Richard E. Gilbert

Proximal Tubulopathy: Prime Moverand Key Therapeutic Target in DiabeticKidney DiseaseDiabetes 2017;66:791–800 | DOI: 10.2337/db16-0796

The current view of diabetic kidney disease, based onmeticulously acquired ultrastructural morphometry andthe utility of measuring plasma creatinine and urinary al-bumin, has been almost entirely focused on the glomer-ulus. While clearly of great importance, changes in theglomerulus are not the major determinant of renal prog-nosis in diabetes and may not be the primary event in thedevelopment of diabetic kidney disease either. Indeed,advances in biomarker discovery and a greater appreci-ation of tubulointerstitial histopathology and the role oftubular hypoxia in the pathogenesis of chronic kidneydisease have given us pause to reconsider the current“glomerulocentric” paradigm and focus attention on theproximal tubule that by virtue of the high energy require-ments and reliance on aerobic metabolism render it par-ticularly susceptible to the derangements of the diabeticstate. Such findings raise important issues for therapeu-tic advances specifically targeting the pathophysiologicalperturbations that develop in this part of the nephron.

The description of diffuse and nodular glomerulosclerosisby Kimmelstiel and Wilson in 1936 (1) set investigationon a course that has since focused primarily on the glo-merulus as a means of understanding the pathogenesis ofdiabetic kidney disease. Changes in glomerular structuresuch as mesangial expansion, reduction in capillary surface,and podocyte loss are undoubtedly major features of dia-betic kidney disease that help differentiate it from otherforms of glomerulonephritis. These findings are, however,juxtaposed with the more recent knowledge that somepatients with advanced disease display neither substantialglomerular pathology nor proteinuria and that kidneyfunction declines well before traditional indicators of kid-ney disease such as microalbuminuria or creatinine-based

estimated glomerular filtration rate (eGFR) decline (2). Inrecognition of these findings, the term diabetic kidneydisease rather than diabetic nephropathy is now commonlyused. On the background of recent advances in the role ofthe proximal tubule as a prime mover in diabetic kidneypathology, this review highlights key recent developments.Published mostly in the general scientific and kidney-specific literature, these advances highlight the pivotalrole this part of the nephron plays in the initiation, pro-gression, staging, and therapeutic intervention in diabetickidney disease. From a pathogenetic perspective, as illus-trated in Fig. 1 and as elaborated on further in this review,tubular hypoxia as a consequence of increased energy de-mands and reduced perfusion combine with nonhypoxia-related forces to drive the development of tubular atrophyand interstitial fibrosis in a vicious cycle that promotesdisease progression in diabetes. These insights offer newopportunities for therapeutic development.

NORMAL AND DISORDERED STRUCTURE

Anatomically, the proximal tubule refers to that part of thenephron that is directly contiguous with the parietal epi-thelium of Bowman’s capsule. Measuring approximately14 mm in length in humans, it consists of three subtly dis-tinct segments. The S1 segment comprises the first two-thirds of the tubule’s early, convoluted component (parsconvoluta); the S2 includes the final portion of the parsconvoluta along with the initial, cortical part of its straightcomponent (pars recta); and the S3 makes up the remain-der of the pars recta as it dives deeply into the cortex andouter medulla (3).

Cells in the S1 segment are characterized by a tall apicalbrush border, prominent basolateral invaginations, exten-sive endocytic-lysosomal apparatus, and abundant, often

Keenan Research Centre for Biomedical Science and Li Ka Shing KnowledgeInstitute of St. Michael’s Hospital, Toronto, Canada

Corresponding author: Richard E. Gilbert, richard.gilbert@utoronto.ca.

Received 30 June 2016 and accepted 2 January 2017.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

Diabetes Volume 66, April 2017 791

PERSPECTIVESIN

DIA

BETES

elongated, mitochondria. In the S2, epithelial cells haveshorter brush borders, less prominent basolateral invagi-nations, and smaller mitochondria, while in the S3 baso-lateral invaginations are absent and mitochondria arefewer (3) (Fig. 2).

The proximal tubule undergoes a range of structuralchanges in diabetes such as tubular atrophy, interstitialfibrosis, and peritubular capillary rarefaction, each of whichcorrelate closely with declining kidney function (4). Addi-tional dysfunction occurs when, as in cystinosis, atrophyoccurs at the critical junction between Bowman’s capsuleand the proximal tubule, giving rise to nonfunctioning

atubular glomeruli (5). Such changes are commonly ob-served in patients with type 1 diabetes with overt protein-uria; Najafian et al. (6) noted that in patients with normalto moderately impaired GFR, 17% of glomeruli were atubularand an additional 51% were attached to atrophic tubules(Fig. 3). Similar findings have also been reported in type2 diabetes, where atubular glomeruli were found in 7%of patients with diabetes, with a further 26% showingglomerulotubular junction abnormalities even in the ab-sence of significant proteinuria so that extent of suchabnormalities correlated inversely with creatinine clear-ance (r = 20.70, P = 0.011) (7).

Figure 1—Proximal tubule and the pathogenesis of diabetic kidney disease. As a consequence of increased consumption, impairedutilization, 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 induceapoptosis and fibrosis in this part of the nephron that together lead to chronic loss of function and a propensity to AKI. Moreover, thediabetes-induced injury to the proximal tubule may in turn lead to glomerular pathology and postglomerular hypoperfusion, while fibroticexpansion of the interstitium compresses and further disrupts the local microvasculature. RAS, renin-angiotensin system.

Figure 2—Transmission electron micrographs of the proximal tubule of the rhesus monkey. The S1 segment (left) shows a typical tallcolumnar cell with numerous elongated mitochondrial profiles (M) enclosed within plications of the basal plasmalemma. Apical system ofvesicles, vacuoles, and dense tubules are well developed. Magnification 39,165. In S2 (center), the brush border is more irregular withoccasional 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. Magnification38,900. Cells in S3 (right) are cuboidaland continue to exhibit a well-developed brush border. Apical dense tubules and apical vacuoles are not as extensive, although small apicalvesicles are abundant. The basement membrane is very thin. Magnification311,000. Reproduced with permission from Tischer et al. (81). AV,apical vacuole; BM, basement membrane; Cs, autophagic vacuole (cytosergresome); TL, tubular lumen.

792 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017

PRIMARY TUBULOPATHY INDUCES SECONDARYGLOMERULAR DISEASE

Glomerular pathology in diabetes occurs as a consequenceof the interaction between resident glomerular endo-thelial, mesangial, and epithelial cells with the diabetic

milieu. In addition, however, recent studies suggest thatthe proximal tubule may also contribute to glomerulop-athy. In their seminal 2013 study, Hasegawa et al. (8)provide evidence of retrograde trafficking between theproximal tubule and the glomerulus, showing that nico-tinamide mononucleotide (NMN) released by proximaltubular epithelial cells diffuses back to the glomerulusto induce podocyte foot process effacement and albumin-uria (9) (Fig. 4). Given the importance of podocyte injurynot only in the development of proteinuria but also in theprogression of glomerulosclerosis and tubuloglomerularjunction pathology (10), the triggering of glomerular pa-thology by the proximal tubule reinforces the primaryimportance of this region in disease development. Weare, however, reminded of the importance of using mul-tiple studies, preferably performed in different laborato-ries using different animal models, to provide confidencefor a new, potentially paradigm-shifting understanding inhow diabetic podocytopathy develops.

Cognizant of the absence of significant albuminuria inmany patients with declining GFR in diabetic kidney dis-ease, other studies show that proximal tubular injury leadsnot only to podocytopathy but also to more extensiveglomerular injury. Using a mouse model of kidney diseasewherein cells of the proximal tubule express the diphtheriatoxin receptor, two research groups induced site-selectiveinjury to the proximal tubule. While recovery occurred aftera single, low dose administration, repeated dosing of toxininduced all the hallmarks of human diabetic kidney diseasewith glomerulosclerosis, interstitial fibrosis, capillary rare-faction, tubular atrophy, proteinuria, and elevated serum

Figure 3—A photomicrograph of an atubular glomerulus showingthat while the glomerular tuft is indistinguishable from other glomer-uli, Bowman’s capsule is markedly thickened and wrinkled at a siteopposite to the vascular pole, where a tubular connection is expectedbut absent. PAS-stained; magnification 3630. Reproduced with per-mission from Najafian et al. (6). ↔, reduplicated Bowman’s capsule;arrowhead, a spindle-shape cell within the reduplicated Bowman’scapsule; arrows, atrophic tubules adjacent to the atubular glomerulus;*periglomerular fibrosis.

Figure 4—Diagram illustrating how proximal tubular injury in diabetes leads to podocyte foot process effacement and albuminuria (9). In diabeticmice (top part of diagram), proximal tubule Sirt1 expression is decreased, leading to a reduction in local (glomerular and tubular) NMN concen-trations that in turn lead to increased Claudin-1 expression in podocytes, which causes foot process effacement and albuminuria. Reproducedwith permission from Nihalani and Susztak (9).

diabetes.diabetesjournals.org Gilbert 793

creatinine (11,12), emphasizing the impact of repeated orcontinuing injurious stimuli such as those of the diabeticmilieu. However, though these studies were not under-taken in diabetic animals, they nevertheless illustrate howintermittent or continuing proximal tubular apoptosis, acommon feature of human diabetic kidney disease (13),may lead secondarily to glomerulosclerosis.

THE CONTIUUM OF ACUTE AND CHRONICKIDNEY DISEASE

The proximal tubule is highly susceptible to ischemia andtoxin-induced injury that result in acute kidney injury(AKI). Indeed, the term acute tubular necrosis was pre-viously used interchangeably with acute renal failure andAKI. In addition to their propensity to develop chronickidney disease (CKD), individuals with diabetes are alsoat much higher risk of AKI (14). While these two disor-ders were previously viewed as distinct, more recent in-formation indicates that they are closely interrelated, sothat patients with CKD are at higher risk of AKI andpatients with AKI are at greater risk of progressing toCKD. Indeed, even with apparent full recovery, AKI maybe followed by maladaptive tubular repair with fibrosis,inflammation, and microvascular rarefaction that lead tothe development of CKD (15–17).

The relationship between episodes of AKI and CKDprogression in diabetes is well supported by epidemiologicaldata. Over a 10-year period, Thakar et al. (18) noted thatAKI was a common event, occurring in 29% of hospitalizedveterans with diabetes. In addition, the study also notedthat each episode of AKI conferred a doubling in the risk ofprogression to CKD stage 4 (eGFR ,30 mL/min/1.73 m2),independent of other covariates associated with diseaseprogression (18). Furthermore, the effect of AKI on kidneyprognosis was “dose dependent,” worsening incrementallywith the number of AKI episodes sustained (Fig. 5). Similarfindings have also been reported in the SURvie, DIAbete de

type 2 et GENEtique (SURDIAGENE) study of individu-als with diabetes. In that study, AKI not only predicted a2.47-fold increase in the likelihood of doubling serumcreatinine or developing end-stage renal disease but wasalso a major predictor of heart failure hospitalization,myocardial infarction, stroke, and cardiovascular deatheven after adjusting for eGFR and albuminuria in mul-tivariate analyses (19).

From an intervention perspective, it is noteworthythat sodium–glucose cotransporter 2 (SGLT2) inhibitorswith their lowering of systemic blood pressure (20) andincrease in afferent arteriolar resistance (21) might, intheory, be expected to increase the likelihood of AKI.However, the reverse relationship was found in the BI10773 (Empagliflozin) Cardiovascular Outcome EventTrial in Type 2 Diabetes Mellitus Patients (EMPA-REGOUTCOME) trial, wherein empagliflozin reduced AKI andacute renal failure (22). These findings, as discussed laterin the section on Na+ transport, suggest that SGLT2 in-hibitor–mediated reduced proximal tubular energy require-ments may have not only increased the kidneys’ resilienceto acute injury (23) but, given their interrelationshipwith AKI, such findings may also explain the reductionin CKD progression and cardiovascular disease noted inthe EMPA-REG OUTCOME trial (22,24).

BIOMARKERS OF PROXIMAL TUBULAR INJURY

The realization that many patients with diabetes and lowGFR do not have significant albuminuria and that GFRdecline frequently precedes the development of micro-albuminuria (25) has led to a vigorous search for alterna-tive or additional biomarkers (26). Among those thatappear relatively specific to proximal tubular epithelial cellsare kidney injury molecule 1 (KIM-1), liver fatty acid bind-ing protein (L-FABP), and N-acetyl-b-D-glucosaminidase(NAG).

KIM-1Several studies have examined urinary KIM-1 in diabetes,showing that while its urinary excretion increases com-mensurately with declining kidney function, it provideslittle additional information on risk or progression beyondconventional markers in either type 1 or type 2 diabetes(27,28). Serum and plasma concentrations may, on theother hand, be more helpful. In type 1 diabetes, serumKIM-1 concentrations continued to predict eGFR lossand risk of end-stage kidney disease in subjects withtype 1 diabetes and proteinuria after adjustment for base-line urinary albumin-to-creatinine ratio, eGFR, and HbA1c(29). In a follow-on study, the same Joslin investigatorsexamined the predictive power of plasma KIM-1 in indi-viduals with type 1 diabetes with normo- and microalbu-minuria whose kidney function, as measured by serumcreatinine and cystatin C, was normal at baseline (30)(Fig. 6). In a multivariate model, plasma KIM-1 remainedstrongly associated with the risk of renal function declineregardless of baseline characteristics, reinforcing the view

Figure 5—Survival to stage 4 CKD (eGFR <30 mL/min/1.73 m2) inpatients with diabetes according to the number of AKI episodesduring hospitalization. No episodes of AKI ($$$), one episode AKI(--), two episodes AKI (—), three or more episodes AKI (—). Repro-duced and adapted with permission from Thakar et al. (18).

794 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017

that proximal tubular injury plays a role in the early func-tion decline in diabetes (30).

L-FABPL-FABP is a 15-kDa protein that, as it names suggests,regulates fatty acid transfer in a range of organs including theproximal tubule (31). An elevated urinary L-FABP concentra-tion provides a rapid and sensitive indicator of AKI riskfollowing cardiac surgery and sepsis that has also been ex-amined as a predictive marker of kidney disease progressionin diabetes. In individuals with type 1 diabetes, an increase inurinary L-FABP precedes the development of microalbumi-nuria and falls with ACE inhibition (32). Further studiesattest to the ability of urinary L-FABP to predict kidneydisease progression and all-cause mortality in type 1 diabetes,independently of urinary albumin excretion (33), with similarability to predict cardio-renal end points in Japanese patientswith type 2 diabetes without overt proteinuria (34). In an-other report, however, the FinnDiane (Finnish Diabetic Ne-phropathy) Study Group found that urinary L-FABP may notimprove risk prediction more than albuminuria but still ad-vocated for further studies be done (35).

NAGThe urinary excretion of NAG, a lysosomal proximaltubular enzyme that is found predominantly in the prox-imal tubule, is also increased in diabetes even in the settingof normoalbuminuria and normal eGFR, consistent withthe view that proximal tubular dysfunction is a measurablecomponent of early diabetic kidney disease (36). Furtherincreases in urinary NAG are noted in the presence ofmicroalbuminuria and moreover, similar to KIM-1, lowerbaseline concentrations of NAG were associated withregression of microalbuminuria over a 2-year period inindividuals with type 1 diabetes (37).

TUBULAR HYPOXIA: A DRIVING FORCE INDIABETIC KIDNEY DISEASE

Receiving ;20% of cardiac output, much of the kidney’shigh O2 requirements are accounted for by the enormousreabsorptive 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 tubularepithelial cells is similar to that at other sites, leading notonly to apoptosis (40) but also to stimulation of both tu-bular cells and resident fibroblasts to elaborate increasedquantities of extracellular matrix by both transforminggrowth factor-b (TGF-b)–dependent and –independentmechanisms (38,41–43). The resultant extracellular matrixexpansion not only increases the diffusion distance for O2

delivery to the parenchyma but also compresses and dis-rupts the local architecture, leading to microvascularrarefaction. This then further aggravates the extent oftubulointerstitial hypoxia, setting up a vicious cyclewhereby fibrosis begets more fibrosis (Fig. 1).

As elaborated in detail below, the proximal tubule’spropensity to hypoxic injury in diabetes can be attributedto three factors: 1) an increase in metabolic activity as aconsequence of the high energy consuming processes ofsodium reabsorption and gluconeogenesis, 2) impaired O2

utilization due to altered substrate delivery and mito-chondrial dysfunction, and 3) reduced O2 due to micro-vascular rarefaction (Fig. 1).

Sodium ReabsorptionThe evolutionary move some 365 million years from thesea onto dry land required substantial changes thatincluded not only adaptation to atmospheric O2 but alsothe ability to avidly reabsorb sodium in this new, compar-atively salt-deficient environment. Indeed, 60% of thekidney’s overall energy consumption is devoted to sodiumreclamation with the proximal tubule responsible for al-most two-thirds, primarily through the activity of thebasal Na+/K+ ATPase, quantified as ouabain-sensitive O2

consumption (44).While sodium–glucose linked transport across the api-

cal membrane of the proximal tubular cell is not of itselfan energy-requiring process, its continuing activity isdependent on the maintenance of the electrochemicalgradient for Na+, generated by Na+/K+ ATPase activity(Fig. 7). Accordingly, the increase in glucose reabsorptivecapacity that develops in diabetes (45) is accompanied bya commensurate demand in Na+/K+ ATPase activity thatwhen measured by ouabain inhibitable O2 consumptionincreases by about 30% in the experimental setting (46).Here, the SGLT1/2 inhibitor phlorizin was shown to ame-liorate the diabetes-induced increase in Na+/K+ ATPase

Figure 6—Incidence of CKD $ stage 3 according to baseline strataof plasma KIM-1 in normoalbuminuric (NA) and microalbuminuric(MA) individuals with type 1 diabetes whose renal function as mea-sured by eGFR and cystatin C was normal at baseline. Reproducedwith permission from Nowak et al. (30). ND, not detectable(<0.2 pg/mL). p-ys, person-years. T1–T3, tertiles of the distributionof detectable values of urinary KIM-1.

diabetes.diabetesjournals.org Gilbert 795

and O2 consumption (46) (Fig. 7). Consistent with thesefindings, the SGLT2 inhibitor dapagliflozin renders proxi-mal tubular epithelial cells resistant to hypoxia-inducedapoptosis, affording protection from ischemia-reperfusioninjury (47). Together, these findings raise the intriguingpossibility that the reduction in both GFR decline andAKI reported in the EMPA-REG OUTCOME trial (22)may be the result of reduced proximal tubular energy re-quirements, the ability of the drug to improve glycemia andmodulate glomerular hemodynamics notwithstanding (23).

In addition to the SGLTs, apical sodium transport in theproximal tubule is mediated by several other carrierproteins including sodium–hydrogen exchanger 3 (NHE3)that, similar to the SGLTs, require Na+/K+ ATPase to main-tain an electrochemical gradient. Increases in NHE3 havebeen reported in human proximal tubular cells exposed tohigh glucose and in animals with streptozotocin-induceddiabetes (48). A similar increase in Na-lactate cotransporteractivity, as a consequence of increased lactate production inthe setting of poor glycemic control (49), has also beenshown to occur in diabetes (50).

GluconeogenesisWhile the proximal tubule reabsorbs glucose, it does notmetabolize any of the enormous load that traverses it,relying on lactate, glutamate, and ketones as alternativesubstrates for energy production (51). Along with hepa-tocytes, proximal tubular epithelial cells are unique intheir ability to undertake gluconeogenesis and export glu-cose into the circulation, although unlike the liver, thekidney is not responsive to glucagon. From an energy

perspective, gluconeogenesis is a demanding process, re-quiring six energy equivalents (4 ATP, 2 GTP) to synthe-size a single molecule of glucose from lactate or pyruvate,contrasting sharply with the 2 molecules of ATP that aregenerated by glycolysis. Indeed, gluconeogenesis is a ma-jor source of the kidney’s ouabain-insensitive O2 usageand energy expenditure, accounting for up to 25% of theenergy needed for sodium reabsorption (50).

In the nondiabetic setting, the liver, by a combina-tion of glycogenolysis (50%) and gluconeogenesis (30%),contributes 80% of glucose released into the circulation,with the remaining 20% derived from renal gluconeogen-esis (52). In the postprandial state when glycogenolysisand hepatic gluconeogenesis are relatively suppressed, denovo glucose synthesis by the kidney accounts for ;60%of endogenous glucose release (52). The extent of renalgluconeogenesis is increased in diabetes where in thefasted state gluconeogenic activity increases approxi-mately threefold such that the kidney releases on average2.21 mmol/kg/min of glucose into the circulation, onlymarginally lower than the liver’s 2.60 mmol/kg/min(53). Postprandial glucose release by the kidney is simi-larly increased in subjects with type 2 diabetes when com-pared with age-, weight-, and sex-matched volunteerswithout diabetes (54).

O2 UtilizationGiven that the proximal tubule’s metabolic activity is al-most entirely oxidative, there is a commensurate reductionin tricarboxylic acid cycle activity and ATP production inthe relatively hypoxic diabetic kidney (55,56). Moreover, by

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

796 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017

requiring more O2 to be consumed for each molecule ofATP generated, the increase in free fatty acids, a compo-nent of the dysglycemic state, may exacerbate the extent ofischemia. This relative energy inefficiency can be quantifiedas the ATP/O2 ratio where glucose has a ratio of 3.17 whilepalmitate has a ratio of 2.83 (57), and while these differ-ences in oxygen consumption may seem modest, they canhave substantial impact when O2 delivery is marginal.

Exacerbating the increased O2 demands in diabetes isthe recent realization that mitochondria, the organellesresponsible for aerobic energy production, are structurallyabnormal and dysfunctional in diabetes (58,59). Indeed,abnormalities of proximal tubular mitochondrial structureand function may be the earliest manifestation of kidneydisease. In the rat, for instance, Coughlan et al. (60) foundevidence of impaired mitochondrial ATP generation andorganelle fragmentation in proximal tubular epithelial cellsas early as 4 weeks after the induction of experimentaldiabetes. That these changes precede increases in urinaryalbumin excretion, abnormal glomerular morphology, oreven elevation of urinary KIM-1 suggests that they maybe primary abnormalities. From a therapeutic point ofview, such findings also raise the possibility of using strat-egies that regulate mitochondrial biogenesis such as thesilent information regulator 1 activators that are currentlyundergoing clinical trial in a range of chronic diseases (61).

O2 SupplyHigh glucose and its downstream effector molecules havelong been known to increase endothelial cell apoptosis incell culture (62,63). Importantly, these changes, recognizedas capillary rarefaction, are also seen in the in vivo settingas a characteristic feature of diabetic kidney disease thatcorrelates with declining kidney function (64–67) (Fig. 8).Though potentially compensated by endothelial cell regen-eration, this reparative process, if anything, is impaired indiabetes (68). As a result, blood supply to the proximaltubule is impaired both by the intrinsic capillary loss withinthe tubulointerstitium described above and a consequenceof glomerular capillary occlusion (64,65).

Therapeutic AngiogenesisGiven the lack of observable capillary loss in animal modelsof diabetic kidney disease, much of the experimental workon therapeutic angiogenesis has relied on nondiabeticmodels such as renal artery stenosis and following subtotalnephrectomy (SNX) that do develop substantial capillaryrarefaction. Here, a number of strategies for reconstitutingthe microcirculation are in development including mesen-chymal stem cells (MSCs), so-called endothelial progenitorcells (EPCs), and extracorporeal shockwave therapy. EPCs,for instance, have been shown to exert both proangiogenicand antifibrotic properties, attenuating capillary loss in thetubulointerstitium and glomerulus as well as preservingkidney function in the SNX rat (69). MSCs, on the otherhand, have a range of potential beneficial effects that inaddition to their angiogenic, immunomodulatory, and anti-inflammatory activities also lower blood glucose in humanswith diabetes by as yet unknown mechanisms (70). Mostrecently, a phase II trial of allogeneic MSCs in patientswith advanced diabetic kidney disease has been reported(NCT01843387). This study compared the effects of twodoses of MSCs in 30 subjects whose baseline GFR was20–50 mL/min/1.73 m2, showing a trend in stabilizingmeasured GFR at 12 weeks when compared with the con-tinuing decline in placebo-treated patients (71).

Perhaps most tantalizing because of its simplicity is thefinding that low-energy extracorporeal shockwave therapy(ESWT) induces angiogenesis. In a double-blind, placebocontrolled study of patients with severe angina, Kikuchiet al. (72) used this technology to improve blood flow andfunction in the ischemic heart, reducing pain scores andimproving left ventricular ejection fraction. Using a sim-ilar strategy in pigs with renal artery stenosis, Zhang et al.(73) reported improvement in microvascular density andtissue oxygenation along with reduced fibrosis and betterkidney function after six sessions of ESWT. In exploringthe mechanisms that underlay these effects, this groupnoted elevated expression of vascular endothelial growthfactor, mainly in proximal tubular cells along with a reduc-tion in TGF-b expression, providing further substance tothe burgeoning exploration of mechanotransduction indisease development and reversal. The effects of ESWT inhuman diabetic kidney disease, however, remain unknown.

NONHYPOXIA-RELATED PROXIMAL TUBULARMECHANISMS

In addition to the hypoxia, several other nonhypoxia-related proximal tubule pathways involved in the develop-ment of diabetic kidney disease have been the subjects ofrecent reviews. These include the now well-documented lo-cal, predominantly proximal tubule–based renin-angiotensinsystem (74), the toxic effects of albumin bound fattyacids (75,76), and the activation of epidermal growth factorreceptor signaling pathways (77). Still more recently, andnot yet the subject of detailed review, is the explorationwhereby diabetic kidney disease, like most forms of CKD,once started, continues to progress inexorably. Among the

Figure 8—Relationship between postglomerular capillary densityand serum creatinine in diabetic kidney disease showing an inversecorrelation (r = 20.73, P < 0.001) in 72 patients. Reproduced andadapted with permission from Bohle et al. (65).

diabetes.diabetesjournals.org Gilbert 797

potential contributors to this process is the recent findingthat organ stiffness, an inevitable consequence of fibrosis,induces further fibrosis. Briefly, the presence of fibrosisleads to tissue stiffness that can be sensed mechanicallyby cells. Rather than dampening the fibrogenic process, thepresence of a stiff matrix seems to induce a positive feed-back cycle to enhance it. The leading contenders in ourcurrent understanding of this process are the mechano-transducing transcription cofactors: Yes-associated protein(YAP) and transcriptional coactivator with PDZ-bindingmotif (TAZ). Together, YAP and TAZ perpetuate TGF-bsignaling intermediates, Smad2/3, to be retained in thenucleus, thereby perpetuating its fibrogenic activity (78).Consistent with diabetes as a profibrotic state, YAP expres-sion and phosphorylation are increased in diabetic mouseproximal tubular cells so that modulating TAZ-YAP andrelated pathways has become an important new targetfor drug development in diabetic kidney disease and otherchronic diseases that are characterized by fibrosis (79).

PROXIMAL TUBULE–TARGETED THERAPEUTICS

The revolution in molecular biology has highlighted theextent of pathophysiological derangements in the diabetickidney that include a broad range of perturbations in epige-netics, protein–protein interactions, transcriptional changes,and posttranslational modifications. Unfortunately, movingthese discoveries into new therapies has been limited in partby the “druggability” of the target, i.e., the likelihood ofbeing able to modulate a target with a small-molecule drug.Indeed,,10% of potential targets are thought to be in thiscategory (80). Antisense technology that mediates specifictarget–directed degradation of mRNA, microRNA (miRNA),and long noncoding RNA (lncRNA), is, however, far lessrestricted. Indeed, this technology has already moved tohuman use with two systemically administered antisenseoligonucleotides (ASOs) approved by the U.S. Food andDrug Administration: mipomersen (Kynamro, Genzyme), aninhibitor of apoprotein B synthesis for the management ofhomozygous familial hypercholesterolemia, and eteplirsen(Exondys 51, Sarepta Therapeutics) for individuals withDuchenne muscular dystrophy that selectively bindsto exon 51 of dystrophin premRNA to restore the openreading frame and enable the production of functionaldystrophin.

Shorter length (12 vs. the usual 18 or longer) second-generation “shortmer” ASOs have comparatively lowerplasma protein binding so that they undergo greater frac-tional clearance through the glomerulus. As a result of theiravid uptake by the brush border of the proximal tubule,these agents are relatively selective for that site. Indeed,this strategy forms the basis for the development of theSGLT2 ASO, ISIS 388626 (Ionis, formerly ISIS), that hascompleted safety, tolerability, and activity studies in type 2diabetes (NCT00836225). Such studies highlight the enor-mous potential for nucleotide-based therapy to pursuenovel, nontraditionally druggable targets to ameliorateproximal tubule pathology in diabetic kidney disease.

CONCLUSION

Although much work needs to be done, substantial datanow support the existence of a diabetes-induced proximaltubulopathy as an early disease event that both predictsand contributes to the development of CKD in diabetes.While not ignoring the glomerulus, directing attention tothe proximal tubule for biomarker development, thera-peutic discovery, and pathophysiological understandingseems prescient.

Acknowledgments. The author regrets that owing to space constraints,much of the excellent work that has been done on this subject could not beincluded.Funding. R.E.G. is the Canada Research Chair in Diabetes Complications, andthis review was supported in part by Canada Research Chairs Program grant950-218644.Duality of Interest. R.E.G. reports having received consulting and lecturefees from Merck, AstraZeneca, Eli Lilly, Boehringer Ingelheim, Mesoblast, andJanssen along with grant funds through his institution from Merck, AstraZeneca,Eli Lilly, and Boehringer Ingelheim. R.E.G. was formerly a shareholder in FibrotechTherapeutics, which was wholly acquired by Shire Pharmaceuticals in 2014. Noother potential conflicts of interest relevant to this article were reported.

References1. Kimmelstiel P, Wilson C. Intercapillary lesions in the glomerulus. Am JPathol 1936;12:83–97, 72. Krolewski AS. Progressive renal decline: the new paradigm of diabeticnephropathy in type 1 diabetes. Diabetes Care 2015;38:954–9623. Fenton RA, Praetorius J. Anatomy of the kidney. In Brenner and Rector’sThe Kidney. Taal MW, Chertow GM, Marsden PA, Skorecki K, Yu SL, Brener BM,Eds. Philadelphia, PA, Elsevier, 20164. Gilbert RE, Cooper ME. The tubulointerstitium in progressive diabetic kidneydisease: more than an aftermath of glomerular injury? Kidney Int 1999;56:1627–16375. Marcussen N. Tubulointerstitial damage leads to atubular glomeruli: sig-nificance and possible role in progression. Nephrol Dial Transplant 2000;15(Suppl. 6):74–756. Najafian B, Kim Y, Crosson JT, Mauer M. Atubular glomeruli and glomer-ulotubular junction abnormalities in diabetic nephropathy. J Am Soc Nephrol2003;14:908–9177. White KE, Marshall SM, Bilous RW. Prevalence of atubular glomeruli in type2 diabetic patients with nephropathy. Nephrol Dial Transplant 2008;23:3539–35458. Hasegawa K, Wakino S, Simic P, et al. Renal tubular Sirt1 attenuates di-abetic albuminuria by epigenetically suppressing Claudin-1 overexpression inpodocytes. Nat Med 2013;19:1496–15049. Nihalani D, Susztak K. Sirt1-Claudin-1 crosstalk regulates renal function.Nat Med 2013;19:1371–137210. Romagnani P, Remuzzi G. Renal progenitors in non-diabetic and diabeticnephropathies. Trends Endocrinol Metab 2013;24:13–2011. Bonventre JV. Can we target tubular damage to prevent renal functiondecline in diabetes? Semin Nephrol 2012;32:452–46212. Grgic I, Campanholle G, Bijol V, et al. Targeted proximal tubule injurytriggers interstitial fibrosis and glomerulosclerosis. Kidney Int 2012;82:172–18313. Kumar D, Robertson S, Burns KD. Evidence of apoptosis in human diabetickidney. Mol Cell Biochem 2004;259:67–7014. Saharfuddin AA, Weisbord SD, Palevsky PM, Molitoris BA. Acute kidney injury.In Brenner and Rector’s The Kidney. Taal MW, Chertow GM, Marsden PA, SkoreckiK, Yu SL, Brener BM, Eds. Philadelphia, PA, Elsevier, 2016, p. 958–101115. Yang L, Besschetnova TY, Brooks CR, Shah JV, Bonventre JV: Epithelial cellcycle arrest in G2/M mediates kidney fibrosis after injury. Nat Med 2010;16:535–543

798 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017

16. Basile DP, Friedrich JL, Spahic J, et al. Impaired endothelial proliferationand mesenchymal transition contribute to vascular rarefaction following acutekidney injury. Am J Physiol Renal Physiol 2011;300:F721–F73317. Lan R, Geng H, Polichnowski AJ, et al. PTEN loss defines a TGF-b-inducedtubule phenotype of failed differentiation and JNK signaling during renal fibrosis.

Am J Physiol Renal Physiol 2012;302:F1210–F122318. Thakar CV, Christianson A, Himmelfarb J, Leonard AC. Acute kidney injuryepisodes and chronic kidney disease risk in diabetes mellitus. Clin J Am SocNephrol 2011;6:2567–257219. Monseu M, Gand E, Saulnier PJ, et al.; SURDIAGENE Study Group. Acutekidney injury predicts major adverse outcomes in diabetes: synergic impact withlow glomerular filtration rate and albuminuria. Diabetes Care 2015;38:2333–234020. Weir MR, Januszewicz A, Gilbert RE, et al. Effect of canagliflozin on bloodpressure and adverse events related to osmotic diuresis and reduced in-

travascular volume in patients with type 2 diabetes mellitus. J Clin Hypertens(Greenwich) 2014;16:875–88221. Cherney DZ, Perkins BA, Soleymanlou N, et al. Renal hemodynamic effect ofsodium-glucose cotransporter 2 inhibition in patients with type 1 diabetes mel-litus. Circulation 2014;129:587–59722. Wanner C, Inzucchi SE, Lachin JM, et al.; EMPA-REG OUTCOME Investi-gators. Empagliflozin and progression of kidney disease in type 2 diabetes.N Engl J Med 2016;375:323–33423. Gilbert RE. SGLT2 inhibitors: b blockers for the kidney? Lancet DiabetesEndocrinol 2016;4:81424. Zinman B, Wanner C, Lachin JM, et al.; EMPA-REG OUTCOME Investigators.Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes. N EnglJ Med 2015;373:2117–212825. Macisaac RJ, Jerums G. Diabetic kidney disease with and without albu-

minuria. Curr Opin Nephrol Hypertens 2011;20:246–25726. Ferguson MA, Waikar SS. Established and emerging markers of kidneyfunction. Clin Chem 2012;58:680–68927. Fufaa GD, Weil EJ, Nelson RG, et al.; Chronic Kidney Disease Biomarkers

Consortium Investigators. Association of urinary KIM-1, L-FABP, NAG and NGALwith incident end-stage renal disease and mortality in American Indians with type2 diabetes mellitus. Diabetologia 2015;58:188–19828. Panduru NM, Sandholm N, Forsblom C, et al.; FinnDiane Study Group.Kidney injury molecule-1 and the loss of kidney function in diabetic nephropathy:

a likely causal link in patients with type 1 diabetes. Diabetes Care 2015;38:1130–113729. Sabbisetti VS, Waikar SS, Antoine DJ, et al. Blood kidney injury molecule-1is a biomarker of acute and chronic kidney injury and predicts progression toESRD in type I diabetes. J Am Soc Nephrol 2014;25:2177–218630. Nowak N, Skupien J, Niewczas MA, et al. Increased plasma kidney injurymolecule-1 suggests early progressive renal decline in non-proteinuric patientswith type 1 diabetes. Kidney Int 2016;89:459–46731. Charlton JR, Portilla D, Okusa MD. A basic science view of acute kidneyinjury biomarkers. Nephrol Dial Transplant 2014;29:1301–131132. Nielsen SE, Sugaya T, Tarnow L, et al. Tubular and glomerular injury indiabetes and the impact of ACE inhibition. Diabetes Care 2009;32:1684–168833. Nielsen SE, Sugaya T, Hovind P, Baba T, Parving HH, Rossing P. Urinaryliver-type fatty acid-binding protein predicts progression to nephropathy in type

1 diabetic patients. Diabetes Care 2010;33:1320–132434. Araki S, Haneda M, Koya D, et al. Predictive effects of urinary liver-type fattyacid-binding protein for deteriorating renal function and incidence of cardio-vascular disease in type 2 diabetic patients without advanced nephropathy.Diabetes Care 2013;36:1248–125335. Panduru NM, Forsblom C, Saraheimo M, et al.; FinnDiane Study Group.Urinary liver-type fatty acid-binding protein and progression of diabetic ne-phropathy in type 1 diabetes. Diabetes Care 2013;36:2077–208336. Fiseha T, Tamir Z. Urinary markers of tubular injury in early diabetic ne-

phropathy. Int J Nephrol 2016;2016:4647685

37. Vaidya VS, Niewczas MA, Ficociello LH, et al. Regression of microalbuminuriain type 1 diabetes is associated with lower levels of urinary tubular injury bio-markers, kidney injury molecule-1, and N-acetyl-beta-D-glucosaminidase. KidneyInt 2011;79:464–47038. Fine LG, Orphanides C, Norman JT. Progressive renal disease: the chronichypoxia hypothesis. Kidney Int Suppl 1998;65:S74–S7839. Laustsen C, Østergaard JA, Lauritzen MH, et al. Assessment of early di-abetic renal changes with hyperpolarized [1-(13) C]pyruvate. Diabetes Metab ResRev 2013;29:125–12940. Khan S, Cleveland RP, Koch CJ, Schelling JR. Hypoxia induces renal tubularepithelial cell apoptosis in chronic renal disease. Lab Invest 1999;79:1089–109941. Fine LG, Norman JT. Chronic hypoxia as a mechanism of progression ofchronic kidney diseases: from hypothesis to novel therapeutics. Kidney Int 2008;74:867–87242. Mimura I, Nangaku M. The suffocating kidney: tubulointerstitial hypoxia inend-stage renal disease. Nat Rev Nephrol 2010;6:667–67843. Orphanides C, Fine LG, Norman JT. Hypoxia stimulates proximal tubular cellmatrix production via a TGF-beta1-independent mechanism. Kidney Int 1997;52:637–64744. Singh P, McDonough AA, Thomson SA. Metabolic basis of solute transport.In Brenner and Rector’s The Kidney. Taal MW, Chertow GM, Marsden PA,Skorecki K, Yu SL, Brener BM, Eds. Philadelphia, PA, Elsevier, 2016, p. 122–14345. DeFronzo RA, Hompesch M, Kasichayanula S, et al. Characterization of renalglucose reabsorption in response to dapagliflozin in healthy subjects and subjectswith type 2 diabetes. Diabetes Care 2013;36:3169–317646. Körner A, Eklöf AC, Celsi G, Aperia A. Increased renal metabolism in di-abetes. Mechanism and functional implications. Diabetes 1994;43:629–63347. Chang YK, Choi H, Jeong JY, et al. Dapagliflozin, SGLT2 inhibitor, attenuatesrenal ischemia-reperfusion injury. PLoS One 2016;11:e015881048. Saad S, Zhang J, Yong R, et al. Role of the EGF receptor in PPARg-mediatedsodium and water transport in human proximal tubule cells. Diabetologia 2013;56:1174–118249. Laustsen C, Lipsø K, Ostergaard JA, et al. Insufficient insulin administrationto diabetic rats increases substrate utilization and maintains lactate production inthe kidney. Physiol Rep 2014;2:e1223350. Cohen JJ. Relationship between energy requirements for Na+ reabsorptionand other renal functions. Kidney Int 1986;29:32–4051. Guder WG, Ross BD. Enzyme distribution along the nephron. Kidney Int1984;26:101–11152. Meyer C, Dostou JM, Welle SL, Gerich JE. Role of human liver, kidney, andskeletal muscle in postprandial glucose homeostasis. Am J Physiol EndocrinolMetab 2002;282:E419–E42753. Meyer C, Stumvoll M, Nadkarni V, Dostou J, Mitrakou A, Gerich J. Abnormalrenal and hepatic glucose metabolism in type 2 diabetes mellitus. J Clin Invest1998;102:619–62454. Meyer C, Woerle HJ, Dostou JM, Welle SL, Gerich JE. Abnormal renal,hepatic, and muscle glucose metabolism following glucose ingestion in type2 diabetes. Am J Physiol Endocrinol Metab 2004;287:E1049–E105655. Blantz RC. Phenotypic characteristics of diabetic kidney involvement. KidneyInt 2014;86:7–956. Zhao L, Gao H, Lian F, Liu X, Zhao Y, Lin D. 1H-NMR-based metabonomicanalysis of metabolic profiling in diabetic nephropathy rats induced by strepto-zotocin. Am J Physiol Renal Physiol 2011;300:F947–F95657. Hütter JF, Schweickhardt C, Piper HM, Spieckermann PG. Inhibition of fattyacid oxidation and decrease of oxygen consumption of working rat heart by4-bromocrotonic acid. J Mol Cell Cardiol 1984;16:105–10858. Zhan M, Brooks C, Liu F, Sun L, Dong Z. Mitochondrial dynamics: regulatorymechanisms and emerging role in renal pathophysiology. Kidney Int 2013;83:568–58159. Zhan M, Usman IM, Sun L, Kanwar YS. Disruption of renal tubular mito-chondrial quality control by Myo-inositol oxygenase in diabetic kidney disease.J Am Soc Nephrol 2015;26:1304–1321

diabetes.diabetesjournals.org Gilbert 799

60. Coughlan MT, Nguyen TV, Penfold SA, et al. Mapping time-course mito-chondrial adaptations in the kidney in experimental diabetes. Clin Sci (Lond)2016;130:711–72061. Haigis MC, Sinclair DA. Mammalian sirtuins: biological insights and diseaserelevance. Annu Rev Pathol 2010;5:253–29562. Baumgartner-Parzer SM, Wagner L, Pettermann M, Grillari J, Gessl A,Waldhäusl W. High-glucose–triggered apoptosis in cultured endothelial cells.Diabetes 1995;44:1323–132763. Ho FM, Liu SH, Liau CS, Huang PJ, Lin-Shiau SY. High glucose-inducedapoptosis in human endothelial cells is mediated by sequential activations ofc-Jun NH(2)-terminal kinase and caspase-3. Circulation 2000;101:2618–262464. Osterby R, Parving HH, Nyberg G, et al. A strong correlation between glo-merular filtration rate and filtration surface in diabetic nephropathy. Diabetologia1988;31:265–27065. Bohle A, Mackensen-Haen S, Wehrmann M. Significance of postglomerularcapillaries in the pathogenesis of chronic renal failure. Kidney Blood Press Res1996;19:191–19566. Eardley KS, Kubal C, Zehnder D, et al. The role of capillary density, mac-rophage infiltration and interstitial scarring in the pathogenesis of human chronickidney disease. Kidney Int 2008;74:495–50467. Lindenmeyer MT, Kretzler M, Boucherot A, et al. Interstitial vascular rare-faction and reduced VEGF-A expression in human diabetic nephropathy. J AmSoc Nephrol 2007;18:1765–177668. Gilbert RE. Endothelial loss and repair in the vascular complications of diabetes:pathogenetic mechanisms and therapeutic implications. Circ J 2013;77:849–85669. Yuen DA, Connelly KA, Advani A, et al. Culture-modified bone marrow cellsattenuate cardiac and renal injury in a chronic kidney disease rat model via anovel antifibrotic mechanism. PLoS One 2010;5:e954370. Skyler JS, Fonseca VA, Segal KR, Rosenstock J; MSB-DM003 Investigators.Allogeneic mesenchymal precursor cells in type 2 diabetes: a randomized, placebo-controlled, dose-escalation safety and tolerability pilot study. Diabetes Care 2015;38:1742–1749

71. Packham DK, Fraser IR, Kerr PG, Segal KR. Allogeneic mesenchymal pre-cursor cells (MPC) in diabetic nephropathy: a randomized, placebo-controlled,dose escalation study. EBioMedicine 2016;12:263–26972. Kikuchi Y, Ito K, Ito Y, et al. Double-blind and placebo-controlled study of theeffectiveness and safety of extracorporeal cardiac shock wave therapy for severeangina pectoris. Circ J 2010;74:589–59173. Zhang X, Krier JD, Amador Carrascal C, et al. Low-energy shockwavetherapy improves ischemic kidney microcirculation. J Am Soc Nephrol 2016;27:3715–372474. Gilbert RE, Advani A. Vasoactive molecules and the kidney. In Brenner andRector’s The Kidney. Taal MW, Chertow GM, Marsden PA, Skorecki K, Yu SL,Brener BM, Eds. Philadelphia, PA, Elsevier, 2016, p. 325–35375. Allison SJ. Fibrosis: dysfunctional fatty acid oxidation in renal fibrosis. NatRev Nephrol 2015;11:6476. Parker MD, Myers EJ, Schelling JR. Na+-H+ exchanger-1 (NHE1) regulationin kidney proximal tubule. Cell Mol Life Sci 2015;72:2061–207477. Forrester SJ, Kawai T, O’Brien S, Thomas W, Harris RC, Eguchi S. Epidermalgrowth factor receptor transactivation: mechanisms, pathophysiology, and po-tential therapies in the cardiovascular system. Annu Rev Pharmacol Toxicol 2016;56:627–65378. Szeto SG, Narimatsu M, Lu M, et al. YAP/TAZ are mechanoregulators ofTGF-b-Smad signaling and renal fibrogenesis. J Am Soc Nephrol. 9 March 2016[Epub ahead of print]79. Santucci M, Vignudelli T, Ferrari S, et al. The Hippo pathway and YAP/TAZ-TEAD protein-protein interaction as targets for regenerative medicine and cancertreatment. J Med Chem 2015;58:4857–487380. Owens J. Target validation: determining druggability. Nat Rev Drug Discov2007;6:18781. Tisher CC, Rosen S, Osborne GB. Ultrastructure of the proximal tubule of therhesus monkey kidney. Am J Pathol 1969;56:469–51782. Chao EC, Henry RR. SGLT2 inhibition–a novel strategy for diabetes treat-ment. Nat Rev Drug Discov 2010;9:551–559

800 Proximal Tubulopathy and Diabetic Kidney Disease Diabetes Volume 66, April 2017