A two-stage method to establish chronic kidney disease (CKD) in the Lewis rat by surgically removing 5/6th of renal mass is described. Combination of the surgical procedure, NOS-inhibition and a high-salt diet leads to a model resembling human CKD, allowing study of causal mechanisms and development of novel therapeutic interventions.
Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using ‘gold standard’ inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.
Due to its progressive nature, ensuing end stage kidney disease, and associated cardiovascular morbidity and mortality, CKD is a growing public health problem1. Slowing CKD progression is therefore a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. The kidney consists of a broad range of different cell types that interact with each other. This complexity cannot be mimicked in vitro.
To study novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well-known experimental model of progressive renal disease, resembling several aspects of human CKD2,3. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is associated with progressive intrarenal capillary loss4, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function5. We used a rat strain (Lewis) that is relatively resistant to development of CKD and therefore we combined removal of 5/6th of renal mass with nitric oxide (NO) depletion6, 7, 8 and a high salt diet9. After arrival and acclimatization, animals receive a NO synthase inhibitor (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy (UNX) with continuation of L-NNA after two days. One week later, subtotal nephrectomy (SNX) i.e. removal of 2/3rds of renal mass is performed on the left side. After SNX, animals are allowed to recover for 2 days followed again by 20 mg/L LNNA in drinking water for a period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. The reason to perform the UNX on the right side and the SNX on the left side is that the renal vessels are longer on the left side which makes it easier to access the kidney without stretching the vessels too much when the kidney is exposed outside the body. In literature, models are described in which the poles of the left kidney are removed first, followed by UNX of the right kidney one week later10,11,12. In our hands this model showed a much more rapid development of renal failure, but also a much larger variation in loss of renal function. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed, characterized by marked reduction in glomerular filtration rate (69%) and effective renal plasma flow (62%)13 hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD, providing a window of opportunity for evaluation of therapeutic interventions.
Surgical removal of 5/6th of renal mass in the Lewis rat, combined with a high-salt diet and temporary NOS inhibition leads to a model of CKD that resembles human CKD and allows study of causal mechanisms and efficacy of therapeutic interventions in CKD.
The 5/6th nephrectomy model is a well-known and extensively described model for CKD. However, simply removing 5/6th of renal mass does not lead to immediate renal failure in all rat strains. We use Lewis rats to study the effects of cell-based…
The authors have nothing to disclose.
We thank Krista den Ouden for her excellent technical assistance. This technique was financially supported by the Dutch Kidney foundation, grant C06.2174. M.C.V. is supported by the Netherlands organisation for Scientific Research (NWO) Vidi-grant 016.096.359.
Reagent | |||
L-NNA | Sigma-aldrich | N5501 | |
Spongostan dental: gel foam pads 1x1x1 cm | Johnson&Johnson | Ms0005 | |
Ethicon Vicryl FS-2S naald 4/0 V392H p/36 | Ethicon | V303H | |
Ethicon Vicryl RB-1+ naald 5/0 V303H p/36 | Ethicon | V392H | |
Buprenorphine (0.3 mg/ml) | Via local pharmacist ordered by Reckitt Benckiser pharmaceuticals | unknown | |
Equipment | |||
Student Tissue Forceps – 1×2 Teeth 12 cm | Fine Science Tools (FST) | 91121-12 | |
Student Standard Pattern Forceps | FST | 91100-12 | |
Mayo Scissors | FST | 14010-15 | |
2X Semken Forceps | FST | 11008-13 | |
Student Iris Scissors | FST | 91460-11 | |
Olsen-Hegar Needle Holder | FST | 12002-14 |