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National Institute of Diabetes and Digestive and Kidney Diseases (E.D.J., S.A.M., J.R.Y., E.K.C., E.A.O., J.E.B., P.G.), National Institutes of Health, Bethesda, Maryland 20892; Amgen (A.M.D.), Thousand Oaks, California 91320; Department of Pediatrics (M.A.T., P.R.B.), University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104; and Department of Medicine (D.B.S., S.O.), University of Cambridge, Addenbrooke’s Hospital, Cambridge CB2 2QQ, United Kingdom
Address all correspondence and requests for reprints to: Edward D. Javor, 10 Center Drive, Room 8D20, Bethesda, Maryland 20892-1770. E-mail: edwardj{at}intra.niddk.nih.gov.
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Abstract |
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionNote Added in ProofReferences |
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In the course of assembling a group of patients for a clinical trial of recombinant leptin administration as a potential therapy for the metabolic and pituitary abnormalities of these syndromes (17, 18), we were struck by the extent of proteinuria exhibited. In the present study, we describe the types of renal diseases thus far elucidated that are associated with these generalized lipodystrophy syndromes. Further, we describe the renal parameters observed in patients who were treated with recombinant leptin over a period of up to 3 yr.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionNote Added in ProofReferences |
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A total of 25 patients with generalized lipodystrophy are presented in this analysis. Seven patients have acquired generalized lipodystrophy (AGL), and 18 patients have congenital generalized lipodystrophy (CGL). The diagnosis of CGL was made clinically based on the presence of lipodystrophy within the first year of life. Patients who had normal fat tissue within the first year of life and subsequently developed generalized fat loss were classified as AGL. Body composition studies on these patients have demonstrated normal lean body mass despite low percent body fat (19). Fifteen patients were female, and 10 were male. They ranged in age from 8–67 yr.
All 25 patients had similar phenotypic features of lipodystrophy. Five patients were excluded because they did not meet entry requirements for the protocol (four due to inadequate metabolic abnormalities and one due to concurrent thyroid cancer), and two patients died while being considered for leptin therapy. The remaining 18 patients initiated recombinant leptin therapy in a protocol designed primarily to study the effects of leptin replacement on glycemic and lipid parameters (17). The study was approved by the institutional review board of the National Institute of Diabetes and Digestive and Kidney Diseases. Informed consent was obtained from the patient or his or her legal guardian. Fifteen of the 18 patients have been followed on recombinant leptin for at least 4 months. Leptin replacement therapy was given as a self-administered, twice-daily sc injection as previously described (17, 19). Patients were seen as inpatients every 4 months for the first year and then every 6 months thereafter. Renal parameters were measured during each visit. Data were collected on a metabolic unit, including 24-h urine collections to ensure completeness. Diabetic and lipid medications were lowered or discontinued if indicated.
Biochemical analyses
Serum leptin levels were determined by immunoassays with the use of a commercial kit (Linco Research, St. Charles, MO) on samples drawn after an overnight fast. Glycosylated hemoglobin (HbA1c) values were measured by ion-exchange HPLC (Bio-Rad Laboratories, Hercules, CA). Serum creatinine and urine creatinine, albumin, and total protein were determined according to standard methods with the use of automated equipment (Beckman, Fullerton, CA). Creatinine clearance was calculated from serum and 24-h urine creatinine values [urine volume (ml) x urine creatinine (mg/dl)]/[time (1440 min) x serum creatinine (mg/dl)] and normalized for 1.73 m2.
Renal biopsies
Routine percutaneous renal biopsies were not included in the original protocol but were performed as deemed clinically indicated for excessive or worsening proteinuria. Two biopsies (NIH-2 and NIH-25) were obtained by the patients’ primary nephrologists before their initial National Institutes of Health (NIH) evaluations. Renal tissue was analyzed at autopsy in two cases (NIH-CGL1 and NIH-CGL3). Light microscopy samples were read by an experienced nephrologist at the NIH and reviewed by the Armed Forces Institute of Pathology, who carried out immunofluorescence and electron microscopy.
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Results |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionNote Added in ProofReferences |
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Twenty-five patients with generalized forms of lipodystrophy were studied (Table 1
). Eighteen patients had CGL, and seven patients had AGL. The median age of the patients was 18 yr (range, 8–67 yr). Resting blood pressures were less than 140/90 mm Hg for all adults and less than 95th percentile adjusted for age, height, and gender for children (20). Twenty of 25 patients had previously diagnosed diabetes, of which 19 had HbA1c more than 7% despite using a variety of medications. There were no consistent abnormalities of autoimmune and inflammatory markers at baseline or after leptin therapy.
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The most common biochemical renal abnormality seen was proteinuria (Table 2 and Fig. 1). The median urinary albumin excretion was 644 mg/24 h (range, 7–9,461 mg/24 h). Twenty-two of 25 patients (88%) had elevated urinary albumin excretions (>30 mg/24 h), and 15 patients (60%) had macroalbuminuria (>300 mg/24 h). The median urinary protein excretion was 1120 mg/24 h (range, 191–11,365 mg/24 h), with 5 patients (20%) having nephrotic range proteinuria (>3,500 mg/24 h). The median protein/creatinine ratio was 0.58 (range, 0.08–13.65). The other notable finding was an elevation of creatinine clearance (Fig. 1), which we presume reflected marked hyperfiltration. The median creatinine clearance was 205 ml/min·1.73m2 (range, 37–452 ml/min·1.73 m2). All values of creatinine clearance, urine albumin, and urine protein were expressed both as raw data and normalized for 1.73 m2 (Table 2).
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Leptin therapy study
Fifteen patients entered an open-label therapeutic study of recombinant leptin for their severe insulin resistance, diabetes, dyslipidemia, and pituitary abnormalities and were observed for 4–36 months. Evidence for the efficacy of this therapy has been previously shown (17, 18). Along with improvements in the metabolic parameters (data not shown), 11 of 15 patients demonstrated a reduction in urinary protein excretion after 4 months of therapy that was maintained thereafter (Fig. 3). This was associated with a reduction in creatinine clearance, presumably reflecting reduction in glomerular hyperfiltration. The following four cases are of notable exception.
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) developed generalized lipodystrophy at age 6 yr in association with type 1 diabetes (low C-peptide and positive islet cell antibody). Over the next few years, she had progressive hypertriglyceridemia and hepatomegaly with evidence of nonalcoholic steatohepatitis. At age 13 yr, she was enrolled into the leptin therapy protocol. Her urinary protein was 2.8 g/24 h with normal serum creatinine and elevated creatinine clearance. She had elevated transaminases and a liver biopsy consistent with autoimmune hepatitis (data not shown) but normal complement levels and undetectable C3 nephritic factor. She was treated with leptin for 1 yr with a decrease in urine protein excretion to 1.5 g/24 h. Her renal function remained normal, her diabetes control improved, and her triglycerides began to fall. However, over the next 2 months, her bilirubin and transaminases began to rise and her albumin was noted to be very low. On readmission, she was found to have massive proteinuria of over 15 g/24 h. Renal biopsy revealed MPGN type 1 (Fig. 2
, B and C). Leptin therapy was discontinued; but over the ensuing 8 months, her massive proteinuria persisted, and her renal function declined.
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) is a 35-yr-old male who developed total lipodystrophy around age 3 yr. When first seen at NIH, his urinary protein excretion was 2.5 g/24 h, and his creatinine clearance was 179 ml/min. Serum BUN and creatinine were normal. He had low C3 with positive C3 nephritic factor. After approximately 6 months of leptin therapy, he developed increased proteinuria, and his serum creatinine began to increase. Leptin therapy was discontinued at 8 months, and a renal biopsy was obtained, demonstrating MPGN type 1. Six months after discontinuing leptin, his proteinuria and elevated serum creatinine have worsened.
Patient NIH-2 (Fig. 4C) is a 17-yr-old female with CGL. She had a baseline urinary protein excretion of 11 g/24 h and a creatinine clearance of 73 ml/min. She was known to have proteinuria since age 11 yr, and a kidney biopsy done 2 yr before initiating leptin therapy revealed focal segmental glomerulosclerosis (FSGS). She was treated with leptin over an 18-month period during which she had a slow, but progressive, deterioration of renal function. There was an initial fall in urinary protein excretion that was not sustained. Leptin therapy was discontinued after 30 months as she approached end-stage renal disease.
Patient NIH-13 (Fig. 4D) was first seen at NIH at age 12 with CGL. She had a urinary protein excretion of 3.3 g/24 h and a creatinine clearance of 242 ml/min. Because of continued nephrotic range proteinuria after 12 months of leptin therapy, she had a kidney biopsy, which revealed FSGS (Fig. 2D). She has now been followed for 16 months on leptin therapy and continues to have the same heavy proteinuria but normal renal function. Leptin therapy has been continued.
Renal biopsy results
We analyzed renal tissue from eight patients (Tables 3 and 4). Six patients had percutaneous biopsies obtained secondary to excessive proteinuria. Of these patients, all three with CGL demonstrated FSGS. Of the patients with AGL, one had FSGS and two had MPGN type 1. The renal tissue analysis of two patients done in conjunction with autopsies revealed diabetic nodular glomerulosclerosis in one and no abnormality in the other.
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionNote Added in ProofReferences |
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The primary purpose of this protocol was to follow metabolic changes in patients with generalized lipodystrophy treated with recombinant leptin. It was only through following this group of patients for 3 yr that we recognized the extent of underlying proteinuria, elevation of creatinine clearance, and renal pathology. We therefore do not have direct measurements of glomerular filtration rate. Although creatinine clearance is not identical to glomerular filtration rate, it has been widely used as its surrogate.
Textbooks of medicine and nephrology describe the relationship between an acquired form of partial lipodystrophy and MPGN type 2 (dense deposit disease), associated with low C3 and elevated C3 nephritic factor. These descriptions are based on numerous case reports (7, 8, 9, 10, 11, 12, 13, 14, 15, 16). In fact, C3 nephritic factor has been implicated as a possible cause for partial lipodystrophy (8).
In contrast, the renal lesion(s) associated with generalized forms of lipodystrophy (congenital or acquired) has remained largely unknown. Forty years ago, Senior and Gellis (3) mentioned proteinuria in two patients with generalized lipodystrophy, but again most of the emphasis was on partial lipodystrophy and renal disease. Three case reports have mentioned diabetes, glomerulosclerosis, and glomerular lipid infiltration in association with generalized lipodystrophy (two congenital, one acquired) (4, 5, 6), and again we were surprised that we could not implicate glomerular lipid infiltration as etiologically related to the renal disease of our patients. This is despite the fact that many of the patients had marked elevation of serum triglyceride levels. Two patients (NIH-1 and NIH-9) had sparsely scattered lipid-ladened macrophages in a minority of glomeruli, unlikely to be pathogenetically relevant to the glomerular proteinuria.
Fifteen of the 25 patients in whom we report baseline data were further followed in a protocol using recombinant leptin to treat their metabolic disorders. We have reported initial results in ameliorating metabolic parameters, including insulin resistance, hyperglycemia, hypertriglyceridemia, and hepatic steatosis (17). We found marked elevation in creatinine clearance, which we interpret as renal hyperfiltration in conjunction with poorly controlled diabetes. In previous studies of obesity or congenital leptin deficiency (17, 18, 19, 21, 22, 23, 24, 25, 26), there has been no mention of renal disease with leptin therapy. In 11 of 15 patients, there was a reduction in proteinuria that coincided with a reduction in creatinine clearance and hence correction of hyperfiltration as a function of leptin therapy.
The other four patients who had a different course are of special note. In two patients with nephrotic range proteinuria at baseline and FSGS, one (NIH-13) remained stable at 1 yr of leptin therapy, and leptin has been continued, whereas the other (NIH-2) progressed toward end-stage renal disease. We assume that the renal disease in these two cases is unrelated to leptin therapy. In the two cases with MPGN, the issue is more complex. Both patients had heavy proteinuria at baseline. One patient (NIH-14) had low C3 with elevated C3 nephritic factor, whereas the other (NIH-9) had a different but strong autoimmune diathesis, including type 1 diabetes. These two patients with underlying renal disease had a marked exacerbation of their renal disease at 6 and 14 months of leptin therapy and no improvement after 6 and 9 months of discontinuing leptin. Thus, in these two patients who had underlying renal disease, we cannot exclude the possibility that leptin therapy was associated with exacerbation of their underlying renal disease.
An additional patient with CGL, not included in this report, had end-stage renal disease of unknown etiology at presentation. She underwent a renal transplant and is the subject of a separate report (27). Leptin therapy was initiated 1 yr after the transplant, and she continues to do well after 1 yr on recombinant leptin therapy.
The association between leptin and renal abnormalities has been investigated using various rodent models. One model studied leptin’s ability to increase expression of TGF-ß. Wolf et al. (28) demonstrated increased expression of TGF-ß1 in cultured rat glomerular endothelial cells incubated with leptin, as well as increased proteinuria and glomerulosclerosis in naïve rats administered leptin for 3 wk. Although leptin administration may be associated with increased TGF-ß and renal disease in rodents, it is yet unclear what relevance it may have in human studies. The above authors suggest that the increased incidence of glomerulosclerosis observed in obese patients in the setting of elevated leptin levels may involve increased expression of TGF-ß. Although FSGS was identified in four of our patients, this hypothesis fails to account for two patients diagnosed with FSGS before receiving leptin (NIH-2 and NIH-25). Additional patients with generalized lipodystrophy diagnosed with forms of glomerulosclerosis (4, 5, 6) had also never received leptin.
Nevertheless, TGF-ß is an intriguing theory in the development of renal disease, particularly FSGS. To be the cause of FSGS in patients with lipodystrophy, increased TGF-ß activity would presumably need to exist before leptin replacement. Whether lipodystrophy is a state of increased TGF-ß activity has not been established. A rodent model suggests that TGF-ß may not only be associated with renal disease but may even be involved in the development of lipodystrophy itself. Transgenic mice designed to overexpress TGF-ß1 in adipose, renal, and hepatic tissues developed lipodystrophy, glomerulosclerosis, and cirrhosis with variable penetrance (29). Ultimately, the roles of TGF-ß in human disease should be further explored.
Another rodent model examined leptin’s role in modulating the immune system. Sanna et al. (30) demonstrated a clear link between leptin presence and onset of experimental autoimmune encephalitis, a Th1-mediated disease, in susceptible mice. Ob/ob mice deficient in leptin were protected against this condition. This was reversed with leptin replacement. Again, the relevance of these rodent studies to the human condition is unclear. Limited data in children with congenital leptin deficiency showed a reduction in CD4+ T cell count that was restored to normal after leptin treatment (23). This was associated with a more robust cytokine response to various in vitro stimuli. Whether leptin replacement can lead to pathological CD4+ activity through Th1 lymphocytes in humans is speculative.
We have described a variety of renal pathologies associated with proteinuria in patients with generalized lipodystrophy. We were surprised by the lack of predominating diabetic nephropathy, despite the prevalence of diabetes and proteinuria. Instead, we identified two distinct pathologies usually associated with other illnesses. The major finding in patients with CGL was FSGS with the exception of one patient with diabetic nephropathy. One patient with AGL (NIH-1) also had FSGS. However, two patients with AGL had MPGN type 1, which has not been previously reported. In contrast, acquired partial lipodystrophy has been associated with MPGN type 2, low C3, and elevated C3-nephritic factor. Only one of the two patients with AGL and MPGN (NIH-14) had this abnormal complement profile. At this point, it is certainly premature to predict renal pathology based on the type of generalized lipodystrophy. Yet, renal disease is clearly a significant component of generalized lipodystrophy. We are unable, however, to provide a mechanistic link for either of these divergent processes. The acquired forms may have an autoimmune basis, but the congenital forms are associated with at least two different genetic mutations (31, 32). Whether these processes have any relevance to the various rodent models of TGF-ß or immune dysfunction remains speculative.
Leptin is a cytokine that is decreased in lipodystrophy, but there are no models to suggest how a deficiency of leptin or any of the other known adipokines may be related to renal disease. Whether physiological replacement could lead to an exacerbation of underlying renal disease in patients, whose metabolic parameters improve on recombinant leptin therapy, remains an important question.
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Note Added in Proof |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionNote Added in ProofReferences |
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Footnotes |
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Received December 15, 2003.
Accepted March 29, 2004.
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