PTH/PTHrP Receptor Mediates Cachexia in Models of Kidney Failure and Cancer

Abstract

Cachexia is a wasting syndrome associated with elevated basal energy expenditure and loss of adipose and muscle tissues. It accompanies many chronic diseases including renal failure and cancer and is an important risk factor for mortality. Our recent work demonstrated that tumor-derived PTHrP drives adipose tissue browning and cachexia. Here, we show that PTH is involved in stimulating a thermogenic gene program in 5/6 nephrectomized mice that suffer from cachexia. Fat-specific knockout of PTHR blocked adipose browning and wasting. Surprisingly, loss of PTHR in fat tissue also preserved muscle mass and improved muscle strength. Similarly, PTHR knockout mice were resistant to cachexia driven by tumors. Our results demonstrate that PTHrP and PTH mediate wasting through a common mechanism involving PTHR, and there exists an unexpected crosstalk mechanism between wasting of fat tissue and skeletal muscle. Targeting the PTH/PTHrP pathway may have therapeutic uses in humans with cachexia.

Keywords: PTHrP; adipose tissue browning; cachexia; cancer; chronic kidney disease; parathyroid hormone (PTH); skeletal muscle atrophy.

Figure 1. 5/6 nephrectomized mice develop cachexia with adipose tissue browning and skeletal muscle atrophy

(A-I) Mice underwent sham or 5/6 nephrectomy (5/6 Nx) surgery and were sacrificed 5 weeks later (n = 5-7). Blood Urea Nitrogen (BUN) (A), plasma PTH levels (B) and body weight (C) were measured. Mice were placed into metabolic cages between post-surgery weeks 2 and 3, when the difference in body weight was small. Oxygen consumption (VO₂) (D) and physical activity (E) were monitored. Fat and muscle tissues were dissected and weighed (F). mRNA levels in iWAT (G), iBAT (H) and gastrocnemius muscle (I) were determined by RT-qPCR. The values are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005. See also Figure S1 and S2.

Figure 2. PTH treatment elevates the thermogenic gene program

(A) Primary cultures of inguinal adipocytes were treated with 10 ng/ml PTHrP(1-34), 10 ng/ml PTH(1-34) or 25 ng/ml PTH(1-84) for 2 hr (n = 3). (B-D) Mice received a single dose of PTH(1-34) (1 mg/kg body weight; SubQ) and were sacrificed 2 hr later (n = 6). Gene expression changes were determined by RT-qPCR. The values are mean ± SEM. *P < 0.05, **P < 0.005, ***P < 0.0005.

Figure 3. The thermogenic gene program is increased in primary hyperparathyroidism patients

(A-D) Subcutaneous and deep cervical fat samples were collected from patients undergoing neck surgeries. Gene expression levels were compared between subcutaneous and deep cervical samples (n = 23) (A-B) or within subcutaneous fat (C) and deep cervical fat (D) of control and primary hyperparathyroidism patients (PHPT) (n = 11-12). mRNA levels were determined by RT-qPCR. Statistics by two-tailed t-test.

Figure 4. PTH and PTHrP are unable to induce thermogenic genes in the absence of PTHR

(A) Primary adipocytes from wild type and mutant cells were treated with 10 ng/ml PTHrP(1-34), 10 ng/ml PTH(1-34) or 10 nM Norepinephrine (NE) for 2 hr (n = 3). (B-D) Mice received a single dose of PTHrP(1-34) (1 mg/kg body weight; SubQ) and were sacrificed 2 hr later (n = 5-6). Gene expression changes were determined by RT-qPCR. Total UCP1 and ERK1/2 protein levels in inguinal fat tissue were measured by western blotting (D). The values are mean ± SEM. (*) refers to differences compared to the control group. (#) refers to differences between the WT and KO groups. *P < 0.05, ***P < 0.0005, ###P < 0.0005. See also Figure S3.

Figure 5. Adipo-PTHR-KO mice are resistant to 5/6 nephrectomy-driven cachexia

(A-F) Mice underwent sham or 5/6 nephrectomy (5/6 Nx) surgery and were sacrificed 5 weeks later (n = 5-6). Blood Urea Nitrogen (BUN) (A) and plasma PTH (B) levels were measured. Mice were placed into metabolic cages between post-surgery weeks 2 and 3, when the difference in body weight was small. Oxygen consumption (VO₂) (C), physical activity (D) and body weight (E) were monitored. Fat and muscle tissues were dissected and weighed (F). The values are mean ± SEM. (*) refers to differences between the Sham and 5/6 Nx groups. (#) refers to differences between the WT-5/6 Nx and KO-5/6 Nx groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005. See also Figure S4.

Figure 6. Adipose tissue browning and skeletal muscle atrophy are suppressed in 5/6 nephrectomized Adipo-PTHR-KO mice

(A-F) Mice underwent sham or 5/6 nephrectomy (5/6 Nx) surgery (n = 5-6). H&E staining of adipose tissues and gastrocnemius muscle were shown (A). mRNA levels in iWAT (B), iBAT (C) and gastrocnemius muscle (F) were measured by RT-qPCR. Total UCP1 and ERK1/2 protein levels in inguinal fat tissue were determined by western blotting (D). Muscle function was analyzed by grip strength (E). The values are mean ± SEM. (*) refers to differences between the Sham and 5/6 Nx groups. (#) refers to differences between the WT-5/6 Nx and KO-5/6 Nx groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05. See also Figure S5.

Figure 7. Adipo-PTHR-KO mice are resistant to LLC tumor-driven cachexia

(A-D) Mice inoculated with LLC cells were sacrificed 16 days later (n = 6). Carcass weight (calculated by subtracting tumor weight from the total weight) (A) and tumor weight (B) were shown. Fat and muscle tissues were dissected and weighed (C). H&E staining of adipose tissues and gastrocnemius muscle were shown (D). (E-G) Mice inoculated with LLC cells were sacrificed 14 days later (n = 4-5). mRNA levels in iWAT (E), iBAT (F) were measured by RT-qPCR. Total UCP1 and ERK1/2 protein levels in inguinal fat tissue were determined by western blotting (G). (H-I) Mice inoculated with LLC cells were sacrificed 16 days later (n = 6). Muscle function was analyzed by grip strength (G). mRNA levels in gastrocnemius muscle (H) were measured by RT-qPCR. The values are mean ± SEM. (*) refers to differences between the LLC and non-tumor-bearing groups. (#) refers to differences between the WT-LLC and KO-LLC groups. *P < 0.05, **P < 0.005, ***P < 0.0005, #P < 0.05, ##P < 0.005, ###P < 0.0005. See also Figure S6.