Research Suggests a Novel Cell-Based Therapy for Chronic Kidney Disease and CKD-Associated Heart Failure


This Nephrology Now Research Showcase is submitted by Darren A. Yuen MD and Richard E. Gilbert MD PhD.   Nephrology Now Research Showcases summarize important bodies of work in clinical or experimental Nephrology.

Background

New therapies are needed for CKD and its cardiovascular complications

Chronic kidney disease (CKD) is a major cause of hospitalization, premature mortality, diminished quality of life and heath care expenditure [1].  Despite its importance and increasing prevalence, little progress in its treatment has been made over the past 20 years, with blood pressure reduction and blockade of the renin-angiotensin system still the mainstays of therapy.

Patients with CKD are not only at high risk of developing heart failure, but frequently have heart failure with preserved left ventricular ejection fraction [2].  These patients with CKD and diastolic dysfunction are a highly prevalent population that is faced with a particularly poor prognosis [2].  Importantly, in contrast to patients with reduced left ventricular ejection fraction, there is no evidence-based treatment for those with preserved ejection fraction in which RCTs failed to show a beneficial effect for either ACE inhibitors [3] or angiotensin receptor blockers [4,5].  Additional therapies are therefore needed.

The importance of fibrovascular injury in mediating progression of renal and cardiac injury in CKD

Renal and cardiac biopsy studies of animals and patients with progressive CKD demonstrate progressive capillary loss and fibrosis in both organs [6,7].  These two inter-related pathological features are thought to contribute to progressive injury and dysfunction in the kidney and heart in CKD [8].

Bone marrow-derived early outgrowth cells (EOCs)The bone marrow harbours novel cell populations with potent tissue protective and regenerative properties [9].  Named for their early appearance in culture (7 – 10 days) when peripheral blood mononuclear cells [10] or whole bone marrow cells [11,12] are grown in endothelial culture medium, early outgrowth cells (EOCs) have been shown to exert potent pro-angiogenic [13,14,15] and anti-fibrotic [12,16] effects in various disease models.

EOCs exert powerful renal and cardiac protective effects in experimental CKD

Intra-venous EOC infusion attenuates renal and cardiac damage and dysfunction

As shown in our Study Design depicted in Figure 1, using the 5/6 subtotal nephrectomy (SNX) rat as a model of progressive experimental CKD that develops diastolic dysfunction, we demonstrated that a single intra-venous infusion of 106 EOCs significantly attenuated the progressive capillary loss and fibrosis that occurs in the kidney and heart of animals with established experimental CKD (Figures 2 – 4) [12].

Figure 1. Study DesignFigure 1. Study Design.

Rats were randomized to either 5/6 subtotal nephrectomy surgery (SNX) to induce the onset of chronic kidney disease or sham surgery. 4 weeks post-surgery, at a time when CKD is established, SNX animals were further randomized to receive: (1) an intra-venous injection of 106 EOCs, (2) an intra-aortic injection of 106 EOCs, or (3) an intra-venous injection of phosphate-buffered saline 12

 

Figure 2. Renal fibrosis is attenuated by EOC therapy in the SNX rat.Figure 2. Renal fibrosis is attenuated by EOC therapy in the SNX rat.

Tubulointerstitial fibrosis was assessed following immunolabelling for Type IV collagen 8 weeks post-surgery. (A – C) Representative cortical tubulointerstitial images. Original magnification x 160. (A) Sham animal. (B) SNX animal. (C) SNX – EOC animal. (D) Type IV collagen % positivity. * p < 0.05 vs. sham operated animals. † p < 0.05 vs. SNX animals. Glomerulosclerosis was assssed on PAS-stained kidney sections at 8 weeks post-surgery. (E – G) Representative glomerular images. Original magnification x 400. (E) Sham animal. (F) SNX animal. (G) SNX – EOC animal. (H) Glomerulosclerosis index (GSI). * p < 0.05 vs. sham operated animals. † p < 0.05 vs. SNX animals {12}

Figure 3. Renal capillary density loss is attenuated by EOC therapy in the SNX rat.Figure 3. Renal capillary density loss is attenuated by EOC therapy in the SNX rat.

(A – C) Kidney sections were immunolabelled with JG-12 antibody, which recognizes a glomerular endothelial cell antigen. Representative JG-12 immunostained images. Original magnification x 400. (A) Sham animal. (B) SNX animal. (C) SNX – EOC animal. (D) % positive area for JG-12 immunostaining per glomerulus. (E – G) Representative fluorescence microangiography (FMA) images of glomeruli. The autofluorescence of the surrounding renal cortex was captured and pseudocolourized red. The green fluorescence of the infused beads outlines microvascular structures. Original magnification x 40. (E) Sham animal. (F) SNX animal. (G) SNX – EOC animal. (H – J) Representative FMA images of peritubular capillaries. Each image is a flattened Z-stack of a 100 m section taken with a confocal microscope. Original magnification x 75. (H) Sham animal. (I) SNX animal. (J) SNX – EOC animal. * p < 0.05 vs. sham operated animals. † p < 0.05 vs. SNX animals 12

Figure 4. Cardiac fibrosis is attenuated by EOC therapy in the SNX rat.Figure 4.  Cardiac fibrosis is attenuated by EOC therapy in the SNX rat. Importantly, these changes were associated with preservation of organ function, as manifested by a reduction in plasma creatinine, urinary protein excretion, and left ventricular (LV) end-diastolic pressure-volume relationship, a measure of LV stiffness (Figures 5 – 6) [12].

 

 

 

Figure 5.  Deterioration of renal function is reduced by intra-vascular EOC therapy in the SNX rat.Figure 5.  Deterioration of renal function is reduced by intra-vascular EOC therapy in the SNX rat.

(A) Plasma creatinine. (B) Urinary protein excretion 12

Figure 6.  Left ventricular stiffness is reduced in SNX rats by EOC therapy.Figure 6.  Left ventricular stiffness is reduced in SNX rats by EOC therapy.

SNX rats develop progressive left ventricular stiffness (diastolic dysfunction) that manifests itself as an increased left ventricular end-diastolic pressure volume relationship (LV EDPVR). This phenotype mimics a common and high risk CKD patient population. Using gold standard invasive cardiac catherization techniques, we measured the pressure and volume within the left ventricle throughout the cardiac cycle under varying preload conditions 8 weeks post-surgery to generate pressure-volume loops. Left ventricular end diastolic pressure volume relationship (LV EDPVR), a measure of passive LV relaxation, is represented by the slope of the tangent of the base of each PV loop (green lines). (A) Sham animal. (B) SNX animal. (C) SNX – EOC animal. (D) Quantitative analysis. * p < 0.05 vs. sham operated animals. † p < 0.05 vs. SNX animals 12

EOCs mediate their benefits from remote locations in the body

 

To test whether the beneficial effects of EOC infusion are dependent upon EOC delivery to the kidney and heart, we compared the effects of intra-arterial versus intra-venous EOC infusion.  Interestingly, both the renal and cardiac tissue protective effects of EOC infusion were observed regardless of route of intravascular administration (Figure 7) [12].

To determine where the EOCs localize post-infusion, we fluorescently labeled the EOCs, and infused them into SNX rats.  Despite the dramatic structural and functional benefits seen upon EOC infusion in the kidney and heart, very few labeled cells were found in either organ, contrasting with their relative abundance in the liver and spleen [Figure 8] [12].

Figure 7. EOC therapy improves renal and cardiac outcomes regardless of route of administration (intravenous vs. intra-arterial).Figure 7.  EOC therapy improves renal and cardiac outcomes regardless of route of administration (intravenous vs. intra-arterial).

(A) Proteinuria. (B) Systolic blood pressure. (C) Glomerulosclerosis. (D) Glomerular endothelial (JG-12) immunostaining. (E) Tubulointerstitial type IV collagen immunostaining. (F) Left ventricular end diastolic pressure-volume relationship. (G) Myocyte cross-sectional area. (H) Cardiac interstitial fibrosis. Abbreviations: SNX – iv: 5/6 nephrectomy (SNX) animal treated with intravenous EOC infusion. SNX – ia: SNX animal treated with intra-arterial EOC infusion. BP: blood pressure. LV EDPVR: left ventricular end diastolic pressure-volume relationship. PSR: picrosirius red 12

Figure 8. Following infusion, EOCs do not localize to the kidney or heart, but rather to the liver.Figure 8.  Following infusion, EOCs do not localize to the kidney or heart, but rather to the liver.

10X6 fluorescently labeled EOCs were infused intra-venously into SNX rats 4 weeks post-surgery, and organs harvested 4 days post-cell infusion to determine EOC localization. EOCs were found in significant numbers in the liver, but not in the kidney or heart. (A – C): Representative confocal microscopy images of kidney, heart, and liver respectively at 4 days post-EOC infusion. Original magnification x 20. (a) Kidney cortex. (b) Heart. (c) Liver. (d) Time course of EOC retention in kidney, heart, and liver. n = 3 animals per time point. * p < 0.05 vs. kidney 12

Conclusion

The results of our studies are the first to demonstrate that EOC infusion may significantly attenuate the renal and cardiac injury that accumulates as CKD progresses.  Given the marked morbidity and mortality associated with CKD, we believe that our data may form the preclinical foundation for clinical trials of EOC therapy for CKD and CKD-associated heart failure.

References

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