5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Summary

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.

Abstract

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.

Introduction

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 CKD^2,3. 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) depletion^{6, 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 later^10,11,12. 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.

Protocol

All experiments are executed in accordance to the animal experimental ethical guide lines of the Utrecht experimental animal committee. The protocol is performed under the guidance and approval of the author’s institution’s animal care and use committee. CKD is induced in male inbred Lewis rats (Charles River, Sulzfeld, Germany) at the age of 8 weeks. Rats are housed under standard conditions in a light-, temperature- and humidity-controlled environment. 1. Surgery Pr…

Representative Results

After subtotal nephrectomy, approximately 1/6th of total renal mass is left. Figure 4 shows the weight of the removed part of the right kidney with mean and standard deviation in two previous experiments. One should keep in mind that in the week after UNX, hypertrophy of the left kidney occurs; indicating that the weight that needs to be removed calculated based on the weight of the right kidney always results in less than 5/6th removal. However, since it is not possible to determine the weight of the le…

Discussion

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…

Materials

			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