Urine RAS Components in Mice and People with Type 1 Diabetes and Chronic Kidney Disease

Articles in PresS. Am J Physiol Renal Physiol (May 3, 2017). doi:10.1152/ajprenal.00074.2017

Title: Urine RAS components in mice and people with type 1 diabetes and chronic kidney disease

Running title: Urine RAS components in diabetic kidney disease

Authors:

Jan Wysocki, MD-PhD, Division of Nephrology and Hypertension, Department of Medicine, The Feinberg

School of Medicine, Northwestern University, Chicago, IL 60611, USA

Anne Goodling, BA. Kidney Research Institute and Division of Nephrology, Department of Medicine, University of Washington, Seattle, WA

Mar Burgaya, Division of Nephrology and Hypertension, Department of Medicine, The Feinberg School of

Medicine, Northwestern University, Chicago, IL 60611, USA

Kathryn Whitlock, MS. Center for Child Health, Behavior and Development, Seattle Children's Research

Institute, Seattle, WA

John Ruzinski, BS. Kidney Research Institute and Division of Nephrology, Department of Medicine, University of Washington, Seattle, WA

Daniel Batlle, MD, Division of Nephrology and Hypertension, Department of Medicine, The Feinberg School of

Medicine, Northwestern University, Chicago, IL 60611

Maryam Afkarian, MD-PhD, Division of Nephrology, Department of Medicine, University of California, Davis,

CA 95616

Corresponding author: Maryam Afkarian, MD-PhD, 4150 V Street, Suite 3500, Sacramento, CA 95817.

Telephone: (916) 734-3774 Fax: (916) 734-7920. Email: [email protected].

Abstract word count: 250

Key words: diabetic kidney disease, renin angiotensin system, angiotensinogen, cathepsin D, angiotensin converting enzyme, angiotensin converting enzyme 2, aminopeptidase-A

2 The pathways implicated in diabetic kidney disease (DKD) are largely derived from animal models. To examine

3 if alterations in renin-angiotensin system (RAS) in humans are concordant with rodent models, we measured

4 concentration of angiotensinogen (AOG), cathepsin D (CTSD), angiotensin converting enzyme (ACE) and

5 ACE2 and enzymatic activities of ACE, ACE2 and aminopeptidase-A in FVB mice 13-20 weeks after treatment

6 with streptozotocin (n=9) or vehicle (n=15) and people with longstanding type 1 diabetes, with (n=37) or

7 without (n=81) DKD. In streptozotocin-treated mice, urine AOG and CTSD were 10.4- and 3.0-fold higher than

8 controls, respectively (p-values <0.001). Enzymatic activities of ACE, ACE2 and APA were 6.2-, 3.2- and 18.8-

9 fold higher, respectively, in diabetic animals (p-values <0.001). Angiotensin II was 2.4-fold higher in diabetic

10 animals (p-value 0.017). Compared to people without DKD, those with DKD had higher urine AOG (170 vs. 15

11 μg/g) and CTSD (147 vs. 31 μg/g). In people with DKD, urine ACE concentration was 1.8-fold higher (1.4 vs.

12 0.8 μg/g in those without DKD), while its enzymatic activity was 0.6-fold lower (1.0 vs. 1.6 x 109 RFU/g in those

13 without DKD). Lower ACE activity, but not ACE protein concentration, was associated with ACE inhibitor

14 (ACEI) treatment. After adjustment for clinical covariates, AOG, CTSD, ACE concentration and ACE activity

15 remained associated with DKD. In conclusion, in mice with streptozotocin-induced diabetes and humans with

16 DKD, urine concentrations and enzymatic activities of several RAS components are concordantly increased,

17 consistent with enhanced RAS activity and greater angiotensin II formation. ACEI use was associated with a

18 specific reduction in urine ACE activity, not ACE protein concentration, suggesting it may be a marker of

19 exposure to this widely-used therapy.

20 INTRODUCTION

21 Diabetic kidney disease is the leading cause of chronic and end-stage kidney disease in the United States and

22 worldwide.(2) Several biological pathways are implicated in the pathogenesis of diabetic kidney disease (DKD).

23 This information is largely obtained from animal models of DKD. However, animal models only partially

24 recapitulate the pathology and pathophysiology of human DKD.(5, 50) As such, the timing and extent of activity

25 of these pathways in the course of human DKD is not known. Given the dearth of research biopsies in human

26 DKD, information on pathway activity in human kidney biopsies obtained throughout the course of DKD is not

27 currently available.

28 Urine is a readily available, non-invasive bio-fluid, which is extensively used in clinical medicine and now

29 increasingly utilized for biomarker discovery.(16) Though formed initially as a filtrate of plasma, urine protein

30 composition is subsequently influenced by secretory and reabsorptive activities of the cells lining the length of

31 the nephron. Urine albumin and total urine protein quantity is a powerful clinical risk stratifier not only for

32 progression of kidney disease but also heart disease and mortality.(36) More recent evidence suggests that

33 urine protein composition may be able to inform on the underlying intrarenal pathogenic mechanisms.(13, 18,

34 21, 57, 59) This potential would open up new diagnostic avenues for the use of urine protein composition as a

35 personalized assay for tailoring treatment of kidney disease to the pathways active in each individual at a given

36 time.

37 We sought to comprehensively examine urine concentrations, and when possible enzymatic activities, of key

38 renin angiotensin system (RAS) components in an animal model of DKD, the FVB mice treated with

39 streptozotocin, as well as people with type 1 diabetes and DKD, compared to those with longstanding type 1

40 diabetes and no DKD. The examined RAS components included angiotensinogen, angiotensin converting

41 enzyme (ACE), ACE2, as well as cathepsin D (CTSD). The latter is a lysosomal protein capable of cleaving

42 angiotensinogen to angiotensin I, enabling ACE-independent angiotensin II generation.(24) In addition, we

43 examined enzymatic activity of aminopeptidase A (APA), which regulates angiotensin II concentration by

44 catalyzing its degradation to angiotensin III.(56) The RAS was selected as an exemplary pathway because it is

45 presumed to be overactive in DKD kidneys and its pharmacological suppression is part of current standards of

46 care. Our findings show concordance between the urine concentration and enzymatic activity of RAS pathway

47 proteins and the known intrarenal pathway activity and between rodent DKD models and human DKD.

48 Furthermore, in humans with DKD, urine ACE activity shows a negative correlation with use of ACE inhibitors

49 that may reflect suppression of ACE activity at the kidney level by these agents.

50 MATERIALS AND METHODS

51 Study cohort

52 People with type 1 diabetes, who were seen in the Diabetes Care Center (University of Washington) for

53 outpatient endocrinology care, were approached for participation in the Kidney Research Institute Diabetic

54 Kidney Disease Repository (University of Washington) and were enrolled into the repository after providing

55 informed consent. Demographic and clinical information was obtained from the electronic medical records and

56 a questionnaire filled by the participants. DKD was defined as either a urine ACR ≥300 mg/g or an eGFR <60

57 mL/min per 1.73 m2 and ACR ≥30 mg/g. People with longstanding diabetes but no evidence of overt DKD were

58 those with ≥30 years of type 1 diabetes, estimated GFR >90 mL/min per 1.73 m2, and ACR <300 mg/g.

59 Demographic and clinical data (age, race, sex, diabetes duration and RAS inhibitor use) were obtained from

60 the electronic medical records and confirmed by patient questionnaires (race, diabetes duration and RAS

61 inhibitor use). Diabetes type was extracted from the clinical notes, as ascertained by the endocrinologists

62 caring for the patients. Hemoglobin A1c and serum creatinine were obtained from the electronic medical

63 records at a time closest to the date of urine sample collection. Glomerular filtration rate was calculated from

64 the serum creatinine using CKD-EPI formula. (26)

65 A random clean-catch, mid-stream urine sample was collected and stored at 4oC after collection, until

66 processed. Urine samples were centrifuged at 4,700g for 15 minutes at 4oC and the supernatant collected,

67 aliquoted and stored at -80oC. The mean (standard deviation, SD) time from sample collection to storage at -

68 80oC was 5.7 (2.0) hours. The use of human samples and data were approved by the Institutional Review

69 Board of the University of Washington.

70 Experimental animals

71 Female FVB mice were acquired from Jackson Laboratories (Bar Harbor, ME). Animals were housed at the

72 Center for Comparative Medicine at Northwestern University Feinberg School of Medicine. Animal care and

73 procedures were approved by the Institutional Animal Care and Use Committee at Northwestern University and

74 in accordance with the NIH Guide for the Care and Use of Laboratory Animals and the institutional, state, and

75 federal guidelines. Streptozotocin (150 mg/kg, Sigma Chemical, St. Louis, MO) dissolved in sodium citrate

76 buffer pH 4.5 (streptozotocin-treated mice) or sodium citrate buffer pH 4.5 alone (non-diabetic vehicle controls)

77 was injected in two intraperitoneal injections to female FVB mice at 19 and 20 weeks of age. Spot urines were

78 collected 13 and 20 weeks after the last streptozotocin or vehicle injection (at 32 and 40 weeks of age,

79 respectively) and stored at -80oC.

80 Laboratory measurements in mouse urine samples

81 Albumin was measured by a quantitative solid-phase sandwich enzyme-Linked immunosorbent assay (ELISA)

82 (Albuwell M, Exocell, Philadelphia, PA). Creatinine was measured using the Jaffe method (Creatinine

83 Companion, Exocell, Philadelphia, PA). AOG was measured using a quantitative solid-phase sandwich ELISA

84 (IBL-America, Minneapolis, MN). CTSD was measured using a sandwich ELISA (OriGene Technologies, Inc,

85 Rockville, MD). ACE activity was measured in a high-throughput ﬂuorimetric assay that uses the substrate

86 hippuryl-L-histidyl-L-leucine, as previously described.(48) The concentration of afﬁnity puriﬁed recombinant

87 testis ACE showed direct linear relationship to the production of L-histidyl-L-leucine within the range of 0–4 10-4

88 nmol ACE. (48) ACE2 activity was determined following incubation with the intramolecularly quenched

89 synthetic ACE2-speciﬁc substrate Mca-APK-Dnp (Anaspec, Fremont, CA) using a method which was validated

90 by spiking recombinant ACE2 protein into ACE2 knockout urines, as described previously.(63) Briefly, urine

91 was added to wells containing a buffer (50 mmol/l 4-morpholineethanesulfonic acid, 300 mmol/l NaCl, 10

92 μmol/l ZnCl2, and 0.01% Triton-X-100, pH 6.5), EDTA-free tablets (Roche, Applied Science, Mannheim,

93 Germany) and 10 μmol/l substrate. Reactions were in duplicate, with one of two wells constituting a negative

94 control. Negative control wells contained the same components, in addition to 10 μmol/l of a speciﬁc ACE2

95 inhibitor, MLN-4760 (Millenium Pharmaceuticals, Cambridge, MA). ACE2 activity was calculated by subtracting

96 negative control values from ﬂuorescence obtained in wells without the specific ACE2 inhibitor.(63)

97 APA activity was measured as previously described (12) and the fluorescence corrected with addition of an

98 APA inhibitor, amastatin (Sigma-Aldrich, St. Louis, MO), at 10-5M concentration to negative control wells. For

99 angiotensin II studies, an aliquot of 100 uL of freshly collected urine was transferred into tubes kept on ice at

100 4oC containing 10x concentrated cocktail of peptidase inhibitors: 25 mM EDTA, 0.44 mM o-phenanthroline, 1

101 mM chloromercuribenzoic acid (PCMB), 120 uM pepstatin A in PBS and mixed thoroughly. The urines with

102 inhibitors were then stored at -80oC until the extraction. Angiotensin peptides were extracted from urines using

103 reverse phase phenyl silica columns (Thermo Scientific Cat.-No. 60108-386, 100 mg) according to the

104 manufacturer’s instructions. Angiotensin II levels were measured using an EIA kit from (Cayman Chemical,

105 Ann Arbor, MI). This assay had less than 0.001% cross-reactivity with Ang(1-7) and 4% cross-reactivity with

106 Ang I (1-10). Cross-reactivity with AngIII (2-8) and, Ang(3-8), were 33% and 36%, respectively, that compares

107 favorably with the corresponding competitive immunoassay.(58) Within-assay coefficients of variation (CV %)

108 were 6.9, 2, 5.6 and 10.2 for 100, 20, 5 and 2 pg/ml of Ang II, respectively. Between-assay coefficients of

109 variation were 6.9, 4.8, 10 and 14.7 for 100, 20, 5 and 2 pgrml of Ang II, respectively.(58)

110 Laboratory measurements in human urine samples

111 Albumin and creatinine were measured in urine samples using an immunoturbidometric assay and the

112 modified rate Jaffe reaction, respectively, using a DXC 600 clinical chemistry analyzer (Beckman Coulter,

113 Indianapolis). Angiotensinogen was measured using a quantitative solid-phase sandwich enzyme-Linked

114 immunosorbent assay (ELISA) distributed by IBL-America (Minneapolis, MN) with a minimum detection limit of

115 30 pg/mL and <0.1% cross-reactivity with human angiotensin I, II, III, or IV, angiotensin (1–9), or angiotensin

116 (1–7). CTSD was measured using a quantitative solid-phase sandwich ELISA distributed by Abcam

117 (Cambridge, MA) with a minimum detection limit of 10 pg/mL and no detectable cross-reactivity with other

118 capthepsins. ACE concentration was measured using a quantitative solid-phase sandwich ELISA distributed by

119 R &D Systems (Minneapolis, MN) with a minimum detection limit of 50 pg/mL and <0.5% cross-reactivity with

120 human ACE2, neprilysin or ECE-2. ACE2 concentration was measured using a quantitative solid-phase

121 sandwich ELISA distributed by IBL-America (Minneapolis, MN) with a minimum detection limit of 10 pg/mL and

122 reported by the manufacturer to have no detectable cross-reactivity with other relevant proteins. ACE, ACE2

123 and APA activities were measured using the same assays as for mouse urine samples (details in previous

124 section).

125 Statistical analysis

126 Distribution of the protein concentrations and enzymatic activities were described using median and

127 interquartile range (IQR). Concentration and enzymatic activity were log2 transformed, and logistic regression

128 was used to assess the association between concentration or enzymatic activity of individual RAS protein

129 components with the DKD status as outcome, after adjusting for age (continuous), gender, race (binary) and

130 diabetes duration (continuous) as covariates. The analysis was performed for urine proteins with and without

131 normalization to urine creatinine. To assess the difference in ACE, ACE2 and CTSD concentration and ACE

132 and ACE2 activity in people with DKD who were or were not on ACE inhibitors, linear regression was used,

133 with the concentration or enzymatic activity of each protein (e.g. ACE or ACE2) as the outcome and ACE

134 inhibitor use as the exposure, after adjustment for age (continuous), gender, race (binary) and diabetes

135 duration (continuous) as covariates. To examine relative associations between RAS proteins and DKD status,

136 logistic regression was again used with the DKD status as outcome, and the exposure consisting of all RAS

137 protein concentrations or activities which were significantly associated with the outcome in individual analyses

138 as above (AOG, CTSD, ACE activity, ACE2 activity and APA activity). This model was also adjusted for age,

139 gender, race and diabetes duration. The nominal significance threshold was set as a two-sided p-value <0.05.

140 All analyses were performed with SAS 9.4 (Carey, NC).

141 RESULTS

142 Urine RAS proteins in experimental DKD

143 Treatment with streptozotocin elicited an increase in glucose and caused mild albuminuria (Table 1). These

144 animals also develop some of the pathologic features of DKD, including glomerular hypertrophy and mesangial

145 matrix expansion, as previously reported.(64) Median urine angiotensin (AOG) concentration normalized for

146 creatinine concentration was significantly higher in mice treated with streptozotocin vs. those treated with

147 vehicle (27.1 vs. 2.6 μg/g Cr, p <0.001). CTSD concentration, normalized to creatinine, was also significantly

148 higher in streptozotocin-treated mice than in controls (96.5 vs 32.0 μg/g Cr, p <0.001) (Table 1, Figure 1).

149 Urine RAS enzymatic activities in experimental DKD

150 The enzymatic activities of three enzymes involved in the formation of angiotensin II (ACE) and its degradation

151 (APA and ACE2) were assessed in urine. Median creatinine-normalized enzymatic activities of ACE, ACE2

152 and APA were all significantly higher in urine of diabetic mice vs. controls (6.2, 3.2, and 18.8-fold, respectively).

153 Urine angiotensin II to creatinine concentration was also higher in diabetic mice vs. controls (2.4-fold, p 0.017)

154 (Table 1, Figure 1).

155 Characteristics of the cohort of patients with type 1 diabetes

156 Compared to people with longstanding type 1 diabetes and no DKD (controls), those with DKD were slightly

157 younger (49 vs. 53 years old), and less likely to be white (78% vs. 98%) and female (24% vs. 54%) (Table

158 2).Diabetic retinopathy was more common in those with DKD than those without DKD (73% vs. 64%,

159 respectively), as was hypertension (97% vs. 47%, respectively). As expected from selection criteria, estimated

160 glomerular filtration rate (eGFR) was markedly lower (median 39 vs. 92 mL/min/1.73 m2) and urine albumin to

161 creatinine ratio (ACR) markedly higher (median 497 vs. 7 mg/g) in people with DKD, as compared to controls.

162 RAS inhibitor use was more prevalent in people with DKD than those without (87% vs. 54%). Diabetes duration

163 was shorter in people with DKD than the controls (mean 34 vs. 39 years) (Table 2).

164 Urine RAS proteins in human DKD

165 Compared with controls, people with DKD had higher median creatinine-normalized urine AOG (170 vs.15

166 μg/g Cr). Median creatinine-normalized urine CTSD concentrations were also markedly higher in people with

167 DKD vs. those without (147 vs. 31 μg/g Cr), as were median creatinine-normalized urine ACE concentrations

168 (1.4 vs. 0.8 μg/g Cr) (Table 3, Figure 2). Creatinine-normalized urine ACE2 concentration was higher in people

169 with than without DKD (medians 4.1 vs. 2.7 μg/g Cr), but this difference was not statistically significant.

170 Urine RAS enzyme activities in human DKD

171 In addition to evaluation of ACE and ACE2 protein concentrations in urine, their enzymatic activities were also

172 quantified in urine. ACE2 and APA activities were higher in people with DKD than in those without DKD (Table

173 3). In contrast, ACE activity was significantly decreased in patients with DKD as compared to those without it.

174 This was attributed to the higher use of ACE inhibitors in this group (see below).

175 Adjusted associations of urine RAS components with DKD in people with type 1 diabetes

176 After adjustment for age, sex, race and diabetes duration, each 2-fold increase in creatinine-normalized urine

177 AOG, CTSD and ACE protein concentrations was significantly associated with 2.7, 6.5 and 2.2-fold higher

178 odds of having DKD, respectively (Table 3). In contrast to urine ACE protein concentration, higher urine ACE

179 activity was significantly associated with lower (0.3-fold) odds of having DKD, most likely due to its association

180 with ACE inhibitor use, as described below.

181 Association between urine ACE2 concentration and DKD status did not withstand adjustment for covariates,

182 whereas enzymatic activities of urine ACE2 and APA showed weaker associations (ORs 1.2 for both) with

183 DKD status (Table 3). The same associations were observed when urine concentrations and activities of these

184 RAS components were not normalized for urine creatinine (Table 4). In a combined model including

185 demographic covariates and those RAS protein concentrations or enzymatic activities which were individually

186 associated with DKD, only urine CTSD protein and urine ACE activity remained significantly associated with

187 DKD status (Table 5).

188 Change in RAS proteins and enzymatic activities with ACE inhibitor use

189 Among people with DKD, urine ACE activity was significantly lower in people who were on ACE inhibitors than

190 those who were not (-335 RFU/hr/mgCr, p-value 0.014), after adjustment for age, gender, race and diabetes

191 duration. In contrast, urine ACE concentration did not vary by ACE inhibitor use (p-value 0.6). Neither did urine

192 CTSD, and ACE2 concentrations or ACE2 activity (p-values 0.6, 0.5 and 0.3, respectively).

193 DISCUSSION

194 This study shows a concordant increase in key urinary RAS components in an experimental model of DKD

195 and in human DKD associated with type 1 diabetes, providing support for activation of the kidney RAS pathway

196 in DKD. Diabetic animals showed increases in urine AOG and CTSD concentrations and ACE activity, all

197 upstream components of the RAS pathway which lead to increased angiotensin II formation. This was shown

198 more directly from the finding of increased urine angiotensin II in the streptozotocin-treated mice. Similarly,

199 people with type 1 diabetes and DKD had higher urine AOG, CTSD, and ACE concentrations than people with

200 longstanding diabetes and no DKD, suggesting a greater capacity for angiotensin II formation, even though this

201 peptide could not be measured directly in human samples. Also of interest was the finding that people with

202 DKD, who were treated with ACE inhibitors, had lower urine ACE activity.

203 RAS activation is generally accepted as an important mechanism of DKD progression.(3, 27, 39) Diabetes per

204 se is associated with an increase in urinary concentration and enzymatic activities of several RAS components

205 (6). Further, an increase in kidney expression and urine concentration of some RAS components has been

206 reported in experimental DKD. Our report adds to the existing work in several ways. First, it supports RAS

207 over-activity in DKD kidneys by providing a uniquely comprehensive assessment of key RAS components in

208 terms of their urine concentrations and enzymatic activities in carefully phenotyped patients with type 1

209 diabetes with and without DKD. Further, we observe an overall congruent pattern of change in the pathway

210 proteins and their activities between experimental and human DKD. Combined assessment of RAS

211 components in animal models and human DKD provides a more complete picture than either setting alone. For

212 example, while experimental DKD arguably presents an earlier disease stage with minimal albuminuria, the

213 human DKD phenotype studied here is a later stage with proteinuria and/or reduced GFR. The observation that

214 urine RAS components are increased in both models makes it less likely that these increases are merely the

215 consequence of co-filtration with albumin through a damaged glomerular sieve. Another example of the

216 strength of combined animal-human analysis is that the experimental DKD provides a glimpse of DKD

217 pathophysiology, which is not modified by treatment, and as such may help decipher the changes treatment

218 makes in pathophysiology of human DKD. An example of this is the observation that while both urine ACE

219 concentration and activity are increased in untreated animal DKD, urine ACE concentration is increased, while

220 its activity in reduced in people with DKD under ACEI treatment.

221 AOG is the substrate for the formation of angiotensin peptides and its overproduction particularly at the kidney

222 level may promote progression of kidney disease.(21, 28, 49) AOG is increased in kidneys of people with DKD

223 (23) and in urine samples from people with type 1 or type 2 diabetes and kidney disease. (1, 20, 22) Urine

224 AOG is strongly correlated with renal angiotensin II and RAS activity.(37, 67) As such, our observed increase

225 in urine AOG in both experimental and human DKD is consistent with increased renal RAS activity.

226 ACE catalyzes conversion of angiotensin I to angiotensin II. ACE expression is increased in renal glomeruli

227 (34, 51, 69) and reduced in tubules of most DKD animal models (51, 65, 68, 69), where ACE protein quantity

228 correlates tightly with its enzymatic activity.(65) In human DKD, however, ACE protein is increased in both

229 glomerular and tubular compartments in kidney biopsies (17, 30, 33, 41) and also increased in urine samples

230 from people with DKD (6, 15, 32). We found increased urine ACE activity in streptozotocin-treated mice and,

231 consistent with ACE overexpression in human kidney biopsies, we found higher urine ACE protein

232 concentration. In contrast, urinary ACE activity was lower in people with DKD than those without. This

233 reduction in ACE activity was associated with treatment with ACE inhibitors. ACE inhibitors are expected to

234 reduce renal ACE activity without influencing ACE protein expression.(47) Consistently, treatment with ACE

235 inhibitors was associated with reduced urine ACE activity, without any change in urine ACE concentration.

236 Furthermore, the ACE inhibitor-associated reduction in ACE activity was specific to ACE in that it did not affect

237 concentration or enzymatic activity of other RAS proteins, such as ACE2 or CTSD. This observation suggests

238 that urine ACE activity may inform on dynamic changes in the kidney ACE activity and as such may be useful

239 for monitoring compliance with, and possibly adequacy of therapeutic response to, ACE inhibitor treatment.

240 Another novel finding of this study is the increase in urine CTSD protein concentration observed in both mice

241 with streptozotocin-induced diabetes and people with DKD. Cathepsins are a family of lysosomal proteases,

242 several members of which (cathepsins D, G and A) can catalyze cleavage of AOG and angiotensin I (Figures

243 1-2), presenting a mechanism for ACE-independent angiotensin II generation.(31, 38, 40, 42, 54, 61) Several

244 pieces of evidence suggest that ACE-independent angiotensin II production occurs and is relevant to renal

245 pathophysiology. First, angiotensin II is only partially blocked with ACE inhibition.(10, 55) In addition, the renal

246 hemodynamic response to angiotensin receptor blockers (ARBs) exceeds the response to ACE inhibitors,

247 suggesting that non-ACE pathways contribute to intrarenal angiotensin II generation.(14) Furthermore, ARBs

248 augment the effect of ACE inhibitors on blood pressure and proteinuria reduction in experimental DKD

249 models,(7, 19, 60) as well as human DKD.(29, 44) There is evidence that cathepsins may contribute to ACE-

250 independent RAS activation in the kidneys. In porcine kidney extracts, cathepsin G-mediated angiotensin-II

251 production is comparable to that of ACE.(45) CTSD expression is induced in experimental CKD models and

252 inhibition of CTSD (but not other cathepsins) improves fibrosis in these models.(9, 11) CTSD is also expressed

253 in human kidneys in both glomerular and tubular compartments and its expression is increased in damaged

254 tubules in DKD.(9) Of interest is the recent finding that podocyte-specific CTSD deficiency leads to proteinuria

255 and ESRD in mice.(66) This, however, is proposed to be due to the non-redundant role of CTSD in

256 autophagy.(66) While the increase in urinary cathepsin may reflect renal processes unrelated to the RAS

257 pathway activity, the presence of large quantities of enzymes that can promote ACE-independent angiotensin

258 II generation suggests the possibility of an ACE-independent mechanism for RAS activation that is novel and

259 worthy of further investigation. If so, urine CTSD concentration may present a biomarker for potential RAS

260 resistance to ACE inhibition and signal the need for additional therapies targeting cathepsin activity.

261 ACE2 is thought to counteract RAS pathway activity by degrading angiotensin I and II to Ang(1-9) and Ang(1-

262 7), respectively, which have opposing biological functions to angiotensin II.(53) In DKD animal models, ACE2

263 protein and enzymatic activity are increased in kidney tubules (8, 25, 46, 63, 65, 68, 69) as well as urine.(6, 8,

264 43, 63) Pharmacologic inhibition (51, 69) or genetic deletion of ACE2 worsens experimental DKD,(62) while

265 ACE2 overexpression within podocytes (35) ameliorates DKD, suggesting that the compensatory ACE2

266 increase in DKD may act to counter RAS pathway activation by helping dispose of angiotensin II. We find an

267 increase in urine ACE2 activity in streptozotocin-treated mice and in people with DKD, consistent with a

268 compensatory increase in kidney ACE2 activity in response to RAS activation in DKD. However, unlike mice,

269 humans with DKD only show a trend towards higher urine ACE2 concentration, which does not attain

270 significance. It is unclear whether this reflects the differences in pathophysiology between animal and human

271 DKD or if it is the result of alterations in the human DKD pathophysiology by treatment.

272 APA, which degrades angiotensin II to angiotensin III, is the primary regulator of degradation for circulating

273 angiotensin II (Figure 1).(56) As such, APA and ACE2 present different mechanisms of countering RAS

274 pathway activation by reducing angiotensin II concentration. Unlike ACE2, APA is abundantly expressed both

275 in tubules and the glomerular compartments of animals with DKD.(52) Thus, urine APA could originate from

276 both glomerular and tubular sources. Here, we report for the first time a concordant increase in urine APA

277 activity in experimental and human DKD. The increase in enzymatic activities of ACE2 and APA in

278 experimental and human DKD can be viewed as compensatory in an effort to enhance angiotensin II

279 degradation and thus attenuate the accumulation of this peptide, which is driven by enhanced formation.

280 However, alternative explanations exist, including shedding of these enzymes, which are plasma membrane

281 proteins, in the urine as a result of kidney damage in DKD.(63)

282 The strengths of this study include a comprehensive analysis of the protein concentrations and enzymatic

283 activities for several RAS pathway components in urine samples from a well-characterized DKD animal model

284 and an accurately ascertained human case-control cohort with DKD, where findings were carefully adjusted for

285 potential confounders. The cross-sectional nature of the study precludes assessing association of the RAS

286 proteins with relevant future outcomes. Also, as DKD was not confirmed by a kidney biopsy, inclusion of non-

287 diabetic CKD among our cases is possible. Another limitation is absence of parallel protein quantification in

288 serum or kidney tissue. Finally, it is worth noting that while widely used and accepted, none of the animal

289 models of DKD, including the STZ-induced model, fully recapitulate the human disease.(4, 5)

290 In summary, in humans with type 1 diabetes and DKD and in the widely-used streptozotocin model of DKD,

291 urine concentrations and enzymatic activities of several RAS components are concordantly increased,

292 consistent with the increased intrarenal RAS activity in DKD. Furthermore, the ACE inhibitor-induced reduction

293 in urine ACE activity suggests its utility as a measure of suppressed intrarenal ACE activity. Lastly, increased

294 urine CTSD concentration in both mice and humans with DKD suggests cathepsins as both a potential

295 mechanism for, and a marker of, ACE-independent RAS pathway activation in DKD.

296 ACKNOWLEDGEMENTS

297 None

298 GRANTS

299 M.A. was supported by grants K23DK089017 and R01DK104706 from the National Institute of Diabetes,

300 Digestive, and Kidney Disease (NIDDK) and the Norman S. Coplon Extramural Grant from Satellite

301 Healthcare. DB was supported by NIDDK grants R01DK104785 and R01DK080089.

302 M.A. and D.B. contributed equally to this paper as senior authors.

303 DISCLOSURES

304 None.

REFERENCES

305 1. Afkarian M, Hirsch IB, Tuttle KR, Greenbaum C, Himmelfarb J, and de Boer IH. Urinary excretion of RAS, BMP, 306 and WNT pathway components in diabetic kidney disease. Physiological reports 2: e12010, 2014. 307 2. Afkarian M, Zelnick LR, Hall YN, Heagerty PJ, Tuttle K, Weiss NS, and de Boer IH. Clinical Manifestations of 308 Kidney Disease Among US Adults With Diabetes, 1988-2014. JAMA 316: 602-610, 2016. 309 3. Brenner BM, Cooper ME, de Zeeuw D, Keane WF, Mitch WE, Parving HH, Remuzzi G, Snapinn SM, Zhang Z, 310 Shahinfar S, and Investigators RS. Effects of losartan on renal and cardiovascular outcomes in patients with type 2 311 diabetes and nephropathy. N Engl J Med 345: 861-869, 2001. 312 4. Breyer MD, Bottinger E, Brosius FC, 3rd, Coffman TM, Harris RC, Heilig CW, Sharma K, and Amdcc. Mouse 313 models of diabetic nephropathy. Journal of the American Society of Nephrology : JASN 16: 27-45, 2005. 314 5. Brosius FC, 3rd, Alpers CE, Bottinger EP, Breyer MD, Coffman TM, Gurley SB, Harris RC, Kakoki M, Kretzler M, 315 Leiter EH, Levi M, McIndoe RA, Sharma K, Smithies O, Susztak K, Takahashi N, Takahashi T, and Animal Models of 316 Diabetic Complications C. Mouse models of diabetic nephropathy. Journal of the American Society of Nephrology : JASN 317 20: 2503-2512, 2009. 318 6. Burns KD, Lytvyn Y, Mahmud FH, Daneman D, Deda L, Dunger DB, Deanfield JE, Dalton RN, Elia Y, Har R, Van 319 JA, Bradley TJ, Slorach C, Hui W, Xiao F, Zimpelmann J, Mertens L, Moineddin R, Reich HN, Sochett EB, Scholey JW, and 320 Cherney DZ. The Relationship between Urinary Renin Angiotensin System Markers, Renal and Vascular Function in 321 Adolescents with Type 1 Diabetes. American journal of physiology Renal physiology ajprenal 00438 02016, 2016. 322 7. Cao Z, Bonnet F, Davis B, Allen TJ, and Cooper ME. Additive hypotensive and anti-albuminuric effects of 323 angiotensin-converting enzyme inhibition and angiotensin receptor antagonism in diabetic spontaneously hypertensive 324 rats. Clinical science 100: 591-599, 2001. 325 8. Chodavarapu H, Grobe N, Somineni HK, Salem ES, Madhu M, and Elased KM. Rosiglitazone treatment of type 2 326 diabetic db/db mice attenuates urinary albumin and angiotensin converting enzyme 2 excretion. PloS one 8: e62833, 327 2013. 328 9. Fox C, Cocchiaro P, Oakley F, Howarth R, Callaghan K, Leslie J, Luli S, Wood KM, Genovese F, Sheerin NS, and 329 Moles A. Inhibition of lysosomal protease cathepsin D reduces renal fibrosis in murine chronic kidney disease. Scientific 330 reports 6: 20101, 2016. 331 10. Fox J, Guan S, Hymel AA, and Navar LG. Dietary Na and ACE inhibition effects on renal tissue angiotensin I and II 332 and ACE activity in rats. The American journal of physiology 262: F902-909, 1992. 333 11. Graciano ML, Cavaglieri Rde C, Delle H, Dominguez WV, Casarini DE, Malheiros DM, and Noronha IL. Intrarenal 334 Renin-Angiotensin system is upregulated in experimental model of progressive renal disease induced by chronic 335 inhibition of nitric oxide synthesis. Journal of the American Society of Nephrology : JASN 15: 1805-1815, 2004. 336 12. Haber PK, Ye M, Wysocki J, Maier C, Haque SK, and Batlle D. Angiotensin-converting enzyme 2-independent 337 action of presumed angiotensin-converting enzyme 2 activators: studies in vivo, ex vivo, and in vitro. Hypertension 63: 338 774-782, 2014. 339 13. He W, Tan RJ, Li Y, Wang D, Nie J, Hou FF, and Liu Y. Matrix metalloproteinase-7 as a surrogate marker predicts 340 renal Wnt/beta-catenin activity in CKD. Journal of the American Society of Nephrology : JASN 23: 294-304, 2012. 341 14. Hollenberg NK, Osei SY, Lansang MC, Price DA, and Fisher ND. Salt intake and non-ACE pathways for intrarenal 342 angiotensin II generation in man. Journal of the renin-angiotensin-aldosterone system : JRAAS 2: 14-18, 2001. 343 15. Hosojima H, Miyauchi E, and Morimoto S. Urinary excretion of angiotensin-converting enzyme in NIDDM 344 patients with nephropathy. Diabetes care 12: 580-582, 1989. 345 16. Hsu CY, Ballard S, Batlle D, Bonventre JV, Bottinger EP, Feldman HI, Klein JB, Coresh J, Eckfeldt JH, Inker LA, 346 Kimmel PL, Kusek JW, Liu KD, Mauer M, Mifflin TE, Molitch ME, Nelsestuen GL, Rebholz CM, Rovin BH, Sabbisetti VS, 347 Van Eyk JE, Vasan RS, Waikar SS, Whitehead KM, Nelson RG, and Consortium CKDB. Cross-Disciplinary Biomarkers 348 Research: Lessons Learned by the CKD Biomarkers Consortium. Clin J Am Soc Nephrol 10: 894-902, 2015. 349 17. Huang XR, Chen WY, Truong LD, and Lan HY. Chymase is upregulated in diabetic nephropathy: implications for 350 an alternative pathway of angiotensin II-mediated diabetic renal and vascular disease. Journal of the American Society of 351 Nephrology : JASN 14: 1738-1747, 2003. 352 18. Ju W, Nair V, Smith S, Zhu L, Shedden K, Song PX, Mariani LH, Eichinger FH, Berthier CC, Randolph A, Lai JY, 353 Zhou Y, Hawkins JJ, Bitzer M, Sampson MG, Thier M, Solier C, Duran-Pacheco GC, Duchateau-Nguyen G, Essioux L,

354 Schott B, Formentini I, Magnone MC, Bobadilla M, Cohen CD, Bagnasco SM, Barisoni L, Lv J, Zhang H, Wang HY, 355 Brosius FC, Gadegbeku CA, Kretzler M, Ercb CPN, and Consortium PK-I. Tissue transcriptome-driven identification of 356 epidermal growth factor as a chronic kidney disease biomarker. Sci Transl Med 7: 316ra193, 2015. 357 19. Kalender B, Ozturk M, Tuncdemir M, Uysal O, Dagistanli FK, Yegenaga I, and Erek E. Renoprotective effects of 358 valsartan and enalapril in STZ-induced diabetes in rats. Acta histochemica 104: 123-130, 2002. 359 20. Kim SS, Song SH, Kim IJ, Yang JY, Lee JG, Kwak IS, and Kim YK. Clinical implication of urinary tubular markers in 360 the early stage of nephropathy with type 2 diabetic patients. Diabetes Res Clin Pract 97: 251-257, 2012. 361 21. Kobori H, Harrison-Bernard LM, and Navar LG. Urinary excretion of angiotensinogen reflects intrarenal 362 angiotensinogen production. Kidney international 61: 579-585, 2002. 363 22. Kobori H, Ohashi N, Katsurada A, Miyata K, Satou R, Saito T, and Yamamoto T. Urinary angiotensinogen as a 364 potential biomarker of severity of chronic kidney diseases. J Am Soc Hypertens 2: 349-354, 2008. 365 23. Lai KN, Leung JC, Lai KB, To WY, Yeung VT, and Lai FM. Gene expression of the renin-angiotensin system in 366 human kidney. Journal of hypertension 16: 91-102, 1998. 367 24. Lavrentyev EN, Estes AM, and Malik KU. Mechanism of high glucose induced angiotensin II production in rat 368 vascular smooth muscle cells. Circulation research 101: 455-464, 2007. 369 25. Leehey DJ, Singh AK, Bast JP, Sethupathi P, and Singh R. Glomerular renin angiotensin system in streptozotocin 370 diabetic and Zucker diabetic fatty rats. Translational research : the journal of laboratory and clinical medicine 151: 208- 371 216, 2008. 372 26. Levey AS, Stevens LA, Schmid CH, Zhang YL, Castro AF, 3rd, Feldman HI, Kusek JW, Eggers P, Van Lente F, 373 Greene T, Coresh J, and Ckd EPI. A new equation to estimate glomerular filtration rate. Ann Intern Med 150: 604-612, 374 2009. 375 27. Lewis EJ, Hunsicker LG, Clarke WR, Berl T, Pohl MA, Lewis JB, Ritz E, Atkins RC, Rohde R, Raz I, and 376 Collaborative Study G. Renoprotective effect of the angiotensin-receptor antagonist irbesartan in patients with 377 nephropathy due to type 2 diabetes. N Engl J Med 345: 851-860, 2001. 378 28. Liu F, Brezniceanu ML, Wei CC, Chenier I, Sachetelli S, Zhang SL, Filep JG, Ingelfinger JR, and Chan JS. 379 Overexpression of angiotensinogen increases tubular apoptosis in diabetes. Journal of the American Society of 380 Nephrology : JASN 19: 269-280, 2008. 381 29. Mann JF, Schmieder RE, McQueen M, Dyal L, Schumacher H, Pogue J, Wang X, Maggioni A, Budaj A, 382 Chaithiraphan S, Dickstein K, Keltai M, Metsarinne K, Oto A, Parkhomenko A, Piegas LS, Svendsen TL, Teo KK, Yusuf S, 383 and investigators O. Renal outcomes with telmisartan, ramipril, or both, in people at high vascular risk (the ONTARGET 384 study): a multicentre, randomised, double-blind, controlled trial. Lancet 372: 547-553, 2008. 385 30. Mezzano S, Droguett A, Burgos ME, Ardiles LG, Flores CA, Aros CA, Caorsi I, Vio CP, Ruiz-Ortega M, and Egido J. 386 Renin-angiotensin system activation and interstitial inflammation in human diabetic nephropathy. Kidney international 387 Supplement S64-70, 2003. 388 31. Miller JJ, Changaris DG, and Levy RS. Conversion of angiotensin I to angiotensin II by cathepsin A isoenzymes of 389 porcine kidney. Biochemical and biophysical research communications 154: 1122-1129, 1988. 390 32. Miyauchi E, Hosojima H, and Morimoto S. Urinary angiotensin-converting enzyme activity in type 2 diabetes 391 mellitus: its relationship to diabetic nephropathy. Acta diabetologica 32: 193-197, 1995. 392 33. Mizuiri S, Hemmi H, Arita M, Ohashi Y, Tanaka Y, Miyagi M, Sakai K, Ishikawa Y, Shibuya K, Hase H, and 393 Aikawa A. Expression of ACE and ACE2 in individuals with diabetic kidney disease and healthy controls. American journal 394 of kidney diseases : the official journal of the National Kidney Foundation 51: 613-623, 2008. 395 34. Moon JY, Jeong KH, Lee SH, Lee TW, Ihm CG, and Lim SJ. Renal ACE and ACE2 expression in early diabetic rats. 396 Nephron Experimental nephrology 110: e8-e16, 2008. 397 35. Nadarajah R, Milagres R, Dilauro M, Gutsol A, Xiao F, Zimpelmann J, Kennedy C, Wysocki J, Batlle D, and Burns 398 KD. Podocyte-specific overexpression of human angiotensin-converting enzyme 2 attenuates diabetic nephropathy in 399 mice. Kidney international 82: 292-303, 2012. 400 36. Ninomiya T, Perkovic V, de Galan BE, Zoungas S, Pillai A, Jardine M, Patel A, Cass A, Neal B, Poulter N, 401 Mogensen CE, Cooper M, Marre M, Williams B, Hamet P, Mancia G, Woodward M, Macmahon S, Chalmers J, and 402 Group AC. Albuminuria and kidney function independently predict cardiovascular and renal outcomes in diabetes. 403 Journal of the American Society of Nephrology : JASN 20: 1813-1821, 2009.

404 37. Nishiyama A, Konishi Y, Ohashi N, Morikawa T, Urushihara M, Maeda I, Hamada M, Kishida M, Hitomi H, 405 Shirahashi N, Kobori H, and Imanishi M. Urinary angiotensinogen reflects the activity of intrarenal renin-angiotensin 406 system in patients with IgA nephropathy. Nephrol Dial Transplant 26: 170-177, 2011. 407 38. Owen CA, and Campbell EJ. Angiotensin II generation at the cell surface of activated neutrophils: novel 408 cathepsin G-mediated catalytic activity that is resistant to inhibition. Journal of immunology 160: 1436-1443, 1998. 409 39. Parving HH, Lehnert H, Brochner-Mortensen J, Gomis R, Andersen S, Arner P, Irbesartan in Patients with Type 410 D, and Microalbuminuria Study G. The effect of irbesartan on the development of diabetic nephropathy in patients with 411 type 2 diabetes. N Engl J Med 345: 870-878, 2001. 412 40. Ramaha A, and Patston PA. Release and degradation of angiotensin I and angiotensin II from angiotensinogen 413 by neutrophil serine proteinases. Archives of biochemistry and biophysics 397: 77-83, 2002. 414 41. Reich HN, Oudit GY, Penninger JM, Scholey JW, and Herzenberg AM. Decreased glomerular and tubular 415 expression of ACE2 in patients with type 2 diabetes and kidney disease. Kidney international 74: 1610-1616, 2008. 416 42. Reilly CF, Tewksbury DA, Schechter NM, and Travis J. Rapid conversion of angiotensin I to angiotensin II by 417 neutrophil and mast cell proteinases. The Journal of biological chemistry 257: 8619-8622, 1982. 418 43. Riera M, Marquez E, Clotet S, Gimeno J, Roca-Ho H, Lloreta J, Juanpere N, Batlle D, Pascual J, and Soler MJ. 419 Effect of insulin on ACE2 activity and kidney function in the non-obese diabetic mouse. PloS one 9: e84683, 2014. 420 44. Rossing K, Jacobsen P, Pietraszek L, and Parving HH. Renoprotective effects of adding angiotensin II receptor 421 blocker to maximal recommended doses of ACE inhibitor in diabetic nephropathy: a randomized double-blind crossover 422 trial. Diabetes care 26: 2268-2274, 2003. 423 45. Rykl J, Thiemann J, Kurzawski S, Pohl T, Gobom J, Zidek W, and Schluter H. Renal cathepsin G and angiotensin II 424 generation. Journal of hypertension 24: 1797-1807, 2006. 425 46. Salem ES, Grobe N, and Elased KM. Insulin treatment attenuates renal ADAM17 and ACE2 shedding in diabetic 426 Akita mice. American journal of physiology Renal physiology 306: F629-639, 2014. 427 47. Schlueter W, Keilani T, and Batlle DC. Tissue renin angiotensin systems: theoretical implications for the 428 development of hyperkalemia using angiotensin-converting enzyme inhibitors. Am J Med Sci 307 Suppl 1: S81-86, 1994. 429 48. Schwager SL, Carmona AK, and Sturrock ED. A high-throughput fluorimetric assay for angiotensin I-converting 430 enzyme. Nature protocols 1: 1961-1964, 2006. 431 49. Singh R, Singh AK, and Leehey DJ. A novel mechanism for angiotensin II formation in streptozotocin-diabetic rat 432 glomeruli. American journal of physiology Renal physiology 288: F1183-1190, 2005. 433 50. Soler MJ, Riera M, and Batlle D. New experimental models of diabetic nephropathy in mice models of type 2 434 diabetes: efforts to replicate human nephropathy. Exp Diabetes Res 2012: 616313, 2012. 435 51. Soler MJ, Wysocki J, Ye M, Lloveras J, Kanwar Y, and Batlle D. ACE2 inhibition worsens glomerular injury in 436 association with increased ACE expression in streptozotocin-induced diabetic mice. Kidney international 72: 614-623, 437 2007. 438 52. Song L, and Healy DP. Kidney aminopeptidase A and hypertension, part II: effects of angiotensin II. Hypertension 439 33: 746-752, 1999. 440 53. Tipnis SR, Hooper NM, Hyde R, Karran E, Christie G, and Turner AJ. A human homolog of angiotensin- 441 converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase. The Journal of 442 biological chemistry 275: 33238-33243, 2000. 443 54. Tonnesen MG, Klempner MS, Austen KF, and Wintroub BU. Identification of a human neutrophil angiotension 444 II-generating protease as cathepsin G. The Journal of clinical investigation 69: 25-30, 1982. 445 55. van Kats JP, Schalekamp MA, Verdouw PD, Duncker DJ, and Danser AH. Intrarenal angiotensin II: interstitial 446 and cellular levels and site of production. Kidney international 60: 2311-2317, 2001. 447 56. Velez JC. The importance of the intrarenal renin-angiotensin system. Nat Clin Pract Nephrol 5: 89-100, 2009. 448 57. Viau A, El Karoui K, Laouari D, Burtin M, Nguyen C, Mori K, Pillebout E, Berger T, Mak TW, Knebelmann B, 449 Friedlander G, Barasch J, and Terzi F. Lipocalin 2 is essential for chronic kidney disease progression in mice and humans. 450 The Journal of clinical investigation 120: 4065-4076, 2010. 451 58. Volland H, Pradelles P, Ronco P, Azizi M, Simon D, Creminon C, and Grassi J. A solid-phase immobilized epitope 452 immunoassay (SPIE-IA) permitting very sensitive and specific measurement of angiotensin II in plasma. J Immunol 453 Methods 228: 37-47, 1999.

454 59. Wada T, Furuichi K, Sakai N, Iwata Y, Yoshimoto K, Shimizu M, Takeda SI, Takasawa K, Yoshimura M, Kida H, 455 Kobayashi KI, Mukaida N, Naito T, Matsushima K, and Yokoyama H. Up-regulation of monocyte chemoattractant 456 protein-1 in tubulointerstitial lesions of human diabetic nephropathy. Kidney international 58: 1492-1499, 2000. 457 60. Wilkinson-Berka JL, Gibbs NJ, Cooper ME, Skinner SL, and Kelly DJ. Renoprotective and anti-hypertensive 458 effects of combined valsartan and perindopril in progressive diabetic nephropathy in the transgenic (mRen-2)27 rat. 459 Nephrol Dial Transplant 16: 1343-1349, 2001. 460 61. Wintroub BU, Klickstein LB, Dzau VJ, and Watt KW. Granulocyte-angiotensin system. Identification of 461 angiotensinogen as the plasma protein substrate of leukocyte cathepsin G. Biochemistry 23: 227-232, 1984. 462 62. Wong DW, Oudit GY, Reich H, Kassiri Z, Zhou J, Liu QC, Backx PH, Penninger JM, Herzenberg AM, and Scholey 463 JW. Loss of angiotensin-converting enzyme-2 (Ace2) accelerates diabetic kidney injury. The American journal of 464 pathology 171: 438-451, 2007. 465 63. Wysocki J, Garcia-Halpin L, Ye M, Maier C, Sowers K, Burns KD, and Batlle D. Regulation of urinary ACE2 in 466 diabetic mice. American journal of physiology Renal physiology 305: F600-611, 2013. 467 64. Wysocki J, Ye M, Khattab AM, Fogo A, Martin A, David NV, Kanwar Y, Osborn M, and Batlle D. Angiotensin- 468 converting enzyme 2 amplification limited to the circulation does not protect mice from development of diabetic 469 nephropathy. Kidney international 2016. 470 65. Wysocki J, Ye M, Soler MJ, Gurley SB, Xiao HD, Bernstein KE, Coffman TM, Chen S, and Batlle D. ACE and ACE2 471 activity in diabetic mice. Diabetes 55: 2132-2139, 2006. 472 66. Yamamoto-Nonaka K, Koike M, Asanuma K, Takagi M, Oliva Trejo JA, Seki T, Hidaka T, Ichimura K, Sakai T, 473 Tada N, Ueno T, Uchiyama Y, and Tomino Y. Cathepsin D in Podocytes Is Important in the Pathogenesis of Proteinuria 474 and CKD. Journal of the American Society of Nephrology : JASN 27: 2685-2700, 2016. 475 67. Yamamoto T, Nakagawa T, Suzuki H, Ohashi N, Fukasawa H, Fujigaki Y, Kato A, Nakamura Y, Suzuki F, and 476 Hishida A. Urinary angiotensinogen as a marker of intrarenal angiotensin II activity associated with deterioration of renal 477 function in patients with chronic kidney disease. Journal of the American Society of Nephrology : JASN 18: 1558-1565, 478 2007. 479 68. Ye M, Wysocki J, Naaz P, Salabat MR, LaPointe MS, and Batlle D. Increased ACE 2 and decreased ACE protein in 480 renal tubules from diabetic mice: a renoprotective combination? Hypertension 43: 1120-1125, 2004. 481 69. Ye M, Wysocki J, William J, Soler MJ, Cokic I, and Batlle D. Glomerular localization and expression of 482 Angiotensin-converting enzyme 2 and Angiotensin-converting enzyme: implications for albuminuria in diabetes. Journal 483 of the American Society of Nephrology : JASN 17: 3067-3075, 2006.

Table 1. RAS pathway components in urine of streptozotocin-treated mice

N p-value Vehicle Streptozotocin Mann- Vehicle STZ Whitney Albumin/Cr (mg/g) 9 15 18.3 (10.8, 39.4) 71.6 (36.0, 104.1) 0.015 Angiotensinogen/Cr (μg/g) 9 15 2.6 (1.8, 5.5) 27.1 (17.5, 46.4) 0.0001 Cathepsin D (μg/g) 9 15 32.0 (24.4, 36.9) 96.5 (67.3, 122.4) <0.001 ACE activity/Cr (109 RFU/g) 9 15 7.1 (6.5, 9.2) 44.2 (39.5, 64.0) <0.0001 ACE2 activity/Cr (106 RFU/hr/g) 9 15 5.2 (4.7, 6.3) 16.5 (12.9, 21.5) <0.0001 APA activity/cr (106 RFU/hr/g) 9 15 2.5 (1.9, 5.6) 47.1 (26.3, 74.6) 0.0001 Angiotensin II/Cr (ng/g) 7 15 30.8 (15.3, 49.6) 74.1 (53.5, 103.9) 0.017 TValues are medians and interquartile ratio (IQR). STZ: Streptozotocin-treated mice; vehicle: control mice treated with vehicle; ACE: angiotensin converting enzyme; APA: aminopeptidase A.

Table 2. Characteristics of the study cohort

No DKD DKD p-value N 81 37 Age, years 53 (9) 49 (12) 0.077 Caucasian, N (%) 75 (93) 29 (78) 0.035 Female, N (%) 44 (54) 9 (24) 0.003 Diabetes duration, years 39 (6) 34 (12) 0.009

RAS inhibitor use, N (%) 46 (57) 32 (87) 0.002

Diabetic retinopathy, N (%) Yes 52 (65) 27 (73) <0.0001 No 22 (27) 0 (0) Unknown 6 (7) 10 (27) Hypertension, N (%) Yes 38 (47) 36 (97) <0.0001 No 42 (52) 0 (0) Unknown 1 (1) 1 (3) Hemoglobin A1c, % 7.5 (7.0, 8.1) 8.3 (7.3, 9.6) 0.003

Glomerular filtration rate 2 92 (82, 102) 39 (33, 53) <0.0001 (mL/min/1.73m ) Urine albumin/cr (mg/g) 7 (5, 12) 497 (122, 1260) <0.0001

Values are mean (standard deviation, SD), median (interquartile range, IQR) or number (%). Glomerular filtration rate was calculated from serum creatinine using the CKD-EPI formula. P-values were obtained using Fisher’s exact test.

Table 3. Association of creatinine-adjusted urine concentration or activity of each RAS pathway component with DKD in people with type 1 diabetes Number No No DKD DKD OR (95% CI) p-value DKD DKD Angiotensinogen/Cr (μg/g) 76 37 15 (8, 24) 170 (18, 597) 2.2 (1.6, 3.3) 9.6 x 10-10 Cathepsin D/Cr (μg/g) 73 37 31 (22, 46) 147 (85, 219) 6.5 (3.5, 14.6) 2.3 x 10-14 ACE/Cr (μg/g) 73 37 0.8 (0.4, 1.4) 1.4 (0.9, 2.5) 2.2 (1.4, 3.9) 3.8 x 10-4 ACE activity/Cr (109 RFU/g) 81 37 1.6 (1.0, 2.1) 1.0 (0.8, 1.2) 0.3 (0.1, 0.6) 4.6 x 10-4 ACE2/Cr (μg/g) 73 37 2.7 (1.7, 4.2) 4.1 (1.6, 5.7) 1.1 (0.8, 1.7) 0.54 ACE2 activity/Cr (106 RFU/hr/g) 81 37 0.3 (0.1, 0.9) 0.4 (0. 2, 0.9) 1.2 (1.0, 1.4) 0.03 APA activity/Cr (106 RFU/hr/g) 81 37 22 (4, 89) 40 (11, 210) 1.2 (1.0, 1.3) 0.05 People with no DKD had eGFR >90 mL/min/1.73m2 and ACR <300 mg/g after >30 years of type 1 diabetes. Those with DKD had either an ACR >300 mg/g or both eGFR <60 mL/min/1.73m2 and ACR >30 mg/g. Values are medians (interquartile ratio, IQR). Odds ratios were obtained from logistic regression models with case-control status as outcome and urine concentration or activity of a single RAS component as exposure. Models were adjusted for age, sex, race and diabetes duration. ACE: angiotensin converting enzyme; APA: aminopeptidase A.

Table 4. Association of urine concentration or activity of each RAS pathway component with DKD in people with type 1 diabetes

N No DKD DKD OR (95% CI) P-values No DKD DKD Angiotensinogen (ng/mL) 59 27 9 (5, 16) 155 (27, 538) 2.1 (1.6, 2.9) 2.0 x 10-9 CTSD (ng/mL) 73 37 18 (11, 35) 99 (63, 135) 3.5 (2.2, 6.4) 1.0 x 10-10 ACE (ng/mL) 73 37 0.5 (0.3, 0.7) 1.1 (0.7, 2.2) 1.9 (1.3, 3.0) 0.001 ACE activity (RFU/μL) 81 37 950 (637, 1446) 736 (585, 872) 0.4 (0.2, 0.7) 0.001 ACE2 (pg/mL) 73 37 1631 (723, 3394) 2810 (1196, 3637) 1.0 (0.8, 1.3) 0.93 ACE2 activity (RFU/hr/μL) 81 37 0.19 (0.02, 0.56) 0.21 (0.13, 0.50) 1.0 (0.8, 1.3) 0.83 APA activity (1000RFU/hr/ μL) 81 37 13 (2, 47) 24 (6, 116) 1.1 (1.0, 1.3) 0.09 Presence and absence of DKD was defined as in Table 3. Values are medians (interquartile ratio, IQR). Odds ratios were obtained from logistic regression models with case-control status as outcome and urine concentration or activity of a single RAS component as exposure. Models were adjusted for age, sex, race and diabetes duration. ACE: angiotensin converting enzyme; APA: aminopeptidase A.

Table 5. Association of urine concentration or activity of all RAS pathway components with DKD in people with type 1 diabetes 484 OR (95% CI) p-value Age, years 0.9 (0.8, 1.0) 0.16 Non-White 1.8 (0.2, 14.8) 0.59 Female 0.5 (0.1, 2.6) 0.41 Diabetes duration, years 1.0 (0.9, 1.0) 0.31 Angiotensinogen/Cr (μg/g) 1.5 (0.9, 2.8) 0.11 Cathepsin D/cr (μg/g) 6.5 (2.7, 22.4) 1.5 x 10-6 ACE activity/Cr (106 RFU/hr/gCr) 0.2 (0.1, 0.6) 0.003 ACE2 activity/Cr (1000 RFU/hr/gCr) 1.0 (0.8, 1.4) 0.98 APA activity/Cr (106 RFU/hr/gCr) 0.9 (0.7, 1.2) 0.36

Controls and cases were defined as in Table 3. Odds ratios were obtained from logistic regression models with case-control status as outcome and the six listed urine RAS component concentrations or activities as exposures. All urine protein concentrations or activities are represented as continuous variables, except urine renin which is binary (detected, not detected). Models were adjusted for age, gender, race and diabetes duration. ACE: angiotensin converting enzyme; APA: aminopeptidase A.

Figure legends

Figure 1. RAS pathway status in experimental DKD, based on urine concentration and enzymatic activity of RAS components.

Figure 2. RAS pathway status in human DKD, based on urine concentration and enzymatic activity of RAS components.

Figure 1. RAAS pathway status in experimental DKD, based on urine concentration and activity of RAAS components. Figure 2. RAAS pathway status in human DKD, based on urine concentration and activity of RAAS components.