/db mice that prevents expression of the 302 amino acid cytoplasmic domain of the leptin receptor which contains conserved signaling motifs for Janus protein tyrosine kinase (Jak), and signal transducers and activators of transcription (STAT) binding. FUNCTION OF LEPTIN AND ITS RECEPTORS The pathway by which leptin regulates energy balance is outlined in Figure 1. Increased caloric intake coupled with decreased caloric expenditure will lead to an expansion of the adipocyte size. This, in some not yet understood way, signals the adipocyte to increase leptin synthesis and release. Leptin enters the circulation, binds to leptin-binding proteins, and is ultimately transported into the cerebrospinal fluid by receptors located on the choroid plexus. Within the hypothalamus, leptin binding to its receptor activates the Jak/STAT signal cascade to activate or 1483

2 1484 Sharma and Considine: Leptin and the kidney Fig. 1. Pathway by which leptin inhibits appetite. Leptin is secreted by adipocytes and interacts with its signaling receptor in the hypothalamus to decrease production of neuropeptide Y. Inhibition of neuropeptide Y decreases hunger, increases activity, and thermogenesis. Fig. 2. Leptin receptors. The long receptor of leptin (OB-Rb) contains an intracellular domain that has a consensus Jak binding site (black segment) and a STAT binding site (hatched segment). The short leptin receptor (OB-Ra) retains the intracellular Jak binding site, however, the STAT binding site is missing. deactivate genes. A major action of leptin on the hypothalamus is to decrease production of neuropeptide Y (NPY) [13]. A reduction in NPY leads to a decrease in appetite and increased energy expenditure, ultimately resulting in weight loss [14]. In general, the circulating leptin concentration is in proportion to the amount of adipose tissue, although absolute levels may be modified by factors such as gender [3, 15]. Changes in adipose tissue mass result in concomitant changes in leptin. Weight loss results in a decrease in leptin and weight gain significantly increases circulating leptin in both humans and rodents [15 18]. In addition, leptin is altered by extremes in energy intake. Serum leptin falls dramatically with short term fasting and is increased 40% by massive overfeeding in humans [17, 19]. Fasting and overfeeding have similar effects on circulating leptin in rodents [18, 20]. However, rodents and humans differ in the leptin response to normal meal intake. Leptin levels do not change acutely following normal meal consumption in humans [15, 20] in contrast to the rapid rise following food intake in rodents [21]. Leptin synthesis is also regulated by several hormones and pharmacologic agents. Insulin and dexamethasone stimulate leptin whereas -adrenergic agonists and testosterone inhibit leptin production [reviewed in greater detail in 13, 14]. The leptin receptor belongs to the class I cytokine receptor family [12]. Five isoforms have been identified [12, 22]; The two major isoforms, Ob-Ra and Ob-Rb, are illustrated in Fig. 2. Observed in all leptin receptor isoforms are the extracellular binding domain consisting of 840 amino acids and the transmembrane region of 34 amino acids. The intracellular domain is variable. Ob-Rb, the long leptin receptor, has a 302 amino acid intracellular domain which contains interaction sites for Jak kinase and STAT proteins, that are characteristic second messengers for cytokine receptor signaling. Ob-Ra, the short leptin receptor, has a 34-aa intracellular domain that retains the Jak binding site but not the STAT site. This receptor is present in almost all tissues examined and it is unknown if it retains functional signaling activity. Large amounts of Ob-Ra are present at the choroid plexus where it acts as a transporter for leptin to cross the blood-brain barrier. One other leptin receptor Ob-Re, lacks a transmembrane domain and thus may function as soluble receptor/binding protein. Leptin is present in the circulation in both bound and free forms. In lean individuals up to 50% of leptin is present in the bound form whereas in obese individuals the majority of leptin is in the free form [23]. This may be due to a limited concentration of leptin binding proteins in the circulation that does not vary with increasing leptin production. The physiologic significance of bound versus free leptin is unknown. Although leptin deficiency leads to obesity in the ob/ob mouse, this is not the case for human obesity. In adults, children, and infants leptin levels are highly correlated with the fat mass [15]. Furthermore, no deleterious mutations in the human leptin receptor have yet been identified [24]. However, of great interest is the recent identification of two related youngsters who manifested severe obesity during their infant years [25]. Both children had normal birth weights (50th percentile) but developed severe intractable obesity by the age of two years with weights twice as great as the 98th percentile. One child required liposuction of lower limb fat in an attempt to improve mobility. A frame-shift mutation was found in their leptin gene which led to a truncated form of the protein that was not secreted by the adipocyte. It remains to be seen whether these individuals respond to exogenous leptin administration. Thus, it is believed that certain rare obesity syndromes that are characterized by the development of obesity at a very early age of onset may be linked to defects in leptin or its receptor. However, in the vast majority of adult-onset obesity it is unlikely that defects in the leptin pathway are solely responsible. In chronic end-stage renal disease, a major issue is not obesity and enhanced appetite but the reverse. Patients often have

3 Sharma and Considine: Leptin and the kidney 1485 anorexia and weight loss that is correlated with significant morbidity and mortality. Several studies have demonstrated that decreased albumin and low protein intake is associated with increased mortality despite adequate dialysis [26 28]. With these issues in mind, several studies have examined the role of the kidney in clearing leptin and have measured leptin levels in patients with end-stage renal failure. LEPTIN CLEARANCE BY THE KIDNEY We postulated that clearance of leptin may be via the kidney in humans. As the leptin molecule is 14 to 16 kda, it would likely be filtered at the glomerulus. To evaluate the role of the kidney in clearance of circulating leptin, we measured levels of leptin in aortic and renal vein plasma from patients with varying degrees of renal function, who were undergoing elective cardiac catheterization. We found a 12% decrease of leptin levels across the kidney bed (aortic leptin, vs. renal vein leptin, ; P 0.005), in adults with intact renal function [29]. Simultaneous measurement of the difference in leptin concentration across the kidney and the renal plasma flow rate enabled the net renal extraction rate to be calculated. There was a mean net renal extraction of 480 ng/min in the 8 patients evaluated. In patients with moderate renal insufficiency (creatinine 2.5 mg/dl; N 5), there was no clearance of leptin across the kidney vascular bed. Urinary levels of leptin were below the detection of our assay, suggesting that leptin was being degraded in the kidney. In support of our findings that the kidney plays a major role in clearing leptin, a recent report of 57 NIDDM patients evaluating determinants of plasma leptin levels [30] using multiple regression analyses found a significant positive correlation between plasma circulating leptin levels and the serum creatinine. Administration of radio-labeled leptin to rats that had undergone bilateral nephrectomy, reduced leptin clearance to 19% of control values [31]. In a separate study by the same investigators, arteriovenous measurement of leptin across the kidney showed a decrease of 25% of circulating endogenous leptin in rats with normal renal function [32]. In addition, leptin was not found in the urine and no leptin metabolites were found in the renal vein, suggesting that leptin was filtered at the glomerulus and likely taken up by proximal tubular cells. Whole body clearance of radiolabeled leptin was estimated at 5.4 ml/min/kg, which was only slightly higher than renal clearance of leptin (5.3 ml/min/kg), indicating that the kidneys account for almost 100% of whole body clearance of leptin. These studies were performed in the fa/fa rats which have a mutation in the long form of the leptin receptor, thus excluding this receptor as playing a major role in renal clearance of leptin. Hence, in both rats and humans, it appears that the kidney is a major site of leptin clearance. LEPTIN ACCUMULATION IN END-STAGE RENAL FAILURE If leptin is primarily cleared by the human kidney, it follows that leptin levels would be elevated in patients with end-stage renal disease (ESRD). Several studies have found such a relationship. In our study of 37 chronic hemodialysis patients, pre-dialysis plasma leptin levels were elevated fourfold as compared to a group of 331 healthy controls ( ng/ml vs ng/ml, respectively; P 0.01) [29]. When corrected for body mass index (BMI), the relationship persisted ( vs , respectively; P 0.005). Very similar results were obtained in an independent study of 141 ESRD patients [33]. Mean values in patients with ESRD of both sexes (males, ng/ml and females, ) were significantly higher (P 0.001) than those obtained in normal volunteers, (normal males, ng/ml and normal females, ng/ml). A separate study found that both hemodialysis and peritoneal dialysis patients had elevated leptin levels [34]. Measurement of pre- and post-hemodialysis levels of leptin failed to demonstrate significant clearance of leptin during hemodialysis with a conventional modified cuprophane membrane [29]. The above studies also demonstrated that circulating leptin in hemodialysis patients primarily existed in the free form, not bound to leptin-binding proteins [29, 34], and was of the same molecular weight as intact leptin [33]. Thus, there does not appear to be an increase in leptin binding proteins nor of leptin degradation products in ESRD patients. Does hyperleptinemia inhibit appetite in patients with renal failure? The answer to this question is not yet known. We found that in our population that leptin levels correlated well with the BMI, although with higher levels than in patients without renal failure. The rate of increase of leptin with BMI was also found to be significantly greater among patients with ESRD as compared to controls in two separate studies [33, 34]. Leptin levels do not appear to correlate with recent changes in weight nor adequacy of dialysis [33]. It thus appears that patients with renal failure have re-adjusted their leptin-stat, and despite high levels of leptin, continue to have a correlation with their BMI. It is possible that due to hyperleptinemia, dialysis patients have less caloric intake at the same BMI than non-dialysis patients; however, this remains to be determined. Based on data that relatively large doses of leptin had to be administered to curtail appetite and induce weight reduction in normal mice [9], it is conceivable that patients with markedly high leptin levels may manifest a relationship between leptin levels and parameters of appetite and nutrition. Interestingly, we found that 5% of our dialysis population had extraordinarily high leptin levels ( 200 ng/ml) that could not be explained purely on the degree of their fat mass [29]. These patients were not on corticosteroids, which may stimulate leptin production directly. We are presently following these patients to determine if serial leptin levels correlate with disturbances in appetite over time. EFFECTS OF LEPTIN ON THE KIDNEY It is interesting to note that the kidney is one of the few extra-neural tissues that express the long-form of the leptin receptor. Northern analysis demonstrates the long form of the leptin receptor mrna (Ob-Rb) in mouse kidney and lung, but not in heart, spleen, liver, skeletal muscle, or testis [35]. Utilizing reverse transcriptase-polymerase chain reaction (RT-PCR), with specific primers for Ob-Rb, a separate study found expression only in mouse kidney and adrenal gland among peripheral tissues examined [36]. In situ analysis with a labeled cdna probe detected Ob-Rb primarily in the medulla of mouse kidney [36]. Hybridization with { 125 I}-leptin of rat kidney found absence of labeling in the cortex; however, there was intense labeling of vascular structures and medullary sites [37]. The functional role of leptin binding sites and leptin receptors in the kidney are unknown. The effects of leptin on kidney function in rats has been studied

4 1486 Sharma and Considine: Leptin and the kidney by several groups. Intravenous administration of leptin for five days to normal rats stimulated a diuresis with urine volumes being twice normal [37]. Administration of leptin directly into the renal artery stimulated a natriuresis but not a kaliuresis suggesting an effect of leptin at the level of the collecting tubule [38]. However, blood pressure was not affected in either study. Sympathetic nerve inflow to the kidney was found to be markedly stimulated by leptin administration [39]. It is possible that leptin has opposing effects on blood pressure by its ability to enhance sympathetic tone to increase blood pressure and promote a natriuresis to lower blood pressure. Other effects of leptin on the kidney remain to be evaluated. Given that the leptin receptor belongs to the class I family of cytokine receptors, the action of leptin may be important in inflammatory disorders of the kidney. The growing study of leptin encompasses many fields including nephrology. Apart from its possible role in appetite regulation in patients with renal insufficiency, it is likely that there are other relationships between leptin and the kidney. The presence of the long form of the leptin receptor in the kidney makes this organ a prime site by which leptin may exert peripheral effects. Studies to evaluate the localization of the leptin receptor in the kidney, a characterization of its signaling pathways in renal cells, and physiologic and pathophysiologic consequences of leptin action on the kidney are intriguing future areas of study that will increase our understanding of this recently discovered hormone. ACKNOWLEDGMENTS This work was supported in part by grants KO8 DK (KS) and R29 DK51140 (RVC) from the National Institutes of Health and a grant from the American Diabetes Association (RVC). We gratefully acknowledge the advice and comments of Dr. Jose Caro. 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