Prime Mover and Key Therapeutic Target in Diabetic Kidney Disease

Diabetes Volume 66, April 2017 791

Richard E. Gilbert

Proximal Tubulopathy: Prime Mover and Key Therapeutic Target in Diabetic Kidney Disease

Diabetes 2017;66:791–800 | DOI: 10.2337/db16-0796

The current view of diabetic kidney disease, based on estimated glomerular filtration rate (eGFR) decline (2). In meticulously acquired ultrastructural morphometry and recognition of these findings, the term diabetic kidney the utility of measuring plasma creatinine and urinary al- disease rather than diabetic nephropathy is now commonly bumin, has been almost entirely focused on the glomer- used. On the background of recent advances in the role of ulus. While clearly of great importance, changes in the the proximal tubule as a prime mover in diabetic kidney DIABETES IN PERSPECTIVES glomerulus are not the major determinant of renal prog- pathology, this review highlights key recent developments. nosis in diabetes and may not be the primary event in the Published mostly in the general scientific and kidney- development of diabetic kidney disease either. Indeed, specific literature, these advances highlight the pivotal advances in biomarker discovery and a greater appreci- role this part of the nephron plays in the initiation, pro- ation of tubulointerstitial histopathology and the role of gression, staging, and therapeutic intervention in diabetic tubular hypoxia in the pathogenesis of chronic kidney kidney disease. From a pathogenetic perspective, as illus- disease have given us pause to reconsider the current trated in Fig. 1 and as elaborated on further in this review, “glomerulocentric” paradigm and focus attention on the proximal tubule that by virtue of the high energy require- tubular hypoxia as a consequence of increased energy de- ments and reliance on aerobic metabolism render it par- mands and reduced perfusion combine with nonhypoxia- ticularly susceptible to the derangements of the diabetic related forces to drive the development of tubular atrophy fi state. Such findings raise important issues for therapeu- and interstitial brosis in a vicious cycle that promotes tic advances specifically targeting the pathophysiological disease progression in diabetes. These insights offer new perturbations that develop in this part of the nephron. opportunities for therapeutic development. NORMAL AND DISORDERED STRUCTURE The description of diffuse and nodular glomerulosclerosis Anatomically, the proximal tubule refers to that part of the by Kimmelstiel and Wilson in 1936 (1) set investigation nephron that is directly contiguous with the parietal epi- on a course that has since focused primarily on the glo- thelium of Bowman’s capsule. Measuring approximately merulus as a means of understanding the pathogenesis of 14 mm in length in humans, it consists of three subtly dis- diabetic kidney disease. Changes in glomerular structure tinct segments. The S1 segment comprises the first two- such as mesangial expansion, reduction in capillary surface, thirds of the tubule’s early, convoluted component (pars and podocyte loss are undoubtedly major features of dia- convoluta); the S2 includes the final portion of the pars betic kidney disease that help differentiate it from other convoluta along with the initial, cortical part of its straight forms of glomerulonephritis. These findings are, however, component (pars recta); and the S3 makes up the remain- juxtaposed with the more recent knowledge that some der of the pars recta as it dives deeply into the cortex and patients with advanced disease display neither substantial outer medulla (3). glomerular pathology nor proteinuria and that kidney Cells in the S1 segment are characterized by a tall apical function declines well before traditional indicators of kid- brush border, prominent basolateral invaginations, exten- ney disease such as microalbuminuria or creatinine-based sive endocytic-lysosomal apparatus, and abundant, often

Keenan Research Centre for Biomedical Science and Li Ka Shing Knowledge © 2017 by the American Diabetes Association. Readers may use this article as Institute of St. Michael’s Hospital, Toronto, Canada long as the work is properly cited, the use is educational and not for proﬁt, and the Corresponding author: Richard E. Gilbert, [email protected]. work is not altered. More information is available at http://www.diabetesjournals .org/content/license. Received 30 June 2016 and accepted 2 January 2017. 792 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017

Figure 1—Proximal tubule and the pathogenesis of diabetic kidney disease. As a consequence of increased consumption, impaired utilization, and reduced delivery of O2, the proximal tubule, by virtue of its high energy requirements and reliance on aerobic metabolism, is susceptible to ischemic injury in diabetes. These pathophysiological disturbances combine with nonischemic mechanisms to induce apoptosis and ﬁbrosis in this part of the nephron that together lead to chronic loss of function and a propensity to AKI. Moreover, the diabetes-induced injury to the proximal tubule may in turn lead to glomerular pathology and postglomerular hypoperfusion, while ﬁbrotic expansion of the interstitium compresses and further disrupts the local microvasculature. RAS, renin-angiotensin system.

elongated, mitochondria. In the S2, epithelial cells have atubular glomeruli (5). Such changes are commonly ob- shorter brush borders, less prominent basolateral invagi- served in patients with type 1 diabetes with overt protein- nations, and smaller mitochondria, while in the S3 baso- uria; Najafian et al. (6) noted that in patients with normal lateral invaginations are absent and mitochondria are to moderately impaired GFR, 17% of glomeruli were atubular fewer (3) (Fig. 2). and an additional 51% were attached to atrophic tubules The proximal tubule undergoes a range of structural (Fig. 3). Similar findings have also been reported in type changes in diabetes such as tubular atrophy, interstitial 2 diabetes, where atubular glomeruli were found in 7% fibrosis, and peritubular capillary rarefaction, each of which of patients with diabetes, with a further 26% showing correlate closely with declining kidney function (4). Addi- glomerulotubular junction abnormalities even in the ab- tional dysfunction occurs when, as in cystinosis, atrophy sence of significant proteinuria so that extent of such occurs at the critical junction between Bowman’scapsule abnormalities correlated inversely with creatinine clear- and the proximal tubule, giving rise to nonfunctioning ance (r = 20.70, P = 0.011) (7).

Figure 2—Transmission electron micrographs of the proximal tubule of the rhesus monkey. The S1 segment (left) shows a typical tall columnar cell with numerous elongated mitochondrial profiles (M) enclosed within plications of the basal plasmalemma. Apical system of vesicles, vacuoles, and dense tubules are well developed. Magnification 39,165. In S2 (center), the brush border is more irregular with occasional skip areas (arrow). Apical vesicles and dense tubules are not as extensively developed, but apical vacuoles are more prominent. The cell is low columnar, and lateral interdigitations with adjacent cells are less complex. Magnification 38,900. Cells in S3 (right) are cuboidal and continue to exhibit a well-developed brush border. Apical dense tubules and apical vacuoles are not as extensive, although small apical vesicles are abundant. The basement membrane is very thin. Magnification 311,000. Reproduced with permission from Tischer et al. (81). AV, apical vacuole; BM, basement membrane; Cs, autophagic vacuole (cytosergresome); TL, tubular lumen. diabetes.diabetesjournals.org Gilbert 793

milieu. In addition, however, recent studies suggest that the proximal tubule may also contribute to glomerulop- athy. In their seminal 2013 study, Hasegawa et al. (8) provide evidence of retrograde trafficking between the proximal tubule and the glomerulus, showing that nico- tinamide mononucleotide (NMN) released by proximal tubular epithelial cells diffuses back to the glomerulus to induce podocyte foot process effacement and albuminuria (9) (Fig. 4). Given the importance of podocyte injury not only in the development of proteinuria but also in the progression of glomerulosclerosis and tubuloglomerular junction pathology (10), the triggering of glomerular pathology by the proximal tubule reinforces the primary importance of this region in disease development. We are, however, reminded of the importance of using mul- Figure 3—A photomicrograph of an atubular glomerulus showing tiple studies, preferably performed in different laborato- that while the glomerular tuft is indistinguishable from other glomer- ries using different animal models, to provide confidence uli, Bowman’s capsule is markedly thickened and wrinkled at a site for a new, potentially paradigm-shifting understanding in opposite to the vascular pole, where a tubular connection is expected how diabetic podocytopathy develops. but absent. PAS-stained; magnification 3630. Reproduced with per- fi mission from Najafian et al. (6). ↔,reduplicatedBowman’scapsule; Cognizant of the absence of signi cant albuminuria in arrowhead, a spindle-shape cell within the reduplicated Bowman’s many patients with declining GFR in diabetic kidney dis- capsule; arrows, atrophic tubules adjacent to the atubular glomerulus; ease, other studies show that proximal tubular injury leads *periglomerular fibrosis. not only to podocytopathy but also to more extensive glomerular injury. Using a mouse model of kidney disease wherein cells of the proximal tubule express the diphtheria toxin receptor, two research groups induced site-selective PRIMARY TUBULOPATHY INDUCES SECONDARY injury to the proximal tubule. While recovery occurred after GLOMERULAR DISEASE a single, low dose administration, repeated dosing of toxin Glomerular pathology in diabetes occurs as a consequence induced all the hallmarks of human diabetic kidney disease of the interaction between resident glomerular endo- with glomerulosclerosis, interstitial fibrosis, capillary rare- thelial, mesangial, and epithelial cells with the diabetic faction, tubular atrophy, proteinuria, and elevated serum

Figure 4—Diagram illustrating how proximal tubular injury in diabetes leads to podocyte foot process effacement and albuminuria (9). In diabetic mice (top part of diagram), proximal tubule Sirt1 expression is decreased, leading to a reduction in local (glomerular and tubular) NMN concentrations that in turn lead to increased Claudin-1 expression in podocytes, which causes foot process effacement and albuminuria. Reproduced with permission from Nihalani and Susztak (9). 794 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017 creatinine (11,12), emphasizing the impact of repeated or type 2 et GENEtique (SURDIAGENE) study of individu- continuing injurious stimuli such as those of the diabetic als with diabetes. In that study, AKI not only predicted a milieu. However, though these studies were not under- 2.47-fold increase in the likelihood of doubling serum taken in diabetic animals, they nevertheless illustrate how creatinine or developing end-stage renal disease but was intermittent or continuing proximal tubular apoptosis, a also a major predictor of heart failure hospitalization, common feature of human diabetic kidney disease (13), myocardial infarction, stroke, and cardiovascular death may lead secondarily to glomerulosclerosis. even after adjusting for eGFR and albuminuria in multivariate analyses (19). THE CONTIUUM OF ACUTE AND CHRONIC From an intervention perspective, it is noteworthy KIDNEY DISEASE that sodium–glucose cotransporter 2 (SGLT2) inhibitors The proximal tubule is highly susceptible to ischemia and with their lowering of systemic blood pressure (20) and toxin-induced injury that result in acute kidney injury increase in afferent arteriolar resistance (21) might, in (AKI). Indeed, the term acute tubular necrosis was pre- theory, be expected to increase the likelihood of AKI. viously used interchangeably with acute renal failure and However, the reverse relationship was found in the BI AKI. In addition to their propensity to develop chronic 10773 (Empagliflozin) Cardiovascular Outcome Event kidney disease (CKD), individuals with diabetes are also Trial in Type 2 Diabetes Mellitus Patients (EMPA-REG at much higher risk of AKI (14). While these two disor- OUTCOME) trial, wherein empagliflozin reduced AKI and ders were previously viewed as distinct, more recent in- acute renal failure (22). These findings, as discussed later formation indicates that they are closely interrelated, so in the section on Na+ transport, suggest that SGLT2 in- that patients with CKD are at higher risk of AKI and hibitor–mediated reduced proximal tubular energy require- patients with AKI are at greater risk of progressing to ments may have not only increased the kidneys’ resilience CKD. Indeed, even with apparent full recovery, AKI may to acute injury (23) but, given their interrelationship be followed by maladaptive tubular repair with fibrosis, with AKI, such findings may also explain the reduction inflammation, and microvascular rarefaction that lead to in CKD progression and cardiovascular disease noted in the development of CKD (15–17). the EMPA-REG OUTCOME trial (22,24). The relationship between episodes of AKI and CKD progression in diabetes is well supported by epidemiological BIOMARKERS OF PROXIMAL TUBULAR INJURY data. Over a 10-year period, Thakar et al. (18) noted that The realization that many patients with diabetes and low AKI was a common event, occurring in 29% of hospitalized GFR do not have significant albuminuria and that GFR veterans with diabetes. In addition, the study also noted decline frequently precedes the development of micro- that each episode of AKI conferred a doubling in the risk of albuminuria (25) has led to a vigorous search for alterna- , 2 progression to CKD stage 4 (eGFR 30 mL/min/1.73 m ), tive or additional biomarkers (26). Among those that independent of other covariates associated with disease appear relatively specific to proximal tubular epithelial cells progression (18). Furthermore, the effect of AKI on kidney are kidney injury molecule 1 (KIM-1), liver fatty acid bind- “ ” prognosis was dose dependent, worsening incrementally ing protein (L-FABP), and N-acetyl-b-D-glucosaminidase with the number of AKI episodes sustained (Fig. 5). Similar (NAG). findings have also been reported in the SURvie, DIAbete de KIM-1 Several studies have examined urinary KIM-1 in diabetes, showing that while its urinary excretion increases com- mensurately with declining kidney function, it provides little additional information on risk or progression beyond conventional markers in either type 1 or type 2 diabetes (27,28). Serum and plasma concentrations may, on the other hand, be more helpful. In type 1 diabetes, serum KIM-1 concentrations continued to predict eGFR loss and risk of end-stage kidney disease in subjects with type 1 diabetes and proteinuria after adjustment for baseline urinary albumin-to-creatinine ratio, eGFR, and HbA1c (29). In a follow-on study, the same Joslin investigators examined the predictive power of plasma KIM-1 in individuals with type 1 diabetes with normo- and microalbuminuria whose kidney function, as measured by serum Figure 5—Survival to stage 4 CKD (eGFR <30 mL/min/1.73 m2)in creatinine and cystatin C, was normal at baseline (30) patients with diabetes according to the number of AKI episodes (Fig. 6). In a multivariate model, plasma KIM-1 remained during hospitalization. No episodes of AKI ($$$), one episode AKI (--), two episodes AKI (—), three or more episodes AKI (—). Repro- strongly associated with the risk of renal function decline duced and adapted with permission from Thakar et al. (18). regardless of baseline characteristics, reinforcing the view diabetes.diabetesjournals.org Gilbert 795

TUBULAR HYPOXIA: A DRIVING FORCE IN DIABETIC KIDNEY DISEASE Receiving ;20% of cardiac output, much of the kidney’s high O2 requirements are accounted for by the enormous reabsorptive functions of the proximal tubule that, in turn, render it particularly vulnerable to hypoxia. Indeed, this vul- nerability forms the basis for the now well-established “chronic hypoxia theory” of CKD elaborated by Fine et al. in 1998 (38) and further refined in subsequent itera- tions by other investigators. In experimental diabetes, principally as a result of increased O2 consumption, kidney cortex pO2 is ;10 mmHg lower than in controls (39). The consequences of hypoxia for proximal tubular epithelial cells is similar to that at other sites, leading not only to apoptosis (40) but also to stimulation of both tu- Figure 6—Incidence of CKD $ stage 3 according to baseline strata bular cells and resident fibroblasts to elaborate increased of plasma KIM-1 in normoalbuminuric (NA) and microalbuminuric quantities of extracellular matrix by both transforming (MA) individuals with type 1 diabetes whose renal function as mea- growth factor-b (TGF-b)–dependent and –independent sured by eGFR and cystatin C was normal at baseline. Reproduced – with permission from Nowak et al. (30). ND, not detectable mechanisms (38,41 43). The resultant extracellular matrix (<0.2 pg/mL). p-ys, person-years. T1–T3, tertiles of the distribution expansion not only increases the diffusion distance for O2 of detectable values of urinary KIM-1. delivery to the parenchyma but also compresses and disrupts the local architecture, leading to microvascular rarefaction. This then further aggravates the extent of that proximal tubular injury plays a role in the early func- tubulointerstitial hypoxia, setting up a vicious cycle tion decline in diabetes (30). whereby fibrosis begets more fibrosis (Fig. 1). As elaborated in detail below, the proximal tubule’s L-FABP propensity to hypoxic injury in diabetes can be attributed L-FABP is a 15-kDa protein that, as it names suggests, to three factors: 1) an increase in metabolic activity as a regulates fatty acid transfer in a range of organs including the consequence of the high energy consuming processes of proximal tubule (31). An elevated urinary L-FABP concentra- sodium reabsorption and gluconeogenesis, 2) impaired O tion provides a rapid and sensitive indicator of AKI risk 2 utilization due to altered substrate delivery and mito- following cardiac surgery and sepsis that has also been ex- chondrial dysfunction, and 3) reduced O due to micro- amined as a predictive marker of kidney disease progression 2 vascular rarefaction (Fig. 1). in diabetes. In individuals with type 1 diabetes, an increase in urinary L-FABP precedes the development of microalbumi- Sodium Reabsorption nuria and falls with ACE inhibition (32). Further studies The evolutionary move some 365 million years from the attest to the ability of urinary L-FABP to predict kidney sea onto dry land required substantial changes that disease progression and all-cause mortality in type 1 diabetes, included not only adaptation to atmospheric O but also independently of urinary albumin excretion (33), with similar 2 the ability to avidly reabsorb sodium in this new, compar- ability to predict cardio-renal end points in Japanese patients atively salt-deficient environment. Indeed, 60% of the with type 2 diabetes without overt proteinuria (34). In an- kidney’s overall energy consumption is devoted to sodium other report, however, the FinnDiane (Finnish Diabetic Ne- reclamation with the proximal tubule responsible for al- phropathy) Study Group found that urinary L-FABP may not most two-thirds, primarily through the activity of the improve risk prediction more than albuminuria but still ad- + + basal Na /K ATPase, quantified as ouabain-sensitive O vocated for further studies be done (35). 2 consumption (44). NAG While sodium–glucose linked transport across the api- The urinary excretion of NAG, a lysosomal proximal cal membrane of the proximal tubular cell is not of itself tubular enzyme that is found predominantly in the prox- an energy-requiring process, its continuing activity is imal tubule, is also increased in diabetes even in the setting dependent on the maintenance of the electrochemical of normoalbuminuria and normal eGFR, consistent with gradient for Na+, generated by Na+/K+ ATPase activity the view that proximal tubular dysfunction is a measurable (Fig. 7). Accordingly, the increase in glucose reabsorptive component of early diabetic kidney disease (36). Further capacity that develops in diabetes (45) is accompanied by increases in urinary NAG are noted in the presence of a commensurate demand in Na+/K+ ATPase activity that microalbuminuria and moreover, similar to KIM-1, lower when measured by ouabain inhibitable O2 consumption baseline concentrations of NAG were associated with increases by about 30% in the experimental setting (46). regression of microalbuminuria over a 2-year period in Here, the SGLT1/2 inhibitor phlorizin was shown to ame- individuals with type 1 diabetes (37). liorate the diabetes-induced increase in Na+/K+ ATPase 796 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017

Figure 7—SGLTs. SGLT1 and SGLT2 mediate the transport of glucose by coupling it with the downhill transport of sodium. While glucose diffuses out basolaterally by facilitative transporters GLUT1 and GLUT2, sodium’s extrusion across the antiluminal membrane into the intercellular ﬂuid requires ATP hydrolysis (upper panel). Adapted with permission from Chao and Henry (82). The lower panels show the effects of the SGLT1/2 inhibitor, phlorizin, on tubular Na/K ATPase activity and O2 extraction in diabetic rats. A-V O2, arteriovenous oxygen difference. Adapted with permission from Körner et al. (46).

and O2 consumption (46) (Fig. 7). Consistent with these perspective, gluconeogenesis is a demanding process, re- findings, the SGLT2 inhibitor dapagliflozin renders proxi- quiring six energy equivalents (4 ATP, 2 GTP) to synthe- mal tubular epithelial cells resistant to hypoxia-induced size a single molecule of glucose from lactate or pyruvate, apoptosis, affording protection from ischemia-reperfusion contrasting sharply with the 2 molecules of ATP that are injury (47). Together, these findings raise the intriguing generated by glycolysis. Indeed, gluconeogenesis is a ma- possibility that the reduction in both GFR decline and jor source of the kidney’s ouabain-insensitive O2 usage AKI reported in the EMPA-REG OUTCOME trial (22) and energy expenditure, accounting for up to 25% of the may be the result of reduced proximal tubular energy re- energy needed for sodium reabsorption (50). quirements, the ability of the drug to improve glycemia and In the nondiabetic setting, the liver, by a combina- modulate glomerular hemodynamics notwithstanding (23). tion of glycogenolysis (50%) and gluconeogenesis (30%), In addition to the SGLTs, apical sodium transport in the contributes 80% of glucose released into the circulation, proximal tubule is mediated by several other carrier with the remaining 20% derived from renal gluconeogen- proteins including sodium–hydrogen exchanger 3 (NHE3) esis (52). In the postprandial state when glycogenolysis that, similar to the SGLTs, require Na+/K+ ATPase to main- and hepatic gluconeogenesis are relatively suppressed, de tain an electrochemical gradient. Increases in NHE3 have novo glucose synthesis by the kidney accounts for ;60% been reported in human proximal tubular cells exposed to of endogenous glucose release (52). The extent of renal high glucose and in animals with streptozotocin-induced gluconeogenesis is increased in diabetes where in the diabetes (48). A similar increase in Na-lactate cotransporter fasted state gluconeogenic activity increases approxi- activity, as a consequence of increased lactate production in mately threefold such that the kidney releases on average the setting of poor glycemic control (49), has also been 2.21 mmol/kg/min of glucose into the circulation, only shown to occur in diabetes (50). marginally lower than the liver’s 2.60 mmol/kg/min Gluconeogenesis (53). Postprandial glucose release by the kidney is simi- While the proximal tubule reabsorbs glucose, it does not larly increased in subjects with type 2 diabetes when compared with age-, weight-, and sex-matched volunteers metabolize any of the enormous load that traverses it, without diabetes (54). relying on lactate, glutamate, and ketones as alternative substrates for energy production (51). Along with hepa- O2 Utilization tocytes, proximal tubular epithelial cells are unique in Given that the proximal tubule’s metabolic activity is al- their ability to undertake gluconeogenesis and export glu- most entirely oxidative, there is a commensurate reduction cose into the circulation, although unlike the liver, the in tricarboxylic acid cycle activity and ATP production in kidney is not responsive to glucagon. From an energy the relatively hypoxic diabetic kidney (55,56). Moreover, by diabetes.diabetesjournals.org Gilbert 797

requiring more O2 to be consumed for each molecule of Therapeutic Angiogenesis ATP generated, the increase in free fatty acids, a compo- Given the lack of observable capillary loss in animal models nent of the dysglycemic state, may exacerbate the extent of of diabetic kidney disease, much of the experimental work ischemia. This relative energy inefficiency can be quantified on therapeutic angiogenesis has relied on nondiabetic as the ATP/O2 ratio where glucose has a ratio of 3.17 while models such as renal artery stenosis and following subtotal palmitate has a ratio of 2.83 (57), and while these differ- nephrectomy (SNX) that do develop substantial capillary ences in oxygen consumption may seem modest, they can rarefaction. Here, a number of strategies for reconstituting have substantial impact when O2 delivery is marginal. the microcirculation are in development including mesen- Exacerbating the increased O2 demands in diabetes is chymal stem cells (MSCs), so-called endothelial progenitor the recent realization that mitochondria, the organelles cells (EPCs), and extracorporeal shockwave therapy. EPCs, responsible for aerobic energy production, are structurally for instance, have been shown to exert both proangiogenic abnormal and dysfunctional in diabetes (58,59). Indeed, and antifibrotic properties, attenuating capillary loss in the abnormalities of proximal tubular mitochondrial structure tubulointerstitium and glomerulus as well as preserving and function may be the earliest manifestation of kidney kidney function in the SNX rat (69). MSCs, on the other disease. In the rat, for instance, Coughlan et al. (60) found hand, have a range of potential beneficial effects that in evidence of impaired mitochondrial ATP generation and addition to their angiogenic, immunomodulatory, and anti- organelle fragmentation in proximal tubular epithelial cells inflammatory activities also lower blood glucose in humans as early as 4 weeks after the induction of experimental with diabetes by as yet unknown mechanisms (70). Most diabetes. That these changes precede increases in urinary recently, a phase II trial of allogeneic MSCs in patients albumin excretion, abnormal glomerular morphology, or with advanced diabetic kidney disease has been reported even elevation of urinary KIM-1 suggests that they may (NCT01843387). This study compared the effects of two be primary abnormalities. From a therapeutic point of dosesofMSCsin30subjectswhosebaselineGFRwas view, such findings also raise the possibility of using strat- 20–50 mL/min/1.73 m2, showing a trend in stabilizing egies that regulate mitochondrial biogenesis such as the measured GFR at 12 weeks when compared with the con- silent information regulator 1 activators that are currently tinuing decline in placebo-treated patients (71). undergoing clinical trial in a range of chronic diseases (61). Perhaps most tantalizing because of its simplicity is the finding that low-energy extracorporeal shockwave therapy O Supply 2 (ESWT) induces angiogenesis. In a double-blind, placebo High glucose and its downstream effector molecules have controlled study of patients with severe angina, Kikuchi long been known to increase endothelial cell apoptosis in et al. (72) used this technology to improve blood flow and cell culture (62,63). Importantly, these changes, recognized function in the ischemic heart, reducing pain scores and as capillary rarefaction, are also seen in the in vivo setting improving left ventricular ejection fraction. Using a sim- as a characteristic feature of diabetic kidney disease that ilar strategy in pigs with renal artery stenosis, Zhang et al. correlates with declining kidney function (64–67) (Fig. 8). (73) reported improvement in microvascular density and Though potentially compensated by endothelial cell regen- tissue oxygenation along with reduced fibrosis and better eration, this reparative process, if anything, is impaired in kidney function after six sessions of ESWT. In exploring diabetes (68). As a result, blood supply to the proximal the mechanisms that underlay these effects, this group tubule is impaired both by the intrinsic capillary loss within noted elevated expression of vascular endothelial growth the tubulointerstitium described above and a consequence factor, mainly in proximal tubular cells along with a reduc- of glomerular capillary occlusion (64,65). tion in TGF-b expression, providing further substance to the burgeoning exploration of mechanotransduction in disease development and reversal. The effects of ESWT in human diabetic kidney disease, however, remain unknown.

NONHYPOXIA-RELATED PROXIMAL TUBULAR MECHANISMS In addition to the hypoxia, several other nonhypoxia- related proximal tubule pathways involved in the development of diabetic kidney disease have been the subjects of recent reviews. These include the now well-documented local, predominantly proximal tubule–based renin-angiotensin system (74), the toxic effects of albumin bound fatty acids (75,76), and the activation of epidermal growth factor receptor signaling pathways (77). Still more recently, and — Figure 8 Relationship between postglomerular capillary density not yet the subject of detailed review, is the exploration and serum creatinine in diabetic kidney disease showing an inverse correlation (r = 20.73, P < 0.001) in 72 patients. Reproduced and whereby diabetic kidney disease, like most forms of CKD, adapted with permission from Bohle et al. (65). once started, continues to progress inexorably. Among the 798 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017 potential contributors to this process is the recent finding CONCLUSION fi that organ stiffness, an inevitable consequence of brosis, Although much work needs to be done, substantial data fi fl fi induces further brosis. Brie y, the presence of brosis now support the existence of a diabetes-induced proximal leads to tissue stiffness that can be sensed mechanically tubulopathy as an early disease event that both predicts fi by cells. Rather than dampening the brogenic process, the and contributes to the development of CKD in diabetes. presence of a stiff matrix seems to induce a positive feed- While not ignoring the glomerulus, directing attention to back cycle to enhance it. The leading contenders in our the proximal tubule for biomarker development, thera- current understanding of this process are the mechano- peutic discovery, and pathophysiological understanding transducing transcription cofactors: Yes-associated protein seems prescient. (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Together, YAP and TAZ perpetuate TGF-b signaling intermediates, Smad2/3, to be retained in the Acknowledgments. The author regrets that owing to space constraints, nucleus, thereby perpetuating its fibrogenic activity (78). much of the excellent work that has been done on this subject could not be Consistent with diabetes as a profibrotic state, YAP expres- included. sion and phosphorylation are increased in diabetic mouse Funding. R.E.G. is the Canada Research Chair in Diabetes Complications, and proximal tubular cells so that modulating TAZ-YAP and this review was supported in part by Canada Research Chairs Program grant related pathways has become an important new target 950-218644. for drug development in diabetic kidney disease and other Duality of Interest. R.E.G. reports having received consulting and lecture chronic diseases that are characterized by fibrosis (79). fees from Merck, AstraZeneca, Eli Lilly, Boehringer Ingelheim, Mesoblast, and Janssen along with grant funds through his institution from Merck, AstraZeneca, PROXIMAL TUBULE–TARGETED THERAPEUTICS Eli Lilly, and Boehringer Ingelheim. R.E.G. was formerly a shareholder in Fibrotech Therapeutics, which was wholly acquired by Shire Pharmaceuticals in 2014. No The revolution in molecular biology has highlighted the other potential conflicts of interest relevant to this article were reported. extent of pathophysiological derangements in the diabetic kidney that include a broad range of perturbations in epige- References netics, protein–protein interactions, transcriptional changes, 1. Kimmelstiel P, Wilson C. Intercapillary lesions in the glomerulus. Am J and posttranslational modifications. Unfortunately, moving Pathol 1936;12:83–97, 7 these discoveries into new therapies has been limited in part 2. Krolewski AS. Progressive renal decline: the new paradigm of diabetic – by the “druggability” of the target, i.e., the likelihood of nephropathy in type 1 diabetes. Diabetes Care 2015;38:954 962 Brenner and Rector’s being able to modulate a target with a small-molecule drug. 3. Fenton RA, Praetorius J. Anatomy of the kidney. In The Kidney. 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