Novel Model of Oxalate Diet-Induced Chronic Kidney Disease in Dahl-Salt-Sensitive Rats

Abstract

Diet-induced models of chronic kidney disease (CKD) offer several advantages, including clinical relevance and animal welfare, compared with surgical models. Oxalate is a plant-based, terminal toxic metabolite that is eliminated by the kidneys through glomerular filtration and tubular secretion. An increased load of dietary oxalate leads to supersaturation, calcium oxalate crystal formation, renal tubular obstruction, and eventually CKD. Dahl-Salt-Sensitive (SS) rats are a common strain used to study hypertensive renal disease; however, the characterization of other diet-induced models on this background would allow for comparative studies of CKD within the same strain. In the present study, we hypothesized that SS rats on a low-salt, oxalate rich diet would have increased renal injury and serve as novel, clinically relevant and reproducible CKD rat models. Ten-week-old male SS rats were fed either 0.2% salt normal chow (SS-NC) or a 0.2% salt diet containing 0.67% sodium oxalate (SS-OX) for five weeks.Real-time PCR demonstrated an increased expression of inflammatory marker interleukin-6 (IL-6) (p < 0.0001) and fibrotic marker Timp-1 metalloproteinase (p < 0.0001) in the renal cortex of SS-OX rat kidneys compared with SS-NC. The immunohistochemistry of kidney tissue demonstrated an increase in CD-68 levels, a marker of macrophage infiltration in SS-OX rats (p < 0.001). In addition, SS-OX rats displayed increased 24 h urinary protein excretion (UPE) (p < 0.01) as well as significant elevations in plasma Cystatin C (p < 0.01). Furthermore, the oxalate diet induced hypertension (p < 0.05). A renin-angiotensin-aldosterone system (RAAS) profiling (via liquid chromatography-mass spectrometry; LC-MS) in the SS-OX plasma showed significant (p < 0.05) increases in multiple RAAS metabolites including angiotensin (1-5), angiotensin (1-7), and aldosterone. The oxalate diet induces significant renal inflammation, fibrosis, and renal dysfunction as well as RAAS activation and hypertension in SS rats compared with a normal chow diet. This study introduces a novel diet-induced model to study hypertension and CKD that is more clinically translatable and reproducible than the currently available models.

Keywords: animal model; chronic kidney disease; uremic cardiomyopathy.

Figure 1

Blood biochemistry analysis (a) of Dahl-Salt-Sensitive rats on oxalate diet (SS-OX) display increased serum albumin (p = 0.0019) (b), increased serum alkaline phosphate (ALP) (p = 0.0002) (c), increased serum amylase (p = 0.0075) (d), increased serum alanine transaminase (ALT) (p = 0.0002) (e), increased blood urea nitrogen (BUN) (p < 0.0001) (f), increased serum phosphorus (p < 0.0001) (g), increased serum creatinine p = 0.0183 (h), and increased serum potassium (p = 0.0057) (i), compared with Dahl-Salt-Sensitive rats on a normal chow diet (SS-NC) (t-test, * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 4–5).

Figure 2

Dahl-Salt-Sensitive rats on an oxalate diet (SS-OX) displayed a marked increase in renal inflammation, pro-fibrotic factors, and immune cell proliferation compared with Dahl-Salt-Sensitive rats on a normal chow diet (SS-NC). SS-OX presented greater levels of pro-inflammatory cytokine IL-6 (p < 0.0001) (a), interstitial inflammation (p < 0.0001) (b), and levels of CD-68 indicating increased macrophage infiltration (p = 0.0001) (c) vs. SS-NC (Magnification: 50 µM, t-test, *** p < 0.001, **** p < 0.0001, n = 10–16).

Figure 3

Dahl-Salt-Sensitive rats on an oxalate diet (SS-OX) displayed an increased presence of fibrosis-related gene tissue inhibitors of metalloproteinase (TIMP-1) (p < 0.0001) (a), increased renal fibrosis (p = 0.0008) (b), and kidney injury indicated by increased protein casts (p = 0.0239) (c) and tubular atrophy (p < 0.0001) (d) compared with Dahl-Salt-Sensitive rats on a normal chow diet (SS-NC) (Magnification: 50 µM, t-test, * p < 0.05, *** p < 0.001, **** p < 0.0001, n = 10–16).

Figure 4

Dahl-Salt-Sensitive rats on an oxalate diet (SS-OX) displayed increased hypertension, glomerular filtration rate, and renin angiotensin aldosterone system (RAAS) activation when compared with Dahl-Salt-Sensitive rats on a normal chow diet (SS-NC) (a) and increased urine protein excretion (UPE) (p = 0.0146) (b), increased levels of levels of cystatin C (p = 0.0064) (c), increased plasma angiotensin (p = 0.0015) (d), and decreased serum aldosterone (p = 0.0068) (e) (t-test, * p < 0.05, ** p < 0.01, n = 3–6).

Figure 5

Cardiac fibrosis and inflammation were markedly elevated in Dahl-Salt-Sensitive rats on an oxalate diet (SS-OX) when compared with Dahl-Salt-Sensitive rats on a normal chow diet (SS-NC). SS-OX rats displayed greater interstitial cardiac inflammation (p = 0.0001, n = 8–16) (a) and increased perivascular cardiac fibrosis (p = 0.0466, n = 4–8) (b) (Magnification: 50 µM, t-test, * p < 0.05, *** p < 0.001).

Figure 6

Dahl-Salt-Sensitive rats on an oxalate diet (SS-OX) presented pathological cardiac hypertrophy and left ventricular dysfunction, indicating uremic cardiomyopathy, when compared with Dahl-Salt-Sensitive rats on a normal chow diet (SS-NC). SS-OX rats displayed cardiac hypertrophy via increased cross-sectional area (CSA) of cardiomyocytes compared with SS-NC rats (p < 0.0001) (a,b). SS-OX rats also displayed increased heart posterior wall thickness (PWTd) (p = 0.0111) (c), increased septal wall thickness (SWTd) (p = 0.0452) (d), and increased relative wall thickness (RWTd) (p = 0.0423) (e) compared with SS-NC models (Magnification: 50 µM, t-test, * p < 0.05, **** p < 0.0001, n = 6–8).

Figure 7

This graphic displays the methods and timeframe used in this study with Dahl-Salt Sensitive (SS) rats fed a normal chow diet (SS-NC) and fed an oxalate diet (SS-OX) to develop the oxalate diet-induced CKD model. The measurements taken after the rats were euthanized 15 weeks later include urine protein excretion, renin–angiotensin–aldosterone-system (RAAS) profiling, blood pressure measurements, kidney collection to assess histological changes in renal architecture, and ultrasound analysis to identify cardiac remodeling.