Age at onset of type 1 diabetes between puberty and 30 years old is associated with increased diabetic nephropathy risk

Diabetic nephropathy is a critical complication of patients with type 1 diabetes, while epidemiological studies were scarce among Asian countries. We conducted a cross-sectional study to identify factors associated with diabetic nephropathy by questionnaires, using student’s t-test, chi-square test, and multivariable logistic regression. Among 898 participants, 16.7% had diabetic nephropathy. Compared with non-diabetic nephropathy patients, the patients with diabetic nephropathy had significantly higher percentage with onset age of type 1 diabetes between puberty and under 30 years old (female ≥ 12 or male ≥ 13 years old to 29 years old), longer diabetes duration, having family history of diabetes and diabetic nephropathy, accompanied with hypertension, hyperlipidemia, or coronary artery disease (CAD). Compared with patients with onset age before puberty, the odds of diabetic nephropathy occurrence increased to 1.61 times in patients with onset age between puberty and under 30 years old (p = 0.012) after adjusting diabetes duration. Age of diabetes onset between puberty and under 30 years old, diabetes duration, HbA1c, hospital admission within 3 years, diabetic retinopathy, hypertension, systolic blood pressure (SBP), triglyceride levels, and use of angiotensin converting enzyme inhibitor (ACEI) and/or angiotensin receptor blockers (ARB) were independent factors associated with diabetic nephropathy Screening for proteinuria is important in daily clinical practice and should be part of diabetes self-management education for patients with type 1 diabetes.

type 1 diabetes 9 .According to the Diabetes Control and Complications Trial (DCCT) cohort, incident reduced eGFR (eGFR < 60 mL/min/1.73m 2 ), increased mean HbA1c, increased mean triglyceride level, older age, and higher systolic blood pressure (SBP) were the most significant risk factors of diabetic nephropathy 10 .
Age at onset of diabetes has been reported to play an important role in classifying different phenotypes of type 1 DM, namely classical childhood-onset type, or latent adult-onset type (latent autoimmune diabetes in adults, LADA) 11 .A nationwide report in United States also demonstrated two peaks of incident type 1 DM, including during their puberty (aged 11-14 years) and early adulthood 12 .However, these two periods may not share the same pathogenesis in triggering diabetic nephropathy.Physiologically, the surge in growth hormone and sex steroids during puberty contributed to insulin resistance, which therefore accelerate type 1 DM onset 13,14 .One study found that patients with type 1 diabetes with onset age during puberty (age 10-14 years) would have higher risk of developing diabetic nephropathy than those with onset age during pre-puberty (aged 0-9 years) 15 .On the other hand, the renal outcome among LADA seemed to be controversial.One nationwide type 1 diabetes cohort revealed the prevalence of end stage kidney disease were higher in older onset group (> 40 years) than childhood-onset (< 20 years) one 16 .However, another recent cohort indicated type 1 DM diagnosed at childhood/ adolescent (age < 20 years) was independently associated with diabetic nephropathy, compared with older onset group (age 30 to 40 years 17 .Since patients diagnosed with LADA not necessarily required insulin injection in the initial period, it should be considered as a mixture of type 1 and type 2 diabetes 18 . Prevention and early detection of diabetic nephropathy is substantial to patients with type 1 diabetes.However, the incidence of type 1 DM in Western Pacific is relatively low, and studies on nephropathy in patients with type 1 diabetes in Asia are scarce.According to International Diabetes Federation (IDF) Atlas report (10th edition) 19 , the incidence of age standardized type 1 diabetes in Taiwan is less than 10.0 per 100,000 population per year, which is almost one-third of United states or most European countries.To study the prevalence and possible associated risk factors of diabetic nephropathy in patients with type 1 diabetes in Taiwan, we conducted a cross-sectional study by obtaining questionnaires from patients with type 1 diabetes who were registered in the Taiwan Diabetes Registry Study of Diabetes Association of the Republic of China (DAROC, Taiwan) from October 2015 to December 2018.

Participants of Taiwan diabetes registry study
The Taiwan Diabetes Registry is a cross-sectional observational study conducted by the Diabetes Association of the Republic of China (Taiwan) to assess the status of healthcare, risk factors, and diabetes-related complications in patients with type 1 and type 2 diabetes.A total of 45 sites (hospitals or primary care clinics) participated in this study and the enrollment of study subjects began in October 2015.The study protocol was approved by the Joint Institutional Review Board and the Institutional Review Board of each hospital (Taichung Veterans General Hospital and National Taiwan University Hospital as representative) participating in this study, (Full detail listed in the Appendix) This study was conducted in accordance with the Declaration of Helsinki and all the participants provided written informed consent in each study subject.In this cross-sectional study, we obtained questionnaires from 1086 patients with type 1 diabetes who were registered in the Taiwan Diabetes Registry Study between October 2015 and December 2018.Because LADA may be the mixture of both type 1 and type 2 diabetes, we excluded those with age of onset over or equal to 30 years old, and 898 participants were recruited finally (Fig. 1).In addition, based on the previous reviews, we divided our groups into pre-puberty and puberty to under 30 group by age at onset of type 1 diabetes.One recent study in Taiwan defined puberty stage as 12 years old in male and 11 years old in female 20 .Because the generation of our cohort is different from the above-mentioned study cohort, after adjustment of age shifting from the above-mentioned cohort (i.e., our cohort is about ten years older than the above-mentioned cohort), we set our puberty group as female ≥ 12 years old and male ≥ 13 years old.Type 1 diabetes was diagnosed in these patients using the Taiwan National Health Insurance Research Database (NHIRD), which has a coverage rate of over 99.9% of the population in Taiwan 21 .Patients diagnosed with type 1 diabetes receiving a certificate for catastrophic illness were recruited in this study.In this questionnaire, each participant's anthropometric, socioeconomic, and biochemical characteristics were collected by the medical staff.Each patient was requested to provide a urine dipstick test to investigate protein (for overt proteinuria) and/or urine albumin-creatinine ratio (UACR, for microalbuminuria) for diagnosis of diabetic nephropathy.The definition of diabetic nephropathy for this study was based on the clinical guidelines for diabetic management in chronic kidney disease (KDIGO 2020) 22 .According to these guidelines, diabetic nephropathy is mainly determined by estimated glomerular filtration rate (eGFR) < 60 (ml/min/1.73m) or persistent albuminuria (UACR ≥ 30 mg/g) 23 .For patients without data on the status of albuminuria, we used protein findings from the urine dipstick test, wherein trace and greater proteinuria (trace to +, ++, +++, and ++++) would fall into albuminuria categories A2 and A3, respectively (A2: microalbuminuria, 30-300 mg/g, dipstick trace to ++, A3: macroalbuminuria, > 300 mg/g, dipstick: +++ to ++++) 23 .According to this urine dipstick conversion model, the sensitivity and specificity for detecting a UACR of ≥ 30 mg/g was 62% and 87.7%, respectively 23 .

Statistical analyses
The student's t-test and chi-square test were used to compare the differences in various continuous and categorical variables between patients with and without diabetic nephropathy, respectively.Multivariate logistic regression was used to identify the diabetic nephropathy associated factors.A two-sided p < 0.05 level was considered statistically significant.All analyses were performed using Stata Statistical Software: Release 14 (StataCorp.2015.College Station, TX: StataCorp LP).

Conference presentation
Parts of this study were presented as oral presentation on March 28, 2021 at the 42nd Annual Meeting of the Endocrine Society and the Diabetes Association of the R.O.C (Taiwan) held in Taipei, Taiwan.Parts of this study were also presented as e-poster at the International Diabetes Federation (IDF) Virtual Congress 2021 held between 6 and 11th, December, 2021.

Associated factors for diabetic nephropathy in patients with type 1 diabetes
To further investigate specific factors associated with diabetic nephropathy in patients with type 1 diabetes, univariate logistic regression was individually applied to the identified statistically significant variables in Table 1.

Discussion
In this registry study, the prevalence of diabetic nephropathy in patients with type 1 diabetes in Taiwan was 16.7%, with a mean diabetes duration of 14.5 years.Longer duration, higher HbA1c, SBP, and plasma triglyceride level, diabetic retinopathy, hospitalization in the past 3 years were independently associated with the presence of diabetic nephropathy in patients with type 1 diabetes.Age at onset of type 1 diabetes between puberty and under 30 years old was also independently associated with higher presence rate of diabetic nephropathy after adjusting diabetes duration.To the best of our knowledge, this is the first Asian-Pacific multicenter diabetes registration study that demonstrated type 1 diabetes diagnosed between puberty and under 30 years old is associated with higher prevalence of diabetic nephropathy.www.nature.com/scientificreports/Compared with our cohort, one prospective study from the UK indicated that the cumulative incidence of microalbuminuria was 25.7% and 50.7% after 10 and 19 years of diabetes, respectively 24 .This difference in incidence may be attributed to both genetic susceptibility and environmental triggers 25,26 .In the recent Genome-Wide Association Study 27 , Caucasian and Chinese populations have been shown to share different type 1 diabetes risk loci, and several environmental factors were identified to affect the incidence of type 1 DM 28 .Another important factor affected diabetic nephropathy risk was the onset age of type 1 diabetes.Patients with type 1 diabetes diagnosed between puberty and under 30 years old had higher odds compared with pre-puberty onset group (OR 95% CI 1.61, 1.11-2.34;p = 0.012) in this study.In our following stepwise multivariate logistic regression model for diabetic nephropathy, patients who diagnosed type 1 diabetes between puberty and under 30 years old also have higher odds for diabetic nephropathy (OR 95% CI 1.56, 1.05-2.34,p = 0.029).Puberty www.nature.com/scientificreports/has been considered as an important but controversial independent risk factor of type 1 diabetic nephropathy.
Several studies demonstrated that albuminuria and variation in renal function during puberty changed more rapidly than those in pre-puberty group 29,30 .Although previous studies showed that patients with type 1 diabetes with onset age during puberty would have higher risk of developing diabetic nephropathy than those with onset age during pre-puberty 15,31,32 , these studies only included patients with type 1 diabetes diagnosed during childhood and adolescence, but the patients with type 1 diabetes between 18 and under 30 years old were not investigated.Recent cohort in Finland showed most type 1 diabetes diagnosed younger than 5 years had lower residual serum C-peptide level and progressed faster to absolute insulin deficiency, and was associated with higher HbA1c, higher percentage with nephropathy, retinopathy, and hypertension than older onset groups 33 .However, in Finland diabetes registry, patients with type 1 diabetes diagnosed between 10 and 14 years old group have the highest relative risk of ESRD (compared to age of onset < 5 years old), suggesting pubertal onset of type 1 diabetes may have an accelerating role in kidney damage 34 .In contrast, a Swedish diabetes registry showed that the risk of ESRD seemed different between gender.The age at onset 20-34 years conferred the highest risk of developing diabetic nephropathy in male, but the highest risk of onset age in female was 10-19 years 35 .Another type 1 diabetes cohort in Korea demonstrated patients with type 1 diabetes diagnosed at childhood/adolescent (age < 20 years) had higher prevalence of diabetes nephropathy than in older onset groups, and the degree of eGFR decrease was more prominent in the childhood/adolescent-onset group than in the older onset group 17 .In contrast, a population-based retrospective cohort study of type 1 diabetes in Hong Kong showed that people with type 1 diabetes diagnosed aged older than 40 years had a higher hazards of end stage renal disease (ESRD) versus those diagnosed aged less than 20 years 16 .The above studies on type 1 diabetes cohort showed different onset age group of type 1 diabetes had different prevalence of diabetic nephropathy.Some reported patients diagnosed type 1 diabetes at younger age had higher prevalence of diabetic nephropathy 17,33 , and one showed type 1 diabetes diagnosed older than 40 years had a higher risk to develop ESRD 16 .This discrepancy among the previous studies and our study may be due to the different genetic backgrounds of ethnic difference, the cohort including LADA with the mixture of type 1 and type 2 diabetes, and the grouping of onset age not according to the puberty.It needs further investigation to elucidate the impact of puberty on the development of diabetic nephropathy and decline rate of eGFR in patients with type 1 diabetes.In our study, we found that the onset of type 1 diabetes between puberty and 30 years old was associated with a higher prevalence of diabetic nephropathy, even after adjusting for disease duration, which supported the hypothesis that post-puberty related hormone disturbance would induce insulin resistance, resulting more contribution in diabetic complications than pre-puberty ones.
From the treatment point of view, more stringent glycemic and blood pressure control, and early detection of microalbuminuria or deterioration of eGFR are especially important in patients with type 1 diabetes diagnosed between puberty and under 30 years old.
Regarding diabetic nephropathy risk factors, our findings were consistent with Diabetes Control and Complications Trial (DCCT) cohort, which increased mean HbA1c, SBP, increased mean triglyceride level and longer diabetes duration were associated with presenting microalbuminuria 10 .According to Taiwan's NHI medical claims (1995-2017), in type 1 diabetes patients with a family history of type 2 diabetes, the adjusted hazard ratio for nephropathy was also higher (HR 95% CI 1.44, 1.27-1.64)compared with those without a family history of type 2 DM 36 .Similarly, type 1 DM patients in this study who had a family history of diabetes had higher odds of having diabetic nephropathy (OR 95% CI 1.91, 1.34-2.72;p < 0.001).In this study, a higher percentage of diabetic nephropathy patients had coexisting CAD and heart failure, which may have contributed to a higher percentage of hospitalization in the past 3 years in the diabetic nephropathy group than in the non-diabetic nephropathy group.Diabetic retinopathy, especially in the proliferative retinopathy group, was also more prevalent in type 1 diabetes patients with diabetic nephropathy in our study.According to the literature, the presence of a preexisting microvascular complication (retinopathy or nephropathy) may contribute to the development of another complication, especially in patients with type 1 diabetes 37 .In the Steno study, which observed 220 patients with type 1 diabetes with and without nephropathy in a 15 year-follow-up study, those developed gross proteinuria were found to have a higher risk of progression to proliferative retinopathy (12% annually) as compared with those without nephropathy (1-2% annually) 38 .In the EURODIAB Complications Study, retinopathy at baseline in type 1 DM patients was positively associated with new-onset microalbuminuria (53.2%) after 15 years, and it was more frequent among proliferative retinopathy group (67.2%) 39 .Chronic hyperglycemia, which is measured by mean blood glucose or HbA1c levels, has been linked to the development and progression of microvascular complications 40 .In this study, we also found that a higher HbA1c level was positively associated with the presence of diabetic nephropathy.In this study, patients with type 1 DM who were taking ACEI or ARB had higher odds for the diagnosis of diabetic nephropathy.Because this registration cohort was a cross-sectional study, only association could be found, but no causal relationship could be determined.This finding may be due to the higher SBP and DBP found in patients with diabetic nephropathy, which led to more patients with diabetic nephropathy receiving ACEI/ARB compared to non-diabetic nephropathy.ACEI and ARB was preferred as first-line therapy to patient with UACR ≥ 30 mg/g to slow down the progression 41 .It is well documented that higher triglyceride and cholesterol levels are associated with a more rapid decline in kidney function 42 .Dyslipidemia may directly affect the kidney by disturbing the renal lipid homeostasis.Further, it indirectly affects the kidneys through systemic inflammation, oxidative stress, and vascular injury 43,44 .A population-based prospective cohort study in Hong Kong showed smoking, increased HbA1c level, hypertension, albuminuria, and dyslipidemia were associated with greater risk of ESRD in patients with youth-onset type 1 diabetes (age at onset of type 1 DM < 20 years old) 45 .The identified risk factors associated with ESRD are similar with the risk factors associated with diabetic nephropathy in patients with type 1 diabetes in our study.However, our study emphasized on the onset age of type 1 diabetes after puberty, instead of only diabetes duration, is associated with higher prevalence of diabetic nephropathy, which was not emphasized in the above study 45  www.nature.com/scientificreports/Some of the strengths of this study are as follows: first, this was the first nationwide registration cohort of type 1 diabetes which emphasized on the impact of onset age of type 1 diabetes on diabetic nephropathy, especially between puberty to under 30 years old.As we excluded LADA (age of onset ≥ 30 years old) group in our statical analysis, the interference of diabetic nephropathy risk factors (more related to type 2 diabetes) was mitigated.Second, detailed information about anthropometric characteristics, family history, comorbidity, diabetes-related complications, medications, and laboratory data were collected, which greatly decreased the bias of making inappropriate inference.
However, our study also had some limitations.First, since this is a cross-sectional study, the efficacy of causal association may be weaker than that of longitudinal cohort studies.Since we lacked exposure period of drugs and baseline kidney function before recruitment, it is difficult to answer the use of ACEi/ARB was before or after diabetic nephropathy noticed, as well as excluding any systemic factors such as systemic lupus erythematosus which may also lead to proteinuria.However, since type 1 diabetes is a relatively young onset disease, we could only assume that the proteinuria was mostly associated with diabetes.Second, 274 (30.5%) of the total patients lacked urine albumin-to-creatinine ratio (UACR), which may strongly influence our diabetic nephropathy classification.Therefore, we applied a conversion equation from urine dipstick protein to UACR for the missing data, which was validated in a meta-analysis 23 .Third, the mean age of menarche could be estimated with a shift of 0.43 years per decade in previous Taiwan study 20 .However, studies referred to puberty onset shift among male are scarce, which the exact cutoff was difficult to be confirmed.Lastly, the total number of participants in the study was approximately 900, which may not be sufficient for extrapolation of our findings to other type 1 diabetes populations with a higher incidence of type 1 diabetes as well as to populations of other ethnicities.Additional limitation of questionnaire including not specifying type 1 or type 2 diabetes in diabetes family history and hospitalization reason were also noted.
In conclusion, we identified several modifiable risk factors of diabetic nephropathy in patients with type 1 diabetes from the nationwide registration of the type 1 diabetes cohort in Taiwan.These included poor glycemic control, high SBP, and high serum triglyceride levels.In addition, we identified that patients with type 1 diabetes diagnosed between puberty and under 30 years old was associated with higher prevalence of diabetic nephropathy in patients with type 1 diabetes.Finally, we found a lower rate of ACEI or ARB use in type 1 DM patients with diabetic nephropathy, which could be a room for improvement to our daily practice.
Since ACEI or ARB prescription is recommended in patient with diabetic nephropathy in both American Diabetes Association (ADA) 41 and Diabetes Association of the Republic of China (DAROC) type 1 diabetes guideline 46 , our government has implemented the policy for screening all diabetic patients at least annually for UACR, and strongly suggest further semi-annually follow-up if the result was abnormal.As there were only 20% of prescription rate of ACEI or ARB and short consultation time for doctors spending on patients in Taiwan (less than 5 min) 47 , enhancing the awareness of the clinicians to regular check proteinuria and a pop-up reminder implemented in electronic medical record (EMR) system would be an applicable solution in raising ACEI/ ARB prescription rate.All the above findings are informative and will be helpful to emphasize the screening for proteinuria in daily clinical practice and diabetes self-management education to improve the quality of care for patients with type 1 diabetes.

Figure 1 .
Figure 1.Flowchart of recruitment of patients with type 1 diabetes (onset age < 30 y/o) in Taiwan Diabetes Registration.

Table 1 .
Anthropometric, socioeconomic, and biochemical characteristics of diabetic nephropathy and nondiabetic nephropathy group in type 1 DM patients in Taiwan Diabetes Registry.GFR Glomerular filtration rate, DM diabetes mellitus, FH Family history, DM diabetes mellitus, CKD Chronic kidney disease, CVD cardiovascular disease, DKA diabetic ketoacidosis, CAD coronary artery disease, HF heart failure, ACEI Angiotensin Converting Enzyme inhibitors, ARB angiotensin receptor blockers, BMI Body mass index, SBP systolic blood pressure, DBP Diastolic blood pressure, HbA1c Hemoglobulin A1c, eGFR estimated Glomerular filtration rate, LDL Low-density lipoprotein, ALT alanine transaminase.

Table 2 .
Association between specific factors and the presence of diabetic nephropathy in patients with type 1 diabetes.DM diabetes mellitus, FH Family history, DM diabetes mellitus, CKD chronic kidney disease, CAD coronary artery disease, ACEI Angiotensin Converting Enzyme inhibitors, ARB angiotensin receptor blockers, SBP systolic blood pressure, DBP Diastolic blood pressure, HbA1c Hemoglobulin A1c.

Table 3 .
Independent factors associated with the presence of diabetic nephropathy in patients with type 1 diabetes using stepwise multivariate logistic regression.HbA1c Hemoglobulin A1c, SBP systolic blood pressure, ACEI Angiotensin Converting Enzyme inhibitors, ARB angiotensin receptor blockers.