Mechanisms of Synergistic Interactions of Diabetes and Hypertension in Chronic Kidney Disease: Role of Mitochondrial Dysfunction and ER Stress

Abstract

Purpose of Review

To discuss the importance of synergistic interactions of diabetes mellitus (DM) and hypertension (HT) in causing chronic kidney disease and the potential molecular mechanisms involved.

Recent Findings

DM and HT are the two most important risk factors for chronic kidney disease (CKD) and development of end-stage renal disease (ESRD). The combination of HT and DM may synergistically promote the progression of renal injury through mechanisms that have not been fully elucidated. Hyperglycemia and other metabolic changes in DM initiate endoplasmic reticulum (ER) stress and mitochondrial (MT) adaptation in different types of glomerular cells. These adaptations appear to make the cells more vulnerable to HT-induced mechanical stress. Excessive activation of mechanosensors, possibly via transient receptor potential cation channel subfamily C member 6 (TRPC6), may lead to impaired calcium (Ca²⁺) homeostasis and further exacerbate ER stress and MT dysfunction promoting cellular apoptosis and glomerular injury.

Summary

The synergistic effects of HT and DM to promote kidney injury may be mediated by increased intraglomerular pressure. Chronic activation of mechanotransduction signaling may amplify metabolic effects of DM causing cellular injury through a vicious cycle of impaired Ca²⁺ homeostasis, mitochondrial dysfunction, and ER stress.

Introduction

Over 15% of adults, or 37 million people, in the USA have chronic kidney disease (CKD) in 2019 [1]. Medicare costs for people with CKD were over $79 billion in 2016, averaging more than $23,000 per person. Unfortunately, the incidence of CKD has continued to increase in the USA and worldwide during the last few decades and has caused a huge economic burden on health care systems [2].

CKD often progresses to end-stage renal disease (ESRD) and greatly increases the risk of cardiovascular diseases. Multiple factors are associated with CKD, such as race, gender, age, and family history. Moreover, smoking, obesity, hypertension (HT), and diabetes mellitus (DM) are major risk factors for kidney disease. Among them, the two most common causes of CKD and ESRD are DM and HT [3].

DM is the leading cause of CKD and ESRD in developed as well as in developing countries, and almost 1 in 3 people with DM have CKD. According to the US Renal Data System (USRDS) data, half of the new ESRD patients in the USA have diabetic nephropathy [4]. HT, even in the absence of DM, accounts for at least 27% of all ESRD in the USA. However, HT is an extremely common comorbid condition in DM, affecting ~ 40–80% of patients with type 2 diabetes (T2D) [5-7]. In T2D, HT is often present as part of the metabolic syndrome of insulin resistance, dyslipidemia, and central obesity [8]. In type 1 diabetes (T1D), HT may reflect the onset of diabetic nephropathy. Thus, most patients with “diabetic” nephropathy actually have DM plus HT as the drivers of their CKD.

The current strategies for treating CKD include control of blood pressure (BP) with angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), as well as control of blood glucose and lipids [9]. However, current treatments only slow, rather than halt, the progression of CKD to ESRD and additional studies are needed to better understand how DM and HT interact to cause CKD [10]. This review focuses on the pathogenesis of kidney injury associated with DM and HT and explores potential molecular mechanisms by which MT dysfunction and ER stress contribute to the progression of CKD.

Synergistic Effects of DM and HT to Promote Kidney Injury

The natural history of CKD with DM has been depicted as a sequence of stages characterized initially by normal or elevated glomerular filtration rate (hyperfiltration) followed by progressive renal function decline to CKD and ultimately ESRD. The causes of hyperfiltration in DM have been extensively studied and may include alteration in tubuloglomerular feedback and increased nitric oxide (NO) [11, 12]. Hyperfiltration and glomerular hypertension may induce early glomerular structural changes such as glomerular basement membrane (GBM) thickening and podocyte foot process extension [13]. The overall structure and coverage of podocytes are largely preserved until BP increases significantly [14]. Although glomerular hyperfiltration initially serves as a compensatory response that permits the maintenance of sodium balance associated with increased tubular reabsorption during hyperglycemia in T1D and obesity in T2D [15, 16], this response may eventually cause glomerular injury and predispose nephrons to irreversible damage, resulting in progressive renal disease and further HT.

Previous studies have shown that superimposition of HT on T1D or T2D produces much more severe kidney injury than DM alone [17••]. Each 10 mmHg increase in mean systolic BP was associated with a 15% increase in the hazard ratio for developing micro- and macroalbuminuria and impaired kidney function, defined as eGFR < 60 ml/min per 1.73 m² or doubling of the blood creatinine level [18]. In fact, increased BP may be a prerequisite for the rapid progression of diabetic nephropathy.

Experimental studies have shown that severe chronic hyperglycemia can cause kidney injury, although renal lesions are often slow to develop in the absence of HT [2]. Also, in many experimental models and in humans with DM, chronic hyperglycemia-induced kidney injury is often associated with obesity and other metabolic abnormalities. For example, in T2D models with leptin receptor mutations such as Zucker fatty rats and db/db mice [19-21], hyperglycemia is accompanied by severe obesity, hyperlipidemia, and HT that may all contribute to kidney injury. Although chemical methods of inducing T1D (e.g., streptozotocin or alloxan) are not complicated by obesity, the amount of kidney injury in these models is often mild despite severe chronic hyperglycemia, and these agents may themselves have toxic effects on the kidneys [22].

To better understand why HT significantly accelerates kidney injury in DM, we developed an HT-diabetic model by applying aorta constriction (AC) between the left and right renal arteries in T2D Goto-Kakizaki (GK) rats [17••]. An important aspect of this model is that both kidneys are exposed to the same levels of hyperglycemia, circulating hormones, and neural influences but the left kidney below the AC has normal to slightly reduced BP, whereas the right kidney above the AC is exposed to elevated BP. Our results demonstrated that combining HT and DM caused much more severe renal dysfunction, as reflected by increases in 24-h urinary albumin excretion, ER stress, MT dysfunction and oxidative stress in the renal cortex, histological injury of glomeruli, and slowly declining glomerular filtration rate (GFR), compared with kidneys exposed to DM or HT alone. The potential mechanisms responsible for this synergistic effect of combined HT and DM to induce kidney injury may be related to enhanced ER stress and MT dysfunction, since kidney injury induced by HT plus DM can be significantly attenuated by the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) [17••]. However, the molecular pathways that initiate ER stress and MT dysfunction and the mediators that promote progression of glomerular injury in the setting of DM plus HT are still unclear and will be the main focus of this review.

Increased Glomerular Capillary Pressure (P_G) when HT Is Combined with DM

Clinically, diabetic nephropathy evolves in a sequence of stages beginning with initial increases in GFR and glomerular hyperfiltration, glomerular hypertrophy, and microalbuminuria [23•]. Hyperglycemia is presumed to contribute to the development of glomerular hyperfiltration and glomerular hypertension through a macula densa feedback mechanism [12]. Reduced delivery of sodium chloride (NaCl) to the macula densa as a consequence of increased proximal co-transport of glucose and sodium may reduce afferent arteriolar resistance and increase P_G and GFR via attenuated tubuloglomerular feedback (TGF) [11, 24, 25]. Also, efferent arteriolar vasoconstriction in response to circulating or locally formed angiotensin II may promote diabetic glomerular hypertension [26, 27]. Recently, Zhang et al. [28] reported that acute hyperglycemia upregulated expression and activity of neuronal nitric oxide synthase 1 (NOS1) in macula densa cells via sodium-glucose cotransporter 1 (SGLT1); increased NO production by NOS1 blunted TGF and promoted glomerular hyperfiltration. This finding is consistent with other studies demonstrating that blockade of macula densa TGF completely abolished glomerular hyperfiltration associated with acute hyperglycemia [12]. When hyperglycemia and hyperfiltration are sustained chronically, there may also be increased nephron size, especially associated with hypertrophy of the proximal tubules and glomerulomegaly.

The hyperfiltration in DM is associated with impaired autoregulation of renal blood flow and GFR which means that when arterial pressure is elevated, there is a greater transmission of the increased pressure to the glomerulus compared with normal kidneys which demonstrate normal autoregulation. In kidneys that show intact autoregulation, HT may not be associated with marked kidney injury since the increased pressure is not transmitted to the glomeruli [29]. However, when renal autoregulation is impaired in non-diabetic experimental models, such as salt-sensitive spontaneously hypertensive stroke-prone (SHRSP) rat or the 5/6 renal ablation model, there is a much lower BP threshold for hypertensive kidney injury [30-32]. These findings suggest that impaired renal autoregulation in hyperfiltration states, such as DM, may lead to much greater transmission of increased pressure to glomeruli and greatly increase the risk for HT-induced kidney injury.

Hyperfiltration in the absence of HT may not be sufficient to cause severe kidney injury. Kidney transplant donors in which filtration rate is increased by 60–70% in the nephrons of the single remaining kidney generally do not have substantially increased risk for developing ESRD in the absence of HT [33, 34].

HT Increases Mechanical Stress in Diabetic Kidney

The high pressure of glomerular capillaries maintains an outward flow of the plasma fluid while causing tensile stress on the capillary wall that is transmitted to the endothelium and podocytes. In parallel, continuous flow of the ultrafiltrate into Bowman’s space generates shear stress on podocytes and parietal epithelial cells [14]. Previous studies have been shown that mechanical forces may promote podocyte detachment under physiologic and pathophysiologic conditions [35-38]. Thus, in CKD, long-lasting intraglomerular mechanical challenges, including glomerular hypertension, hyperfiltration, hypertrophy, and outflow of glomerular filtrate from subpodocyte spaces, may promote progressive podocyte loss through detachment from the GBM, and which ultimately cause complete loss of podocytes in advanced CKD with overt proteinuria [39, 40]. Transduction of mechanical forces into biochemical signals requires “mechanosensor” proteins. These proteins can sense mechanical forces and induce biochemical changes of the whole mechanosensory complex, including ion channels and anchor proteins that provide cell-cell or cell-matrix linkage [41, 42•].

Role of Ion Channels in Mechanotransduction and Kidney Injury

Transient Receptor Potential Cation Channel Subfamily C Member 6 (TRPC6)

TRPC6 is highly selective for Ca²⁺ over other cations and has been implicated in Ca²⁺-dependent processes in the peripheral vasculature, kidney, and heart [43]. There is evidence that TRPC6 may interact with nephrin, podocin, and mechanosensitive Ca²⁺-activated potassium channels (BKCa), permitting TRPC6 to function as part of a mechanosensory complex and allowing Ca²⁺ influx to modulate nephrin signaling and cytoskeletal dynamics [44]. However, whether TRPC6 channels are directly or indirectly gated by mechanical factors is still controversial [45, 46]. TRPC6 can be activated in response to G protein-mediated signaling cascades associated with phospholipase C activation [47, 48]. TRPC6 activation may also be enhanced by the combination of receptor binding and mechanical stimulation [49] and has been reported to play an important role in mediating kidney injury in DM [50, 51]. Other studies have suggested that activation of TRPC6 channels is linked to renal injury and may contribute to the development of CKD [52]. Whether TRPC6 activation mediates diabetic-HT renal injury, however, is still unclear.

Piezo Channels

Piezo proteins are pore-forming subunits of ion channels that open in response to mechanical stimuli, allowing positively charged ions, including Ca²⁺, to flow into the cell. Piezo ion channels (Piezo1 and Piezo2) were discovered as mechanosensor proteins by Coste et al. [53] in 2010 and have been established as important mechanosensitive cation channels in mammals [54]. Piezo channels may have crucial roles in vascular mechanotransduction processes. Piezo1 is expressed in the blood vessels and appears to be important for sensing blood flow-associated shear stress and blood vessel development [55-57], whereas Piezo 2 mediates touch, proprioception, airway stretching, and lung inflation [58, 59]. Furthermore, mutations in Piezo genes have been linked to several hereditary human diseases that involve mechanotransduction [60]. Although Piezo1 has been found on renal glomeruli as well as tubules in the cortex and medulla [61], so far, little is known about the expression and function of Piezo channels in podocytes.

P2X Purinoceptor 4 (P2X4)

P2X4 gene belongs to the family of purinoceptors for ATP and acts as a ligand-gated ion channel. P2X4R mRNA and protein have been detected in a variety of renal cell types, including glomerular mesangial, epithelial, and tubular cells, especially proximal tubular cells [62, 63]. P2X4 receptors have been implicated in the regulation of ATP-mediated cell death, synaptic strengthening, and activation of the inflammasome in response to injury [64, 65]. P2X4 is involved in ATPinduced Ca²⁺ influx [66] and flow-mediated vasodilatation through NO release, affecting BP and vascular remodeling [67]. Forst et al. [45] showed that P2X4 channels are mechanical sensors in podocytes, and pharmacological blockade of P2X4 channels not only abrogated mechanically stimulated Ca²⁺ influx but also prevented actin reorganization of in vitro stretched podocytes.

The response to mechanical stress may be largely dependent on the glomerular cell type. Previous studies have shown that podocytes lose stress fibers and reorganize the actin cytoskeleton with excessive mechanical stretch [68]. However, endothelial or mesangial cells may develop increased actin stress fibers in response to increased mechanical stress [38]. Defining the mechanosensor and their signal transduction pathway in different cell types and understanding how the adaptive responses to increased mechanical stress are converted into maladaptive responses and cell injury will help us better understand the pathogenesis of CKD.

Role of MT Dysfunction in Diabetic-Hypertensive Renal Injury

MT are often described as the “powerhouses” of cells and are responsible for synthesis of adenosine triphosphate (ATP) and supply of energy to cells. They also participate in many other biological functions in cells, including generation of reactive oxygen species (ROS) and regulation of cell apoptosis [69]. Given the high energy and oxygen requirements of the kidney, MT and kidney function are intimately linked. Hence, dysfunctional MT are increasingly postulated to be central to the development and progression of diabetic CKD [70]. The main pathophysiological changes associated with MT dysfunction include increased ROS generation, impaired ATP synthesis, elevated cytoplasmic Ca²⁺ concentrations, dysregulated energy metabolism, and activation of proteases, endonucleases, and apoptosis. Although these changes have been shown in experimental animal models of CKD and in patients with CKD [71], time-dependent studies to reveal the changes in MT function at different stages of CKD associated with DM and HT have not been performed.

Mitochondrial Transition from Compensating to Decompensating in DM

Diabetes is a state of nutrient excess with higher glucose and free fatty acids (FFA) in the circulation but impaired glucose utilization by many tissues such as skeletal muscle and liver. In diabetic kidneys, renal tubular cells may have increased glucose metabolic rate since they are reabsorbing glucose at the maximal rate with increased activity of the Na⁺-dependent glucose transporter 2 (SGLT2) [72]. In contrast, podocytes lack SGLT and take up glucose via insulin-dependent glucose transporter 4 (GLUT4) [73], and may develop cellular injury. Factors like advanced glycation end products (AGEs), fructose 6-phosphate/hexosamine pathway, and increased cellular FFA levels may contribute to diabetic podocyte injury [74].

In both T1D and T2D, hyperglycemia and other metabolic factors contribute to metabolic imbalance and disease initiation. MT actively adapt to these metabolic changes in an attempt to balance the energy supply with energy demand. For example, studies in diabetic humans or animal models have shown that hyperglycemia and hyperlipidemia induce PGC-1 α expression to increase MT biogenesis and MT oxidative capacity in response to chronic hyperglycemia. [75] However, this adaptation is not harmless since it is also accompanied by reduced respiratory efficiency, increased proton leakage, uncoupling, and ROS generation.

Besides the oxidative phosphorylation changes, the rates of MT fusion and fission are increased and altered towards more fission (fragmentation). Mitophagy as a cellular protection mechanism is activated to degrade MT with decreased membrane potential and impaired MT electron transfer capability. Overall, in the early stages of DM, the kidney MT are working in a state of increased metabolic rate with highly activated biogenesis processes. Renal ATP production in DM can be normal or slightly increased, although MT-derived ROS production is increased and electron transfer efficiency is reduced [76]. The adaptation of MT during early DM may make the kidneys more vulnerable when extra energy demands are superimposed (e.g., due to increased sodium reabsorption or HT-induced mechanical stress). How MT function transitions from compensating to decompensating as CDK progresses, especially when HT is added to existing DM, is still poorly understood.

Role of ER Stress in Diabetic-Hypertensive Renal Injury

Several lines of evidence suggest that ER stress plays a major role in the development and progression of CKD. Pathophysiological states that increase the demand for protein folding or that disrupt normal folding processes result in the accumulation of misfolded proteins in the ER and cause ER stress. ER stress activates a cellular protective mechanism called unfolded protein response (UPR). The UPR is important for maintaining normal ER function and facilitates recovery from stress and may protect against additional stresses. By contrast, sustained or prolonged ER stress may be cytotoxic, leading to apoptosis. Hyperglycemia, proteinuria, AGEs, and FFA have all been reported as inducers of ER stress and UPR in diabetic kidneys [77]. A large body of evidence suggests that activation of the ER stress pathway in different glomerular cell types is associated with the onset and progression of CKD [78].

Glomerular Endothelial Cell Injury and ER Stress

Endothelial cell injury is a central event in the development of diabetic vascular diseases. Prolonged ER stress contributes to endothelial dysfunction. Studies in cultured endothelial cells and animal models have provided insights into the molecular mechanisms linking the induction of ER stress to endothelial cell dysfunction [79-81]. Hyperglycemia-induced ER stress has also been closely linked to various aspects of endothelial cell dysfunction in patients with diabetes. Chen et al. [82] reported that high glucose-induced ER stress in retinal endothelial cells results in activation of the PERK–eIF2α-ATF4 pathway. However, there are currently only a few reports on the role of ER stress in glomerular endothelial cells in DM.

Podocyte Injury and ER Stress

Podocytes are highly specialized and terminally differentiated glomerular epithelial cells, and their dysfunction causes defective glomerular filtration, resulting in the onset of proteinuria [83, 84]. Podocytes are thought to be highly susceptible to ER stress due to their high protein-folding capacity in the ER [85]. Studies show that high glucose concentrations can directly induce ER stress and apoptosis in podocytes [86]. AGEs and FFAs are critically involved in the pathogenesis of DM and can induce ER stress in podocyte, leading to the induction of ER chaperone BiP and proapoptotic transcription factor CHOP, which promotes apoptotic cell injury [87, 88].

Interaction of MT Dysfunction and ER Stress in DM

MT and ER are important organelles and nutrient sensors, and their dysfunction has been extensively and independently implicated in metabolic diseases [89]. However, the UPR during ER stress and MT dysfunction are not two distinct pathways without interactions. Studies showed that the UPR pathway in the ER also regulates mitochondrial metabolic status, indicating the pathophysiological significance of organelle crosstalk between the ER and MT via the UPR pathway [90]. In recent years, it has become obvious that communication among organelles also is conducted through direct interactions at MTassociated membranes (MAMs) in order to exchange metabolites and Ca²⁺ [91]. The role of mitochondria in many Ca²⁺ signaling pathways depends on close interactions with the ER Ca²⁺ storage and the formation of ER–MT contact sites. For instance, opening of inositol 1,4,5-trisphosphate (IP3) receptors in the ER membrane allows MT to be exposed to high Ca²⁺ concentration that is necessary to induce Ca²⁺ accumulation through the low-affinity mitochondrial calcium uniporter (MCU) complex [92].

Taken together, UPR and MT function influence each other reciprocally through MAMs by (1) MT dysfunction that may enhance ER stress and UPR by reduced ATP generation and (2) UPR during ER stress that could induce mitophagy to clear stress-damaged MT and regulate MT bioenergetics such as ATP generation. The interaction of MT dysfunction and ER stress may generate a vicious cycle that promotes cell injury through amplified ROS generation and the activation of the pro-apoptotic pathway [93].

Mechanical Stress-Induced Ca²⁺ Influx May Exacerbate ER Stress and MT Dysfunction and Induce Apoptosis in DM

As discussed previously, mechanical stress may induce cell Ca²⁺ influx from the extracellular fluid through mechanosensitive ion channels and through ER release of Ca²⁺. Under normal physiological conditions, there is a tight coordination between ER Ca²⁺-release and changes in Ca²⁺ influx enabling proper Ca²⁺ signaling in response to physiological stimuli (e.g., Ca²⁺-induced ATP generation in MT). In pathological conditions, such as in DM or HT, Ca²⁺ influx may be enhanced transiently as an adaptation to the metabolic changes. However, chronic and excessive Ca²⁺ influx induced by mechanical stress may further exacerbate ER stress by depletion of ER Ca²⁺ storage and increased cytoplasmic and MT Ca²⁺ concentrations, which will eventually induce cell apoptosis [94-96] (Fig. 1).

Fig. 1.

Diabetes (DM) and hypertension (HT) synergistically amplify glomerular injury through mitochondrial (MT) dysfunction and ER stress. Diabetes alone may induce MT adaptation to altered metabolic balance (dashed line). This adaptation may make cells more vulnerable to secondary injury from HT. When HT combines with existing diabetes, a transition of MT function from compensating to decompensating may further impair mitochondrial bioenergetics and induce ER stress and apoptosis (solid line). This synergistic effect may be mediated by HT-induced mechanical stretch which activates mechanosensitive ion channels and is amplified by the interaction of MT dysfunction, ER stress, and impaired Ca²⁺ homeostasis

The initiation step of the intrinsic pathways of apoptosis involves the release of apoptosome components, such as cytochrome C, from the MT. Organelle fragmentation following mitochondrial permeability transition pore (MPTP) opening also plays a key role in this process [97•]. Despite the lack of a mechanistic understanding, numerous data reveal that the most important trigger for MPTP opening is Ca²⁺, which acts in conjunction with a variety of apoptotic signals in living cells [98]. Cytoplasmic Ca²⁺ increases were also shown to directly induce MT pro-apoptotic morphological modifications. Indeed, calcineurin-dependent translocation of mitochondrial fission 1 protein (FIS1) triggers mitochondrial fission and hence cytochrome C release [99]. The role of Ca²⁺ signals in apoptosis is further reinforced by the demonstration that anti-apoptotic proteins such as B cell lymphoma 2 (BCL-2) lower ER Ca²⁺ levels and reduce cytosolic and MT Ca²⁺ responses to extracellular stimuli by increasing ER Ca²⁺ leak [100]. Overall, a general consensus has emerged that MT Ca²⁺ loading exerts a permissive role, allowing several toxic challenges to cause the release of caspase cofactors from the organelle, resulting in apoptotic cell death. At the same time, prolonged MPTP opening leads to the complete collapse of the membrane potential and Ca²⁺ discharge, which results in the complete loss of MT function and apoptotic cell death.

Outlook

MT dysfunction and ER stress are present in diabetic kidney injury. However, the temporal and causative relationships between functional and morphological changes of MT and ER in the establishment and progression of renal injury in the diabetic-hypertensive environment are unclear. Below are some critical questions that will need to be addressed for a better understanding of the pathophysiology of CKD:

Question 1. How do mechanosensitive ion channels, including TRPC6 channels, activated by HT contribute to the development of MT dysfunction in diabetes?

Question 2. What is the role of MT in the progression of kidney dysfunction and what is the mechanistic relationship of morphological distortion and bioenergetic dysfunction?

Question 3. What cellular changes exacerbate the damage caused by MT dysfunction and ER stress that drive cellular maladaptation and lead to kidney dysfunction?

Conclusions

1. DM and HT are the two most important risk factors for CKD and the development of ESRD. HT and DM interact synergistically to promote the progression of the renal injury.

2. The hyperfiltration in DM is associated with impaired renal autoregulation which allows increased arterial pressure to be more readily transmitted to the glomerulus and induce cell injury through mechanotransduction pathways.

3. Ion channels including TRPC6, Piezo, and P2X4 may have important roles in the transduction of the mechanical stress to downstream cellular responses by enhancing Ca²⁺ influx.

4. Hyperglycemia and HT may induce ER stress and MT dysfunction in different glomerular cell types. MT adaptation to the metabolic environment changes may make the cells more vulnerable to hypertension-induced mechanical stress. Impaired Ca^{2 +} homeostasis, ER stress, and MT dysfunction may synergistically promote cellular apoptosis and contribute to the development of glomerular injury.