SMALL VESSELS, BIG ROLE: RENAL MICROCIRCULATION AND PROGRESSION OF RENAL INJURY

Introduction

Chronic kidney disease (CKD) is a growing health problem. Data from the US Renal Data System Reports show that the number of patients enrolled in end-stage renal disease (ESRD) Medicare-funded programs increased a staggering 60-fold during the last 4 decades^1-4 and now consumes a significant portion of the health-care budget. Despite the magnitude of resources dedicated to treatment of chronic renal disease and the substantial improvements in the quality of dialysis and co-adjuvant therapeutic strategies, these patients experience significant reductions in their quality of life, increased morbidity, and higher mortality. Furthermore, a recent projection shows that the incidence and prevalence of CKD may continue to increase in coming decades⁵. This somber scenario emphasizes the need for a better understanding of underlying mechanisms of renal injury and the development of novel interventions and strategies to slow the onset and progression of CKD.

Microvascular (MV) networks are dynamic anatomical units that are tightly balanced to provide nutrition and remove waste products in order to meet the metabolic and functional demands of each tissue. In the kidney, the glomerular and peritubular capillaries also command glomerular filtration, tubular reabsorption, and recirculation of body fluids, nutrients, hormones, and other substances to the body^{6, 7}. Endothelial dysfunction as well as functional and structural rarefaction⁸ of the renal microvessels play a prominent role in inducing renal injury associated with major cardiovascular risk factors such as hypertension, dyslipidemia, diabetes, and atherosclerosis. Furthermore, a defective renal microcirculation is a universal pathological feature in CKD that progresses as CKD evolves and compromise both the renal nutrition and renal function^{6, 7}.

The current review will focus on the role of MV disease in the progression of renal injury. It will discuss the involvement of MV disease as both cause and consequence of pathological mechanisms affecting the kidney. Finally, I will also discuss the potential of therapeutic interventions to protect the renal microvasculature using clinically available and experimental treatments. Although promising^9-15, the success of targeted MV therapies may depend on how severe and extensive the MV damage is and mainly, whether glomeruli are lost or still viable, narrowing the window of opportunity to change the progressive nature of CKD/ESRD.

Epidemiology, major causes, and pathological contributors to chronic kidney disease

The emergent population of CKD/ESRD patients in recent years is linked to dramatic increases in obesity and obesity-associated cardiovascular risk factors such as lipid abnormalities, atherosclerosis, diabetes, and hypertension. Although these comorbid conditions may develop separately, they often co-exist and may potentiate injury of target organs, including the kidneys, in an additive or synergistic fashion^{7, 16, 17}.

Recent reports from the Centers for Disease Control¹⁸, the National Institutes of Health¹⁹, and the World Health Organization²⁰ show that obesity has more than doubled since 1980. Currently, 68.8% of adults in the U.S. are overweight or obese¹⁹ (with a slightly higher prevalence in women- 40.4 vs. 35%²¹), with 6-8% of them having extreme obesity (body/mass index, BMI, over 40). Furthermore, about 33% of the U.S children and adolescents are overweight and over 18% are obese^{18, 19}. Unfortunately, the prevalence (and consequently, impact) of obesity will likely continue to increase unless these trends can be reversed, since approximately 80% of obese children may become obese adults with the associated increased susceptibility to develop cardiovascular, metabolic, and kidney diseases.

Dyslipidemia and atherosclerosis are parts of a chronic and systemic inflammatory process that compromises the function and structure of small and large vessels. They are frequently associated with chronic renal disease and may serve as cause and as consequence of CKD. Not only by the build-up of atherosclerotic plaques, but also at preceding stages of vascular fatty streaks or microscopic lipid accumulation, dyslipidemia may promote vascular dysfunction and early remodeling in several vascular beds including the kidneys. Indeed, MV and glomerular dysfunction may precede the onset and represent the silent phase of chronic renal disease^{22, 23}. Experimental studies indicate that diet-induced lipid abnormalities leading to renal endothelial dysfunction, intrarenal inflammation, fibrosis, and a significant vascular dysfunction, damage, and remodeling^22-25 on renal vasculature. Furthermore, recent studies showed that dyslipidemia superimposed on experimental renal artery stenosis can not only accelerate renal MV dysfunction and remodeling, but also MV loss, underscoring ample deleterious effects of atherogenic factors on the renal parenchyma^{23, 26}.

The major causes of CKD and ESRD are diabetes and hypertension, which lead to progressive MV damage and loss and set the stage for evolving renal injury^27-30. Hypertension is the second leading cause of CKD/ESRD in the US after diabetes, is estimated to affect 1 billion people worldwide, and is responsible of 9 million deaths per year³¹. The recent SPRINT study demonstrated that aggressive control of blood pressure decreases risk for cardiovascular events and even death³², which underscores the pathophysiological importance of hypertension and its priority for treatment. The effect of high blood pressure on the renal vessels is a major driving force for the early development of vascular remodeling in both large and small vessels, which precedes, predicts, and significantly contributes to development of overt renal abnormalities^33-35. Diabetes is the number one cause of CKD/ESRD and has almost quadrupled in the last three decades, affecting 422 million adults worldwide. These staggering numbers are largely due to the rise in type 2 diabetes, which accounts for up to 95% of the 25.8 million cases of diabetes in the US and is driven by overweight and obesity³⁶. The development and mechanisms of diabetes-induced renal injury and progression towards diabetic nephropathy have been reviewed and discussed elsewhere^{37, 38} and are beyond the scope of this review. However, it is important to emphasize that, as in hypertension, renal MV abnormalities in diabetes also lead and foresee the deterioration of renal function, underscoring the central role of MV disease in development and progression of renal injury^{27, 29}.

Life expectancy has significantly increased in the past 50 years³⁹. Aging carries a physiological decline in renal function which may be accelerated or aggravated by comorbid conditions. Indeed, with time the kidney shows an age-associated reduction of function disclosed by a progressive (but widely variable) decline in glomerular filtration rate (GFR) and renal blood flow (RBF), which is possibly driven by reduced renal bioavailability of NO and often associates with different degrees of renal parenchymal damage⁴⁰. Importantly, most of these changes are driven by functional and structural changes of the renal microcirculation at the pre- and post-glomerular level, an increase in glomerular capillary hydraulic pressure, and parenchymal changes that lead to loss of renal mass, MV remodeling, and tubule-interstitial fibrosis^{41, 42}. Although such changes do not always translate into CKD and may not require interventions in a disease-free individual, they can increase the susceptibility of the aging kidney to acute kidney injury and/or make it more labile for the development of CKD^{40, 41}.

The renal microvascular network

Progressive ramifications from the main renal artery branch into interlobar, arcuate, and interlobular arteries towards the smaller branching afferent arterioles leading to the glomerular capillaries where fluid and solutes are filtered (except for plasma proteins). Then, the distal ends of the glomerular capillaries converge to form the efferent arterioles, which are followed by a second capillary network, the peritubular capillaries, which are key components for filtration, secretion, and reabsorption of minerals and removal of waste from the filtered blood that will be excreted by urine. On the venous side, the small veins run in parallel to the arterioles to subsequently form the interlobular, arcuate, interlobar, and renal vein, which leaves the kidney beside the renal artery and ureter. This unique vascular network deals with about 1.1 l/min of the cardiac output and only 10% of the delivered oxygen is normally sufficient to satisfy the renal metabolic demands⁶. Thus, the majority of the workload on the renal MV networks is to maintain body homeostasis, which underscores the importance of renal function in health and disease.

The intra-renal MV network may be disrupted from a functional or a structural angle. MV rarefaction is defined as a reduction of available vessels which can be divided in functional (the vessels are anatomically present but with a deficient or absent perfusion) and structural (anatomical reduction in the number of vessels in the tissue) rarefaction⁸. A study by Bohle⁴³ et al demonstrated in biopsies of patients with chronic renal disease from different etiologies a significant inverse relationship between the number and area of the cortical tubular capillaries and their serum creatinines, showing that MV rarefaction develops in human CKD and negatively correlates with renal dysfunction.

It is important to emphasize that these two forms of MV rarefaction are not mutually exclusive, can co-exist, and can be parts of a progressive process that goes from dysfunction to loss of microvessels. A potential bridge between functional and structural rarefaction could be defined as MV remodeling, in which anatomical changes in the MV wall develops, progressively contributes to disrupt MV function, and may lead towards MV loss. Finally, renal MV rarefaction may also develop as a physiological event in the aging kidney, and is suggested as a major determinant in the decreased RBF and GFR⁴⁴ associated with age. Therefore, since smaller arterioles and capillaries are primary targets of acute or chronic insults^{11, 12, 45, 46} and their damage may impact on renal hemodynamics, function, and progression of renal injury, MV rarefaction (functional and structural, physiological or pathological) plays an important role in progression of renal injury regardless of the primary etiology.

Mechanisms of kidney injury driven by MV rarefaction

Work in progress: MV endothelial dysfunction

Renal MV endothelial dysfunction is a central mechanism driving functional rarefaction as it may contribute to the transition from functional to structural rarefaction. It results from abnormal function of endothelial cells combined with reduced availability of substances that are produced by or act on the endothelium to determine vascular tone, permeability, fluid balance, and cell proliferation^{47, 48}. Endothelial dysfunction develops early in cardiovascular and renal disease and is a consequence and a contributor for the development and progression of hypertension, diabetes, atherosclerosis, and chronic heart and renal pathologies^49-52.

A central player in endothelial dysfunction is deficiency of nitric oxide (NO), a gaseous molecule that controls vascular tone and regulates inflammatory and coagulant properties of the endothelium (beyond the scope of this review). A reduced availability of NO may be the result of altered production from major sources such as endothelial NO synthase (eNOS) or augmented NO removal. Studies from Goligorsky et al^{53, 54} demonstrated that inhibition of NOS and deficiency of NO are powerful pro-fibrotic stimuli that increase endothelial to mesenchymal transdifferentiation and may contribute to MV endothelial dysfunction and subsequent MV rarefaction. Furthermore, an altered production by eNOS can also result from a lack of a key cofactor such as tetra-hydrobiopterin, which results in eNOS uncoupling and switches eNOS from production of NO to generation of reactive oxygen species (ROS)⁵⁵. In turn, ROS decrease renal MV bioavailability of NO through ROS-mediated quenching effects⁵⁶. Factors that favor deleterious ROS-NO interactions in the kidney include excessive activation of the reninangiotensin-aldosterone (RAAS) and endothelin (ET) systems^{55, 57}. These systems play important physiological roles in controlling renal hemodynamics but are often up-regulated in cardiovascular and renal disease and contribute to vasoconstriction and endothelial dysfunction directly and via stimulation of ROS production. Increased ROS may in turn stimulate production and activation of redox-sensitive pro-inflammatory and pro-fibrotic factors in the kidney, such as nuclear factor kappa (NFk)B, tumor necrosis factor (TNF)-α, transforming growth factor (TGF)-β, or connective tissue growth factor (CTGF), to name a few, which are often involved in development and progression of renal injury^{22-24, 58}. Thus, ROS-mediated reduction in renal NO takes center stage in pathological effects associated with endothelial dysfunction and may promote vasoconstriction, vascular inflammation and tissue damage⁵⁹, which in turn further reduce NO bioavailability and perpetuates a vicious circle.

Work in progress: From MV constriction to MV remodeling

In parallel, a dysfunctional or damaged endothelium may lead to a sustained renal vasoconstriction which may lead to inadequate intrarenal nutrition as well as compromised renal hemodynamics and function. A prolonged vasoconstriction of intra-renal microvessels may lead to inward remodeling, a structural MV alteration that is a prominent mechanism for development and progression of renal damage in hypertension and renal ischemia^60-62. Our previous studies in a model of chronic renovascular hypertension and renovascular disease demonstrated that progressive renal injury associates with significant intra-renal MV remodeling, disclosed by increased MV media-to-lumen ratio, perivascular fibrosis, and decreased MV diameter^{23, 60, 63}. Such changes in the kidney microvasculature were mainly observed in those vessels under 200 μm in diameter, which suggest that pre-and post-glomerular microvessels are susceptible targets and likely contributors to development of renal injury.

A combination of a sustained vasoconstriction and a pro-injurious milieu likely pave the way for progression of functional rarefaction towards MV remodeling and eventually loss of the smaller vessels, which may contribute to further renal injury. Furthermore, a recent study demonstrated that renal endothelial cells show a distinctly poor endogenous proliferative ability, which may play a role in the limited intrinsic regenerative capacity of renal capillaries⁶⁴ and may significantly contribute to the progressive nature of renal MV rarefaction. However, studies have also shown that therapeutic interventions using different compounds such as clinically available antioxidant vitamins, statins, ET receptor or RAAS blockers, or experimental agents can preserve or recover endothelial function, improve renal hemodynamics, and reduce the development and progression of renal injury in hypertension, diabetes, atherosclerosis, acute and chronic renal injury ^{4, 12, 24, 45, 65-70}. It is important to emphasize that such improvements are often accompanied by attenuated remodeling of the renal MV architecture, underscoring the notion that MV disease is an evolving process that, if unattended, actively participates on the progression of renal damage. Moreover, MV remodeling may also diminish the efficacy of therapeutic interventions⁶¹, suggesting a potential tipping point of MV damage that could limit recovery and lead the progression towards irreversible tissue damage and loss of function.

Renal MV loss: cause and consequence of progressive renal damage

Loss of the microvasculature in any tissue or organ may reflect the final stage of progressive loss of MV homeostasis paired with disruption of healing mechanisms. Indeed, plasticity of the renal microcirculation to adapt to a new environment or to generate from preexisting vasculature as needed are important characteristics that may be lost as diseases progress, as can be observed in atherosclerosis²⁵, ischemia^{12, 63}, or diabetes-induced renal injury²⁹.

One of the pivotal players for maintenance and repair of MV networks everywhere, including the kidney, is vascular endothelial growth factor (VEGF). This pro-angiogenic cytokine play important roles in the kidney that go beyond vasculo-protective effects^{3, 71}.VEGF is highly ubiquitous and the renal cells are sources and targets of this cytokine. Major sources of renal VEGF are tubular epithelial cells and podocytes, and major targets are endothelial cells and podocytes as well, suggesting renal autocrine and paracrine effects^{3, 72, 73}.

Progressive dysfunction, damage, and loss of endothelial cells, and glomeruli and peritubular MV drop-out paired with a marked reduction of VEGF expression have been described in clinical⁷⁴ and experimental settings^{15, 75-77}. Altered expression and availability of renal VEGF coupled with MV abnormalities has been demonstrated in CKD, renal ischemia, early diabetic-induced renal injury, and diabetic nephropathy^{11, 27, 29, 78}. The decreased renal bioavailability likely results from loss of renal sources VEGF (e.g. proximal tubular cells, podocytes^{3, 11}), altered VEGF-upstream signaling⁶³ and possibly, disruption of post-translational mechanisms of VEGF¹². The decrease in renal VEGF also impacts on the VEGF receptors-mediated downstream angiogenic signaling, as shown by the blunted expression of renal angiopoietins, Akt and ERK ½, which are prominent mediators of endothelial cell survival, proliferation, and maturation of newly generated vessels⁹. Furthermore, insufficient or reduced renal VEGF may also have a major impact on preceding steps that involved mobilization and homing of cell progenitors towards MV repair, proliferation, and tissue healing¹³. Indeed, a decreased in renal VEGF also drives the blunted expression of stromal-derived factor 1 (SDF-1), angiopoietins, and Oct-4, which are prominent factors involved in progenitor cell biology that are all recovered after improving VEGF signaling^{9, 13, 15}. Therefore, in a context of blunted renal bioavailability of VEGF, the impetus for MV proliferation and repair is severely diminished, leading to a reduction in renal MV density and may become a central progressive mechanism of renal parenchymal damage.

The renal MV network can also be disturbed or reduced by other mechanisms. Hepatocyte growth factor (HGF) is a pleiotropic cytokine with distinct renoprotective roles, as has been demonstrated in diabetes-induced renal injury, acute, and chronic renal ischemia^{10, 79, 80}. HGF promotes tissue healing by stimulating mobilization of cell progenitors, by interactions with VEGF⁸¹ to promote MV proliferation and repair, and by counteracting renal inflammation, fibrosis, and apoptosis via TGF-β, NFkB, and Bax/BcL inhibition^{10, 80, 82-84}. Reduced bioavailability of this factor significantly accelerates development of MV rarefaction, renal inflammation and fibrosis^{10, 80, 83} supporting an important role of HGF to protect the renal parenchyma and preserve the renal microvasculature.

Renal fibrosis is the common pathway of advanced renal disease that is closely related to MV rarefaction. A recent study from Ehling et al using 3 different mouse models of progressive renal disease showed that abnormalities in the renal MV function and structure, such as reductions in MV diameter and increased MV tortuousity, develop early and may precede and contribute to development of renal fibrosis, supporting a prominent role of MV abnormalities as a possible universal mechanism for progression of tissue damage⁸⁵. Fibrosis is the loss of functional tissue that is replaced by non-functional scarred tissue that reflects a marked imbalance between extra-cellular matrix (ECM) production and degradation towards ECM accumulation. The renal accumulation of ECM in turn may induce powerful effects on renal MV development, proliferation, and function⁸⁶ directly and via increasing anti-angiogenic products. Indeed, the ECM is a rich source of factors that may diminish MV proliferation and repair such as endostatin, a potent inhibitor of angiogenesis and VEGF and a prominent contributor to MV rarefaction in CKD⁸⁷. Another ECM-related anti-angiogenic factor that may contribute to renal MV loss is angiostatin⁹, a potent pro-apoptotic and anti-VEGF factor that can contribute to development of tubular and interstitial damage by inducing capillary fragility and dropout^{88, 89}. These anti-angiogenic factors may interfere with VEGF and reduce MV repair and proliferation, which may negatively impact renal tissue healing and functional recovery independent of the initial insult. In depth discussion of molecular mechanisms of renal fibrosis are beyond the scope of this review and readers are suggested to consult published literature^{90, 91}.

Another pathway to renal MV loss is apoptosis or programed cell death, a prominent mechanism by which podocytes³, renal endothelial and epithelial cells may be killed when facing acute or chronic ischemia and a contributor for the progression of vascular dropout and tubular injury^{92, 93}. An ischemic/hypoxic renal milieu may stimulate apoptosis and thus exacerbate renal injury via the intrinsic (caspase-dependent) and extrinsic (caspase-independent) pathways^{3, 4, 94}. Apoptosis of renal endothelial cells could be driven and enhanced by a reduction in bioavailability of angiogenic factors such as VEGF or HGF, which not only stimulates migration and proliferation, but also promotes cell survival via powerful anti-apoptotic effects^95-97. In turn, ischemic-induced apoptosis of podocytes may contribute to the loss of renal VEGF³, which may further accelerate endothelial cell death and reduce compensatory renal MV proliferation and repair. Our recent studies in a swine model of chronic renal artery stenosis showed that a reduced expression of these factors in the ischemic kidney parallels increased apoptotic activity and apoptotic cells, progressive MV rarefaction, and evolving renal dysfunction and damage^{4, 11, 66}. Thus, apoptosis may serve as an important contributor for renal MV endothelial cell damage and loss, development of MV rarefaction, and may increase the risk for progression of renal injury. However, unlike necrosis, apoptosis is an energy-dependent mechanism of cell death and the extent of its pathophysiological role in MV damage and loss may be context-dependent and determined by the severity of the initial renal insult and development of renal disease⁹⁴.

Therapeutic strategies: is protection of renal microvessels feasible?

Severity of MV damage and loss may define the limits between reversible and irreversible renal injury, and success in restoring kidney function may depend on how far MV rarefaction has progressed (Figure 1). The different degrees of MV dysfunction and damage, which may in turn depend on the extent of the initial insult or length of the disease, may offer an opportunity for therapeutic targeting of renal MV disease to stimulate MV repair, peritubular and glomerular capillary regrow. Evidence from our laboratory supports the notion that renal functional and structural damage could be ameliorated by targeted interventions that reverses renal MV rarefaction ^{9, 11, 15} (Figure 2). However, such approach may need to be initiated before the entire glomerulus is lost. It is possible that generation of new vessels that shunt preexisting damaged ones and stimulation of MV repair may contribute to restoration of blood flow and to improve GFR in partly damaged or hibernated but recoverable nephrons^{98, 99}.

Figure 1.

Schematic overview of the progressive nature of chronic kidney disease (CVD) and contributions of microvascular (MV) rarefaction to the process.

Regardless of the etiology (e.g. hypertension, diabetes mellitus, atherosclerosis, obesity), CKD universally associates with MV rarefaction. A progressive damage of the microcirculation deteriorates renal hemodynamics and perfusion, leading to a progressive loss of filtration, tubular function, and development of fibrosis, which in turn serves as a feedback mechanism that may further accelerates progression of CKD towards irreversible renal injury.

Therapeutic interventions (grey box, clinically available and/or experimental) to protect the renal MV architecture and function (remodeling, rarefaction, regeneration, repair) may be more effective to improve renal function and slow the progression of CKD if applied before severe reductions in renal blood flow and nephron loss (above red dotted lines).

Figure 2.

Representative picture showing renal fibrosis (top, x20) and 3D micro-CT reconstruction of the renal MV architecture (bottom), tomographically isolated microvessels (yellow arrow, bottom), and cross sections of microfilm-perfused kidneys (orange arrow, bottom, stereo-microscopy, x20) in normal, stenotic kidney, and stenotic kidney after VEGF therapy.

The stenotic kidney has a significant MV loss, increased MV tortuousity, MV remodeling, and fibrosis after 10 weeks of renal artery stenosis. A single intra-renal administration of VEGF after 6 weeks of renal artery stenosis largely reversed most of these changes (quantified 4 weeks later). Visibly perfused glomeruli correlates with changes in density of renal MV.

G: glomeruli; CT: computerized tomography

Reproduced and modified with notification from: Chade A.R., Am J Physiol Regul Integr Comp Physiol 300: R783-R790, 2011.

MV protection or targeted renal MV angiogenesis strategies are not yet fully developed and therefore current therapies have not focused on preventing rarefaction of the renal microcirculation. This section will discuss the effects of clinically-available and experimental therapies and their effects on renal MV function.

Direct and indirect MV protection: anti-hypertensive, anti-diabetic, and lipid-lowering drugs

Diabetes, hypertension, and lipid abnormalities are major causes of CKD. Therapeutic agents often used in these diseases display effects on MV function, remodeling, and/or even proliferation which seem to be a “pleiotropic” effect of these compounds. Previous studies demonstrated that insulin¹⁰⁰, angiotensin receptor blockers¹⁰¹, or statins⁶⁰ play important roles in regulating endothelial function, vascular tone, and tissue perfusion by preserving or stimulating production of NO in endothelium and MV perfusion in different organs. Furthermore, other studies showed that the effects of these agents may also extend to attenuating MV remodeling and even promoting MV proliferation in several vascular beds like in the heart and kidney^{60, 68, 102, 103}. Therefore, part of the renoprotective effects of these agents may result from preserving renal MV function and integrity, directly or indirectly, as well as from controlling risk factors such as hyperglycemia, high blood pressure, and dyslipidemia.

For example, studies showed that insulin, beyond glucose control, may stimulate NO generation and pro-angiogenic activity in endothelial cells (e.g. migration, tube formation¹⁰⁴), which may be a prominent mechanism in insulin-mediated healing actions in different tissues^{105, 106} that seems to be independent of VEGF signaling¹⁰⁷. Similar stimulatory effects on angiogenesis has been described in experimental settings, both in vitro and in vivo, for new generation of clinically available oral anti-diabetic drugs such as sitagliptin¹⁰⁸ and for glucagon-like peptide-1 (GLP-1) agonists in a dose-dependent manner¹⁰⁹. On the other hand, metformin has been suggested to improve MV density in experimental stroke by increasing VEGF expression and activity¹¹⁰, although these effects may be short-term and are disputed by other studies suggesting net anti-angiogenic action of metformin¹¹¹. However, the use of metformin or GLP-1 agonists is limited by deterioration of renal function in diabetic patients, thus its effects on the renal MV function and structure, if any, may be limited and warrants additional studies^{112, 113}. Furthermore, renal vasculo-protective effects of sitagliptin have not been yet determined.

Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers (ARBs) are among the first line of anti-hypertensive drugs. Beyond blood pressure control, studies have shown that ARBs can have an impact upon a number of other physiological processes such as angiogenesis. In the kidney, studies using a porcine model of renal disease showed that ARB therapy results in improved renal hemodynamics, decreased oxidative stress (possibly driven by increase in NO bioavailability), and improved MV density and angiogenic signaling, implying distinct effects on renal MV functional and structural rarefaction^{68, 114}. However, the exact role of ARBs in angiogenesis is still controversial. Indeed, studies have reported pro- or anti-angiogenic effects driven by modulation of VEGF signaling, inflammation cytokines, and apoptosis that seems to depend on the tissue environment, disease state, and animal model investigated. In depth discussion of these controversies are beyond the scope of this review and the readers are encouraged to consult excellent literature¹¹⁵.

ET-1 receptor blockers, currently used to treat pulmonary hypertension, also showed renoprotective actions. ET-1 is one of the most powerful vasoconstrictors, exerts its effects through specific ET-A and –B receptors, and plays important roles in controlling ET-1 renal clearance, normal renal blood flow, and sodium handling¹¹⁶. These effects were the major impetus for early attempts to use ET-1 receptor blockers as anti-hypertensive drugs¹¹⁶. Emerging research demonstrated that blockade of the ET-A receptors also led to significant renoprotection. Experimental studies showed that chronic blockade of the ET-A (but not ET-B) receptors significantly reduces renal oxidative stress, inflammation, fibrosis, and preserves podocyte integrity, accompanied by recovery of the renal MV architecture and function (partly by improving renal VEGF and HGF expression and signaling), and improved renal hemodynamics regardless of the etiology^{3, 65, 66, 70, 117, 118}. The potential for ET-A blockade-induced renoprotection is underscored by recent evidence from clinical studies showing that ET-A antagonism may reduce renal injury, proteinuria (a marker of MV damage^{119, 120}), arterial stiffness, and cardiovascular risk in CKD from different etiologies^{121, 122}, which supports experimental data demonstrating renal MV protection. Whether effects of ET-A blockade on the renal microcirculation are independent or the result of the overall improvements in renal function and injury are still unclear. The results of the ongoing SONAR clinical trial (NCT01858532), which aims to determine the efficacy of ET-A antagonism for renoprotection in diabetic nephropathy may help determine whether ET-A blockade could serve as an additional strategy to recover the kidney.

Potential novel therapies

Cell-based therapies

Circulating and resident progenitor cells are stimulated when injury develops, resulting in proliferation, differentiation, and homing of these cells in damaged tissues. These complex events are key steps of the endogenous healing mechanisms in the body. However, the severity or chronicity of the initial insult may overwhelm these mechanisms and make them insufficient to reverse organ injury. Thus, there is still a major need for development of new cell-based therapeutic strategies.

The kidney has limited regenerative capacity and no effective treatment has been developed to prevent progression of CKD to end-stage kidney disease, independent of the initial insult. The underlying mechanisms of this distinct deficiency are not entirely clear and are likely multifactorial. For example, renal healing may be blunted by a reduced availability of progenitor cells in a context of chronic diseases, as frequently observed in disorders such as hypertension or diabetes^123-125. In turn, an uremic milieu can induce reduction and dysfunction of cell progenitors of endothelial or mesenchymal origin¹²⁶.

The use of bone marrow or adipose-derived mesenchymal stem cell therapy is gaining momentum. Growing evidence supports its feasibility and potential application for cardiovascular and renal therapy since these pluripotent cells are rich sources of cytokines and growth factors that can promote tissue healing and slow or halt the progression of tissue damage. Administration of exogenous stem cells could prevent and promote renal recovery via modulation of the immune system, release of paracrine factors, and production of microvesicles with powerful anti-inflammatory and anti-fibrotic effects^{127, 128}.

Our recent study in a model of chronic renovascular disease showed that renal chemotactic signaling to attract cell progenitors may be defective, which in the context of a reduced or dysfunctional circulating progenitors could contribute to the progressive nature of renal injury¹³. This imbalance may indeed lead to MV rarefaction since a major effect of cell therapy is stimulation of MV repair and proliferation. Indeed, previous studies demonstrated that intra-renal endothelial¹⁵ and mesenchymal¹²⁹ stem cell therapy can efficiently improve MV density, stimulate MV repair and proliferation, and restore MV function in experimental renovascular disease. The MV improvements were followed by significant recovery of renal function and decreased renal inflammation and fibrosis^{13, 15, 129} despite a relatively low renal retention and incorporation of the cells into renal structures, suggesting stimulation of powerful autocrine and paracrine actions of these cells to promote recovery. These effects are possibly driven by cell-derived secretion of growth factors (e.g. VEGF) and vesicles that modulate angiogenesis, inflammation, fibrosis, and other cell pathways to facilitate tissue repair^{130, 131}. More studies are needed to further understand the definitive underlying mechanisms and breadth of actions of cell therapy. Nevertheless, this is a promising strategy that aims to recover endogenous mechanisms of tissue healing (largely via MV proliferation and repair), which may be overwhelmed or deficient in the sick kidney and contribute to its poor renal regenerative capacity.

Angiogenic cytokines

The potential use of angiogenic cytokines to recover ischemic tissues has been attempted in peripheral vascular disease, wound healing, hind-limb and myocardial ischemia, in experimental and clinical settings^132-136 as recently reviewed¹³⁷. Strategies using angiogenic cytokines or stem cell therapies are significantly related since progenitor cells are major sources of growth factors as their proliferation, homing, and stimulation of neovascularization and tissue repair are significantly driven by such growth factors^{138, 139}. Like with cell therapy, the basic principle is propelling neovascularization and MV repair to recuperate adequate blood supply needed to sustain tissue function. Unlike cell therapy, the potential effects of angiogenic cytokines may not primarily counteract inflammation or fibrosis, although by improving tissue ischemia, recovering tissue perfusion and organ function, they may ameliorate those injurious mechanisms as well.

CKD is universally associated with renal MV rarefaction which likely contribute to its progressive nature (Figure 1). The importance of MV integrity for renal function and body homeostasis is underscored by undesired effects of anti-angiogenic strategies, which are at the fore-front for the treatment of various forms of cancer. These strategies mainly aim on VEGF inhibition, and major collateral effects are hypertension and renal injury, disclosed by proteinuria and glomerular injury^{140, 141}, which support the importance of both VEGF and MV integrity for the kidney. On the other hand, elegant studies by Basile et al and Kang et al were among the first to demonstrate the feasibility of using VEGF administration as a tool to ameliorate renal injury in the remnant kidney model, aging, and in acute kidney injury^{42, 45, 76, 77}. VEGF therapy in these models reduced MV rarefaction, which was associated with a significant reduction in overall renal damage. A disruption of VEGF signaling has been suggested as a potential link between MV rarefaction and fibrogenesis via modulation of endothelial-pericyte cross-talk¹⁴². Similarly, our studies in a swine model of chronic renovascular disease demonstrated that progression of renal damage develops in a context of blunted biovailability of VEGF, which accompanies progressive MV rarefaction in cortex and medulla, loss of renal function, and development of renal fibrosis, which were all significantly reversed after VEGF therapy^{36, 37} (Figure 2) Although VEGF is a cytokine of a relatively short half-life (a few minutes), our studies using intra-renal administration of VEGF (from human origin) demonstrated that long-term improvements occur after a single administration. Indeed, these effects were persistent up to 4 weeks after administration and functionally consequential since they were followed by a recovery of renal function as well, indicating that the new vessels were functional. The long-term effects are possibly driven by stimulation of circulating and resident cell progenitors (author's unpublished observations) that initiate and boost angiogenesis, MV repair, and tissue healing. Furthermore, VEGF therapy in this model restored its downstream signaling since factors that closely interact with VEGF to promote endothelial cell survival and vascular maturation such as Akt, endothelial nitric oxide synthase, and angiopoietins were also improved^{11, 12}.

Evidence on the importance of renal bioavailability of VEGF and the integrity of the VEGF pathway for the kidney in health and disease is abundant and supports prominent roles of this cytokine to preserve the renal MV networks^{12, 45}, promote MV repair and proliferation^{11, 12}, and protect podocyte integrity^{3, 9}. However, some studies suggest that overexpression or abundance of VEGF may also be deleterious for the kidney, although it may depend on the disease model, the stage of the disease, species, and isoform of VEGF^143-145. Polycystic kidney disease^{71, 145} and diabetes^{146, 147} are examples of the potential dual effect of VEGF depending on experimental conditions. Hence, carefully designed additional studies in selected models of renal disease may be needed to determine renal VEGF levels and signaling that may maintain a healthy glomerular structure and function, should this intervention be considered for clinical applications.

Another important angiogenic and anti-fibrotic cytokine that has been use for renal therapy is HGF. A few studies have shown that recovery of HGF signaling (by exogenous HGF therapy or genetic manipulations^{10, 80, 83}) associates with a significant reduction in MV remodeling and MV endothelial inflammation without significant effects on neovascularization¹⁰, but associated with significant improvements in renal function. These studies may suggest powerful effects of HGF therapy are on the existing renal microvasculature by promoting MV repair and decreasing MV remodeling¹⁰.

Thus, pre-clinical evidence supports the possibility of VEGF or HGF therapy for the kidney as a promising strategy to reverse or slow down renal injury via restoration of angiogenesis, MV repair, and function. However, their therapeutic potential should be confirmed and additional studies are needed to determine the safety of these interventions since they have the potential of inducing aberrant vascular growth or favor the progression of tumors without a careful selection of the patients.

Renal therapeutic angiogenesis using drug delivery vectors

CKD represents a major challenge to any therapy due to the limitations of renal clearance and increased risks for toxic effects. Thus, efficacy of renal therapies may depend on high concentrations of therapeutic agents in the kidney to achieve desired effects, which paired with a limited renal function result in uncertainties regarding beneficial effects, outcomes, and toxicity.

Improving drug delivery to the kidney using renal-targeted therapeutics is still an under-developed area. Current efforts in our laboratory aim to apply renal therapeutic angiogenesis using non-immunogenic protein-based carriers derived from elastin (elastin-like polypeptides, ELP) that can stabilize attached small molecule and peptide therapeutics^{9, 148}. The plasticity of these vectors allowed the tagging of drugs by relatively simple molecular biology techniques. Furthermore, ELPs display a higher affinity for kidney tissue than other organs^{9, 148}, making them ideal candidates for renal targeted therapies. We recently developed and characterized a construct built on an ELP carrier fused to VEGF⁹. The ELP-VEGF construct greatly accumulates in the kidney and may target renal endothelial and tubular epithelial cells of different species (e.g. rodents, swine, humans), and showed a prolonged circulating time compared to unbound VEGF. We showed that a single intra-renal administration of ELP-VEGF restored renal hemodynamics, function, and attenuated renal injury to a greater extent than unconjugated VEGF, which underscored a higher efficacy of this novel bio-engineered compound. Notably, these effects were accompanied by the significant recovery of the cortical and medullary MV density, remodeling, and function compared to controls, possibly driven by restored expression of VEGF and downstream mediators⁹.

The ELP carrier can be modified to enhance their tissue targeting and penetration properties. Our recent work¹⁴⁸ demonstrated that an ELP modified at its N-terminus with a cyclic, seven amino acid kidney-targeting peptide (KTP) significantly increases renal targeting and accumulation, which again was independent of the species and further demonstrate a unique property of these drug-delivery vectors. Furthermore, the C-terminus of the ELP construct was also modified with a cysteine residue for tracer conjugation and to allow the addition of a protein or therapeutic agent of choice, which is part of our ongoing efforts and future studies. The plasticity of ELP for attachment of any class of therapeutics unravels the possibility of applying ELP technology for targeted treatment of MV abnormalities that are present in acute or chronic renal diseases of different etiologies that can be mimic in different animal models. The distinct properties of these constructs may also open avenues for application of ELP technologies for targeted interventions beyond VEGF or therapeutic angiogenesis. The road ahead is long but this strategy holds promise.

Conclusion and perspectives

The complexity of CKD and ESRD has been a major burden for therapeutic strategies. The growing number of patients has been paralleled with an expanded variety of therapeutic opportunities developed thanks to relentless research efforts. However, the possibility of reversing or halting the progressive nature of chronic renal disease is still a challenging task. The vasculature is the core for the survival and function of every organ, and in the kidney is not only necessary for their own nutrition but also for the normal function of the rest of the body.

Targeting the renal microvasculature offers a therapeutic niche to improve current treatments. However, it is likely that the window of opportunity to protect the renal microvasculature with beneficial consequences for the kidney is small. Success in restoring kidney function may depend on how far MV rarefaction has progressed since MV repair and potential regrow of peritubular and possibly glomerular capillary loops needs to be attempted and achieved before the entire glomerulus is lost (Figure 1). Promising results using cell-based therapies, angiogenic cytokines, and bioengineered compounds suggest that MV repair, neovascularization, and protection of the existing renal microvasculature is feasible and functionally consequential, although some limitations to their therapeutic application remain and need to be resolved (Table 1). Thus, efforts are needed to solidify these results and define the timeframe of MV interventions to allow the translation towards application of renal targeted MV therapies, which may offer a novel therapeutic opportunity to the growing number of renal patients.

Table 1.

Summary of advantages and major limitations of cell-based and angiogenic therapies to target the renal microcirculation and potentially treat chronic renal disease

Intervention	Advantages	Major limitations
Endothelial progenitor cells126, 149	1) Relatively easy to obtain from bone marrow or from circulation. 2) Available for collection, expansion in vitro, differentiation into endothelial phenotype, and re-injection (autologous therapy). 3) Paracrine stimulation of vascular repair, angiogenesis, and anti-inflammatory effects. 4) May differentiate into mature endothelial cells and incorporate into the damaged endothelium to replace or support existing endothelial cells.	1) Still in pre-clinical stage and some clinical trials. 2) Methods to enhance survival and longevity of cells prior to delivery are still not standardized. 3) Autologous EPCs are difficult to isolate and expand (must be isolated from peripheral blood, cultured for several weeks, large amounts of peripheral blood required).
Mesenchymal stem cells126, 150,131, 151, 152	1) Easy to obtain from different sources such as adipose tissue, bone marrow, skin, skeletal muscle, and umbilical cord. 2) Easily expanded in vitro within a relatively short period of time (compared to endothelial progenitor cells). 3) Amenable for re-injection (autologous therapy) 4) Allogeneic therapy may be possible 5) Paracrine proangiogenic effects, anti-inflammatory, immunomodulatory, and anti-fibrotic effects. 6) Safe: approved by the US FDA for treatment of steroid-resistant graft-versus-host disease.	1) Methods used for cell isolation, selection, sorting, conditions for expansion in vitro, number of passages in vitro, and assessment are still not standardized. 2) Relatively large size of the cells may cause micro-infarcts. 3) FDA approved for only one clinical application.
Angiogenic cytokines137	1) Pre-clinical and some pilot clinical studies showed feasibility and efficacy in treatment of peripheral vascular disease, myocardial and hind-limb ischemia. 2) Pre-clinical evidence suggests feasibility of VEGF or HGF administration for intra-renal therapy.	1) Early and limited pre-clinical evidence supporting their application for renal therapy. 2) Potential for off-target effects (e.g. abnormal vascular growth in other organs) 3) May favor the progression of tumors.
Bioengineered compounds (Drug delivery vectors –ELP- for VEGF delivery to the kidneys)9, 148	1) In vitro evidence shows enhanced uptake of the construct by human renal cells. 2) Pre-clinical evidence supports their use for renal targeted delivery of VEGF after intra-renal or systemic administration. 3) Pre-clinical evidence suggests renal effects only without “spill over” of the VEGF constructs or binding to other organs. 4) Pre-clinical evidence shows that the renal targeting is independent of the species tested (rodents, swine). 5) Pre-clinical efficacy shows that ELP-VEGF constructs are more efficient than unconjugated VEGF to protect the renal microvasculature, recover renal function, and reduce renal damage.	1) Early and limited pre-clinical evidence supporting their application for renal therapy

VEGF: Vascular endothelial growth factor; HGF: hepatocyte growth factor; ELP: elastin-like polypeptides.